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  1. Chen, John, Novick, Richard P. "Phage-mediated intergeneric transfer of toxin genes," Science 2009 Jan 2;323(5910):139-41.   (MEDL:19119236 PMID: 19119236 #J0159421)    

    Because bacteriophages generally parasitize only closely related bacteria, it is assumed that phage-mediated genetic exchange occurs primarily within species. Here we report that staphylococcal pathogenenicity islands, containing superantigen genes, and other mobile elements transferred to Listeria monocytogenes at the same high frequencies as they transfer within Staphylococcus aureus. Several staphylococcal phages transduced L. monocytogenes but could not form plaques. In an experiment modeling phage therapy for bovine mastitis, we observed pathogenicity island transfer between S. aureus and L. monocytogenes in raw milk. Thus, phages may participate in a far more expansive network of genetic information exchange among bacteria of different species than originally thought, with important implications for the evolution of human pathogens.

  2. Cisar, EA, Geisinger, E, Muir, TW, Novick, RP. "Symmetric signalling within asymmetric dimers of the Staphylococcus aureus receptor histidine kinase AgrC," Molecular microbiology 2009 OCT;74(1):44-57.   (ISI:000270129300004 #J0177358)    

    P>Virulence in Staphylococcus aureus is largely under control of the accessory gene regulator (agr) quorum-sensing system. The AgrC receptor histidine kinase detects its autoinducing peptide (AIP) ligand and generates an intracellular signal resulting in secretion of virulence factors. Although agr is a well-studied quorum-sensing system, little is known about the mechanism of AgrC activation. By co-immunoprecipitation analysis and intermolecular complementation of receptor mutants, we showed that AgrC forms ligand-independent dimers that undergo trans-autophosphorylation upon interaction with AIP. Remarkably, addition of specific AIPs to AgrC mutant dimers with only one functional sensor domain caused symmetric activation of either kinase domain despite the sensor asymmetry. Furthermore, mutant dimers involving one constitutive protomer demonstrated ligand-independent activity, irrespective of which protomer was kinase deficient. These results demonstrate that signalling through either individual AgrC protomer causes symmetric activation of both kinase domains. We suggest that such signalling across the dimer interface may be an important mechanism for dimeric quorum-sensing receptors to rapidly elicit a response upon signal detection.

  3. Geisinger, Edward, Muir, Tom W, Novick, Richard P. "agr receptor mutants reveal distinct modes of inhibition by staphylococcal autoinducing peptides," Proceedings of the National Academy of Sciences of the United States of America 2009 Jan 27;106(4):1216-21.   (MEDL:19147840 PMID: 19147840 #J0158591)    

    Through the agr quorum-sensing system, staphylococci secrete unique autoinducing peptides (AIPs) and detect their concentration via the AgrC transmembrane receptor, coordinating local bacterial population density with global changes in gene expression. Unique AIP and AgrC variants exist within and between species, and although autologous interactions lead to agr activation, heterologous interactions usually lead to cross-inhibition, resulting in natural quorum-sensing interference. To gain insight into the mechanisms responsible for these phenomena at the level of the receptor, we used random mutagenesis to isolate variants of Staphylococcus aureus AgrC-I with constitutive activity. Constitutive mutations in the sensor domain of the receptor were localized to the last transmembrane helix, whereas those in the histidine kinase domain were mostly clustered to a region near the phosphorylation site histidine. Analysis of these mutants with a range of noncognate AIPs revealed that inhibition is manifested by inverse agonism in certain heterologous pairings and by neutral antagonism in others. In addition, we isolated and characterized an AgrC sensor domain mutant with dramatically broadened activation specificity and reduced sensitivity to inhibition, identifying a single amino acid as a critical determinant of ligand-mediated inhibition. These results suggest that certain noncognate AIPs stabilize an inhibitory receptor conformation that may be a critical feature of the ligand-receptor interaction not initially appreciated in previous analyses of agr inhibition.

  4. Selva L., Viana D., Regev-Yochay G., Trzcinski K., Corpa J.M., Lasa I., Novick R.P., Penades J.R.. "Killing niche competitors by remote-control bacteriophage induction," Proceedings of the National Academy of Sciences of the United States of America 2009;106(4):1234-1238.   (EMBASE:2009052053 #J0158710)    

    A surprising example of interspecies competition is the production by certain bacteria of hydrogen peroxide at concentrations that are lethal for others. A case in point is the displacement of Staphylococcus aureus by Streptococcus pneumoniae in the nasopharynx, which is of considerable clinical significance. How it is accomplished, however, has been a great mystery, because H2O2 is a very well known disinfectant whose lethality is largely due to the production of hyperoxides through the abiological Fenton reaction. In this report, we have solved the mystery by showing that H 2O2 at the concentrations typically produced by pneumococci kills lysogenic but not nonlysogenic staphylococci by inducing the SOS response. The SOS response, a stress response to DNA damage, not only invokes DNA repair mechanisms but also induces resident prophages, and the resulting lysis is responsible for H2O2 lethality. Because the vast majority of S. aureus strains are lysogenic, the production of H 2O2 is a very widely effective antistaphylococcal strategy. Pneumococci, however, which are also commonly lysogenic and undergo SOS induction in response to DNA-damaging agents such as mitomycin C, are not SOS-induced on exposure to H2O2. This is apparently because they are resistant to the DNA-damaging effects of the Fenton reaction. The production of an SOS-inducing signal to activate prophages in neighboring organisms is thus a rather unique competitive strategy, which we suggest may be in widespread use for bacterial interference. However, this strategy has as a by-product the release of active phage, which can potentially spread mobile genetic elements carrying virulence genes. copyright 2009 by The National Academy of Sciences of the USA.

  5. Ubeda, Carles, Olivarez, Nicholas P, Barry, Peter, Wang, Huaibin, Kong, Xiangpeng, Matthews, Avery, Tallent, Sandra M, Christie, Gail E, Novick, Richard P. "Specificity of staphylococcal phage and SaPI DNA packaging as revealed by integrase and terminase mutations," Molecular microbiology 2009 Apr;72(1):98-108.   (MEDL:19347993 PMID: 19347993 #J0165195)    

    SaPI1 and SaPIbov1 are chromosomal pathogenicity islands in Staphylococcus aureus that carry tst and other superantigen genes. They are induced to excise and replicate by certain phages, are efficiently encapsidated in SaPI-specific small particles composed of phage virion proteins and are transferred at very high frequencies. In this study, we have analysed three SaPI genes that are important for the phage-SaPI interaction, int (integrase) terS (phage terminase small subunit homologue) and pif (phage interference function). SaPI1 int is required for SaPI excision, replication and packaging in a donor strain, and is required for integration in a recipient. A SaPI1 int mutant, following phage induction, produces small SaPI-specific capsids which are filled with partial phage genomes. SaPIbov1 DNA is efficiently packaged into full-sized phage heads as well as into SaPI-specific small ones, whereas SaPI1 DNA is found almost exclusively in the small capsids. TerS, however, determines DNA packaging specificity but not the choice of large versus small capsids. This choice is influenced by SaPIbov1 gene 12, which prevents phage DNA packaging into small capsids, and which is also primarily responsible for interference by SaPIbov1 with phage reproduction.

  6. Adhikari, Rajan P, Novick, Richard P. "Regulatory organization of the staphylococcal sae locus," Microbiology 2008 Mar;154(Pt 3):949-59.   (MEDL:18310041 PMID: 18310041 #J0137550)    

    This paper describes an investigation of the complex internal regulatory circuitry of the staphylococcal sae locus and the impact of modifying this circuitry on the expression of external genes in the sae regulon. The sae locus contains four genes, the saeR and S two-component signalling module (TCS), and saeP and Q, two upstream genes of hitherto unknown function. It is expressed from two promoters, P(A)sae, which transcribes only the TCS, and P(C)sae, which transcribes the entire locus. A bursa aurealis (bursa) transposon insertion in saeP in a derivative of Staphylococcus aureus NCTC 8325 has a profound effect on sae function. It modifies the activity of the TCS, changing the expression of many genes in the sae regulon, even though transcription of the TCS (from P(A)sae) is not interrupted. Moreover, these effects are not due to disruption of saeP since an in-frame deletion in saeP has essentially no phenotype. The phenotype of S. aureus strain Newman is remarkably similar to that of the saeP : : bursa and this similarity is explained by an amino acid substitution in the Newman saeS gene that is predicted to modify profoundly the signalling function of the protein. This concurrence suggests that the saeP : : bursa insertion affects the signalling function of saeS, a suggestion that is supported by the ability of an saeQR clone, but not an saeR clone, to complement the effects of the saeP : : bursa insertion.

  7. Geisinger, Edward, George, Elizabeth A, Muir, Tom W, Novick, Richard P. "Identification of ligand specificity determinants in AgrC, the Staphylococcus aureus quorum-sensing receptor," Journal of biological chemistry 2008 Apr 4;283(14):8930-8.   (MEDL:18222919 PMID: 18222919 #J0137970)    

    Activation of the agr system, a major regulator of staphylococcal virulence, is initiated by the binding of a specific autoinducing peptide (AIP) to the extracellular domain of AgrC, a classical receptor histidine protein kinase. There are four known agr specificity groups in Staphylococcus aureus, and we have previously localized the determinant of AIP receptor specificity to the C-terminal half of the AgrC sensor domain. We have now identified the specific amino acid residues that determine ligand activation specificity for agr groups I and IV, the two most closely related. Comparison of the AgrC-I and AgrC-IV sequences revealed a set of five divergent residues in the region of the second extracellular loop of the receptor that could be responsible. Accordingly, we exchanged these residues between AgrC-I and AgrC-IV and tested the resulting constructs for activation by the respective AIPs, measuring activation kinetics with a transcriptional fusion of blaZ to the principal agr promoter, P3. Exchange of all five residues caused a complete switch in receptor specificity. Replacement of two of the AgrC-IV residues by the corresponding residues in AgrC-I caused the receptor to be activated by AIP-I nearly as well as the wild type AgrC-I receptor. Replacement of two different AgrC-I residues by the corresponding AgrC-IV residues broadened receptor recognition specificity to include both AIPs. Various types of intermediate activity were observed with other replacement mutations. Preliminary characterization of the AgrC-I-AIP-I interaction suggests that ligand specificity may be sterically determined.

  8. George, Elizabeth A, Novick, Richard P, Muir, Tom W. "Cyclic peptide inhibitors of staphylococcal virulence prepared by Fmoc-based thiolactone peptide synthesis," Journal of the American Chemical Society 2008 Apr 9;130(14):4914-24.   (MEDL:18335939 PMID: 18335939 #J0137968)    

    Virulence factor production in Staphylococcus aureus is largely under the control of the accessory gene regulator (agr) quorum sensing system. There are four agr groups, all of which exhibit bacterial interference: each agr type synthesizes a cyclic autoinducing peptide (AIP) with a distinct sequence that activates its cognate AgrC receptor and inhibits activation of others. To better understand inhibitory AIP-AgrC interactions, we aimed to identify the minimal molecular determinants required to inhibit both non-cognate and cognate receptors. This minimization of the AIP pharmacophore also may have therapeutic relevance as the use of native AIPs to block virulence of non-cognate agr strains can prevent the establishment of an infection in vivo. We synthesized and evaluated the inhibitory activities of 10 AIP derivatives based on a truncated AIP analogue that inhibits all four agr types. To carry out the rapid, parallel synthesis of these peptides, we employed a new linker for Fmoc-based thioester peptide synthesis. Our results identify key structural elements that are necessary for AgrC inhibition and reveal key differences between non-cognate and cognate inhibitory requirements.

  9. Mwakingwe, A, Ting, LM, Hochman, S, Chen, J, Novick, R, Sinnis, P, Kim, K. "REAL-TIME IN VIVO IMAGING OF LIVER STAGES OF PLASMODIUM YOEL [Abstract]," American journal of tropical medicine & hygiene 2008 DEC;79(6):273-273.   (ISI:000261644601293 #J0157472)    

  10. Novick, Richard P. "Medicine. Combating impervious bugs [comment]," Science 2008 Feb 15;319(5865):910-1.   (MEDL:18276877 PMID: 18276877 #J0133944)    

  11. Novick, R. "MRSA: RIP?," Scientist 2008 FEB;22(2):14-15.   (ISI:000252530200004 #J0133561)    

  12. Novick, Richard P, Geisinger, Edward. "Quorum sensing in staphylococci," Annual review of genetics 2008;42:541-64.   (MEDL:18713030 PMID: 18713030 #J0157883)    

    The staphylococcal agr locus encodes a quorum sensing (QS) system that controls the expression of virulence and other accessory genes by a classical two-component signaling module. Like QS modalities in other Gram-positive bacteria, agr encodes an autoactivating peptide (AIP) that is the inducing ligand for AgrC, the agr signal receptor. Unlike other such systems, agr variants have arisen that show strong cross-inhibition in heterologous combinations, with important evolutionary implications. Also unlike other systems, the effector of global gene regulation in the agr system is a major regulatory RNA, RNAIII. In this review, we describe the functions of the agr system's elements, show how they interact to bring about the regulatory response, and discuss the role of QS in staphylococcal pathobiology. We conclude with the suggestion that agr autoactivation, unlike classical enzyme induction, can occur under suboptimal conditions and can distinguish self from non-self by inducing an exclusive and coordinated population wide response.

  13. Shopsin, Bo, Drlica-Wagner, Alex, Mathema, Barun, Adhikari, Rajan P, Kreiswirth, Barry N, Novick, Richard P. "Prevalence of agr dysfunction among colonizing Staphylococcus aureus strains," Journal of infectious diseases 2008 Oct 15;198(8):1171-4.   (MEDL:18752431 PMID: 18752431 #J0159390)    

    Mutations in the staphylococcal virulence regulator gene agr frequently occur during Staphylococcus aureus infection. Whether agr-defective strains are fit for colonization, an important prerequisite for infection, is unknown. Screening by means of assays to detect delta-hemolysin activity and agr autoinducing peptide production indicated that 15 ( approximately 9%) of 160 healthy human subjects were colonized with an agr-defective strain or a mixture of agr-positive and -defective S. aureus strains. The presence of identical agr-defective strains in family members suggests that these strains are transmissible. Additionally, carriage of an agr-defective strain was associated with hospitalization, raising the possibility that such strains may be selected in a nosocomial setting.

  14. Tormo, Maria Angeles, Ferrer, Maria Desamparados, Maiques, Elisa, Ubeda, Carles, Selva, Laura, Lasa, Inigo, Calvete, Juan J, Novick, Richard P, Penades, Jose R. "Staphylococcus aureus pathogenicity island DNA is packaged in particles composed of phage proteins," Journal of bacteriology 2008 Apr;190(7):2434-40.   (MEDL:18223072 PMID: 18223072 #J0137969)    

    Staphylococcus aureus pathogenicity islands (SaPIs) have an intimate relationship with temperate staphylococcal phages. During phage growth, SaPIs are induced to replicate and are efficiently encapsidated into special small phage heads commensurate with their size. We have analyzed by amino acid sequencing and mass spectrometry the protein composition of the specific SaPI particles. This has enabled identification of major capsid and tail proteins and a putative portal protein. As expected, all these proteins were phage encoded. Additionally, these analyses suggested the existence of a protein required for the formation of functional phage but not SaPI particles. Mutational analysis demonstrated that the phage proteins identified were involved only in the formation and possibly the function of SaPI or phage particles, having no role in other SaPI or phage functions.

  15. Traber, Katrina E, Lee, Elsie, Benson, Sarah, Corrigan, Rebecca, Cantera, Mariela, Shopsin, Bo, Novick, Richard P. "agr function in clinical Staphylococcus aureus isolates," Microbiology 2008 Aug;154(Pt 8):2265-74.   (MEDL:18667559 PMID: 18667559 #J0152283)    

    The accessory gene regulator (agr) of Staphylococcus aureus is a global regulator of the staphylococcal virulon, which includes secreted virulence factors and surface proteins. The agr locus is important for virulence in a variety of animal models of infection, and has been assumed by inference to have a major role in human infection. Although most human clinical S. aureus isolates are agr(+), there have been several reports of agr-defective mutants isolated from infected patients. Since it is well known that the agr locus is genetically labile in vitro, we have addressed the question of whether the reported agr-defective mutants were involved in the infection or could have arisen during post-isolation handling. We obtained a series of new staphylococcal isolates from local clinical infections and handled these with special care to avoid post-isolation mutations. Among these isolates, we found a number of strains with non-haemolytic phenotypes owing to mutations in the agr locus, and others with mutations elsewhere. We have also obtained isolates in which the population was continuously heterogeneous with respect to agr functionality, with agr(+) and agr(-) variants having otherwise indistinguishable chromosomal backgrounds. This finding suggested that the agr(-) variants arose by mutation during the course of the infection. Our results indicate that while most clinical isolates are haemolytic and agr(+), non-haemolytic and agr(-) strains are found in S. aureus infections, and that agr(+) and agr(-) variants may have a cooperative interaction in certain types of infections.

  16. Ubeda, Carles, Maiques, Elisa, Barry, Peter, Matthews, Avery, Tormo, Maria Angeles, Lasa, Inigo, Novick, Richard P, Penades, Jose R. "SaPI mutations affecting replication and transfer and enabling autonomous replication in the absence of helper phage," Molecular microbiology 2008 Feb;67(3):493-503.   (MEDL:18086210 PMID: 18086210 #J0137971)    

    The SaPIs are chromosomal islands in staphylococci and other Gram-positive bacteria that carry genes for superantigens, virulence factors, resistance and certain metabolic functions. They have intimate relationships with certain temperate phages involving phage-induced excision, replication and efficient packaging in special small-headed infective phage-like particles, resulting in very high transfer frequencies. They generally contain 18-22 ORFs. We have systematically inactivated each of these ORFs and determined their functional groupings. In other reports, we have shown that five are involved in excision/integration, replication and packaging. In this report, we summarize the mutational analysis and focus on two key ORFs involved in regulation of the SaPI excision-replication-packaging cycle vis-a-vis phage induction. These two genes are divergently transcribed and define the major transcriptional organization of the SaPI genome. One of them, stl, encodes a master repressor, possibly analogous to the standard cI phage repressor. Mutational inactivation of this gene results in SaPI excision and replication in the absence of any inducing phage. This replicated SaPI DNA is not packaged; however, since the capsid components are provided by the helper phage. We have not yet ascertained any specific function for the second putative regulatory gene, though it is highly conserved among the SaPIs.

  17. Adhikari, Rajan P, Arvidson, Staffan, Novick, Richard P. "A nonsense mutation in agrA accounts for the defect in agr expression and the avirulence of Staphylococcus aureus 8325-4 traP::kan," Infection & immunity 2007 Sep;75(9):4534-40.   (MEDL:17606604 PMID: 17606604 #J0130345)    

    TraP is a triply phosphorylated staphylococcal protein that has been hypothesized to be the mediator of a second Staphylococcus aureus quorum-sensing system, 'SQS1,' that controls expression of the agr system and therefore is essential for the organism's virulence. This hypothesis was based on the loss of agr expression and virulence by a traP mutant of strain 8325-4 and was supported by full complementation of both phenotypic defects by the cloned traP gene in strain NB8 (Y. Gov, I. Borovok, M. Korem, V. K. Singh, R. K. Jayaswal, B. J. Wilkinson, S. M. Rich, and N. Balaban, J. Biol. Chem. 279:14665-14672, 2004), in which the wild-type traP gene was expressed in trans in the 8325-4 traP mutant. We initiated a study of the mechanism by which TraP activates agr and found that the traP mutant strain used for this and other recently published studies has a second mutation, an adventitious stop codon in the middle of agrA, the agr response regulator. The traP mutation, once separated from the agrA defect by outcrossing, had no effect on agr expression or virulence, indicating that the agrA defect accounts fully for the lack of agr expression and for the loss of virulence attributed to the traP mutation. In addition, DNA sequencing showed that the agrA gene in strain NB8 (Gov et al., J. Biol. Chem., 2004), in contrast to that in the agr-defective 8325-4 traP mutant strain, had the wild-type sequence; further, the traP mutation in that strain, when outcrossed, also had no effect on agr expression.

  18. Chen, John, Novick, Richard P. "svrA, a multi-drug exporter, does not control agr," Microbiology 2007 May;153(Pt 5):1604-8.   (MEDL:17464075 PMID: 17464075 #J0132454)    

    The Staphylococcus aureus svrA gene was identified in a signature-tagged mutagenesis screen for Tn917 insertions attenuated for mouse virulence, and subsequently found to be defective in agr expression. Its attenuation of virulence was attributed to its failure to express the agr regulon. In addition to the Tn917 insertion in svrA, the original svrA mutant strain (P6C63) has an adventitious frame-shift in agrC, which results in truncation of the AgrC peptide. Separation of the svrA mutation from the agrC frame-shift revealed that svrA has no detectable affect on agr activation, as assessed by exoprotein profiles and the production of haemolytic toxins. These results indicate that svrA does not play a role in Staphylococcus aureus infections via an agr-mediated pathway.

  19. Maiques, Elisa, Ubeda, Carles, Tormo, Maria Angeles, Ferrer, Maria Desamparados, Lasa, Inigo, Novick, Richard P, Penades, Jose R. "Role of staphylococcal phage and SaPI integrase in intra- and interspecies SaPI transfer," Journal of bacteriology 2007 Aug;189(15):5608-16.   (MEDL:17545290 PMID: 17545290 #J0137973)    

    SaPIbov2 is a member of the SaPI family of staphylococcal pathogenicity islands and is very closely related to SaPIbov1. Typically, certain temperate phages can induce excision and replication of one or more of these islands and can package them into special small phage-like particles commensurate with their genome sizes (referred to as the excision-replication-packaging [ERP] cycle). We have studied the phage-SaPI interaction in some depth using SaPIbov2, with special reference to the role of its integrase. We demonstrate here that SaPIbov2 can be induced to replicate by different staphylococcal phages. After replication, SaPIbov2 is efficiently encapsidated and transferred to recipient organisms, including different non-Staphylococcus aureus staphylococci, where it integrates at a SaPI-specific attachment site, att(C), by means of a self-coded integrase (Int). Phages that cannot induce the SaPIbov2 ERP cycle can transfer the island by recA-dependent classical generalized transduction and can also transfer it by a novel mechanism that requires the expression of SaPIbov2 int in the recipient but not in the donor. It is suggested that this mechanism involves the encapsidation of standard transducing fragments containing the intact island followed by int-mediated excision, circularization, and integration in the recipient.

  20. Novick, RP, Ubeda, C. "Comparative genetics and replication of the mobile pathogenicity islands of the Staphylococci [Abstract]," Plasmid 2007 MAR;57(2):214-215.   (ISI:000245374100080 #J0125508)    

  21. Novick, Richard P, Subedi, Abhignya. "The SaPIs: mobile pathogenicity islands of Staphylococcus," Chemical immunology & allergy 2007;93:42-57.   (MEDL:17369699 PMID: 17369699 #J0127043)    

    The SaPIs are 15- to 17-kb mobile pathogenicity islands in staphylococci. They usually carry two or more superantigens and are responsible for most superantigen-related human diseases, especially staphylococcal toxic shock syndrome. SaPIs are extremely common in Staphylococcus aureus, with all but one of the sequenced genomes containing one or more. The SaPIs have a highly conserved overall genome organization, parallel to that of typical temperate phages. Each occupies a specific chromosomal site from which it is induced to excise and replicate by one or more specific staphylococcal phages. Following replication, the SaPI DNA is efficiently encapsidated into infectious small-headed phage-like particles, resulting in extremely high transfer frequencies..

  22. Shopsin, B., Creanga, D. L., Riesman, A., Hogan, P., Thompson, C. A., Kunkel, M. J., Mathema, B., Novick, R. P., Kreiswirth, B. N.. "Bacterial Predictors of Outcome in Hospital-Acquired Pneumonia (HAP) Due to Methicillin-Resistant Staphylococcus aureus," Program & abstracts (Interscience Conference on Antimicrobial Agents & Chemotherapy) 2007;47(8):62-62.   (BIOSIS:PREV200900083073 #J0180870)    

  23. Subedi, Abhignya, Ubeda, Carles, Adhikari, Rajan P, Penades, Jose R, Novick, Richard P. "Sequence analysis reveals genetic exchanges and intraspecific spread of SaPI2, a pathogenicity island involved in menstrual toxic shock," Microbiology 2007 Oct;153(Pt 10):3235-45.   (MEDL:17906123 PMID: 17906123 #J0132484)    

    SaPIs are a family of homologous phage-related pathogenicity islands in staphylococci that carry superantigen and other virulence genes, and are responsible for a wide variety of superantigen-related diseases. SaPIs are induced to excise and replicate by particular staphylococcal phages and are encapsidated in infectious, small-headed, phage-like particles, which are transmitted at very high frequency among staphylococcal strains and species. SaPI2 is a prototypical member of this family that was identified in a typical menstrual toxic shock syndrome (TSS) strain of Staphylococcus aureus, the so-called Harrisburg strain, and found to be mobilizable by typing phage 80. Most menstrual TSS strains belong to a highly uniform agr group III clone of electrophoretic type (ET) 41, and this study was undertaken to determine whether such strains typically carry SaPI2, and whether it has spread beyond the ET41 clone. We report here the complete sequence of SaPI2, describe its relation to other known SaPIs, and show that it, or a very similar element, is carried by most ET41 strains but that it has disseminated to other strains that have also been implicated in TSS. We show additionally, that SaPIs are widespread among the staphylococci and that most TSS strains carry two or more, including SaPI2.

  24. Ubeda, Carles, Barry, Peter, Penades, Jose R, Novick, Richard P. "A pathogenicity island replicon in Staphylococcus aureus replicates as an unstable plasmid," Proceedings of the National Academy of Sciences of the United States of America 2007 Sep 4;104(36):14182-8.   (MEDL:17693549 PMID: 17693549 #J0130662)    

    The SaPIs are 14- to 17-kb mobile pathogenicity islands in staphylococci that carry genes for superantigen toxins and other virulence factors and are responsible for the toxic shock syndrome and other superantigen-related diseases. They reside at specific chromosomal sites and are induced by certain bacteriophages to initiate an excision-replication-packaging program, resulting in their incorporation into small infective phage-like particles. These are responsible for very high transfer frequencies that often equal and sometimes exceed the plaque-forming titer of the inducing phage. The ability of the SaPIs to replicate autonomously defines them as individual replicons and, like other prokaryotic replicons, they possess replicon-specific initiation functions. In this paper, we report identification of the SaPI replication origin (ori) and replication initiation protein (Rep), which has helicase as well as initiation activity. The SaPI oris are binding sites for the respective Rep proteins and consist of multiple oligonucleotide repeats in two sets, flanking an AT-rich region that may be the site of initial melting. Plasmids containing the rep-ori complex plus an additional gene, pri, can replicate autonomously in Staphylococcus aureus but are very unstable, probably because of defective segregation.

  25. Ubeda, Carles, Maiques, Elisa, Tormo, Maria Angeles, Campoy, Susana, Lasa, Inigo, Barbe, Jordi, Novick, Richard P, Penades, Jose R. "SaPI operon I is required for SaPI packaging and is controlled by LexA," Molecular microbiology 2007 Jul;65(1):41-50.   (MEDL:17581119 PMID: 17581119 #J0137972)    

    Transfer of Staphylococcus aureus pathogenicity islands (SaPIs) is directly controlled by the cellular repressor LexA. We have found that transcription of the SaPIbov1 operon I is repressed by LexA and is therefore SOS-induced. Two copies of the LexA binding site consensus (Cheo box) are present in the 5' region of this operon, at the same location in all of 15 different SaPIs analysed. Both of these boxes bind LexA protein. Furthermore, replacement of the chromosomal lexA with a non-cleavable mutant LexA (G94E) greatly diminished expression of SaPIbov1 operon I and differentially reduced the production of SaPI transducing particles in comparison with the production of plaque-forming particles. In concordance with this finding, deletion of operon I blocked the formation of SaPI transducing particles but had no effect on replication of the island. Operon I contains a gene encoding a homologue of the phage terminase small subunit plus two other genes that direct the assembly of the small sized SaPIbov1 capsids. Interestingly, mutations affecting the latter two genes were not defective in SaPI transfer, but rather encapsidated the island in full-sized phage heads, which would have to contain a multimeric SaPI genome.

  26. Geisinger, Edward, Adhikari, Rajan P, Jin, Ruzhong, Ross, Hope F, Novick, Richard P. "Inhibition of rot translation by RNAIII, a key feature of agr function," Molecular microbiology 2006 Aug;61(4):1038-48.   (MEDL:16879652 PMID: 16879652 #J0120772)    

    RNAIII is a 514 nt regulatory RNA that is the effector molecule of the staphylococcal agr quorum-sensing system, regulating a large set of virulence and other accessory genes at the level of transcription. RNAIII was discovered nearly 20 years ago and we long ago hypothesized that it would function by regulating the synthesis or activity of one or more intermediary transcription factors. We have finally confirmed this hypothesis, showing that Staphylococcus aureus RNAIII regulates the synthesis of a major pleiotropic transcription factor, Rot, by blocking its translation. RNAIII has a complex secondary structure with several stable hairpins that have highly C-rich end loops, unusual in an AT-rich organism. We noted that these loops are complementary to two G-rich stem loops of the rot mRNA translation initiation region (TIR). Pairing of the complementary RNAs would be predicted to occlude the rot Shine-Dalgarno (SD) site and to block rot translation. Through a combination of transcriptional and translational fusions and Northern and Western blot hybridization analyses, we show that RNAIII does, indeed, block rot translation. Through alterations in the C-rich loops of RNAIII and the G-rich loops of rot, we show that the sequences of these loops are critical for inhibition of rot translation and suggest that this inhibition is affected by pairing between the complementary stem loops, followed by the cleavage of rot mRNA. We propose that the RNAIII-rot mRNA interaction plays a key role in agr regulation of staphylococcal virulence.

  27. Maiques E, Ubeda C, Campoy S, Salvador N, Lasa I, Novick RP, Barbe J, Penades JR. "beta-lactam antibiotics induce the SOS response and horizontal transfer of virulence factors in Staphylococcus aureus," Journal of bacteriology 2006 Apr;188(7):2726-9.   (MEDL:16547063 PMID: 16547063 #J0114099)    

    Antibiotics that interfere with DNA replication and cell viability activate the SOS response. In Staphylococcus aureus, the antibiotic-induced SOS response promotes replication and high-frequency horizontal transfer of pathogenicity island-encoded virulence factors. Here we report that beta-lactams induce a bona fide SOS response in S. aureus, characterized by the activation of the RecA and LexA proteins, the two master regulators of the SOS response. Moreover, we show that beta-lactams are capable of triggering staphylococcal prophage induction in S. aureus lysogens. Consequently, and as previously described for SOS induction by commonly used fluoroquinolone antibiotics, beta-lactam-mediated phage induction also resulted in replication and high-frequency transfer of the staphylococcal pathogenicity islands, showing that such antibiotics may have the unintended consequence of promoting the spread of bacterial virulence factors.

  28. Traber K, Novick R. "A slipped-mispairing mutation in AgrA of laboratory strains and clinical isolates results in delayed activation of agr and failure to translate delta- and alpha-haemolysins," Molecular microbiology 2006 Mar;59(5):1519-30.   (MEDL:16468992 PMID: 16468992 #J0114477)    

    agr is a global regulator of staphylococcal virulence and other accessory gene functions, especially including the haemolysins. Lack of haemolysin production therefore generally represents a defect in agr function. An example of this is Staphylococcus aureus strain RN4220, a widely used laboratory strain that carries a nitrosoguanidine (MNNG)-induced mutation enabling it to accept DNA from Escherichia coli and other bacteria. We show here that the non-haemolytic phenotype of RN4220 is caused by an extra A residue in a run of seven As at the 3' end of agrA (agrA-8A). This causes a frameshift that results in the addition of three amino acyl residues to the C-terminal end of the protein. The 8A mutation does not inactivate the agr locus, but rather delays agr activation by 2-3 h, which results in failure to translate alpha- and delta-haemolysins, and hence, in a non-haemolytic phenotype. This mutation turned out not to be an adventitious consequence of MNNG mutagenesis, but rather had arisen in RN450, the immediate parent of RN4220. RN450 had become haemolytically heterogeneous in storage, and its non-haemolytic variants had the 8A mutation. The same mutation was also identified in a clinical isolate in which a non-haemolytic variant had arisen during the course of infection. Haemolytic activity in the mutant laboratory strains could be restored by the addition of auto-inducing peptide (AIP) early in growth, indicating that delayed production of RNAIII is responsible for the failure to translate alpha- and delta-haemolysins. Discovery of the 8A mutation has revealed the basis of the dissociation between agr activity and the non-haemolytic phenotype of RN4220, and has solved the long-standing mystery of the variable non-haemolytic phenotype of its immediate parent, RN450. The occurrence of this mutation in a clinical isolate indicates that it is not simply a laboratory phenomenon, and may represent a naturally occurring mechanism for the modulation of agr activity.

  29. Yao J, Zhong J, Fang Y, Geisinger E, Novick RP, Lambowitz AM. "Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll.LtrB group II intron splicing," RNA 2006 Jul;12(7):1271-81..   (MEDL:16741231 PMID: 16741231 #J0114881)    

    We show that a targetron based on the Lactococcus lactis Ll.LtrB group II intron can be used for efficient chromosomal gene disruption in the human pathogen Staphylococcus aureus. Targetrons expressed from derivatives of vector pCN37, which uses a cadmium-inducible promoter, or pCN39, a derivative of pCN37 with a temperature-sensitive replicon, gave site-specific disruptants of the hsa and seb genes in 37%-100% of plated colonies without selection. To disrupt hsa, an essential gene, we used a group II intron that integrates in the sense orientation relative to target gene transcription and thus could be removed by RNA splicing, enabling the production of functional HSa protein. We show that because splicing of the Ll.LtrB intron by the intron-encoded protein is temperature-sensitive, this method yields a conditional hsa disruptant that grows at 32 degrees C but not 43 degrees C. The temperature sensitivity of the splicing reaction suggests a general means of obtaining one-step conditional disruptions in any organism. In nature, temperature sensitivity of group II intron splicing could limit the temperature range of an organism containing a group II intron inserted in an essential gene.

  30. Adhikari RP, Novick RP. "Subinhibitory cerulenin inhibits staphylococcal exoprotein production by blocking transcription rather than by blocking secretion," Microbiology 2005 Sep;151(Pt 9):3059-69.   (MEDL:16151216 PMID: 16151216 #J0114101)    

    Cerulenin is an antibiotic that inhibits fatty acid synthesis by covalent modification of the active thiol of the chain-elongation subtypes of beta-ketoacyl-acyl carrier protein synthase. It also inhibits other processes that utilize essential thiols. Cerulenin has been widely reported to block protein secretion at sub-MIC levels, an effect that has been postulated to represent interference with membrane function through interference with normal fatty acid synthesis. This study confirms the profound reduction in extracellular proteins caused by low concentrations of the antibiotic, and shows by Northern blot hybridization that this reduction is due to interference with transcription. By exchanging promoters between entB, a gene that is inhibited by cerulenin, and entA, a gene that is not, it was also shown that the antibiotic does not block secretion. Subinhibitory concentrations of cerulenin were also found to block transcriptional activation of at least two regulatory determinants, agr and sae, that function by signal transduction. Interference with the activation of these and other regulatory determinants probably accounts for much of the inhibitory effect on exoprotein production of sub-MIC concentrations of cerulenin.

  31. George, EA, Wright, JS, Novick, RP, Muir, TW. "Synthesis of dimeric quorum sensing peptides to probe virulence in S. aureus [Abstract]," Biopolymers 2005 AUG;80(4):541-541.   (ISI:000229901200262 #J0105414)    

  32. Ji G, Pei W, Zhang L, Qiu R, Lin J, Benito Y, Lina G, Novick RP. "Staphylococcus intermedius produces a functional agr autoinducing peptide containing a cyclic lactone," Journal of bacteriology 2005 May;187(9):3139-50.   (MEDL:15838041 PMID: 15838041 #J0114103)    

    The agr system is a global regulator of accessory functions in staphylococci, including genes encoding exoproteins involved in virulence. The agr locus contains a two-component signal transduction module that is activated by an autoinducing peptide (AIP) encoded within the agr locus and is conserved throughout the genus. The AIP has an unusual partially cyclic structure that is essential for function and that, in all but one case, involves an internal thiolactone bond between a conserved cysteine and the C-terminal carboxyl group. The exceptional case is a strain of Staphylococcus intermedius that has a serine in place of the conserved cysteine. We demonstrate here that the S. intermedius AIP is processed by the S. intermedius AgrB protein to generate a cyclic lactone, that it is an autoinducer as well as a cross-inhibitor, and that all of five other S. intermedius strains examined also produce serine-containing AIPs.

  33. Novick RP. "Interrupters on the bacterial party line," Nature Chemical Biology 2005 Nov;1(6):321-2.   (MEDL:16408068 PMID: 16408068 #J0114100)    

  34. Novick, RP, Wright, JS. "Quorum sensing and interference by bacterial autoinducing peptides [Abstract]," Biopolymers 2005 AUG;80(4):490-490.   (ISI:000229901200029 #J0105413)    

  35. Sakoulas G, Eliopoulos GM, Fowler VG Jr, Moellering RC Jr, Novick RP, Lucindo N, Yeaman MR, Bayer AS. "Reduced susceptibility of Staphylococcus aureus to vancomycin and platelet microbicidal protein correlates with defective autolysis and loss of accessory gene regulator (agr) function," Antimicrobial agents & chemotherapy 2005 Jul;49(7):2687-92.   (MEDL:15980337 PMID: 15980337 #J0114102)    

    Loss of agr function, vancomycin exposure, and abnormal autolysis have been linked with both development of the GISA phenotype and low-level resistance in vitro to thrombin-induced platelet microbicidal proteins (tPMPs). We examined the potential in vitro interrelationships among these parameters in well-characterized, isogenic laboratory-derived and clinical Staphylococcus aureus isolates. The laboratory-derived S. aureus strains included RN6607 (agrII-positive parent) and RN6607V (vancomycin-passaged variant; hetero-GISA), RN9120 (RN6607 agr::tetM; agr II knockout parent), RN9120V (vancomycin-passaged variant), and RN9120-GISA (vancomycin passaged, GISA). Two serial isolates from a vancomycin-treated patient with recalcitrant, methicillin-resistant S. aureus (MRSA) endocarditis were also studied: A5937 (agrII-positive initial isolate) and A5940 (agrII-defective/hetero-GISA isolate obtained after prolonged vancomycin administration). In vitro tPMP susceptibility phenotypes were assessed after exposure of strains to either 1 or 2 mug/ml. Triton X-100- and vancomycin-induced lysis profiles were determined spectrophotometrically. For agrII-intact strain RN6607, vancomycin exposure in vitro was associated with modest increases in vancomycin MICs and reduced killing by tPMP, but no change in lysis profiles. In contrast, vancomycin exposure of agrII-negative RN9120 yielded a hetero-GISA phenotype and was associated with defects in lysis and reduced in vitro killing by tPMP. In the clinical isolates, loss of agrII function during prolonged vancomycin therapy was accompanied by emergence of the hetero-GISA phenotype and reduced tPMP killing, with no significant change in lysis profiles. An association was identified between loss of agrII function and the emergence of hetero-GISA phenotype during either in vitro or in vivo vancomycin exposure. In vitro, these events were associated with defective lysis and reduced susceptibility to tPMP. The precise mechanism(s) underlying these findings is the subject of current investigations.

  36. Ubeda C, Maiques E, Knecht E, Lasa I, Novick RP, Penades JR. "Antibiotic-induced SOS response promotes horizontal dissemination of pathogenicity island-encoded virulence factors in staphylococci," Molecular microbiology 2005 May;56(3):836-44.   (MEDL:15819636 PMID: 15819636 #J0114104)    

    Although mobile genetic elements have a crucial role in spreading pathogenicity-determining genes among bacterial populations, environmental and genetic factors involved in the horizontal transfer of these genes are largely unknown. Here we show that SaPIbov1, a Staphylococcus aureus pathogenicity island that belongs to the growing family of these elements that are found in many strains, is induced to excise and replicate after SOS induction of at least three different temperate phages, 80alpha, phi11 and phi147, and is then packaged into phage-like particles and transferred at high frequency. SOS induction by commonly used fluoroquinolone antibiotics, such as ciprofloxacin, also results in replication and high-frequency transfer of this element, as well as of SaPI1, the prototypical island of S. aureus, suggesting that such antibiotics may have the unintended consequence of promoting the spread of bacterial virulence factors. Although the strains containing these prophages do not normally contain SaPIs, we have found that RF122-1, the original SaPIbov1-containing clinical isolate, contains a putative second pathogenicity island that is replicated after SOS induction, by antibiotic treatment, of the prophage(s) present in the strain. Although SaPIbov1 is not induced to replicate after SOS induction in this strain, it is transferred by the antibiotic-activated phages. We conclude that SOS induction by therapeutic agents can promote the spread of staphylococcal virulence genes.

  37. Wright JS 3rd, Traber KE, Corrigan R, Benson SA, Musser JM, Novick RP. "The agr radiation: an early event in the evolution of staphylococci," Journal of bacteriology 2005 Aug;187(16):5585-94.   (MEDL:16077103 PMID: 16077103 #J0105664)    

    Agr is a global regulatory system in the staphylococci, operating by a classical two-component signaling module and controlling the expression of most of the genes encoding extracellular virulence factors. As it is autoinduced by a peptide, encoded within the locus, that is the ligand for the signal receptor, it is a sensor of population density or a quorum sensor and is the only known quorum-sensing system in the genus. agr is conserved throughout the staphylococci but has diverged along lines that appear to parallel speciation and subspeciation within the genus. This divergence has given rise to a novel type of interstrain and interspecies cross-inhibition that represents a fundamental aspect of the organism's biology and may be a predominant feature of the evolutionary forces that have driven it. We present evidence, using a newly developed, luciferase-based agr typing scheme, that the evolutionary divergence of the agr system was an early event in the evolution of the staphylococci and long preceded the development of the nucleotide polymorphisms presently used for genotyping. These polymorphisms developed, for the most part, within different agr groups; mobile genetic elements appear also to have diffused recently and, with a few notable exceptions, have come to reside largely indiscriminately within the several agr groups.

  38. Wright JS 3rd, Jin R, Novick RP. "Transient interference with staphylococcal quorum sensing blocks abscess formation [Journal Article D]," Proceedings of the National Academy of Sciences of the United States of America 2005 Feb 1;102(5):1691-6.   (MEDL:15665088 PMID: 15665088 #J0082035)    

    The staphylococcal virulon is controlled largely by the agr locus, a global accessory gene regulator that is autoinduced by a self-coded peptide (AIP) and is therefore a quorum sensor. The agr locus has diverged within and between species, giving rise to AIP variants that inhibit heterologous agr activation, an effect with therapeutic potential against Staphylococcus aureus: a single dose of an inhibitory AIP blocks the formation of an experimental murine abscess. As the AIP is unstable at physiological pH, owing to its essential thiolactone bond, its single-dose efficacy seems paradoxical, which has led us to analyze the in vivo kinetics of agr activation and the consequences of its blockage by a heterologous AIP. Initially, the infecting bacteria grow rapidly, achieving sufficient population density within the first 3 h to activate agr, and then enter a neutrophil-induced metabolic eclipse lasting for 2-3 d, followed by agr reactivation concomitantly with the development of the abscess. The inhibitory AIP prevents agr expression only during its short in vivo lifetime, suggesting that the agr-induced and therefore quorum-dependent synthesis of virulence factors shortly after infection is necessary for the subsequent development of the abscess lesion and bacterial survival. We confirm this finding by showing that a sterile agr+ supernatant causes a sterile abscess similar to the septic abscess caused by live bacteria. These results may provide a biological rationale for regulation of virulence factor expression by quorum sensing rather than by response to specific host signals.

  39. Charpentier E, Anton AI, Barry P, Alfonso B, Fang Y, Novick RP. "Novel cassette-based shuttle vector system for gram-positive bacteria," Applied & environmental microbiology 2004 Oct;70(10):6076-85.   (MEDL:15466553 PMID: 15466553 #J0076201)    

    Our understanding of staphylococcal pathogenesis depends on reliable genetic tools for gene expression analysis and tracing of bacteria. Here, we have developed and evaluated a series of novel versatile Escherichia coli-staphylococcal shuttle vectors based on PCR-generated interchangeable cassettes. Advantages of our module system include the use of (i) staphylococcal low-copy-number, high-copy-number, thermosensitive and theta replicons and selectable markers (choice of erythromycin, tetracycline, chloramphenicol, kanamycin, or spectinomycin); (ii) an E. coli replicon and selectable marker (ampicillin); and (iii) a staphylococcal phage fragment that allows high-frequency transduction and an SaPI fragment that allows site-specific integration into the Staphylococcus aureus chromosome. The staphylococcal cadmium-inducible P(cad)-cadC and constitutive P(blaZ) promoters were designed and analyzed in transcriptional fusions to the staphylococcal beta-lactamase blaZ, the Vibrio fischeri luxAB, and the Aequorea victoria green fluorescent protein reporter genes. The modular design of the vector system provides great flexibility and variety. Questions about gene dosage, complementation, and cis-trans effects can now be conveniently addressed, so that this system constitutes an effective tool for studying gene regulation of staphylococci in various ecosystems.

  40. Lyon GJ, Novick RP. "Peptide signaling in Staphylococcus aureus and other Gram-positive bacteria," Peptides 2004 Sep;25(9):1389-403.   (MEDL:15374643 PMID: 15374643 #J0114106)    

    There are two basic types of bacterial communication systems--those in which the signal is directed solely at other organisms and those in which the signal is sensed by the producing organism as well. The former are involved primarily in conjugation; the latter in adaptation to the environment. Gram-positive bacteria use small peptides for both types of signaling, whereas Gram-negative bacteria use homoserine lactones. Since adaptation signals are autoinducers the response is population-density-dependent and has been referred to as 'quorum-sensing'. Gram-negative bacteria internalize the signals which act upon an intracellular receptor, whereas Gram-positive bacteria use them as ligands for the extracellular receptor of a two-component signaling module. In both cases, the signal activates a complex adaptation response involving many genes.

  41. Mangold M, Siller M, Roppenser B, Vlaminckx BJ, Penfound TA, Klein R, Novak R, Novick RP, Charpentier E. "Synthesis of group A streptococcal virulence factors is controlled by a regulatory RNA molecule," Molecular microbiology 2004 Sep;53(5):1515-27.   (MEDL:15387826 PMID: 15387826 #J0114105)    

    The capacity of pathogens to cause disease depends strictly on the regulated expression of their virulence factors. In this study, we demonstrate that the untranslated mRNA of the recently described streptococcal pleiotropic effect locus (pel), which incidentally contains sagA, the structural gene for streptolysin S, is an effector of virulence factor expression in group A beta-haemolytic streptococci (GAS). Our data suggest that the regulation by pel RNA occurs at both transcriptional (e.g. emm, sic, nga) and post-transcriptional (e.g. SpeB) levels. We could exclude the possibility that the pel phenotype was linked to a polar effect on downstream genes (sagB-I). Remarkably, the RNA effector is regulated in a growth phase-dependent fashion and we provide evidence that pel RNA expression is induced by conditioned media.

  42. Tseng JC, Levin B, Hurtado A, Yee H, Perez de Castro I, Jimenez M, Shamamian P, Jin R, Novick RP, Pellicer A, Meruelo D. "Systemic tumor targeting and killing by Sindbis viral vectors," Nature biotechnology 2004 Jan;22(1):70-7.   (MEDL:14647305 PMID: 14647305 #J0069678)    

    Successful cancer gene therapy requires a vector that systemically and specifically targets tumor cells throughout the body. Although several vectors have been developed to express cytotoxic genes via tumor-specific promoters or to selectively replicate in tumor cells, most are taken up and expressed by just a few targeted tumor cells. By contrast, we show here that blood-borne Sindbis viral vectors systemically and specifically infect tumor cells. A single intraperitoneal treatment allows the vectors to target most tumor cells, as demonstrated by immunohistochemistry, without infecting normal cells. Further, Sindbis infection is sufficient to induce complete tumor regression. We demonstrate systemic vector targeting of tumors growing subcutaneously, intrapancreatically, intraperitoneally and in the lungs. The vectors can also target syngeneic and spontaneous tumors in immune-competent mice. We document the anti-tumor specificity of a vector that systemically targets and eradicates tumor cells throughout the body without adverse effects.

  43. Weinrick B, Dunman PM, McAleese F, Murphy E, Projan SJ, Fang Y, Novick RP. "Effect of mild acid on gene expression in Staphylococcus aureus," Journal of bacteriology 2004 Dec;186(24):8407-23.   (MEDL:15576791 PMID: 15576791 #J0076522)    

    During staphylococcal growth in glucose-supplemented medium, the pH of a culture starting near neutrality typically decreases by about 2 units due to the fermentation of glucose. Many species can comfortably tolerate the resulting mildly acidic conditions (pH, approximately 5.5) by mounting a cellular response, which serves to defend the intracellular pH and, in principle, to modify gene expression for optimal performance in a mildly acidic infection site. In this report, we show that changes in staphylococcal gene expression formerly thought to represent a glucose effect are largely the result of declining pH. We examine the cellular response to mild acid by microarray analysis and define the affected gene set as the mild acid stimulon. Many of the genes encoding extracellular virulence factors are affected, as are genes involved in regulation of virulence factor gene expression, transport of sugars and peptides, intermediary metabolism, and pH homeostasis. Key results are verified by gene fusion and Northern blot hybridization analyses. The results point to, but do not define, possible regulatory pathways by which the organism senses and responds to a pH stimulus.

  44. Wirth T, Wang X, Linz B, Novick RP, Lum JK, Blaser M, Morelli G, Falush D, Achtman M. "Distinguishing human ethnic groups by means of sequences from Helicobacter pylori: lessons from Ladakh," Proceedings of the National Academy of Sciences of the United States of America 2004 Apr 6;101(14):4746-51.   (MEDL:15051885 PMID: 15051885 #J0114107)    

    The history of mankind remains one of the most challenging fields of study. However, the emergence of anatomically modern humans has been so recent that only a few genetically informative polymorphisms have accumulated. Here, we show that DNA sequences from Helicobacter pylori, a bacterium that colonizes the stomachs of most humans and is usually transmitted within families, can distinguish between closely related human populations and are superior in this respect to classical human genetic markers. H. pylori from Buddhists and Muslims, the two major ethnic communities in Ladakh (India), differ in their population-genetic structure. Moreover, the prokaryotic diversity is consistent with the Buddhists having arisen from an introgression of Tibetan speakers into an ancient Ladakhi population. H. pylori from Muslims contain a much stronger ancestral Ladakhi component, except for several isolates with an Indo-European signature, probably reflecting genetic flux from the Near East. These signatures in H. pylori sequences are congruent with the recent history of population movements in Ladakh, whereas similar signatures in human microsatellites or mtDNA were only marginally significant. H. pylori sequence analysis has the potential to become an important tool for unraveling short-term genetic changes in human populations.

  45. Wright JS 3rd, Lyon GJ, George EA, Muir TW, Novick RP. "Hydrophobic interactions drive ligand-receptor recognition for activation and inhibition of staphylococcal quorum sensing," Proceedings of the National Academy of Sciences of the United States of America 2004 Nov 16;101(46):16168-73.   (MEDL:15528279 PMID: 15528279 #J0076174)    

    Two-component systems represent the most widely used signaling paradigm in living organisms. Encoding the prototypical two-component system in Gram-positive bacteria, the staphylococcal agr (accessory gene regulator) operon uses a polytopic receptor, AgrC, activated by an autoinducing peptide (AIP), to coordinate quorum sensing with the global synthesis of virulence factors. The agr locus has undergone evolutionary divergence, resulting in the formation of several distinct inter- and intraspecies specificity groups, such that most cross-group AIP-receptor interactions are mutually inhibitory. We have exploited this natural diversity by constructing and analyzing AgrC chimeras generated by exchange of intradomain segments between receptors of different agr groups. Functional chimeras fell into three general classes: receptors with broadened specificity, receptors with tightened specificity, and receptors that lack activation specificity. Testing of these chimeric receptors against a battery of AIP analogs localized the primary ligand recognition site to the receptor distal subdomain and revealed that the AIPs bind primarily to a putative hydrophobic pocket in the receptor. This binding is mediated by a highly conserved hydrophobic patch on the AIPs and is an absolute requirement for interactions in self-activation and cross-inhibition of the receptors. It is suggested that this recognition scheme provides the fundamental basis for agr activation and interference.

  46. Novick RP. "Autoinduction and signal transduction in the regulation of staphylococcal virulence," Molecular microbiology 2003 Jun;48(6):1429-49.   (MEDL:22676861 PMID: 12791129 #J0053242)    

    The accessory genes of Staphylococcus aureus, including those involved in pathogenesis, are controlled by a complex regulatory network that includes at least four two-component systems, one of which, agr, is a quorum sensor, an alternative sigma factor and a large set of transcription factors, including at least two of the superantigen genes, tst and seb. These regulatory genes are hypothesized to act in a time- and population density-dependent manner to integrate signals received from the external environment with the internal metabolic machinery of the cell, in order to achieve the production of particular subsets of accessory/virulence factors at the time and in quantities that are appropriate to the needs of the organism at any given location. From the standpoint of pathogenesis, the regulatory agenda is presumably tuned to particular sites in the host organism. To address this hypothesis, it will be necessary to understand in considerable detail the regulatory interactions among the organism's numerous controlling systems. This review is an attempt to integrate a large body of data into the beginnings of a model that will hopefully help to guide research towards a full-scale test.

  47. Novick RP. "Mobile genetic elements and bacterial toxinoses: the superantigen-encoding pathogenicity islands of Staphylococcus aureus," Plasmid 2003 Mar;49(2):93-105.   (MEDL:22613824 PMID: 12726763 #J0053280)    

    It is a remarkable observation that virtually all bacterial toxins associated with specific clinical conditions (toxinoses) are encoded by mobile (and therefore variable) genetic elements. Remarkably, these rarely, if ever, carry determinants of antibiotic resistance. Examples are the toxins responsible for diphtheria, anthrax, tetanus, botulism, cholera, toxic shock, scarlet fever, exfoliative dermatitis, food poisoning, travelers' diarrhea, shigella dysentery, necrotizing pneumonia, and others. A recently discovered example of this phenomenon is the family of related staphylococcal pathogenicity islands encoding superantigens (SAgs). These are 15-20kb elements that occupy constant positions in the chromosomes of toxigenic strains, and are characterized by certain phage-related features, namely genes encoding integrases, helicases, and terminases, and the presence of flanking direct repeats. The prototype, SaPI1 of Staphylococcus aureus, encodes TSST-1 plus two newly described SAgs, SEK and SEL. Other members of the family encode enterotoxins B (SaPI3) and C (SaPI4), plus at least two other SAgs each. SaPI1 and SaPI2, also encoding TSST-1, are excised and induced to replicate by certain staphylococcal phages, and are then encapsidated at high efficiency into phage-like infectious particles with heads about 1/3 the size of the helper phage heads, commensurate with the sizes of the respective genomes. This results in transfer frequencies of the order of 10(8)/ml, and is presumably responsible for the spread of these elements as well as for their acquisition in the first place. In the absence of a helper phage, these two islands are highly stable; neither excision, loss, or transfer occurs at detectable frequency. Several general implications of this phenomenon will be discussed. One is that the determinants of these toxins have been imported from other species and therefore are not components of the basic genome of the extant producing organisms. This raises the question of the biological (adaptive?) roles of these toxins. Another is that the toxin-carrying units can spread among different (though probably related) species. An interesting question is that of the biological basis for the separation of toxin and resistance determinants.

  48. Novick RP, Jiang D. "The staphylococcal saeRS system coordinates environmental signals with agr quorum sensing," Microbiology 2003 Oct;149(Pt 10):2709-17.   (MEDL:22885464 PMID: 14523104 #J0053055)    

    sae is a two-component signal transduction system in Staphylococcus aureus that regulates the expression of many virulence factors at the transcriptional level and appears to act synergistically with agr in some cases. In this study, the interactions between sae and agr have been characterized in some detail. It was found that the sae locus is larger and more complex than originally envisioned, in that it is expressed from several promoters, giving rise to four or five transcripts, at least three of which are initiated upstream of saeRS and contain two additional reading frames, here designated saeP and saeQ, which are likely to have important roles in sae function. The upstream transcripts are induced during exponential phase concomitantly with the onset of RNAIII synthesis and their induction requires the agr effector, RNAIII, but is blocked by several environmental signals that override the effects of RNAIII. saeR is also required for the induction of these transcripts, so that the sae locus contains an autoinduction circuit. It is suggested that sae is downstream of agr in the exoprotein activation pathway (and also epistatic with agr), that it coordinates the effects of environmental signals with the agr quorum-sensing system, and therefore that it is a key intermediary in the overall regulatory strategy by which S. aureus senses and responds to its environment.

  49. Sakoulas G, Eliopoulos GM, Moellering RC Jr, Novick RP, Venkataraman L, Wennersten C, DeGirolami PC, Schwaber MJ, Gold HS. "Staphylococcus aureus accessory gene regulator (agr) group II: is there a relationship to the development of intermediate-level glycopeptide resistance?," Journal of infectious diseases 2003 Mar 15;187(6):929-38.   (MEDL:12660939 PMID: 12660939 #J0114108)    

    We previously determined that all 6 Staphylococcus aureus strains with confirmed intermediate-level resistance to glycopeptides (glycopeptide intermediate S. aureus [GISA]) from the United States that we tested belonged to accessory gene regulator (agr) group II. In the present study, we found that 56% of surveyed bloodstream methicillin-resistant S. aureus isolates (n = 148) at our hospital were agr group II, whereas only 24% of methicillin-susceptible S. aureus isolates (n = 33) were agr group II (P = .001). Population analysis of genetically engineered agr-null and parent wild-type strains of groups I, II, and IV revealed that, when agr function is lost, the agr group II knockout S. aureus was most likely to develop glycopeptide heteroresistance after growth in 1 microg/mL but not 16 microg/mL vancomycin. This strain was unique in showing decreased autolysis after growth in these conditions. This study suggests that some S. aureus strains have an intrinsic survival advantage under a glycopeptide selective pressure, which is possibly related to reduced autolysis after exposure to subinhibitory concentrations of glycopeptide.

  50. Dufour P, Jarraud S, Vandenesch F, Greenland T, Novick RP, Bes M, Etienne J, Lina G. "High genetic variability of the agr locus in Staphylococcus species," Journal of bacteriology 2002 Feb;184(4):1180-6.   (MEDL:11807079 PMID: 11807079 #J0114112)    

    The agr quorum-sensing and signal transduction system was initially described in Staphylococcus aureus, where four distinct allelic variants have been sequenced. Western blotting suggests the presence of homologous loci in many other staphylococci, and this has been confirmed for S. epidermidis and S. lugdunensis. In this study we isolated agr-like loci from a range of staphylococci by using PCR amplification from primers common to the six published agr sequences and bracketing the most variable region, associated with quorum-sensing specificity. Positive amplifications were obtained from 14 of 34 staphylococcal species or subspecies tested. Sequences of the amplicons identified 24 distinct variants which exhibited extensive sequence divergence with only 10% of the nucleotides absolutely conserved on multiple alignment. This variability involved all three open reading frames involved in quorum sensing and signal transduction. However, these variants retained several protein signatures, including the conserved cysteine residue of the autoinducing peptide, with the exception of S. intermedius of pigeon origin, which contained a serine in place of cysteine at this position. We discuss hypotheses on the mode of action and the molecular evolution of the agr locus based on comparisons between the newly determined sequences.

  51. Lyon GJ, Wright JS, Muir TW, Novick RP. "Key determinants of receptor activation in the agr autoinducing peptides of Staphylococcus aureus," Biochemistry 2002 Aug 6;41(31):10095-104.   (MEDL:12146974 PMID: 12146974 #J0114109)    

    Staphylococcal pathogenesis is regulated by a two-component quorum-sensing system, agr, activated upon binding of a self-coded autoinducing peptide (AIP) to the receptor-histidine kinase, AgrC. The AIPs consist of a thiolactone macrocyle and an exocyclic 'tail', both of which are important for function. In this report, characterization of the unique AIPs from the four known agr specificity groups of Staphylococcus aureus has been completed, along with analysis of cross-group inhibition of AgrC activation by each of the four AIPs. The following conclusions have been drawn: (i) The native thiolactone macrocyle and tail are necessary and sufficient for full activation by the AIPs, whereas the AIP-I macrocycle alone is a partial agonist. (ii) The native N-terminus is less critical, as that of AIP-I can be modified without affecting bioactivity, although that of AIP-III cannot. (iii) The ring and tail may function differently in different AIPs. Thus the group I and IV AIPs differ at a single (endocyclic) residue, which is the determinant of AIP specificity for these two groups and is essential for function. A similarly critical residue in AIP-II, however, is exocyclic. (iv) Cross-inhibition is more tolerant of sequence and structural diversity than is activation, suggesting that the AIPs interact differently with cognate than with heterologous receptors. (v) Chimeric peptides, in which the tails and macrocycles are switched, do not activate and instead inhibit receptor activation. These data suggest a model in which activation and inhibition involves different binding orientations within the ligand binding pocket of each receptor.

  52. Lyon GJ, Wright JS, Christopoulos A, Novick RP, Muir TW. "Reversible and specific extracellular antagonism of receptor-histidine kinase signaling," Journal of biological chemistry 2002 Feb 22;277(8):6247-53.   (MEDL:11733525 PMID: 11733525 #J0114113)    

    Staphylococcal pathogenesis is regulated by a two-component quorum-sensing system, agr, activated by a self-coded autoinducing peptide (AIP). The agr system is widely divergent and is unique in that variant AIPs cross-inhibit agr activation in heterologous combinations. Cross-inhibition, but not self-activation, is widely tolerant of structural diversity in the AIPs so that these two processes must involve different mechanisms of interaction with the respective receptors. Herein, we have utilized this naturally occurring antagonism to demonstrate that both activation and inhibition are reversible and that activators and inhibitors interact at a common site on the receptor. These results suggest that molecules designed to compete with natural agonists for binding at receptor-histidine kinase sensor domains could represent a general approach to the inhibition of receptor-histidine kinase signaling.

  53. Romero-Gallo J, Perez-Perez GI, Novick RP, Kamath P, Norbu T, Blaser MJ. "Responses of endoscopy patients in Ladakh, India, to Helicobacter pylori whole-cell and Cag A antigens," Clinical & diagnostic laboratory immunology 2002 Nov;9(6):1313-7.   (MEDL:22301452 PMID: 12414766 #J0047754) Full Text Link!   

    Although Helicobacter pylori is a cosmopolitan colonizer of the human stomach, the responses among persons in remote populations from whom H. pylori was cultured have not been studied. We report on studies of 189 persons in the Ladakh region of India in whom serum immunoglobulin G responses to H. pylori whole-cell and Cag A antigens were measured. H. pylori was isolated from 68 of these patients. An H. pylori whole-cell antigen derived from Ladakhi strains outperformed a similar antigen from U.S. strains, as determined by antigen-specific enzyme-linked immunosorbent assays. In total, 95% of the population was seropositive, including individuals responding only to the Cag A antigen. Correlation with culture results showed that these were true positives and, therefore, that the H. pylori whole-cell serology was falsely negative in some cases. In addition to establishing a collection of H. pylori isolates from a remote area in the world, we show that use of H. pylori whole-cell and Cag A serology together increases the sensitivity for the detection of colonization.

  54. Sakoulas G, Eliopoulos GM, Moellering RC Jr, Wennersten C, Venkataraman L, Novick RP, Gold HS. "Accessory gene regulator (agr) locus in geographically diverse Staphylococcus aureus isolates with reduced susceptibility to vancomycin," Antimicrobial agents & chemotherapy 2002 May;46(5):1492-502.   (MEDL:11959587 PMID: 11959587 #J0114111)    

    The majority of infections with glycopeptide intermediate-level resistant Staphylococcus aureus (GISA) originate in biomedical devices, suggesting a possible increased ability of these strains to produce biofilm. Loss of function of the accessory gene regulator (agr) of S. aureus has been suggested to confer an enhanced ability to bind to polystyrene. We studied agr in GISA, hetero-GISA, and related glycopeptide-susceptible S. aureus isolates. All GISA strains from diverse geographic origins belong to agr group II. All GISA strains were defective in agr function, as demonstrated by their inability to produce delta-hemolysin. Hetero-GISA isolate A5940 demonstrated a nonsense mutation in agrA that was not present in a pulsed-field gel electrophoresis-indistinguishable vancomycin-susceptible isolate from the same patient. Various other agr point mutations were noted in several clinical GISA and hetero-GISA isolates. A laboratory-generated agr-null strain demonstrated a small but reproducible increase in vancomycin heteroresistance after growth in vitro in subinhibitory concentrations of vancomycin. This was not seen in the isogenic agr group II parent strain in which agr was intact. The in vitro bactericidal activity of vancomycin was attenuated in the agr-null strain compared to the parent strain. These findings imply that compromised agr function is advantageous to clinical isolates of S. aureus toward the development of vancomycin heteroresistance, perhaps through the development of vancomycin tolerance.

  55. Vojtov N, Ross HF, Novick RP. "Global repression of exotoxin synthesis by staphylococcal superantigens," Proceedings of the National Academy of Sciences of the United States of America 2002 Jul 23;99(15):10102-7.   (MEDL:22133747 PMID: 12110733 #J0053914)    

    Virulent Staphylococcus aureus strains typically produce and secrete large quantities of many extracellular proteins involved in pathogenesis. Such strains cause the classical staphylococcal lesion--local tissue destruction and aggressive inflammation accompanied by the massive influx of polymorphonuclear leukocytes, leading to the formation of pus. Most strains causing toxic shock syndrome, however, produce and secrete very small quantities of most exoproteins although they elaborate high levels of toxic shock syndrome toxin-1 (TSST-1). These strains cause local infections that are remarkably apurulent although potentially fatal owing to the superantigen. We have analyzed this disparity and have found that TSST-1 itself is a negative global regulator of exoprotein gene transcription. TSST-1 not only represses most exoprotein genes but determines its own high expression level by autorepression. We report also that a second superantigen, enterotoxin B, has similar regulatory properties.

  56. Zhang L, Gray L, Novick RP, Ji G. "Transmembrane topology of AgrB, the protein involved in the post-translational modification of AgrD in Staphylococcus aureus," Journal of biological chemistry 2002 Sep 20;277(38):34736-42.   (MEDL:12122003 PMID: 12122003 #J0114110)    

    The accessory gene regulator (agr) of Staphylococcus aureus is the central regulatory system that controls the gene expression for a large set of virulence factors. This global regulatory locus consists of two transcripts: RNAII and RNAIII. RNAII encodes four genes (agrA, B, C, and D) whose gene products assemble a quorum sensing system. RNAIII is the effector of the Agr response. Both the agrB and agrD genes are essential for the production of the autoinducing peptide, which functions as a signal for the quorum sensing system. In this study, we demonstrated the transmembrane nature of AgrB protein in S. aureus. A transmembrane topology model of AgrB was proposed based on AgrB-PhoA fusion analyses in Escherichia coli. Two hydrophilic regions with several highly conserved positively charged amino acid residues among various AgrBs were found to be located in the cytoplasmic membrane as suggested by PhoA-AgrB fusion studies. However, this finding is inconsistent with the putative transmembrane profile of AgrB by computer analysis. Furthermore, we detected an intermediate peptide of processed AgrD from S. aureus cells expressing AgrB and a 6 histidine-tagged AgrD. These results provide direct evidence that AgrB is involved in the proteolytic processing of AgrD. We speculate that AgrB is a novel protein with proteolytic enzyme activity and a transporter facilitating the export of the processed AgrD peptide.

  57. Herbert S, Barry P, Novick RP. "Subinhibitory clindamycin differentially inhibits transcription of exoprotein genes in Staphylococcus aureus," Infection & immunity 2001 May;69(5):2996-3003.   (MEDL:21189213 PMID: 11292717 #J0023171) Full Text Link!   

    It has long been known that certain antibiotics, at subinhibitory concentrations, differentially inhibit the synthesis of alpha-hemolysin and other staphylococcal virulence factors. In this report, we show that subinhibitory clindamycin (SBCL) eliminates production of nearly all exoproteins by Staphylococcus aureus but has virtually no effect on cytoplasmic proteins. The effect was abolished by a gene conferring resistance to macrolides-lincosamides-streptogramin B, showing that differential inhibition of protein synthesis is responsible; remarkably, however, subinhibitory clindamycin blocked production of several of the individual exoprotein genes, including spa (encoding protein A), hla (encoding alpha-hemolysin), and spr (encoding serine protease), at the level of transcription, suggesting that the primary effect must be differential inhibition of the synthesis of one or more regulatory proteins. In contrast to earlier reports, however, we found that subinhibitory clindamycin stimulates synthesis of coagulase and fibronectin binding protein B, also at the level of transcription. agr and sar expression was minimally affected by subinhibitory clindamycin. These effects varied from strain to strain and do not seem to be responsible for the effects of subinhibitory clindamycin on the overall exoprotein pattern.

  58. Jin R, Novick RP. "Role of the double-strand origin cruciform in pT181 replication," Plasmid 2001 Sep;46(2):95-105.   (MEDL:21476060 PMID: 11591135 #J0028862)    

    pT181 is a small rolling-circle plasmid from Staphylococcus aureus whose initiator protein, RepC, melts the plasmid's double-strand origin (DSO) and extrudes a cruciform involving IR II, a palindrome flanking the initiation nick site. We have hypothesized that the cruciform is required for initiation, providing a single-stranded region for the assembly of the replisome (R. Jin et al., 1997, EMBO J. 16, 4456-4566). In this study, we have tested the requirement for cruciform extrusion by disrupting the symmetry of the IR II palindrome or by increasing its length. The modified DSOs were tested for replication with RepC in trans. Rather surprisingly, disruption of the IR II symmetry had no detectable effect on replication or on competitivity of the modified DSO, though plasmids with IR II disrupted were less efficiently relaxed than the wild type by RepC. However, in conjunction with IR II disruption, modification of the tight RepC binding site IR III blocked replication. These results define two key elements of the pT181 initiation mechanism--the IR II conformation and the RepC binding site (IR III)--and they indicate that pT181 replication initiation is sufficiently robust to be able to compensate for significant modifications in the configuration of the DSO.

  59. Novick RP, Schlievert P, Ruzin A. "Pathogenicity and resistance islands of staphylococci," Microbes & infection 2001 Jun;3(7):585-94.   (MEDL:21311995 PMID: 11418332 #J0023123) Full Text Link!   

    Variable genetic elements including plasmids, transposons and prophages are involved in pathogenesis and antibiotic resistance, and are an important component of the staphylococcal genome. This review covers a set of newly described variable chromosomal elements, pathogenicity and resistance islands, carrying superantigen and resistance genes, especially toxic shock and methicillin resistance, respectively.

  60. Orwin PM, Leung DY, Donahue HL, Novick RP, Schlievert PM. "Biochemical and biological properties of Staphylococcal enterotoxin K," Infection & immunity 2001 Jan;69(1):360-6.   (MEDL:11119525 PMID: 11119525 #J0114115)    

    Staphylococcus aureus is an important human pathogen which is implicated in a wide variety of diseases. Major determinants of the virulence of this organism include extracellular virulence factors. Staphylococcal enterotoxins (SEs) are important causative agents in staphylococcal toxic shock syndrome and food poisoning. Our study identified a novel enterotoxin, SEK, and examined its biochemical and biological properties. SEK had a molecular weight of 26,000 and an experimentally determined pI of between 7.0 and 7.5. SEK was secreted by clinical isolates of S. aureus. We demonstrated that SEK had many of the biological activities associated with the SEs, including superantigenicity, pyrogenicity, the ability to enhance the lethal effect of endotoxin, and lethality in a rabbit model when administered by subcutaneous miniosmotic pump. Recombinant SEK was shown to stimulate human CD4(+) and CD8(+) T cells in a Vbeta-specific manner; T-cells bearing Vbeta 5.1, 5.2, and 6.7 were significantly stimulated to proliferate.

  61. Ruzin A, Lindsay J, Novick RP. "Molecular genetics of SaPI1--a mobile pathogenicity island in Staphylococcus aureus," Molecular microbiology 2001 Jul;41(2):365-77.   (MEDL:11489124 PMID: 11489124 #J0114114)    

    The Staphylococcus aureus gene for toxic shock toxin (tst) is carried by a 15 kb mobile pathogenicity island, SaPI1, that has an intimate relationship with temperate staphylococcal phage 80alpha. During phage growth, SaPI1 is excised from its unique chromosomal site, attC, replicates autonomously, interferes with phage growth, and is efficiently encapsidated into special small phage heads commensurate with its size. Upon transfer to a recipient organism, SaPI1 integrates at attC by means of a self-coded integrase. One or more phage functions are required for excision, autonomous replication and encapsidation of the element and, thus, the overall relationship between SaPI1 and 80alpha is similar to that between coliphages P4 and P2. Among other staphylococcal phages tested, only phi13 interacts with SaPI1, inducing excision but not replication or transfer of the element.

  62. Jarraud S, Lyon GJ, Figueiredo AM, Gerard L, Vandenesch F, Etienne J, Muir TW, Novick RP. "Exfoliatin-producing strains define a fourth agr specificity group in Staphylococcus aureus," Journal of bacteriology 2000 Nov;182(22):6517-22.   (MEDL:11053400 PMID: 11053400 #J0114117)    

    The staphylococcal virulon is activated by the density-sensing agr system, which is autoinduced by a short peptide (autoinducing peptide [AIP]) processed from a propeptide encoded by agrD. A central segment of the agr locus, consisting of the C-terminal two-thirds of AgrB (the putative processing enzyme), AgrD, and the N-terminal half of AgrC (the receptor), shows striking interstrain variation. This finding has led to the division of Staphylococcus aureus isolates into three different agr specificity groups and to the division of non-aureus staphylococci into a number of others. The AIPs cross-inhibit the agr responses between groups. We have previously shown that most menstrual toxic shock strains belong to agr specificity group III but that no strong clinical identity has been associated with strains of the other two groups. In the present report, we demonstrate a fourth agr specificity group among S. aureus strains and show that most exfoliatin-producing strains belong to this group. A striking common feature of group IV strains is activation of the agr response early in exponential phase, at least 2 h earlier than in strains of the other groups. This finding raises the question of the biological significance of the agr autoinduction threshold.

  63. Lyon GJ, Mayville P, Muir TW, Novick RP. "Rational design of a global inhibitor of the virulence response in Staphylococcus aureus, based in part on localization of the site of inhibition to the receptor-histidine kinase, AgrC," Proceedings of the National Academy of Sciences of the United States of America 2000 Nov 21;97(24):13330-5.   (MEDL:11087872 PMID: 11087872 #J0114116)    

    Two-component signaling systems involving receptor-histidine kinases are ubiquitous in bacteria and have been found in yeast and plants. These systems provide the major means by which bacteria communicate with each other and the outside world. Remarkably, very little is known concerning the extracellular ligands that presumably bind to receptor-histidine kinases to initiate signaling. The two-component agr signaling circuit in Staphylococcus aureus is one system where the ligands are known in chemical detail, thus opening the door for detailed structure-activity relationship studies. These ligands are short (8- to 9-aa) peptides containing a thiolactone structure, in which the alpha-carboxyl group of the C-terminal amino acid is linked to the sulfhydryl group of a cysteine, which is always the fifth amino acid from the C terminus of the peptide. One unique aspect of the agr system is that peptides that activate virulence expression in one group of S. aureus strains also inhibit virulence expression in other groups of S. aureus strains. Herein, it is demonstrated by switching the receptor-histidine kinase, AgrC, between strains of different agr specificity types, that intragroup activation and intergroup inhibition are both mediated by the same group-specific receptors. These results have facilitated the development of a global inhibitor of virulence in S. aureus, which consists of a truncated version of one of the naturally occurring thiolactone peptides.

  64. Novick RP. "Sortase: the surface protein anchoring transpeptidase and the LPXTG motif," Trends in microbiology 2000 Apr;8(4):148-51.   (MEDL:20219642 PMID: 10754567 #J0009047) Full Text Link!   

  65. Ruzin A, Novick RP. "Equivalence of lauric acid and glycerol monolaurate as inhibitors of signal transduction in Staphylococcus aureus," Journal of bacteriology 2000 May;182(9):2668-71.   (MEDL:20225874 PMID: 10762277 #J0009036) Full Text Link!   

    Glycerol monolaurate (GML) inhibits the expression of virulence factors in Staphylococus aureus and the induction of vancomycin resistance in Enterococcus faecalis, presumably by blocking signal transduction. Although GML is rapidly hydrolyzed by bacteria, one of the products, lauric acid, has identical inhibitory activity and is metabolized much more slowly. At least four distinct GML-hydrolyzing activities are identified in S. aureus: the secreted Geh lipase, residual supernatant activity in a geh-null mutant strain, a novel membrane-bound esterase, and a cytoplasmic activity.

  66. Mayville P, Ji G, Beavis R, Yang H, Goger M, Novick RP, Muir TW. "Structure-activity analysis of synthetic autoinducing thiolactone peptides from Staphylococcus aureus responsible for virulence," Proceedings of the National Academy of Sciences of the United States of America 1999 Feb 16;96(4):1218-23.   (MEDL:9990004 PMID: 9990004 #J0104599)    

    The synthesis of virulence factors and other extracellular proteins responsible for pathogenicity in Staphylococcus aureus is under the control of the agr locus. A secreted agr-encoded peptide, AgrD, processed from the AgrD gene product, is known to be an effector of self-strain activation and cross-strain inhibition of the agr response. Biochemical analysis of AgrD peptides isolated from culture supernatants has suggested that they contain an unusual thiol ester-linked cyclic structure. In the present work, chemical synthesis is used to confirm that the mature AgrD peptides contain a thiolactone structure and that this feature is absolutely necessary for full biological activity. The AgrD synthetic thiolactone peptides exhibited biological activity in vivo in a mouse protection test. Structure-activity studies have allowed key aspects of the peptide structure involved in the differential activation and inhibition functions to be identified. Accordingly, we propose a model for activation and inhibition of the agr response in which the former, but not the latter, involves specific acylation of the agr transmembrane receptor, AgrC.

  67. Novick RP, Muir TW. "Virulence gene regulation by peptides in staphylococci and other Gram-positive bacteria," Current opinion in microbiology 2(1):40-5, 1999 Feb.   (MEDL:99158949 PMID: 10047551 #J0002527) Full Text Link!   

    In staphylococci, autoinducing peptides activate agr. a global regulator of the expression of genes encoding virulence factors and other exoproteins. During the past year, there have been major advances in the structure-function analysis of these peptides and the regulation of a virulence factor by an autoinducing peptide in pneumococci has been demonstrated.

  68. Goldstein, BP, Wei, J, Greenberg, K, Novick, R. "Activity of nisin against Streptococcus pneumoniae, in vitro, a mouse infection model - J Antimicrob Chemother 1998; 42 : 277-278," Journal of antimicrobial chemotherapy 1998 AUG;42(2):277-278.   (ISI:000075476000030 #J0094075)    

  69. Lina G, Jarraud S, Ji G, Greenland T, Pedraza A, Etienne J, Novick RP, Vandenesch F. "Transmembrane topology and histidine protein kinase activity of AgrC, the agr signal receptor in Staphylococcus aureus," Molecular microbiology 28(3):655-62, 1998 May.   (MEDL:98294055 PMID: 9632266 #J0002954) Full Text Link!   

    The agr P2 operon in Staphylococcus aureus codes for the elements of a density-sensing cassette made up of a typical two-component signalling system and its corresponding inducer. It is postulated that the autoinducer, a post-translationally modified octapeptide generated from the AgrD peptide, interacts with a receptor protein, coded by agrC, to transmit a signal via AgrA regulating expression of staphylococcal virulence genes through expression of agr RNA III. We show by analysis of PhoA fusions that AgrC is a transmembrane protein, and confirm using Western blotting that a 46 kDa protein corresponding to AgrC is present in the bacterial membrane. This protein is autophosphorylated on a histidine residue only in response to supernatants from an agr+ strain, and can also respond to the purified native octapeptide. A recombinant fusion protein where most of the N-terminal region of AgrC is replaced by the Escherichia coli maltose-binding protein is also autophosphorylated in response to stimulation by agr+ supernatants or purified octapeptide. We conclude that AgrC is the sensor molecule of a typical two-component signal system in S. aureus, and that the ligand-binding site of AgrC is probably located in the third extracellular loop of the protein.

  70. Lindsay JA, Ruzin A, Ross HF, Kurepina N, Novick RP. "The gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus," Molecular microbiology 29(2):527-43, 1998 Jul.   (MEDL:98385824 PMID: 9720870 #J0002955) Full Text Link!   

    Tst, the gene for toxic shock syndrome toxin-1 (TSST-1), is part of a 15.2 kb genetic element in Staphylococcus aureus that is absent in TSST-1-negative strains. The prototype, in RN4282, is flanked by a 17 nucleotide direct repeat and contains genes for a second possible superantigen toxin, a Dichelobacter nodosus VapE homologue and a putative integrase. It is readily transferred to a recA recipient, and it always inserts into a unique chromosomal copy of the 17 nucleotide sequence in the same orientation. It is excised and circularized by staphylococcal phages phi13 and 80alpha and replicates during the growth of the latter, which transduces it at very high frequency. Because of its site and orientation specificity and because it lacks other identifiable phage-like genes, we consider it to be a pathogenicity island (PI) rather than a transposon or a defective phage. The tst element in RN4282, near tyrB, is designated SaPI1. That in RN3984 in the trp region is only partially homologous to SaPI1 and is excised by phage 80 but not by 80alpha. It is designated SaPI2. These PIs are the first in any gram-positive species and the first for which mobility has been demonstrated. Their mobility may be responsible for the spread of TSST-1 production among S. aureus strains.

  71. Novick RP. "Contrasting lifestyles of rolling-circle phages and plasmids," Trends in biochemical sciences 23(11):434-8, 1998 Nov.   (MEDL:99069864 PMID: 9852762 #J0002526) Full Text Link!   

    The rolling-circle mechanism of DNA replication is used by small prokaryotic genomes, such as single-stranded phages and plasmids. However, phages and plasmids have adapted the rolling-circle mechanism differently to suit their contrasting biological needs. The phi X174 phage uses a monomeric initiator protein catalytically, displays incomplete termination and recycles the initiator protein, in order to mass-produce phage progeny. By contrast, to control replication precisely, the pT181 plasmid uses a dimeric initiator protein stochiometrically, completes termination and inactivates the initiator after each replication cycle. The phi X174 phage and the pT181 plasmid represent paradigmatic adaptations of the rolling-circle mechanism and could provide models for other replicons.

  72. Ruzin A, Novick RP. "Glycerol monolaurate inhibits induction of vancomycin resistance in Enterococcus faecalis," Journal of bacteriology 1998 Jan;180(1):182-185.   (MEDL:98083075 PMID: 9422612 #J0003131) Full Text Link!   

    Glycerol monolaurate (GML) is a surfactant that has been found to inhibit the post-exponential phase activation of virulence factor production and the induction of beta-lactamase in Staphylococcus aureus. It has been suggested that signal transduction is the most probable target for GML (S. J. Projan, S. Brown-Skrobot, P. M. Schlievert, F. Vandenesch, and R. P. Novick, J. Bacteriol. 176:4204-4209, 1994). We found that GML suppresses growth of vancomycin-resistant Enterococcus faecalis on plates with vancomycin and blocks the induction of vancomycin resistance, which involves a membrane-associated signal transduction mechanism, either at or before initiation of transcription. Given the surfactant nature of GML and the results of previous experiments, we suggest that GML blocks signal transduction. In contrast, GML has no effect on the induction of erythromycin-inducible macrolide resistance in S. aureus, which does not involve signal transduction.

  73. Ji G, Beavis R, Novick RP. "Bacterial interference caused by autoinducing peptide variants," Science 276(5321):2027-30, 1997 Jun 27.   (MEDL:97342847 PMID: 9197262 #J0002120) Full Text Link!   

    The synthesis of virulence factors and other extracellular proteins by Staphylococcus aureus is globally controlled by the agr locus, which encodes a two-component signaling pathway whose activating ligand is an agr-encoded autoinducing peptide. The cognate peptides produced by some strains inhibit the expression of agr in other strains, and the amino acid sequences of peptide and receptor are markedly different between such strains, suggesting a hypervariability-generating mechanism. Cross-inhibition of gene expression represents a type of bacterial interference that could be correlated with the ability of one strain to exclude others from infection or colonization sites, or both.

  74. Jin R, Rasooly A, Novick RP. "In vitro inhibitory activity of RepC/C*, the inactivated form of the pT181 plasmid initiation protein, RepC," Journal of bacteriology 1997 Jan;179(1):141-7.   (MEDL:97136614 PMID: 8981991 #J0010109) Full Text Link!   

    pT181 is a Staphylococcus aureus rolling circle plasmid that regulates its replication by controlling the synthesis of its dimeric initiator protein RepC/C and by inactivating the protein following its use in replication (A. Rasooly and R. P. Novick, Science 262:1048-1050, 1993). This inactivation consists of the addition of an oligonucleotide, representing several nucleotides immediately 3' to the initiation nick site, to the active site tyrosine of one of the two subunits, generating a heterodimer, RepC/C*. Previous results suggested that the inactive form was metabolically stable and was present at a much higher level than the active form (A. Rasooly and R. P. Novick, Science 262:1048-1050, 1993). In the present study we have measured total RepC antigen as a function of plasmid copy number and have analyzed the interaction of the two forms. We find that pT181-containing staphylococci contain approximately one RepC dimer per plasmid copy over a 50-fold range of copy numbers. This is consistent with previous measurements of the rate of RepC synthesis, which suggested that one RepC dimer is synthesized per replication event (J. Bargonetti, P.-Z. Wang and R. P. Novick, EMBO J. 12:3659-3667, 1993). The RepC/C* heterodimer, which is inactive for replication, is a competitive inhibitor of the replication and the topoisomerase-like and cruciform-enhancing activities of the native protein. These results suggest that the inactive form may have a specific regulatory role in vivo. Since the known plasmid-determined controls, which maintain a constant plasmid copy number, are designed to ensure the synthesis of one RepC/C dimer per plasmid replication event, it is difficult to envision any role for yet another negative regulator of replication. Conceivably, under conditions where the initiator is overproduced, such as in the absence of the normal antisense regulation of initiator production, RepC/C* could serve as a fail-safe means of preventing autocatalytic replication.

  75. Jin R, Fernandez-Beros ME, Novick RP. "Why is the initiation nick site of an AT-rich rolling circle plasmid at the tip of a GC-rich cruciform?," EMBO journal 1997 Jul 16;16(14):4456-4466.   (MEDL:97392476 PMID: 9250690 #J0002122) Full Text Link!   

    pT181 and other closely related rolling circle plasmids have the nicking site for initiation of replication between the arms of a GC-rich inverted repeat sequence adjacent to the binding site for the dimeric initiator protein. Replication is initiated by the initiator-induced extrusion of this sequence as a cruciform, creating a single-stranded region for nicking by the protein. Nicking is followed by assembly of the replisome without relaxation of the secondary structure. Following termination, the initiator protein is released with a short oligonucleotide attached to one subunit, which prevents it from being recycled, a necessary feature of the plasmid's replication control system. The modified initiator can cleave single-stranded substrates and can nick and relax supercoiled plasmid DNA weakly. Although it can bind to its recognition sequence in the leading strand origin, the modified protein cannot induce cruciform extrusion, and it is proposed that this inability is the key to understanding the biological rationale for having the nicking site at the tip of a cruciform: the need to provide the functional initiator with a catalytic advantage over the modified one sufficient to offset the numerical advantage and metabolic stability of the latter.

  76. Jin R, Zhou X, Novick RP. "The inactive pT181 initiator heterodimer, RepC/C, binds but fails to induce melting of the plasmid replication origin," Journal of biological chemistry 1996 Dec 6;271(49):31086-91.   (MEDL:97094870 PMID: 8940104 #J0010123) Full Text Link!   

    Staphylococcus aureus plasmid pT181 replicates via a rolling circle mechanism. The synthesis of the pT181 initiator protein (RepC) is regulated by antisense RNAs, and RepC is inactivated after usage by the attachment of an oligonucleotide to one of its subunits. The inactivated heterodimeric RepC/C* has been shown be unable to initiate replication in vitro (Rasooly, A., and Novick, R. P. (1993) Science 262, 1048-1050). The inactive RepC/C* has been found to be very stable and constitute about 90-95% of the total RepC antigen inside the cell. We studied the specific interaction of the RepC/C and RepC/C* complex with the pT181 double strand origin. The results indicated that RepC/C and RepC/C* footprint supercoiled DNA differently although their footprints on linear DNA are similar; we also find that RepC/C is able to enhance cruciform extrusion while RepC/C* cannot. RepC/C* binds and bends the double strand origin much more weakly than does RepC/C. These results suggest that the attached oligonucleotide induces a conformational change in the RepC/C* molecule that is responsible for its lack of activity.

  77. Murray DL, Earhart CA, Mitchell DT, Ohlendorf DH, Novick RP, Schlievert PM. "Localization of biologically important regions on toxic shock syndrome toxin 1," Infection & immunity 1996 Jan;64(1):371-4.   (MEDL:8557369 PMID: 8557369 #J0114119)    

    Toxic shock syndrome toxin 1 (TSST-1) contains a long central alpha helix that forms the base of two grooves on opposite sides of the molecule. Previous studies indicated that residues 132, 135, and 140 along the back of the central alpha helix are important in the biological activities. We made mutations of additional central alpha-helix residues exposed along this groove on the back of TSST-1. The proteins were purified, shown not to have gross alteration in structure, and tested for both superantigenicity and ability to elicit lethal TSS, using the superantigenicity, likely to because of alteration in T-cell receptor binding. Mutants H135A, Q136A, and E132K/ Q136K lost the ability to induce lethal TSS. The mutant Q136A was most increasing because it was superantigenic, yet nonlethal.

  78. Novick, Richard. "Mycobacteria : growth, metabolism, and molecular biology," In: Tuberculosis / Rom, William; Garay, Stuart M (eds)    1st ed.. Boston : Little Brown, 1996. p.?-?    (offline #C0002766)

  79. Witcher KJ, Novick RP, Schlievert PM. "Modulation of immune cell proliferation by glycerol monolaurate," Clinical & diagnostic laboratory immunology 1996 Jan;3(1):10-3.   (MEDL:8770497 PMID: 8770497 #J0114118)    

    Previous studies have shown that glycerol monolaurate (GML), a surfactant commonly used in a wide variety of food and cosmetic products, inhibits the production of a variety of exotoxins by group A streptococci and staphylococci. Given the highly lipophilic nature of the structure of GML, it is suspected that the surfactant exerts its toxin inhibition effects via interaction with the cell membrane. The present study attempted to characterize some of the potential targets of GML action using the model system of lymphocyte activation. Results from murine splenocytes show that GML stimulates proliferation at concentrations between 10(-5) and 5 micrograms/ml/5 x 10(5) splenocytes. At concentrations greater than 5 micrograms/ml, GML inhibited lymphocyte proliferation and blocked the proliferative effects of the lymphocyte mitogens phorbol myristate acetate and concanavalin A and the potent T-cell mitogen toxic shock syndrome toxin-1. Studies using purified immune cell subsets indicated that GML at a concentration of 0.1 microgram/ml optimally induced proliferation of T cells but did not affect B cells. At higher concentrations, GML inhibited the toxic shock syndrome toxin-1 mitogenic effects on T cells, but did not inhibit the lipopolysaccharide-induced stimulation of B cells, suggesting that GML preferentially affects the T-cell population. GML-induced proliferation was blocked by the immunosuppressive drug cyclosporin A, suggesting that GML may be exerting its T-cell-proliferative effects along the calcium-dependent inositol phospholipid signal transduction pathway.

  80. Balaban N, Novick RP. "Autocrine regulation of toxin synthesis by Staphylococcus aureus," Proceedings of the National Academy of Sciences of the United States of America 1995 Feb 28;92(5):1619-23.   (MEDL:7533297 PMID: 7533297 #J0104156)    

    Staphylococcus aureus is a major human pathogen causing diseases which range from minor skin infection to endocarditis and toxic shock syndrome. The pathogenesis of S. aureus is due primarily to the production of toxic exoproteins, whose synthesis is controlled by a global regulatory system, agr. We show here that agr is autoinduced by a proteinaceous factor produced and secreted by the bacteria and that it is inhibited by a peptide produced by an exoprotein-deficient S. aureus mutant strain. The inhibitor, RIP, competes with the activator, RAP, and may be a mutational derivative. Our results suggest two possible approaches, independent of antibiotics, to the control of S. aureus infections. RIP may prove useful as a direct inhibitor of virulence and RAP as a vaccine against the expression of agr-induced virulence factors; either could interfere with the ability of the bacteria to establish and maintain an infection.

  81. Balaban N, Novick RP. "Translation of RNAIII, the Staphylococcus aureus agr regulatory RNA molecule, can be activated by a 3'-end deletion," FEMS microbiology letters 1995 Nov 1;133(1-2):155-61.   (MEDL:8566701 PMID: 8566701 #J0114120)    

    RNAIII, an RNA molecule shown to encode delta-hemolysin and independently to regulate toxin synthesis in Staphylococcus aureus, is transcribed at the mid-exponential phase of growth, while its target genes are activated 2 h later, at the post-exponential phase of growth. We show here that the translation of RNAIII to the 26-amino acid peptide delta-hemolysin is delayed by 1 h, and that this delay is abolished when the 3'-end of this molecule is deleted. We suggest that structural changes of RNAIII to a translatable form of the molecule precede its regulation of target gene expression.

  82. Ji G, Beavis RC, Novick RP. "Cell density control of staphylococcal virulence mediated by an octapeptide pheromone," Proceedings of the National Academy of Sciences of the United States of America 1995 Dec 19;92(26):12055-9.   (MEDL:8618843 PMID: 8618843 #J0104373)    

    Some bacterial pathogens elaborate and secrete virulence factors in response to environmental signals, others in response to a specific host product, and still others in response to no discernible cue. In this study, we have demonstrated that the synthesis of Staphylococcus aureus virulence factors is controlled by a density-sensing system that utilizes an octapeptide produced by the organism itself. The octapeptide activates expression of the agr locus, a global regulator of the virulence response. This response involves the reciprocal regulation of genes encoding surface proteins and those encoding secreted virulence factors. As cells enter the postexponential phase, surface protein genes are repressed by agr and secretory protein genes are subsequently activated. The intracellular agr effector is a regulatory RNA, RNAIII, whose transcription is activated by an agr-encoded signal transduction system for which the octapeptide is the ligand.

  83. Nesin M, Projan SJ, Kreiswirth B, Bolt Y, Novick RP. "Molecular epidemiology of Staphylococcus epidermidis blood isolates from neonatal intensive care unit patients," Journal of hospital infection 1995 Oct;31(2):111-21.   (MEDL:8551017 PMID: 8551017 #J0114121)    

    Twelve episodes of Staphylococcus epidermidis bacteraemia occurred within three months in a neonatal intensive care unit. Plasmid profiles and Southern blot hybridization with five different probes were used to determine whether an endemic strain of S. epidermidis could be identified among the contemporary isolates. It was concluded that this methodology was satisfactory for differentiation between isolates of coagulase-negative staphylococci: fifteen isolates were divided in eight groups indicating that there was no single endemic strain causing the outbreak.

  84. Novick RP, Projan SJ, Kornblum J, Ross HF, Ji G, Kreiswirth B, Vandenesch F, Moghazeh S. "The agr P2 operon: an autocatalytic sensory transduction system in Staphylococcus aureus," Molecular & general genetics 1995 Aug 30;248(4):446-58.   (MEDL:7565609 PMID: 7565609 #J0114122)    

    The synthesis of virulence factors and other exoproteins in Staphylococcus aureus is controlled by the global regulator, agr. Expression of secreted proteins is up-regulated in the postexponential growth phase, whereas expression of surface proteins is down-regulated by agr. The agr locus consists of two divergent operons, transcribed from neighboring but non-overlapping promoters, P2 and P3. The P2 operon sequence, reported here, contains 4 open reading frames, agrA, C, D, and B, of which A and C appear to encode proteins of a classical 2-component signal transduction pathway. The P3 operon specifies a 0.5 kb transcript, RNA III, which is the actual effector of the agr response, and, incidentally, encodes the agr-regulated peptide delta-hemolysin. Transcriptional fusions have shown that both P2 and P3 are agr sensitive (function in an agr+ but not in an agr- background) and deletion analysis has shown that all four of the P2 ORFs are involved; agrA and agrC seem to be absolutely required for the transcriptional activation of the agr locus, whereas agrB and agrD seem to be partially required. Since transcription of P2 requires P2 operon products, the P2 operon is autocatalytic, and is thus admirably suited to the need for rapid production of exoproteins at a time when overall growth is coming to a halt.

  85. Lebeau C, Vandenesch F, Greenland T, Novick RP, Etienne J. "Coagulase expression in Staphylococcus aureus is positively and negatively modulated by an agr-dependent mechanism," Journal of bacteriology 1994 Sep;176(17):5534-6.   (MEDL:8071233 PMID: 8071233 #J0114882)    

    Expression of staphylocoagulase by agr+ Staphylococcus aureus depends on the growth phase, being maximal during exponential growth and decreasing sharply postexponentially, while an agr-deleted strain continuously expresses an intermediate level of coagulase. Therefore, coagulase expression appears to be both positively and negatively modulated by an agr-dependent mechanism.

  86. Murray DL, Prasad GS, Earhart CA, Leonard BA, Kreiswirth BN, Novick RP, Ohlendorf DH, Schlievert PM. "Immunobiologic and biochemical properties of mutants of toxic shock syndrome toxin-1," Journal of immunology 1994 Jan 1;152(1):87-95.   (MEDL:8254210 PMID: 8254210 #J0114126)    

    Toxic shock syndrome (TSS) is a multisystem illness caused mainly by Staphylococcus aureus producing TSS toxin-1 (TSST-1). A variant of TSST-1 has been isolated from ovine mastitis S. aureus. This toxin, TSST-ovine (TSST-O) is only weakly T cell mitogenic, is nonpyrogenic, does not enhance endotoxin shock, and does not cause TSS in the miniosmotic pump model. The sequence of the ovine gene (tstO) differs from the TSST-1 gene (tstH) by 14 nucleotides that change seven amino acids in the mature protein of which two are in the C-terminal half. A gene fusion containing half of both tstH and tstO was made and cloned into S. aureus. The fusion protein contained the two C-terminal amino acid differences that are in TSST-O at residues 132 and 140. The fusion protein was not T cell mitogenic and did not elicit TSS in two rabbit models. Additional experiments used mutagenesis to change the lysine residue at position 132 of TSST-O to glutamate (TSST-OK132E), as exists in TSST-1, and to change the lysine residue of the human-ovine fusion at position 132 to glutamate (TSST-11140T). Both mutants were pyrogenic, enhanced endotoxin shock, and caused TSS in the miniosmotic pump model. However, the proteins were only partially T cell mitogenic. The restoration of lethality of TSST-O and the human-ovine fusion by changing the lysine to glutamate, as exists in TSST-1, indicates that residue 132 is important in lethality. The failure to regenerate complete T cell mitogenicity of the same mutants indicates that residues 132 and 140 are important for that activity.

  87. Projan SJ, Brown-Skrobot S, Schlievert PM, Vandenesch F, Novick RP. "Glycerol monolaurate inhibits the production of beta-lactamase, toxic shock toxin-1, and other staphylococcal exoproteins by interfering with signal transduction," Journal of bacteriology 1994 Jul;176(14):4204-9.   (MEDL:8021206 PMID: 8021206 #J0114124)    

    Glycerol monolaurate (GML) is a naturally occurring surfactant that is used widely as an emulsifier in the food and cosmetics industries and is generally regarded as lacking in important biological activities. The recent observation that it inhibits the production of staphylococcal toxic shock toxin-1 (P. M. Schlievert, J. R. Deringer, M. H. Kim, S. J. Projan, and R. P. Novick, Antimicrob. Agents Chemother. 36:626-631, 1992) is therefore rather surprising and raises the interesting question of how such a compound might interact with cells. In this report, we show that GML inhibits the synthesis of most staphylococcal toxins and other exoproteins and that it does so at the level of transcription. We find that GML blocks the induction but not the constitutive synthesis of beta-lactamase, suggesting that it acts by interfering with signal transduction.

  88. Rasooly A, Projan SJ, Novick RP. "Plasmids of the pT181 family show replication-specific initiator protein modification," Journal of bacteriology 1994 Apr;176(8):2450-2453.   (MEDL:94209250 PMID: 8157616 #J0000917)    

    The rolling circle plasmids of Staphylococcus aureus regulate their replication by controlling initiator (Rep) protein synthesis. It was demonstrated recently that the pT181 initiator protein RepC is inactivated during pT181 replication by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC* (A. Rasooly and R. P. Novick, Science, 262:1048-1050). We establish here that this initiator modification occurs with four other members of the pT181 family and that it occurs in Bacillus subtilis as well as S. aureus. These results suggest that Rep conversion to Rep* is probably universal among plasmids of the pT181 family and is not host dependent.

  89. Rasooly A, Wang PZ, Novick RP. "Replication-specific conversion of the Staphylococcus aureus pT181 initiator protein from an active homodimer to an inactive heterodimer," EMBO journal 1994 Nov 1;13(21):5245-51.   (MEDL:95045418 PMID: 7957090 #J0010855)    

    The Staphylococcus aureus rolling circle plasmid pT181 regulates its replication by controlling the synthesis of its initiator protein RepC. RepC is inactivated during pT181 replication by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC*. We analyzed RepC and RepC* in four classes of mutants: plasmid copy number mutants, two classes of RepC mutants affecting different portions of the protein and oriC (origin) mutants. We have found that in the cell with wild-type RepC there are similar relative amounts of RepC and RepC*, regardless of copy number, and that the conversion of RepC to RepC* is replication dependent. Genetic and biochemical evidence is presented that RepC functions as a dimer and that during replication the RepC homodimer is converted to the RepC/RepC* heterodimer.

  90. Vandenesch F, Lebeau C, Bes M, McDevitt D, Greenland T, Novick RP, Etienne J. "Coagulase deficiency in clinical isolates of Staphylococcus aureus involves both transcriptional and post-transcriptional defects," Journal of medical microbiology 1994 May;40(5):344-9.   (MEDL:8176721 PMID: 8176721 #J0114125)    

    The molecular basis of the non-expression of coagulase was investigated for 14 coagulase-negative isolates of Staphylococcus aureus obtained from different clinical samples. These isolates had typical S. aureus characteristics such as production of clumping factor, DNAase and protein A, but, with one exception, failed to produce detectable amounts of alpha-haemolysin. All 14 strains had DNA homologous to the coagulase gene (coa), but a coa-specific transcript was found in only seven of them. alpha-Haemolysin mRNA was detected in only eight strains without direct correlation to coa-mRNA expression. Thus, coagulase and alpha-haemolysin deficiencies in S. aureus may involve either transcriptional or post-transcriptional alterations although additional regulatory factors may influence the expression of both genes.

  91. Bargonetti J, Wang PZ, Novick RP. "Measurement of gene expression by translational coupling: effect of copy mutations on pT181 initiator synthesis," EMBO journal 1993 Sep;12(9):3659-67.   (MEDL:8253088 PMID: 8253088 #J0114130)    

    We have prepared and analyzed two types of gene fusion between the replication initiator gene, repC, and the reporter gene, blaZ, in order to investigate the relationship between pT181 plasmid copy number and RepC initiator protein production. A series of pT181 copy mutant plasmids, with copy numbers ranging from 70 to 800 copies per cell, were analyzed. In one type of gene fusion used in this study, blaZ was translationally coupled to the C-terminal end of the repC coding sequence such that native forms of both proteins were produced. This gene fusion arrangement, which permitted monitoring of RepC production (as BlaZ activity) by plasmids using the protein for their own replication, demonstrated a linear relationship, with one exception, between RepC production and plasmid copy number over a 20-fold range. In the second type of fusion, blaZ was translationally fused to the C-terminal end of repC. As the translational fusion did not produce active RepC protein, the fusion-containing pT181 derivatives were maintained in a strain which provided RepC in trans, and were thus analyzed at constant copy number. In contrast to previous analyses of this type, our translational fusion constructs expressed repC at levels proportional to the copy numbers of the plasmids from which the fusions were prepared. Using these data, we have calculated a minimum figure for the number of RepC molecules synthesized per replication event.

  92. Earhart CA, Prasad GS, Murray DL, Novick RP, Schlievert PM, Ohlendorf DH. "Growth and analysis of crystal forms of toxic shock syndrome toxin 1," Proteins 1993 Nov;17(3):329-34.   (MEDL:8272430 PMID: 8272430 #J0114129)    

    Native toxic shock syndrome toxin 1 (TSST-1) purified from Staphylococcus aureus has been crystallized in four different forms. The highest resolution data (2.05 A) was collected from orthorhombic crystals belonging to the space group C222(1). The unit cell dimensions are a = 108.7 A, b = 177.5 A, c = 97.6 A. Rotation function analysis of this form indicates that there is trimer of toxin molecules in the asymmetric unit with a local 3-fold axis parallel to the crystallographic c axis. Crystals of a double mutant of TSST-1 have been grown which has a single molecule in the asymmetric unit and diffract to 1.9 A. The space group is P2(1) with unit cell parameters of a = 44.4 A, b = 34.0 A, c = 55.2 A, beta = 93.0 degrees.

  93. Kreiswirth B, Kornblum J, Arbeit RD, Eisner W, Maslow JN, McGeer A, Low DE, Novick RP. "Evidence for a clonal origin of methicillin resistance in Staphylococcus aureus," Science 1993 Jan 8;259(5092):227-30.   (MEDL:8093647 PMID: 8093647 #J0114132)    

    Soon after methicillin was introduced into clinical practice in the early 1960s, resistant strains of Staphylococcus aureus (MRSA) appeared, bearing a newly acquired resistance gene, mecA, that encodes a penicillin binding protein, PBP2a. MRSA have spread throughout the world, and an investigation of the clonality of 472 isolates by DNA hybridization was performed. All 472 isolates could be divided into six temporally ordered mecA hybridization patterns, and three of these were subdivided by the chromomosomal transposon Tn554. Each Tn554 pattern occurred in association with one and only one mecA pattern, suggesting that mecA divergence preceded the acquisition of Tn554 in all cases and therefore that mecA may have been acquired just once by S. aureus.

  94. Novick RP, Ross HF, Projan SJ, Kornblum J, Kreiswirth B, Moghazeh S . "Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule," EMBO journal 1993 Oct;12(10):3967-3975.   (MEDL:94009003 PMID: 7691599 #J0004375)    

    The production of most toxins and other exoproteins in Staphylococcus aureus is controlled globally by a complex polycistronic regulatory locus, agr. Secretory proteins are up-regulated by agr whereas surface proteins are down-regulated. agr contains two divergent promoters, one of which directs the synthesis of a 514 nucleotide (nt) transcript, RNAIII. In this report, we show that the cloned RNAIII determinant restores both positive and negative regulatory functions of agr to an agr-null strain and that the RNA itself, rather than any protein, is the effector molecule. RNAIII acts primarily on the initiation of transcription and, secondarily in some cases, at the level of translation. In these cases, translation and transcription are regulated independently. RNAIII probably regulates translation directly by interacting with target gene transcripts and transcription indirectly by means of intermediary protein factors.

  95. Prasad GS, Earhart CA, Murray DL, Novick RP, Schlievert PM, Ohlendorf DH. "Structure of toxic shock syndrome toxin 1," Biochemistry 1993 Dec 21;32(50):13761-6.   (MEDL:8268150 PMID: 8268150 #J0114127)    

    The three-dimensional structure of toxic shock syndrome toxin 1 (TSST-1) from Staphylococcus aureus has been determined and refined to an R value of 0.226 for data between 8- and 2.5-A resolution. Overall, the structure of TSST-1 is similar to that of another superantigen, staphylococcal enterotoxin B (SEB). The key differences between these molecules are in the amino termini and in the degree to which a long central helix is covered by surface loops. The region around the carboxyl end of this central helix is proposed to govern the superantigenic properties of TSST-1. An adjacent region along this helix is proposed to be critical in the ability of TSST-1 to induce toxic shock syndrome.

  96. Projan S, Mandal S, Moghazeh S, Novick R. "Glycerol monolaurate inhibits production of Staphylococcus aureus beta-lactamase by blocking induction [Abstract]," Abstracts of the ... general meeting of the American Society for Microbiology 1993;?:16.   (offline #J0145343)    

  97. Rasooly A, Novick RP. "Replication-specific inactivation of the pT181 plasmid initiator protein," Science 1993 Nov 12;262(5136):1048-50.   (MEDL:8235621 PMID: 8235621 #J0114128)    

    Replication of the Staphylococcus aureus plasmid pT181, which occurs by the rolling circle mechanism, is accompanied by the covalent attachment of a approximately 12-residue oligodeoxy-nucleotide to one subunit of the dimeric plasmid-coded initiator protein, RepC. This oligonucleotide represents the plasmid sequence immediately 3' to the initiating nick site. The resulting heterodimeric protein lacks the topoisomerase and replication activities of unmodified RepC, suggesting that the regulation of plasmid DNA replication requires post-replicational inactivation of the initiator protein as well as control of its synthesis.

  98. Vandenesch F, Projan SJ, Kreiswirth B, Etienne J, Novick RP. "Agr-related sequences in Staphylococcus lugdunensis," FEMS microbiology letters 1993 Jul 15;111(1):115-22.   (MEDL:8359673 PMID: 8359673 #J0114131)    

    Sequences related to the Staphylococcus aureus accessory gene regulator (agr) were demonstrated in S. lugdunensis by Southern blot analysis of 13 strains and sequencing of the S. lugdunensis agr-like locus (agr-sl). Northern blot analysis of cellular RNA revealed the presence of a transcript having homology with the agr-P3 transcript (RNAIII) for three of the six strains tested. The three strains containing this transcript produce a hemolysin with phenotypic properties similar to that of S. aureus delta-hemolysin. Nevertheless, unlike agr-P3 from S. aureus, agr-sl does not encode any potential peptide homologous to S. aureus delta-hemolysin, suggesting that the hemolytic activity detected in S. lugdunensis is encoded elsewhere and may be controlled by agr-sl.

  99. Wang PZ, Projan SJ, Henriquez V, Novick RP. "Origin recognition specificity in pT181 plasmids is determined by a functionally asymmetric palindromic DNA element," EMBO journal 1993 Jan;12(1):45-52.   (MEDL:8428593 PMID: 8428593 #J0114133)    

    The leading strand replication origin of pT181 plasmids consists of two adjacent inverted repeat elements (IR-II and IR-III), which are involved in origin recognition by the initiator (Rep) protein. The conserved core element, IR-II, which contains the initiation nick site, is induced by Rep to form a cruciform structure, probably the primary substrate for the initiation of rolling circle replication. The divergent repeat, IR-III, constitutes the determinant of origin recognition specificity. We show here that the distal arm of IR-III is not required for sequence-specific recognition, whereas the proximal arm and central region are required. Since the initiator is dimeric, we presume that it binds symmetrically to IR-III. A unique type of DNA-protein interaction is proposed, in which the lack of sequence requirement for the distal arm is a consequence of binding to the adjacent IR-II, which thereby polarizes the stringency of binding to the two arms of IR-III. In addition, genetic evidence indicates that both the spacing and the phasing of IR-II to IR-III are crucial for function and that the central segment of IR-III may serve to position the two flanking half-sites for optimal interaction of Rep with IR-III.

  100. Lee PK, Kreiswirth BN, Deringer JR, Projan SJ, Eisner W, Smith BL, Carlson E, Novick RP, Schlievert PM. "Nucleotide sequences and biologic properties of toxic shock syndrome toxin 1 from ovine- and bovine-associated Staphylococcus aureus," Journal of infectious diseases 1992 Jun;165(6):1056-63.   (MEDL:1583323 PMID: 1583323 #J0114135)    

    Toxic shock syndrome toxin (TSST) 1 was purified from ovine (TSST-ovine) and bovine (TSST-bovine) mastitis-associated Staphylococcus aureus. These toxins were previously reported to have molecular weights identical to that of human TSST-1. However, TSST-ovine was reported as having an isoelectric point (pI) of 8.5, whereas TSST-bovine has the same pI (7.2) as TSST-1. Nucleotide sequence analysis revealed that TSST-bovine was identical to TSST-1 and that TSST-ovine had 14 nucleotide differences that changed 9 amino acid residues. Only 1 nucleotide difference, at position 514, was predicted to cause an amino acid charge difference, as glutamic acid at position 132 of TSST-1 was changed to lysine in TSST-ovine. Like TSST-1, TSST-ovine was mitogenic, but unlike TSST-1, it was not pyrogenic, was unable to enhance endotoxic shock, and was unable to induce TSS in a rabbit model. Also, TSST-ovine was less reactive to certain monoclonal antibodies raised against TSST-1.

  101. Projan SJ, Novick RP. "cis-inhibitory elements in the pT181 replication system," Plasmid 1992 Mar;27(2):81-92.   (MEDL:1615066 PMID: 1615066 #J0114137)    

    We report here the existence of a pair of sequence elements in plasmid cointegrates that together block the function of pT181 plasmid replication origins in cis. The study is an outgrowth of the use of plasmid pE194 as a vector for the analysis of the pT181 replication system. We have observed that whereas the isolated pT181 replication origin is fully functional when cloned to pE194, it is inactive when the entire pT181 plasmid genome is cloned. This cis-inhibition is relieved by deletion of all or part of the pE194 palA element or of the pT181 countertranscript promoter. The inhibitory effect of pE194 palA is independent of distance and orientation, whereas the inhibitory effect of the countertranscript promoter is lost when the promoter is moved to a distance of 1.5 kb from the replication origin or inverted in situ. We found that the cis-inhibited pT181 origin expresses origin-specific (Inc3B) incompatibility, which involves competition for the initiator protein. This finding suggests that the cis-inhibited origin binds the initiator protein and therefore that the inhibition affects a step in the initiation process subsequent to initiator binding.

  102. Regassa LB, Novick RP, Betley MJ. "Glucose and nonmaintained pH decrease expression of the accessory gene regulator (agr) in Staphylococcus aureus," Infection & immunity 1992 Aug;60(8):3381-8.   (MEDL:1639506 PMID: 1639506 #J0114134)    

    The effect of glucose on accessory gene regulator (agr) expression in Staphylococcus aureus was examined. agr is a global regulator that affects the expression of numerous genes, including those for some factors implicated in virulence, such as toxic shock syndrome toxin 1, alpha-hemolysin, and protein A. The agr locus determines two divergent transcripts, designated RNAII and RNAIII. RNAII contains four open reading frames (agrABCD), and RNAIII encodes delta-hemolysin. The mechanisms responsible for agr-mediated regulation are not well understood, but it appears that the RNAIII transcript plays a central role in the regulation of a number of target genes, including those for alpha-hemolysin (hla), beta-hemolysin (hlb), protein A (spa), and staphylococcal enterotoxin B (seb+). In this study, S. aureus cultures were grown either in a shake flask system with a complex medium or in a fermentor system with a completely defined medium in which the pH and glucose concentration were maintained. Northern (RNA) blot analysis revealed that a dramatic reduction in agr expression was apparent only when the cultures contained glucose and when the pH was 5.5 or was not maintained. The effect of glucose on two agr target genes, sec+ and hla, was also studied. Glucose-containing cultures produced less sec+ and hla mRNAs at maintained pH (6.5). In addition, the glucose effect on sec+ and hla was enhanced under conditions that inhibited agr expression (i.e., pH 5.5 or a nonmaintained pH).

  103. Schlievert PM, Deringer JR, Kim MH, Projan SJ, Novick RP. "Effect of glycerol monolaurate on bacterial growth and toxin production," Antimicrobial agents & chemotherapy 1992 Mar;36(3):626-31.   (MEDL:1622174 PMID: 1622174 #J0114136)    

    Glycerol monolaurate (GML) is a naturally occurring surfactant that has potential use as an additive to tampons and wound dressings to reduce the incidence of certain bacterial toxin-mediated illnesses. In vitro studies were undertaken to evaluate the effect of GML on the growth of and toxin production by potentially pathogenic bacteria. GML inhibited the growth of clinical isolates of group A, B, F, and G streptococci at concentrations of 10 to 20 micrograms/ml. Exotoxin production, including that of pyrogenic exotoxins and hemolysins, was reduced by concentrations of GML that were below those inhibitory for growth as well as growth inhibitory. The growth of Staphylococcus aureus strains from patients with toxic shock syndrome and scalded skin syndrome was inhibited or delayed in the presence of 100 to 300 micrograms of GML per ml. Growth inhibition by GML could be overcome by the production of lipase. S. aureus elaboration of hemolysin, toxic shock syndrome toxin 1, and exfoliative toxin A was inhibited at GML concentrations below those necessary to inhibit growth. Results similar to those for S. aureus were obtained in tests of S. hominis. Escherichia coli growth and Salmonella minnesota growth were unaffected by GML, but an S. minnesota Re mutant was susceptible to growth-inhibitory activity. Endotoxin release into the medium from E. coli cells was also unaffected by GML, but the release or activity of E. coli hemolysin was increased by GML. Streptococcal pyrogenic endotoxin A production by an E. coli clone was not affectd by GML. These studies indicate that GML is effective in blocking or delaying the production of exotoxins by pathogenic gram-positive bacteria.

  104. Wang PZ, Projan SJ, Henriquez V, Novick RP. "Specificity of origin recognition by replication initiator protein in plasmids of the pT181 family is determined by a six amino acid residue element," Journal of molecular biology 1992 Jan 5;223(1):145-58.   (MEDL:1731066 PMID: 1731066 #J0114138)    

    We have investigated the specificity of replication origin recognition by the initiator proteins of a set of six closely related Staphylococcus aureus plasmids, the pT181 family. These plasmids replicate by an asymmetric rolling-circle mechanism using plasmid-coded initiators that nick the replication origins and form a phosphotyrosine bond at the 5' nick terminus. Five of the plasmids are in different incompatibility groups and their initiator proteins do not cross-complement the cloned origins of any but their own plasmid. One pair is weakly incompatible and their initiator proteins and origins do cross-complement for replication in vivo. This pattern of cross-reactivity led to the prediction that the determinant of specificity would correspond to a homologously positioned set of six residues in the C-terminal domain of the protein, some 80 residues away from the active site tyrosine, that are divergent for all of the compatible plasmids and identical for the incompatible pair. Site-directed mutagenesis was used to exchange these six residues among three pairs of plasmids and these exchanges brought about the predicted switching of origin recognition specificity. Single substitution within this six residue set reduced or eliminated the activity of the protein but did not alter the origin recognition specificity. These six and flanking residues cannot form an amphipathic alpha-helix nor do they conform to the classical helix-turn-helix or other known DNA binding motifs. A novel type of interaction is suggested in which the protein binds to its recognition site, bends and melts the DNA, and causes or enhances the extrusion of an adjacent cruciform containing the nick site. This configuration would juxtapose the nicking target and the active site tyrosine residue and would unwind the highly G + C-rich replication origin.

  105. Lee PK, Deringer JR, Kreiswirth BN, Novick RP, Schlievert PM. "Fluid replacement protection of rabbits challenged subcutaneous with toxic shock syndrome toxins," Infection & immunity 1991 Mar;59(3):879-84.   (MEDL:1997438 PMID: 1997438 #J0114142)    

    Toxic shock syndrome toxin 1 (TSST-1) and streptococcal pyrogenic exotoxin A (SPE A) belong to a family of pyrogenic toxins produced by Staphylococcus aureus and Streptococcus pyogenes, respectively. Both toxins are responsible for causing toxic shock syndrome (TSS) and related illnesses, clinically characterized by multiorgan involvement. The most severe TSS symptom is acute hypotension and shock after the initial febrile response. In this study, we examined possible mechanisms of shock development in TSS, particularly the role of T-cell proliferation, endotoxin enhancement by toxins, and capillary leakage. American Dutch belted rabbits, with subcutaneously implanted miniosmotic pumps filled with either TSST-1 or SPE A, served as the animal model. For both TSST-1 and SPE A-treated rabbits, administration of cyclosporin A prevented toxin-induced T-cell proliferation but failed to protect the rabbits. Polymyxin B treatment of rabbits, to neutralize endogenous endotoxin, partially protected rabbits from challenge with either exotoxin; two of six rabbits survived on day 2 when treated with only TSST-1, whereas six of six animals survived after challenge with TSST-1 and polymyxin B. Similarly, with SPE A-treated rabbits, only 1 of 10 animals without polymyxin B treatment survived on day 8, but 4 of 6 rabbits survived on day 8 when given polymyxin B. Fluid replacement was successful in preventing lethality. Twelve of 14 rabbits survived when given TSST-1 with fluid, and all rabbits treated with SPE A and fluid survived. Finally, by using miniosmotic pumps, staphylococcal exfoliative toxin A and concanavalin A were administered to rabbits in an attempt to induce lethality. These two T-cell mitogens caused T-cell proliferation but failed to induce lethality in rabbits. The data suggest that toxin interactions causing vascular leakage and to some extent endotoxin enhancement are of major importance in development of hypotension and shock in TSS. It appears that T-cell proliferation may not contribute significantly to the induction of shock and death.

  106. Novick RP. "Genetic systems in staphylococci," Methods in enzymology 1991;204:587-636.   (MEDL:1658572 PMID: 1658572 #J0114144)    

  107. Novick RP. "In memoriam. Royston C. Clowes (1921-1989)," Plasmid 1991 Jan;25(1):1-2.   (MEDL:2034720 PMID: 2034720 #J0114143)    

  108. Vandenesch F, Kornblum J, Novick RP. "A temporal signal, independent of agr, is required for hla but not spa transcription in Staphylococcus aureus," Journal of bacteriology 1991 Oct;173(20):6313-20.   (MEDL:1717437 PMID: 1717437 #J0114139)    

    Staphylococcus aureus exoprotein expression is controlled by a global regulon known as agr. This system activates transcription of some target genes and represses transcription of others. Target genes expressed postexponentially such as alpha-hemolysin (hla) are activated by agr; target genes expressed during exponential phase such as protein A (spa) are repressed by agr. A unique feature of the agr system is that this transcriptional regulation is mediated by a 517-nucleotide transcript, RNAIII. While it is clear that agr differentially regulates the expression of exponential and postexponential exoproteins, the precise role of agr in the temporal control of these events has not yet been explored. In this report, we examine the effects of expressing RNAIII, the agr regulator, under the control of the inducible beta-lactamase (bla) promoter at different times in the growth cycle. We confirm previous results showing that agr is required for postexponential-phase expression of hla and further show that a separate postexponential-phase signal independent of agr function is also needed for activation of hla transcription. We also show that in an agr mutant transcription of spa occurs throughout the growth cycle, is inhibited immediately upon induction of RNAIII, and is thus indifferent to the postexponential signal required for hla activation.

  109. Wang PZ, Henriquez VB, Projan SJ, Iordanescu S, Novick RP. "The effect of plasmid copy number mutations on pT181 replication initiator protein expression," Plasmid 1991 May;25(3):198-207.   (MEDL:1924557 PMID: 1924557 #J0114141)    

    Previous studies have shown that plasmid pT181 controls its replication by countertranscript-mediated regulation of the rate of synthesis of the pT181 initiator, RepC. In this study, the relation has been studied between plasmid copy number and RepC synthesis for a series of pT181 copy number mutants. For each mutant plasmid, the repC coding sequence along with its 5' regulatory region was translationally fused to the beta-lactamase structural gene on a vector plasmid unrelated to pT181. By means of these constructs, the effect of regulatory mutations on the initiator synthesis could be measured at constant copy number. With one exception, the mutant control regions showed elevated beta-lactamase activity in comparison to the wild-type. However, the relative increase was not very well correlated with the copy number of the corresponding mutant plasmid. The possibility is considered that factors such as DNA secondary structure may have important ancillary effects on the regulation mechanism.

  110. Wang PZ, Projan SJ, Novick RP. "Nucleotide sequence of beta-lactamase regulatory genes from staphylococcal plasmid pI258," Nucleic acids research 1991 Jul 25;19(14):4000.   (MEDL:1861992 PMID: 1861992 #J0114140)    

  111. Highlander SK, Novick RP. "Mutational and physiological analyses of plasmid pT181 functions expressing incompatibility," Plasmid 1990 Jan;23(1):1-15.   (MEDL:1693440 PMID: 1693440 #J0114148)    

    Plasmid pT181 is a small multicopy plasmid from Staphylococcus aureus that belongs to incompatibility group 3 and expresses two distinct types of incompatibility, Inc3A and Inc3B. Inc3A incompatibility is expressed by the primary replication control determinant, copA, which specifies two small transcripts, RNA I and RNA II, that jointly inhibit the synthesis of the rate-limiting initiator protein, RepC. Inc3B incompatibility is expressed by the leading strand replication origin and is due to competition for RepC. The copA region from each of 11 different pT181 copy number mutants was cloned onto the pT181-compatible vector, pE194, and tested for its ability to inhibit the replication of pT181 and its copy number mutants. The pT181 replication origin was also cloned and tested for its ability to inhibit the replication of the same plasmids. In general copA mutations that alter the production or sequence of RNA I and RNA II greatly reduced or completely eliminated Inc3A activity. Unlike the wild-type, all of the copy mutants were resistant to Inc3B inhibition. The separately cloned wild-type copA and ori regions each reduced the copy number of pT181 in proportion to their gene dosage, but neither blocked replication completely. It is proposed that the cloned Inc determinants cause incompatibility by interfering with the plasmid's copy correction mechanism; this interference destabilizes the plasmid even under conditions where its average copy number is not greatly reduced.

  112. McCollister BD, Kreiswirth BN, Novick RP, Schlievert PM. "Production of toxic shock syndrome-like illness in rabbits by Staphylococcus aureus D4508: association with enterotoxin A," Infection & immunity 1990 Jul;58(7):2067-70.   (MEDL:2365451 PMID: 2365451 #J0114146)    

    Staphylococcus aureus D4508, obtained from a patient with nonmenstrual toxic shock syndrome (TSS), produced enterotoxin A while not making other known enterotoxins or TSS toxin 1. Concentrated culture fluids of the organism, administered subcutaneously in miniosmotic pumps, induced TSS-like symptoms (four of six animals succumbed). Identical culture fluids pretreated with anti-enterotoxin A serum failed to induce symptoms except for fever (none of six animals succumbed). Purified staphylococcal enterotoxin A also had the ability to induce TSS-like symptoms. These data suggest that enterotoxin A is the major TSS-associated toxin made by strain D4508.

  113. Noirot P, Bargonetti J, Novick RP. "Initiation of rolling-circle replication in pT181 plasmid: initiator protein enhances cruciform extrusion at the origin," Proceedings of the National Academy of Sciences of the United States of America 1990 Nov;87(21):8560-4.   (MEDL:2236066 PMID: 2236066 #J0114145)    

    Plasmid pT181 DNA secondary structures have been analyzed in vitro by nuclease S1 digestion and in vivo by bromoacetaldehyde treatment. A cruciform structure occurring at the pT181 replication origin in vitro is greatly enhanced by the binding of the plasmid-encoded initiator protein RepC. In vivo a DNA secondary structure also existed in the replication origin. Its frequency of formation was correlated with efficiency of RepC utilization. These data suggest that cruciform extrusion at the origin is involved in initiation of pT181 rolling-circle replication. A neighboring DNA structure influences the conformation of the origin in vivo.

  114. Norton SD, Schlievert PM, Novick RP, Jenkins MK. "Molecular requirements for T cell activation by the staphylococcal toxic shock syndrome toxin-1," Journal of immunology 1990 Mar 15;144(6):2089-95.   (MEDL:2313089 PMID: 2313089 #J0114147)    

    The activation of Ag-specific, Ia molecule-restricted, TCR V beta 3+ T cell clones by staphylococcal toxic shock syndrome toxin-1 (TSST-1), was investigated. The results show that although Ag- and TSST-1-induced activation of T cell clones both require TCR expression and similar biologic activation signals, the Ia molecule requirement for TSST-1 recognition was much less stringent than that observed for antigenic peptide recognition. In addition, T cell clones recognized TSST-1 without processing by APC. These results suggest that the ability of TSST-1 to polyclonally activate T cells is dependent on TCR recognition of the intact toxin molecule bound to a nonpolymorphic region(s) of the Ia molecule resulting in the same activation events induced by Ag recognition.

  115. Novick, Richard P. Molecular biology of the staphlococci    New York : VCH Publishers, 1990. xxvii, 639 p. ; 24 cm. .   (offline #B0001406)

  116. Bohach GA, Kreiswirth BN, Novick RP, Schlievert PM. "Analysis of toxic shock syndrome isolates producing staphylococcal enterotoxins B and C1 with use of southern hybridization and immunologic assays," Reviews of infectious diseases 1989 Jan-Feb;11 Suppl 1:S75-81; discussion S81-2.   (MEDL:2494691 PMID: 2494691 #J0114154)    

    A study was undertaken to evaluate the production of enterotoxin B (Ent B), EntC1, and toxic shock syndrome toxin 1 (TSST-1) by isolates of Staphylococcus aureus from patients with toxic shock syndrome (TSS) and from a variety of other sources. Levels of toxin in culture supernatants were measured by a quantitative immunodiffusion assay. Most vaginal TSS isolates produced TSST-1, either alone or with EntC1. However, strains that produced EntB or EntC1 but did not express TSST-1 were commonly isolated from patients with nonmenstrual TSS; EntB was usually produced alone or--rarely--with EntC1. These results were confirmed by probing DNA from representative isolates with an internal probe of the EntC1 gene (entC1). Extensive sequence homology between entC1 and entB enabled detection of both genes under conditions of high stringency. The genomic location of entC1 in strains producing both EntC1 and TSST-1 varied little but was dependent on the mammalian host. In contrast, the genomic location of entC1 or entB in strains producing EntC1 or EntB alone was variable. These results suggest that these genes are contained on mobile elements.

  117. Gennaro ML, Iordanescu S, Novick RP, Murray RW, Steck TR, Khan SA. "Functional organization of the plasmid pT181 replication origin," Journal of molecular biology 1989 Jan 20;205(2):355-62.   (MEDL:2926812 PMID: 2926812 #J0114151)    

    Replication of the staphylococcal plasmid pT181 is initiated at the origin (ori) with the introduction of a site-specific nick by the plasmid-encoded initiator protein RepC. Deletion analysis showed that a sequence of about 70 base-pairs is required for full ori function, including the ability to compete with a co-resident wild-type origin for the trans-acting RepC protein. A shorter sequence of 43 base-pairs is sufficient for origin function in the absence of competition. Single and double point mutations within these 43 base-pairs were used to determine the sequence requirement for replication within the minimal origin. Deletion mutants and point mutants were tested in replication and competition assays in vivo and in vitro, and in a RepC-mediated nicking assay.

  118. Kreiswirth BN, Projan SJ, Schlievert PM, Novick RP. "Toxic shock syndrome toxin 1 is encoded by a variable genetic element," Reviews of infectious diseases 1989 Jan-Feb;11 Suppl 1:S83-8; discussion S88-9.   (MEDL:2564693 PMID: 2564693 #J0114153)    

    The primary cause of toxic shock syndrome is toxic shock syndrome toxin 1 (TSST-1), a 22,049-dalton exotoxin. Approximately 20% of Staphylococcus aureus isolates produce TSST-1; the production of this toxin is therefore a variable genetic trait. The TSST-1 gene and its flanking sequences are found on a genetic element that is present in TSST-1-positive isolates and absent in TSST-1-negative isolates. Preliminary sequence data and Southern hybridization experiments with the cloned flanking sequences have provided evidence that the TSST-1 element is 4-7 kilobases in size. Hybridization analysis of whole-cell DNA from two genetically mapped TSST-1-positive strains has demonstrated that the TSST-1 element has at least two chromosomal locations. This finding suggests that the element is mobile. Biotyping of 75 TSST-1-positive isolates showed that the large majority were tryptophan-negative, and Southern hybridization analysis of whole-cell DNA from these isolates revealed a common blotting pattern--an observation suggesting that these strains are clonal.

  119. Novick RP, Iordanescu S, Projan SJ, Kornblum J, Edelman I. "pT181 plasmid replication is regulated by a countertranscript-driven transcriptional attenuator," Cell 1989 Oct 20;59(2):395-404.   (MEDL:2478296 PMID: 2478296 #J0114149)    

    pT181 is the prototype of a family of staphylococcal plasmids that regulate their replication by means of antisense RNAs (countertranscripts) that block expression of the plasmid-coded initiator protein. In this paper, we show that the pT181 countertranscripts induce premature termination (attenuation) of the initiator mRNA by promoting the formation of a termination-causing hairpin just 5' to the initiator start codon. In the absence of the countertranscripts, an upstream sequence, the preemptor, pairs with the proximal arm of the terminator hairpin, preventing termination and permitting transcription of the initiator gene. This system thus differs from the classical attenuators in that attenuation is driven by antisense RNAs rather than by tRNA-induced stalling of ribosomes.

  120. Novick RP. "Staphylococcal plasmids and their replication," Annual review of microbiology 1989;43:537-65.   (MEDL:2679362 PMID: 2679362 #J0114152)    

  121. Projan SJ, Kornblum J, Kreiswirth B, Moghazeh SL, Eisner W, Novick RP. "Nucleotide sequence: the beta-hemolysin gene of Staphylococcus aureus," Nucleic acids research 1989 Apr 25;17(8):3305.   (MEDL:2726469 PMID: 2726469 #J0114150)    

  122. Chu MC, Kreiswirth BN, Pattee PA, Novick RP, Melish ME, James JF. "Association of toxic shock toxin-1 determinant with a heterologous insertion at multiple loci in the Staphylococcus aureus chromosome," Infection & immunity 1988 Oct;56(10):2702-8.   (MEDL:2843468 PMID: 2843468 #J0114156)    

    Most Staphylococcus aureus strains associated with toxic shock syndrome and producing toxic shock syndrome toxin 1 (TSST-1) require tryptophan because of a genetic defect in tryptophan biosynthesis. The association between TSST-1 production and tryptophan auxotrophy was not correlated with the phage type, the colonization site, or the disease status of the patient from whom the isolate came. Protoplast fusion and transformation mapping located the genetic determinant of TSST-1 production (tst) very close to the trp operon in such strains and very close to tyrB in a Trp+ TSST-1+ strain. Southern blot hybridization of ClaI-restricted chromosomal DNA with a tst-specific probe revealed a common homologous segment in all of the Trp+ strains with tst linked to tyrB. These results confirmed that the tst determinant in Trp- strains is located at one site, whereas in Trp+ TSST-1+ strains the determinant is located elsewhere on the S. aureus chromosome. It is suggested that the TSST-1 determinant is associated with the insertion of a transposonlike segment into several sites on the S. aureus chromosome.

  123. Gennaro ML, Novick RP. "An enhancer of DNA replication," Journal of bacteriology 1988 Dec;170(12):5709-17.   (MEDL:3192513 PMID: 3192513 #J0114155)    

    cmp, a nucleotide sequence element in the plasmid pT181 of Staphylococcus aureus, acts as an enhancer of DNA replication. When cmp is present on an unrelated vector along with the pT181 origin of replication, it increases the ability of the linked pT181 origin to compete with a coresident pT181 plasmid for the initiator protein RepC. cmp is contained within a 156-base-pair segment, and its deletion from pT181 reduces by twofold the frequency of plasmid replication under derepressed conditions. The enhancer sequence contains a locus of DNA bending, and enhancer activity decreases with distance from the replication origin.

  124. Kornblum JS, Projan SJ, Moghazeh SL, Novick RP. "A rapid method to quantitate non-labeled RNA species in bacterial cells," Gene 1988;63(1):75-85.   (MEDL:2454872 PMID: 2454872 #J0114161)    

    We have developed a rapid method to quantitate specific bacterial RNA species. The method measures the steady-state level of RNA, produces a linear response over more than a 16-fold range of RNA concentration, and can be used for Staphylococcus aureus, Escherichia coli and Bacillus subtilis. In this method, a sheared whole-cell lysate of approx. 7 x 10(8) organisms, prepared as for plasmid screening, is separated on agarose, blotted to a nitrocellulose filter, hybridized with a radiolabeled DNA probe, and autoradiographed. The RNA species are quantitated by counting the radioactive bands on the filter. We have applied the method to the measurement of mRNA induction of the genes encoding beta-lactamase, ermC rRNA methylase, and the alpha-complementing fragment of beta-galactosidase. Upon induction, a ten-fold increase in the mRNA for each gene was observed. The peak mRNA level occurred after 30 min for beta-lactamase, 20 min for beta-galactosidase, and 5 min for the ermC rRNA methylase.

  125. Majumder S, Novick RP. "Intermediates in plasmid pT181 DNA replication," Nucleic acids research 1988 Apr 11;16(7):2897-912.   (MEDL:3368310 PMID: 3368310 #J0114159)    

    Staphylococcus aureus plasmid pT181 is thought to replicate via an asymmetric rolling-circle mechanism. By studying pulse labeled replicative intermediates, here we report that pT181 replication involves: (1) a post-replicative hypersupercoiled monomer and (2) a partially replicated intermediate which lacks superhelicity but is unlike a typical rolling-circle intermediate in that only nascent strands of less than unit length are released by alkali denaturation. A model for pT181 replication is proposed to accommodate this apparent discrepancy.

  126. Murphy BG, Kreiswirth BN, Novick RP, Schlievert PM. "Localization of a biologically important epitope on toxic-shock-syndrome toxin-1," Journal of infectious diseases 1988 Sep;158(3):549-55.   (MEDL:2457635 PMID: 2457635 #J0114157)    

    A monoclonal antibody, designated B-14, inhibits the nonspecific T lymphocyte mitogenicity of toxic-shock-syndrome toxin-1 (TSST-1), and the antibody binds to an internal cyanogen bromide (CNBr) fragment (Mr, 14,000) of the toxin. The epitope recognized by B-14 was further localized to include a decapeptide at the NH2-terminus of the CNBr fragment. The decapeptide inhibited the ELISA and western blot reactivity of B-14 with TSST-1, although it was approximately 10,000-fold less effective than the native toxin. The peptide also inhibited the capacity of B-14 to block TSST-1-induced mitogenicity. A conjugate, consisting of decapeptide4-ovalbumin, was used to hyperimmunize three rabbits. Serum from these rabbits reacted specifically with intact TSST-1 in ELISA and western blots and partially neutralized toxin mitogenicity; however, the serum did not prevent fever and enhancement of susceptibility to endotoxin shock typically seen in rabbits after administration of TSST-1.

  127. Peng HL, Novick RP, Kreiswirth B, Kornblum J, Schlievert P. "Cloning, characterization, and sequencing of an accessory gene regulator (agr) in Staphylococcus aureus," Journal of bacteriology 1988 Sep;170(9):4365-72.   (MEDL:2457579 PMID: 2457579 #J0114158)    

    We have previously identified a gene in Staphylococcus aureus, agr, whose activity is required for high-level post-exponential-phase expression of a series of secreted proteins. In this paper, we describe the cloning of this gene in Escherichia coli by using an inserted transposon (Tn551) as a cloning probe. The cloned gene, consisting of a 241-codon open reading frame containing the site of the transposon insertion, was recloned to an S. aureus vector, pSK265, and shown to be functional in S. aureus. Activity was evaluated by determinations of alpha-hemolysin, beta-hemolysin, and toxic shock syndrome toxin-1 production in early-stationary-phase cultures. The cloned gene showed considerable variation with respect to different exoproteins and different host strains compared with the chromosomal agr determinant; this variation could not be attributed to the higher copy number of the cloned gene and probably reflects inapparent subtleties of the regulatory system.

  128. Projan SJ, Moghazeh S, Novick RP. "Nucleotide sequence of pS194, a streptomycin-resistance plasmid from Staphylococcus aureus," Nucleic acids research 1988 Mar 25;16(5):2179-87.   (MEDL:3357770 PMID: 3357770 #J0114160)    

    pS194 is a naturally occurring Staphylococcus aureus plasmid encoding streptomycin resistance. The plasmid has a copy number of about 25 per cell, and belongs to the inc5 incompatibility group. The nucleotide sequence of pS194 has been determined and consists of 4397 base pairs including four open reading frames potentially encoding proteins of greater than 100 amino acids. All four of these reading frames are on the same coding strand. The first reading frame, repE, encodes a 38 kd protein specifically required for pS194 replication. The second open reading frame, str, encodes a 34 kd polypeptide required for streptomycin resistance, probably a streptomycin adenylyltransferase. The third potential polypeptide, rlx, would be 37 kd and is probably required for relaxation complex formation and plasmid mobilization by conjugative plasmids. The fourth, orfD, overlapping the rlx reading frame, is potentially 27 kd, and may also be involved in mobilization.

  129. Gennaro ML, Kornblum J, Novick RP. "A site-specific recombination function in Staphylococcus aureus plasmids," Journal of bacteriology 1987 Jun;169(6):2601-10.   (MEDL:3584064 PMID: 3584064 #J0114165)    

    All known small staphylococcal plasmids possess one or two recombination sites at which site-specific cointegrate formation occurs. One of these sites, RSA, is present on two small multicopy plasmids, pT181 and pE194; it consists of 24 base pairs of identity in the two plasmids, the 'core,' flanked by some 50 base pairs of decreasing homology. Here we show that recombination at RSA is recA independent and is mediated by a plasmid-encoded, trans-acting protein, Pre (plasmid recombination). Pre-mediated recombination is site specific in that it occurs within the core sequence of RSA in a recA1 host. Recombination also occurs between two intramolecular RSA sites. Unlike site-specific recombination systems encoded by other plasmids, Pre-RSA is not involved in plasmid maintenance.

  130. Gruss AD, Ross HF, Novick RP. "Functional analysis of a palindromic sequence required for normal replication of several staphylococcal plasmids," Proceedings of the National Academy of Sciences of the United States of America 1987 Apr;84(8):2165-9.   (MEDL:3104910 PMID: 3104910 #J0114168)    

    Most small multicopy antibiotic-resistance plasmids of Staphylococcus aureus contain a major axis of hyphenated dyad symmetry (palA) that is required for normal replication and stability, although located outside of the minimal replicon. Rearrangements affecting palA cause plasmid instability, a marked reduction in copy number, and the accumulation of large quantities of strand-specific circular single-stranded plasmid DNA. In view of the recent observation that pT181 initiates replication by a nick and 3'-extension mechanism (S. Khan, personal communication), it is suggested that these plasmids replicate by an asymmetric rolling-circle mechanism in which the displaced plus strand remains single stranded until palA is exposed, forming a hairpin that serves as the lagging strand origin.

  131. Highlander SK, Novick RP. "Plasmid repopulation kinetics in Staphylococcus aureus," Plasmid 1987 May;17(3):210-21.   (MEDL:2442785 PMID: 2442785 #J0114167)    

    We have analyzed the kinetic route by which the indirectly controlled Staphylococcus aureus plasmid, pT181, responds to and corrects fluctuations in copy number. The kinetics of copy number correction from low to steady-state levels (termed repopulation) were determined using two different methods of copy number reduction. Thermosensitive replication (Tsr) mutants of pT181 were grown at nonpermissive temperatures to lower copy number and then shifted to a permissive temperature to allow repopulation. After the downshift, both wild-type and copy mutant plasmids, with active inhibitors, exhibited a burst of exponential replication that resulted in a two- to threefold overshoot of normal steady-state copy numbers. This was followed by inhibition of replication and eventual reestablishment of the steady-state replication rate. Similar replication kinetics were observed when these plasmids were introduced into naive cells by high-frequency transduction. By contrast, a pT181 copy mutant with a nonfunctional inhibitor-target regulation did not overshoot its steady-state copy number, but instead repopulated asymptotically. These results suggest that at low copy numbers, pT181 and its derivatives replicate at near-maximal rates and overshoot prior to the establishment of an inhibitory concentration of repressor. The maximal replication rate is independent of the plasmid's cop genotype. As the copy number increases, inhibitor accumulates and eventually reduces the replication rate. In the absence of an active inhibitor, the steady-state copy number is established at a level that must be limited by some other invariant factor.

  132. Kreiswirth BN, Handley JP, Schlievert PM, Novick RP. "Cloning and expression of streptococcal pyrogenic exotoxin A and staphylococcal toxic shock syndrome toxin-1 in Bacillus subtilis," Molecular & general genetics 1987 Jun;208(1-2):84-7.   (MEDL:3112526 PMID: 3112526 #J0114166)    

    The genes encoding streptococcal pyrogenic exotoxin type A (SPE A) and staphylococcal toxic shock syndrome toxin-1 (TSST-1) were stably cloned and expressed in Bacillus subtilis. In the non-pathogenic Bacillus background, the recombinant speA clone expressed 32-fold more SPE A than the native streptococcus, and similarly, the recombinant plasmid harboring tst expressed 4-fold more TSST-1 in Bacillus than in the native Staphylococcus aureus. The Bacillus-derived products were secreted into the culture fluid, were resistant to proteolytic degradation and their biological activities mimicked native preparations.

  133. Kreiswirth BN, Schlievert PM, Novick RP. "Evaluation of coagulase-negative staphylococci for ability to produce toxic shock syndrome toxin 1," Journal of clinical microbiology 1987 Oct;25(10):2028-9.   (MEDL:3117846 PMID: 3117846 #J0114163)    

    A large and diverse group of coagulase-negative staphylococci were assayed for the ability to produce toxic shock syndrome toxin 1 (TSST-1) by immunological reactivity, and whole-cell DNAs from 33 of these strains were hybridized with a TSST-1-specific gene probe. None of the strains tested, including isolates that have been reported as TSST-1+, produced the exotoxin, and no DNA homology was found with the gene probe.

  134. Novick RP. "Plasmid incompatibility," Microbiological reviews 1987 Dec;51(4):381-95.   (MEDL:3325793 PMID: 3325793 #J0114162)    

  135. Recsei PA, Gruss AD, Novick RP. "Cloning, sequence, and expression of the lysostaphin gene from Staphylococcus simulans," Proceedings of the National Academy of Sciences of the United States of America 1987 Mar;84(5):1127-31.   (MEDL:3547405 PMID: 3547405 #J0114170)    

    A 1.5-kilobase-pair fragment of DNA that contains the lysostaphin gene from Staphylococcus simulans and its flanking sequences has been cloned and completely sequenced. The gene encodes a preproenzyme of Mr 42,000. The NH2-terminal sequence of the preproenzyme is composed of a signal peptide followed by seven tandem repeats of a 13-amino acid sequence. Conversion of prolysostaphin to the mature enzyme occurs extracellularly in cultures of S. simulans and involves removal of the NH2-terminal portion of the proenzyme that contains the tandem repeats. The high degree of homology of the repeats suggests that they have arisen by duplication of a 39-base-pair sequence of DNA. In S. simulans, the lysostaphin gene is present on a large beta-lactamase plasmid.

  136. Wang PZ, Novick RP. "Nucleotide sequence and expression of the beta-lactamase gene from Staphylococcus aureus plasmid pI258 in Escherichia coli, Bacillus subtilis, and Staphylococcus aureus," Journal of bacteriology 1987 Apr;169(4):1763-6.   (MEDL:3104315 PMID: 3104315 #J0114169)    

    The structural gene for beta-lactamase in the Staphylococcus aureus plasmid pI258 was cloned into a Staphylococcus aureus-Bacillus subtilis-Escherichia coli shuttle vector, pWN101, and the nucleotide sequence of the gene was determined. pWN101 was structurally stable and the beta-lactamase gene was expressed efficiently from its native promoter and ribosome-binding site in all three hosts.

  137. Wang PZ, Projan SJ, Leason KR, Novick RP. "Translational fusion with a secretory enzyme as an indicator," Journal of bacteriology 1987 Jul;169(7):3082-7.   (MEDL:3496329 PMID: 3496329 #J0114164)    

    A novel type of translational fusion system has been developed by using a secretory protein, staphylococcal beta-lactamase, as an indicator. The beta-lactamase structural gene was modified to provide N-terminal extensions of 13 and 162 amino acids, and in both cases, the fusion protein was processed and the mature active enzyme was secreted; thus, the expression of a particular upstream gene can be analyzed by monitoring the beta-lactamase activity.

  138. Blomster-Hautamaa DA, Novick RP, Schlievert PM. "Localization of biologic functions of toxic shock syndrome toxin-1 by use of monoclonal antibodies and cyanogen bromide-generated toxin fragments," Journal of immunology 1986 Dec 1;137(11):3572-6.   (MEDL:3782792 PMID: 3782792 #J0114171)    

    Monoclonal antibodies (mAb) against toxic shock syndrome toxin-1 (TSST-1) were generated that block two of the most important biologic activities of TSST-1, nonspecific T lymphocyte mitogenicity and the suppression of immunoglobulin synthesis. Fourteen hybridomas producing antibody against TSST-1 were isolated independently. The culture supernatant and ascitic fluids from each were analyzed to determine the mAb isotypes. Seven of the mAb were IgG1, and the remaining seven were IgM; all the mAb had kappa light chains. Immunoglobulin was partially purified from hybridoma-generated ascitic fluid by ammonium sulfate precipitation and tested for the ability to block TSST-1-induced mitogenicity and immunosuppression. Three mAb (all IgG1) were shown to block both the toxin-induced mitogenicity and the suppression. None of the mAb tested inhibited just one of the two toxin activities. The neutralizing mAb were then used in Western analysis with previously mapped cyanogen bromide (CNBr)-generated toxin fragments to localize the aforementioned biologic functions. The Western blot analysis showed that the mitogenic and the suppressive functions of TSST-1 were located on a 14,000 dalton internal CNBr fragment.

  139. Blomster-Hautamaa DA, Kreiswirth BN, Kornblum JS, Novick RP, Schlievert PM. "The nucleotide and partial amino acid sequence of toxic shock syndrome toxin-1," Journal of biological chemistry 1986 Nov 25;261(33):15783-6.   (MEDL:3782090 PMID: 3782090 #J0114173)    

    The nucleotide sequence of toxic shock syndrome toxin-1 (TSST-1) has been determined. In addition, one-third of the predicted amino acid sequence was confirmed by amino acid sequence analysis of cyanogen bromide-generated TSST-1 protein fragments. The DNA sequencing results identified a 708-base pair open reading frame starting with an ATG, 7 base pairs downstream from a Shine-Dalgarno sequence, and terminating at a UAA stop codon. Amino acid analysis of the intact protein defined the NH2 terminus of the mature protein and located the cleavage point for the signal peptide (Ala/Ser). The signal peptide contained the first 40 amino acids and had characteristic structural similarities with other bacterial signal peptides. The coding sequence of the mature protein was 585 base pairs (194 amino acids) in length, and the molecular weight of the predicted protein was 22,049. This is in good agreement with the previously reported molecular weight of TSST-1 (22,000), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NH2-terminal amino acid sequence analysis performed on isolated TSST-1 CNBr fragments determined the position of the peptides in the TSST-1 sequence and verified the predicted amino acid sequence in those positions. Computer analyses of the amino acid sequence showed that TSST-1 has little or no sequence homology with biologically related toxins, streptococcal pyrogenic exotoxin A, and staphylococcal enterotoxins B and C.

  140. Blomster-Hautamaa DA, Kreiswirth BN, Novick RP, Schlievert PM. "Resolution of highly purified toxic-shock syndrome toxin 1 into two distinct proteins by isoelectric focusing," Biochemistry 1986 Jan 14;25(1):54-9.   (MEDL:3954993 PMID: 3954993 #J0114179)    

    Highly purified toxic-shock syndrome toxin 1 (TSST-1) was prepared by differential precipitation with ethanol and resolubilization in water followed by successive electrofocusing in pH gradients of 3-10, 6-8, and 6.5-7.5. TSST-1, thus isolated, migrated as two distinct protein bands with isoelectric points of 7.08 (TSST-1a) and 7.22 (TSST-1b). When tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both toxins migrated as homogeneous bands with molecular weights of 22 000. The gel bands were visualized by silver staining. The two toxins have nearly identical amino acid compositions and are immunologically identical as shown by Ouchterlony reactivity against TSST-1 hyperimmune serum. TSST-1a and TSST-1b have the same biological activities as TSST-1: the capacity to induce fever, enhancement of host susceptibility to lethal endotoxin shock, nonspecific T lymphocyte mitogenicity, and suppression of immunoglobulin M synthesis against sheep erythrocytes. These two proteins have been isolated from several different TSS-associated Staphylococcus aureus strains. The data suggest that the differences in isoelectric point result either from the presence of a cofactor or from alternative conformations. Since only two bands appear, microheterogeneity as a result of deamination or acetylation is unlikely.

  141. Gennaro ML, Novick RP. "cmp, a cis-acting plasmid locus that increases interaction between replication origin and initiator protein," Journal of bacteriology 1986 Oct;168(1):160-6.   (MEDL:3759903 PMID: 3759903 #J0114176)    

    pT181, a 4.4-kilobase multicopy plasmid of Staphylococcus aureus, encodes a trans-acting initiator protein, RepC, which was rate limiting for replication. Deletions in a 500-base-pair region of the plasmid external to the minimal replicon decreased the ability of the plasmid to compete with a coexisting incompatible plasmid. These deletions, which define a region called cmp (for competition), appeared to affect the interaction of RepC and the plasmid origin of replication. However, in the homoplasmid state the deletions affected neither copy number nor plasmid stability. The Cmp phenotype is orientation independent, and cmp defects could not be complemented in trans.

  142. Kornblum J, Hartman BJ, Novick RP, Tomasz A. "Conversion of a homogeneously methicillin-resistant strain of Staphylococcus aureus to heterogeneous resistance by Tn551-mediated insertional inactivation," European Journal of clinical microbiology 1986 Dec;5(6):714-8.   (MEDL:3026802 PMID: 3026802 #J0114172)    

    Plasmid pRN3208, thermosensitive for replication, and carrying the erythromycin transposon Tn551, was used for insertional inactivation of methicillin resistance in a highly and homogeneously resistant strain of Staphylococcus aureus. Two kinds of insertionally inactivated cells were obtained. Cultures of the major class contained highly methicillin resistant cells with a frequency of about 10(-3) to 10(-4), produced DNA with methicillin resistance transforming activity, and also produced penicillin binding protein 2a, the 78 kd low affinity penicillin binding protein characteristic of methicillin resistant Staphylococcus aureus, in apparently normal quantities. The single member of class B had no detectable methicillin resistant cells (less than 10(-8)) with an MIC greater than 1 micrograms/ml, contained no DNA with methicillin resistant transforming activity and no penicillin binding protein 2a. The data suggest that in the class A cells insertional inactivation did not affect the structural gene(s) of methicillin resistance but a regulatory locus or loci needed for the homogeneous expression of resistance.

  143. Kreiswirth BN, Kravitz GR, Schlievert PM, Novick RP. "Nosocomial transmission of a strain of Staphylococcus aureus causing toxic shock syndrome," Annals of internal medicine 1986 Nov;105(5):704-7.   (MEDL:3021039 PMID: 3021039 #J0114175)    

    A strain of Staphylococcus aureus producing toxic shock syndrome toxin-1 was repeatedly isolated from the nares of a neurosurgeon. This strain was identical to strains cultured from two of his patients who developed toxic shock syndrome after laminectomy. The relatedness of the isolates was shown by Southern blot hybridization analyses using chromosomal transposons as probes. This approach should be considered, in addition to standard bacteriologic techniques, as an effective method to analyze the relatedness of nosocomial isolates.

  144. Levy, Stuart B.; Novick, Richard P. Antibiotic resistance genes : ecology, transfer, and expression    Cold Spring Harbor, N.Y. : Cold Spring Harbor Laboratory, 1986. xvii, 436 p. : ill. ; 24 cm (Banbury report ; 24).   (MEDCAT:b104804a #B0000078)

  145. Manch-Citron JN, Gennaro ML, Majumder S, Novick RP. "RepC is rate limiting for pT181 plasmid replication," Plasmid 1986 Sep;16(2):108-15.   (MEDL:3462754 PMID: 3462754 #J0114177)    

    The effect on pT181 plasmid replication of the concentration of the plasmid-coded initiator protein, RepC, has been analyzed. In one type of experiment, plasmid replication was found to stop immediately after the addition of an inhibitory concentration of chloramphenicol (Cm) to growing cultures. Chromosomal replication showed the slow turnoff that is usual for Cm inhibition. Because plasmid replication rate is determined autogenously, no host factor can be rate limiting, suggesting that the specific factor affected is Rep C. In another type of experiment, we constructed a translational fusion between the repC coding sequence and a translationally inducible Cm-acetylase gene, cat-86, using pUB110 as the carrier replicon. The fusion plasmid showed an eightfold amplification of its own copy number and a similar amplification of a co-resident pT181 plasmid upon Cm induction. The amplified plasmids did not show autocatalytic runaway replication but rather established stable elevated copy numbers, indicating the existence of a secondary level of regulation. These results suggest that RepC is rate limiting for pT181 replication and support the hypothesis that pT181 replication is regulated at the level of RepC synthesis. The nature of the secondary regulation is unknown.

  146. Novick RP, Edelman I, Lofdahl S. "Small Staphylococcus aureus plasmids are transduced as linear multimers that are formed and resolved by replicative processes," Journal of molecular biology 1986 Nov 20;192(2):209-20.   (MEDL:2951524 PMID: 2951524 #J0114174)    

    The molecular processes involved in the transduction of small staphylococcal plasmids by a generalized transducing phage, phi 11, have been analysed. The plasmids are transduced in the form of linear concatemers containing only plasmid DNA; plasmid-initiated replication is required for their generation but additive interplasmid recombination is not. Concatemers are probably generated by the interaction of one or more phage functions with replicating plasmid DNA. Insertion of any restriction fragment of the phage into the plasmid causes an approximately 10(5)-fold increase in transduction frequency, regardless of the size or genetic content of the fragment. The resulting transducing particles (Hft particles) contain mostly pure linear concatemers composed of tandem repeats of the plasmid::phage chimera, and their production requires active plasmid-initiated replication. The high frequency of transduction is a consequence of homologous recombination between the linear chimeric and phage concatemers, which has the effect of introducing an efficient pac site into the former. Following introduction into lysogenic recipient bacteria, the transducing DNA is first converted to the supercoiled form, then processed to monomers by a mechanism that requires the active participation of the plasmid replication system.

  147. Projan SJ, Novick RP. "Incompatibility between plasmids with independent copy control," Molecular & general genetics 1986 Aug;204(2):341-8.   (MEDL:3020371 PMID: 3020371 #J0114178)    

    Incompatibility between autonomous plasmids has been attributed, for the most part, to interaction between plasmids' negative control systems and/or partitioning systems. In this report it is shown that indirectly regulated plasmids with non-interactive negative control systems are incompatible on the basis of their shared initiator protein. This principle was demonstrated for a family of Staphylococcus aureus plasmids whose copy number is regulated by inhibitory RNAs that control the production of a rate-limiting, trans-active, initiator protein. We have constructed a pair of plasmids that have the same regulation systems and different initiator proteins and another pair with different regulation systems and the same initiators. Both of these pairs of plasmids were shown to be incompatible.

  148. Recsei P, Kreiswirth B, O'Reilly M, Schlievert P, Gruss A, Novick RP. "Regulation of exoprotein gene expression in Staphylococcus aureus by agar," Molecular & general genetics 1986 Jan;202(1):58-61.   (MEDL:3007938 PMID: 3007938 #J0114180)    

    Insertion of the erythromycin-resistance transposon Tn551 into the Staphylococcus aureus chromosome at a site which maps between the purB and ilv loci has a pleiotrophic effect on the production of a number of extracellular proteins. Production of alpha, beta and delta hemolysin, toxic shock syndrome toxin (TSST-1) and staphylokinase was depressed about fifty-fold while protein A production was elevated twenty-fold. Hybridization analysis showed that the defect in expression of TSST-1 and alpha hemolysin was at the transcriptional level. Inability of the mutant strain to express either a cloned TSST-1 gene or the chromosomal gene indicates that the transposon has inactivated a trans-active positive control element. This element has been designated agr for accessory gene regulator.

  149. de Azavedo JC, Foster TJ, Hartigan PJ, Arbuthnott JP, O'Reilly M, Kreiswirth BN, Novick RP. "Expression of the cloned toxic shock syndrome toxin 1 gene (tst) in vivo with a rabbit uterine model," Infection & immunity 1985 Oct;50(1):304-9.   (MEDL:4044040 PMID: 4044040 #J0114181)    

    Toxic shock syndrome (TSS) toxin 1 (TSST1) is produced by strains of Staphylococcus aureus associated with TSS. Purified TSST1 induces in rabbits a shock-like illness with many features similar to TSS in humans. These symptoms were also induced by TSST1-producing bacteria in diffusion chambers implanted in the rabbit uterus. Naturally occurring TSST1+ strains and a TSST1- strain harboring a pE194-derived plasmid carrying the cloned TSST1 determinant tst gave the same symptoms. TSST1- strains and a TSST1- strain carrying a pE194-tst plasmid with a deletion of the tst gene had no effect in rabbits. The results with the plasmid-carrying TSST1+ and TSST1- strains, which were isogenic apart from tst, show that the toxin is responsible for the illness in rabbits and suggest that it is a major factor in the pathogenesis of TSS.

  150. Kumar CC, Novick RP. "Plasmid pT181 replication is regulated by two countertranscripts," Proceedings of the National Academy of Sciences of the United States of America 1985 Feb;82(3):638-42.   (MEDL:2579377 PMID: 2579377 #J0114183)    

    A transcription map of the replication control region of the Staphylococcus aureus plasmid pT181 has been constructed. Two major leftward transcripts, RNA III and RNA IV, start at positions 339 and 413, respectively. These two RNAs can serve as mRNAs for a plasmid-specific replication protein RepC. Two short rightward transcripts, RNA I and RNA II, approximately 85 and 150 nucleotides long, respectively, start at position 246. These rightward transcripts (referred to as countertranscripts) do not appear to be translated but act directly as negative regulators of plasmid replication, probably by interfering with translation of the RepC mRNAs. There is no significant base sequence homology among the countertranscripts of pT181, ColE1, and R1/NR1/R6-5, suggesting that the structural parallelism has risen by convergent molecular evolution.

  151. Novick RP, Murphy E. "MLS-resistance determinants in Staphylococcus aureus and their molecular evolution," Journal of antimicrobial chemotherapy 1985 Jul;16 Suppl A:101-10.   (MEDL:3932297 PMID: 3932297 #J0114182)    

    This paper describes the genetic phenomenology of resistance to macrolide-lincosamide-streptogramin B antibiotics (MLSr) in Staphylococcus aureus and attempts to place this phenomenology in a broad evolutionary context. As antibiotic resistance in general and MLS resistance in particular are typical variable traits in bacteria of clinical interest, we shall begin by introducing the concept of variable genetic traits, as outlined in Figure 1. Variable traits are those that are expressed by some strains of a given species but not by others--in comparison to constant traits which are always present as part of the standard genetic make-up of the species and have constant chromosomal locations. Variable traits are often associated with variable and mobile genetic elements and it is suggested that, in general, they are not likely to have evolved as such in the species in which they are found. Rather, they will most probably have evolved as constant (chromosomal) traits in other species and acquired genetic mobility much later as a rare occurrence in that species. These rarely occurring mobile variants would then spread horizontally within a range of new species. The MLSr determinants in Gram-positive bacteria would appear to represent a classic example of this process. Their remarkable variability will be described as the extant end-point of the process and a probable evolutionary pathway will be traced back to the streptomycetes which are a likely primary source.

  152. Novick RP, Projan SJ, Kumar CC, Carleton S, Gruss A, Highlander SK, Kornblum J. "Replication control for pT181, an indirectly regulated plasmid," Basic life sciences 1985;30:299-320.   (MEDL:2990414 PMID: 2990414 #J0114185)    

    PT181 is a fully sequenced Staphylococcus aureus plasmid whose size is 4,437 bp. It specifies tetracycline resistance and has a copy number of about 22 per cell in exponentially growing cultures. The functional organization of the pT181 replicon is centered around the coding sequence for a 35-kd protein, RepC, that is absolutely required for replication of the plasmid. The replication origin is contained within the repC coding sequence and the region immediately 5' to the RepC start is involved in control of the plasmid replication rate. PT181 replication is controlled at the level of RepC synthesis by a negative regulatory system that is functionally similar to that of the Co1E1 and IncFII plasmids of Escherichia coli. The pT181 control circuit involves 2 short transcripts, RNA I and RNA II, that are transcribed from the region specifying the 5' end of the untranslated repC mRNA leader and in the opposite direction. These are referred to as countertranscripts. The countertranscripts regulate RepC synthesis by a mechanism that probably involves interaction with the repC mRNA leader in a manner that interferes with translation. Both of the countertranscripts seem to be necessary for normal replication control; their separate roles remain unclear. Unlike plasmids of the Co1E1 and IncFII groups, plasmids such as Co1E1 are considered to have direct regulation of replication because the inhibitory element of the copy control circuit directly inhibits the initiation of replication. Plasmids such as pT181 are considered to have indirect regulation of replication because the product of the regulated step, RepC, is trans-active. Plasmids of the IncFII type are considered to have direct regulation of replication because the product of the regulated step, RepA is cis-active The analysis of pT181 replication physiology has illustrated 2 important differences between directly and indirectly regulated plasmids: a) for directly regulated plasmids, copy mutants specifying a normal inhibitor substance but an inactive target site exclude the wild-type or recessive mutants by directly interfering with their replication. Analogous mutants of indirectly regulated plasmids coexist readily with the wild-type and all mutants (although they do manifest segregational incompatibility) because the Rep protein is always shared by all plasmids in the cell, regardless of its source. b) Mutations of directly regulated plasmids in the region where target transcript and countertranscript overlap may give rise to totally new incompatibility groups because they engender independently self-correcting copy pools.(ABSTRACT TRUNCATED AT 400 WORDS).

  153. Projan SJ, Kornblum J, Moghazeh SL, Edelman I, Gennaro ML, Novick RP. "Comparative sequence and functional analysis of pT181 and pC221, cognate plasmid replicons from Staphylococcus aureus," Molecular & general genetics 1985;199(3):452-64.   (MEDL:2993795 PMID: 2993795 #J0114184)    

    The nucleotide sequence of pC221, a 4.6 kb Staphylococcus aureus plasmid is presented. The replication region of the plasmid is identified and compared with the corresponding region of pT181, a compatible but related plasmid. Both plasmids encode trans-active replicon-specific initiator proteins, RepC for pT181 and RepD for pC221. Plasmid replication rate is controlled by regulation of the rate of synthesis of the initiator protein by means of inhibitory 5' countertranscripts. Key elements of the control system are closely conserved between the two plasmids whereas less critical elements show extensive divergence. Overall architecture is also conserved, suggesting functional parallelism. The replication origin for both plasmids is contained within the N-terminal region of the initiator protein coding sequence; the two coding sequences are highly homologous but have two important areas of divergence, one within the origin region, the other near the C-terminus. In vivo recombinants between the two plasmids isolated previously (Iordanescu 1979) have crossover points within the initiator gene, between the two divergent regions. The recombinant plasmids have hybrid initiator proteins and are defective for replication, requiring the simultaneous presence of the parental plasmid from which their origin is derived. They are able to complement replication-defective mutants of the other parental plasmid, suggesting that the recognition specificity of the hybrid initiator protein resides in its C-terminal end and that the specific recognition site for the protein corresponds to the divergent region within the origin.

  154. Betley MJ, Lofdahl S, Kreiswirth BN, Bergdoll MS, Novick RP. "Staphylococcal enterotoxin A gene is associated with a variable genetic element," Proceedings of the National Academy of Sciences of the United States of America 1984 Aug;81(16):5179-83.   (MEDL:6089183 PMID: 6089183 #J0114188)    

    The genetic determinant of Staphylococcus aureus enterotoxin A (SEA) has been cloned in pBR322 in Escherichia coli and found to be expressed and secreted into the periplasmic space in that organism. The SEA gene (entA) is within a 2.5-kilobase-pair HindIII fragment that is part of a discrete genetic element 8-12 kilobase pairs in length. This entA element has a standard chromosomal location [between the purine (pur) and isoleucine-valine (ilv) markers] in most S. aureus strains. In some strains it is unlinked to pur-ilv. However, its internal structure is conserved at different locations. Some naturally occurring SEA-nonproducer (EntA-) strains lack the entire entA element, and one instance of its spontaneous loss is reported. Other naturally occurring strains have EntA- structural variants of the element at the same pur-ilv location at which the intact element is most commonly found. Some of these strains are EntA-, others are EntA+; the latter have a second, unlinked copy of the element containing their functional entA gene. These results suggest that entA is associated with a structurally unstable, possibly mobile, discrete genetic element.

  155. Carleton S, Projan SJ, Highlander SK, Moghazeh SM, Novick RP. "Control of pT181 replication II. Mutational analysis," EMBO journal 1984 Oct;3(10):2407-14.   (MEDL:6437809 PMID: 6437809 #J0114187)    

    We describe the isolation and analysis of mutations affecting the regulation of Staphylococcus aureus plasmid pT181 replication. Previous results suggested that regulation is achieved by control of the synthesis of RepC, a plasmid-coded replication protein and that the primary negative control element is CopA RNA, which consists of two transcripts that are complementary to the 5' region of the repC mRNA leader. CopA inhibition probably involves a base pairing interaction with the complementary region of the RepC mRNA leader which would facilitate the formation of a downstream stem-loop in the leader that occludes the repC ribosome binding site. RepC is freely diffusible so that regulation of pT181 replication is indirect. Both CopA RNA-sensitive (recessive) and -insensitive (dominant) mutants were isolated. The recessives have defects in CopA RNA structure or activity, the dominants have defects in the site of action (target) of the inhibitor. Some dominants were located within the copA coding sequence. These therefore affect the structure of CopA RNA as well as that of its target. Other dominant mutations mapped outside of the copA gene and therefore produced wild-type CopA RNA. In contrast to directly regulated plasmids, pT181 copy mutants producing wild-type inhibitor could be co-maintained with the wild-type plasmid and mutational changes in inhibitor-target specificity did not change incompatibility specificity.

  156. Kreiswirth BN, O'Reilly M, Novick RP. "Genetic characterization and cloning of the toxic shock syndrome exotoxin," Survey & synthesis of pathology research 1984;3(1):73-82.   (MEDL:6438758 PMID: 6438758 #J0114190)    

  157. Novick RP, Adler GK, Projan SJ, Carleton S, Highlander SK, Gruss A, Khan SA, Iordanescu S. "Control of pT181 replication I. The pT181 copy control function acts by inhibiting the synthesis of a replication protein," EMBO journal 1984 Oct;3(10):2399-405.   (MEDL:6499834 PMID: 6499834 #J0114186)    

    pT181 is a fully sequenced 4.4-kb 20 copy Tcr plasmid from Staphylococcus aureus. Its replication system involves a unique unidirectional origin embedded in the coding sequence for a plasmid-determined protein, RepC, that is required for initiation. When joined to a 55 copy carrier plasmid, pE194, pT181 excludes autonomous isologous replicons by inhibiting their replication. Two types of spontaneous pT181 copy mutants have been isolated, one that eliminates sensitivity to this inhibition and another that does not. A spontaneous 180-bp deletion, delta 144, eliminates both the inhibitory activity and sensitivity to it. This deletion increases copy number by 50-fold and RepC production by at least 10-fold. It is located directly upstream from the repC coding sequence and the deletion-bearing plasmid supports the replication of inhibitor-sensitive plasmids in cells containing active inhibitor. This effect is probably due to the overproduction of RepC by the delta 144 plasmid. On the basis of these results, it is suggested that RepC synthesis is negatively controlled by an inhibitor that is encoded directly upstream from the repC coding sequence and acts as a tareget set in the same region. It is likely, therefore, that pT181 replication rate is determined by the level of RepC.

  158. Novick RP, Projan SJ, Rosenblum W, Edelman I. "Staphylococcal plasmid cointegrates are formed by host- and phage-mediated general rec systems that act on short regions of homology," Molecular & general genetics 1984;195(1-2):374-7.   (MEDL:6092862 PMID: 6092862 #J0114191)    

    Cointegrates involving pairs of compatible staphylococcal plasmids can be isolated either by co-selection during transduction (Novick et al. 1981) or by selection for survival at the restrictive temperature of a thermosensitive, replication defective plasmid in the presence of a stable one. Cointegrates are formed by recombination at two specific sites, RSA and RSB. RSB is present on each of six plasmids analyzed, namely pT181, pE194, pC194, pS194, pUB110, and pSN2, and RSA is present on two of these, pT181 and pE194. In this communication, it is shown that the RS represent short regions of homology (RSA is some 70 bp in length and RSB is about 30) embedded in largely non-homologous contexts and that the crossovers take place within these homologous regions. The pT181 and pE194 RSA sequences contain several mismatches which permit the localization of the crossover events to several different sites within the overall RS segment. The recombination system involved is therefore general (homology-specific) rather than site-specific (sequence-specific). Mismatches included within the crossover region are always corrected to the pT181 configuration. The cointegrates are therefore formed by a relatively efficient general rec system that recognizes short regions of homology and gives rise to Holliday junctions that probably involve very short heteroduplex overlaps. The sequence results are consistent with asymmetric single-strand invasion of a contralateral gap with nucleotide conversion by copying. It is noted that RSB has substantial homology with the par sequence of plasmid pSC101, suggesting that it may be involved in plasmid partitioning.

  159. Projan SJ, Novick RP. "Reciprocal intrapool variation in plasmid copy numbers: a characteristic of segregational incompatibility," Plasmid 1984 Jul;12(1):52-60.   (MEDL:6494316 PMID: 6494316 #J0114189)    

    An experimental analysis of the concept that incompatible plasmids occupy a common intracellular pool from which copies are drawn at random for replication and assortment is presented. Intrapool variations in an incompatible heteroplasmid strain are inevitable and it is shown that these variations can be exploited by differential selection to amplify one plasmid at the expense of the other. Constant overall copy number is demonstrated for isogenic wild-type replicons and also for isogenic copy mutants whose copy numbers are so great that segregational incompatibility cannot be measured. In the test system used, that of the Staphylococcus aureus plasmid pT181, the rate of replication is probably determined by the availability of a trans-active initiator protein, RepC. In heteroplasmid strains containing wild-type and dominant copy mutant plasmids, although intrapool variation occurs, the total copy number is not constant but varies as a consequence of selection for or against the mutant plasmid. This is because all of the RepC is synthesized from the mutant plasmid (the wild-type is hyper-repressed) and therefore the selection affects the supply of RepC at the same time that it affects the copy number of the plasmid. None of these effects are seen with single plasmids or with compatible pairs.

  160. Christie PJ, Davidson JN, Novick RP, Dunny GM. "Effects of tylosin feeding on the antibiotic resistance of selected gram-positive bacteria in pigs," American journal of veterinary research 1983 Jan;44(1):126-8.   (MEDL:6824216 PMID: 6824216 #J0114195)    

    The effect of tylosin on macrolide resistance of gram-positive bacteria of pigs was determined. After an initial base-line period during which the pigs were given antibiotic-free feed, 1 group of 8 pigs was given tylosin feed (100 g/US ton of feed), and a 2nd group of 7 was given antibiotic-free feed. Samples were taken at 2- to 3-week intervals. For each pig, rectal, skin, and nasal swab samples were collected for enumeration of fecal streptococci and skin and nasal staphylococci. Percentages of macrolide resistant organisms of each group were tabulated on the basis of colony counts from antibiotic free and erythromycin-containing plates. After the introduction of tylosin into the feed of 1 group, a clear difference between the 2 groups with respect to the macrolide resistance of their gram-positive microflora was observed. The data indicate that tylosin feeding results in an increase in macrolide resistance of the bacterial flora of pigs.

  161. Khan SA, Novick RP. "Complete nucleotide sequence of pT181, a tetracycline-resistance plasmid from Staphylococcus aureus," Plasmid 1983 Nov;10(3):251-9.   (MEDL:6657777 PMID: 6657777 #J0114192)    

    pT181 is a naturally occurring Staphylococcus aureus plasmid, encoding inducible resistance to tetracycline. The plasmid has a copy number of about 20 per cell, and belongs to the incompatibility group inc3. The complete nucleotide sequence of pT181 has been determined and consists of 4437 bp. The nucleotide sequence contains 69.8% A-T and 30.2% G-C pairs. pT181 was found to contain four open reading frames capable of coding for polypeptides containing more than 50 amino acids. All the putative polypeptides are coded by one strand. The molecular weights of the four putative polypeptides are (in daltons): A, 37,500; B, 35,000; C, 23,000, and D, 18,000. Polypeptide A corresponds to the repC protein, earlier shown to be specifically required for pT181 replication. Polypeptide B (and possibly polypeptide D) are involved in tetracycline resistance. No role has yet been established for polypeptide C; deletion of the coding sequence for the C polypeptide has no detectable effect on any property of the pT181 plasmid. A region consisting of about 1200 bp contains information for the replication and copy number control of this plasmid. The sequencing results are discussed in relation to the replication properties and tetracycline resistance associated with the pT181 plasmid.

  162. Kreiswirth BN, Lofdahl S, Betley MJ, O'Reilly M, Schlievert PM, Bergdoll MS, Novick RP. "The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage," Nature 1983 Oct 20-26;305(5936):709-12.   (MEDL:6226876 PMID: 6226876 #J0114193)    

    Toxic shock syndrome (TSS) is a complex of generalized symptoms caused by a local staphylococcal infection, and a circulating toxin is thought to be involved. Indeed, nearly 100% of TSS isolates produce an exoprotein, TSSE, that is thought to have an aetiological role on the basis of positive animal tests (refs 1,2 and F. Quimby, personal communication) and human serological data. Although the precise role of TSSE in TSS remains unclear (E. Kass, personal communication), no other staphylococcal factor has been implicated. Our preliminary studies of the genetics of TSSE production failed to demonstrate plasmid or phage involvement or linkage with known chromosomal genes (ref. 4 and B.N.K. et al., unpublished data); however, Schutzer et al. have found that most TSS strains harbour prophages with common plating characteristics and suggest that the toxin(s) involved in TSS are transmitted by lysogenic conversion. We show here that TSSE is not demonstrably transferred by lysogeny; moreover, we have cloned the gene and found that the cloned product is serologically and biologically indistinguishable from the native protein, and that the TSSE determinant is associated with a larger DNA segment that is absent or rearranged in TSSE- strains.

  163. Projan SJ, Carleton S, Novick RP. "Determination of plasmid copy number by fluorescence densitometry," Plasmid 1983 Mar;9(2):182-90.   (MEDL:6344110 PMID: 6344110 #J0114194)    

    A simple and reliable method for the determination of plasmid copy numbers by direct fluorescence densitometry of ethidium bromide-stained electrophoretic gels was developed. In developing the method, the following parameters were evaluated and controlled: plasmid DNA trapping in the linear chromosomal DNA, staining-destaining kinetics for ethidium bromide, linearity of the fluorescence response, and the effect of the molecular topology of DNA on ethidium bromide binding to DNA in agarose.

  164. Khan SA, Adler GK, Novick RP. "Functional origin of replication of pT181 plasmid DNA is contained within a 168-base-pair segment," Proceedings of the National Academy of Sciences of the United States of America 1982 Aug;79(15):4580-4.   (MEDL:6956881 PMID: 6956881 #J0114196)    

    We have used a recently developed in vitro replication system from Staphylococcus aureus to determine the origin and direction of replication of pT181 plasmid DNA. The origin was located to within 168 base pairs by two methods: (i) sequential labeling of restriction endonuclease fragments after synchronous initiation in vitro in the presence of various amounts of dideoxy-TTP and (ii) by constructing in vitro deletions of pT181 DNA close to the origin of replication and testing for their ability to replicate in vitro pT181 plasmid was found to replicate unidirectionally and anticlockwise, as the map is conventionally drawn. The nucleotide sequence of the region containing the origin of replication has been determined and found to be partially or entirely contained within the coding sequence for the repC protein, which is uniquely required for pT181 plasmid replication. Preliminary evidence suggesting that pT181 replicates by a rolling circle mechanism is discussed.

  165. Khan SA, Novick RP. "Structural analysis of plasmid pSN2 in Staphylococcus aureus: no involvement in enterotoxin B production," Journal of bacteriology 1982 Feb;149(2):642-9.   (MEDL:7056699 PMID: 7056699 #J0114200)    

    Earlier studies have suggested the involvement of a small 1.3-kilobase plasmid, pSN2, in the production of enterotoxin D by certain Staphylococcus aureus strains. On the basis of extensive biochemical studies on pSN2, including the determination of its coding properties and its primary nucleotide sequence, we conclude that this plasmid is not in act involved in enterotoxin B production in S. aureus: although the toxin genes are apparently chromosomal, it is probable that they are part of a special genetic system such as a hitchhiking transposon.

  166. Kreiswirth BN, Novick RP, Schlievert PM, Bergdoll M. "Genetic studies on Staphylococcal strains from patients with toxic shock syndrome," Annals of internal medicine 1982 Jun;96(6 Pt 2):974-7.   (MEDL:6212007 PMID: 6212007 #J0114198)    

    Thirteen isolates of Staphylococcus aureus that produce the toxic shock syndrome exotoxin were screened to identify and characterize this specific determinant and understand its role in pathogenicity. These stains belong to phage group I, are sensitive to phage 29, and are similar with respect to their resistance to cadmium, arsenate, and penicillin. These three resistances, commonly found on plasmids in many strains of S. aureus, were not plasmid-associated in 13 toxic shock strains. The cadmium and arsenate resistances were cotransferred both in transduction and in protoplast fusion; penicillin resistance was unlinked. The toxic syndrome exotoxin gene was not linked to any of these three traits. We suggest that the trait is borne by a special genetic element that acts as a heterologous chromosomal insertion and is independent of the cadmium-arsenate linkage group or the penicillinase-determinant. The genetic properties of extracellular proteins in S. aureus are reviewed as possible models for the acquisition or expression of this toxic shock antigen.

  167. Novick RP, Adler GK, Majumder S, Khan SA, Carleton S, Rosenblum WD, Iordanescu S. "Coding sequence for the pT181 repC product: a plasmid-coded protein uniquely required for replication," Proceedings of the National Academy of Sciences of the United States of America 1982 Jul;79(13):4108-12.   (MEDL:6287465 PMID: 6287465 #J0114197)    

    pT181 is a 4.4-kilobase plasmid from Staphylococcus aureus specifying tetracycline resistance and present in about 20 copies per cell. The existence of a diffusible pT181 product required for plasmid replication has been proposed on the basis of trans-complementable thermosensitive mutants defective in plasmid maintenance (phenotype Tsr). In this report, the Tsr mutants are shown to have primary replication defects, and the genetic complementation data are confirmed biochemically. All of five mutations are in a single cistron, the repC cistron; interruption of the plasmid DNA molecule at any of three neighboring restriction sites inactivates repC function. Analysis of the DNA sequence in this region reveals an open reading frame of 939 base pairs which encodes the repC product, a 313-amino acid protein. pT181 replication has been demonstrated in cell-free extracts to require specifically a pT181-coded protein of approximately the same size, and it is proposed that this protein is, indeed, the repC product. Preliminary evidence is discussed suggesting that the pT181 replication rate is controlled at the level of synthesis of the repC protein.

  168. Polak J, Novick RP. "Closely related plasmids from Staphylococcus aureus and soil bacilli," Plasmid 1982 Mar;7(2):152-62.   (MEDL:7079389 PMID: 7079389 #J0114199)    

  169. Khan SA, Carleton SM, Novick RP. "Replication of plasmid pT181 DNA in vitro: requirement for a plasmid-encoded product," Proceedings of the National Academy of Sciences of the United States of America 1981 Aug;78(8):4902-6.   (MEDL:6946436 PMID: 6946436 #J0114202)    

    PT181 is a naturally occurring 4.5-kilobase Staphylococcus aureus plasmid encoding resistance to tetracycline. The plasmid has a copy number of about 20 per cell; a mutant, cop-608, that has a copy number of 800-1000 has been isolated. A cell-free extract has been developed that carries out complete replication of this plasmid. Extracts made from a strain containing the mutant have much greater replication activity than do extracts of strains containing pT181. In an extract from which endogenous DNA has been removed, DNA synthesis is dependent upon the addition of exogenous plasmid DNA. The replication system is specific for pT181 and related plasmids but it is inactive with other S. aureus plasmids. Furthermore, pT181 DNA does not replicate in extracts made from plasmid-negative strains or strains containing other plasmids. The results suggest that a specific plasmid-encoded substance is required for the replication of pT181 DNA.

  170. Krolewski JJ, Murphy E, Novick RP, Rush MG. "Site-specificity of the chromosomal insertion of Staphylococcus aureus transposon Tn554," Journal of molecular biology 1981 Oct 15;152(1):19-33..   (MEDL:82170454 PMID: 6279864 #J0019211)    

  171. Murphy E, Phillips S, Edelman I, Novick RP. "Tn554: isolation and characterization of plasmid insertions," Plasmid 1981 May;5(3):292-305.   (MEDL:6267632 PMID: 6267632 #J0114204)    

  172. Novick RP. "The development and spread of antibiotic-resistant bacteria as a consequence of feeding antibiotics to livestock," Annals of the New York Academy of Sciences 1981;368:23-59.   (MEDL:7020539 PMID: 7020539 #J0114205)    

  173. Novick RP, Khan SA, Murphy E, Iordanescu S, Edelman I, Krolewski J, Rush M. "Hitchhiking transposons and other mobile genetic elements and site-specific recombination systems in Staphylococcus aureus," Cold Spring Harbor symposia on quantitative biology 1981;45 Pt 1:67-76.   (MEDL:6271492 PMID: 6271492 #J0114206)    

  174. Novick RP, Iordanescu S, Surdeanu M, Edelman I. "Transduction-related cointegrate formation between Staphylococcal plasmids: a new type of site-specific recombination," Plasmid 1981 Sep;6(2):159-72.   (MEDL:6458059 PMID: 6458059 #J0114201)    

  175. Silver S, Budd K, Leahy KM, Shaw WV, Hammond D, Novick RP, Willsky GR, Malamy MH, Rosenberg H. "Inducible plasmid-determined resistance to arsenate, arsenite, and antimony (III) in escherichia coli and Staphylococcus aureus," Journal of bacteriology 1981 Jun;146(3):983-96.   (MEDL:7016838 PMID: 7016838 #J0114203)    

    Plasmids in both Escherichia coli and Staphylococcus aureus contain an 'operon' that confers resistance to arsenate, arsenite, and antimony(III) salts. The systems were always inducible. All three salts, arsenate, arsenite, and antimony(III), were inducers. Mutants and a cloned deoxyribonucleic acid fragment from plasmid pI258 in S. aureus have lost arsenate resistance but retained resistances to arsenite and antimony, demonstrating that separate genes are involved. Arsenate-resistant arsenite-sensitive S. aureus plasmid mutants were also isolated. In E. coli, plasmid-determined arsenate resistance and reduced uptake were additive to that found with chromosomal arsenate resistance mutants. Arsenate resistance was due to reduced uptake of arsenate by the induced plasmid-containing cells. Under conditions of high arsenate, when some uptake could be demonstrated with the induced resistant cells, the arsenate was rapidly lost by the cells in the absence of extracellular phosphate. Sensitive cells retained arsenate under these conditions. When phosphate was added, phosphate-arsenate exchange occurred. High phosphate in the growth medium protected cells from arsenate, but not from arsenite or antimony(III) toxicity. We do not know the mechanisms of arsenite or antimony resistance. However, arsenite was not oxidized to less toxic arsenate. Since cell-free medium 'conditioned' by prior growth to induced resistant cells with toxic levels of arsenite or antimony(III) retained the ability to inhibit the growth of sensitive cells, the mechanism of arsenite and antimony resistance does not involve conversion of AsO2- or SbO+ to less toxic forms or binding by soluble thiols excreted by resistant cells.

  176. el Solh N, Fouace JM, Shalita Z, Bouanchaud DH, Novick RP, Chabbert YA. "Epidemiological and structural studies of Staphylococcus aureus R plasmids mediating resistance to tobramycin and streptogramin," Plasmid 1980 Jul;4(1):117-20.   (MEDL:6821499 PMID: 6821499 #J0114209)    

  177. Khan SA, Novick RP. "Terminal nucleotide sequences of Tn551, a transposon specifying erythromycin resistance in Staphylococcus aureus: homology with Tn3," Plasmid 1980 Sep;4(2):148-54.   (MEDL:6100928 PMID: 6100928 #J0114208)    

    The erythromycin resistance determinant of Staphylococcus aureus plasmid pI258 resides on a 5.3 kb transposon, Tn551. We have determined DNA sequences surrounding the junctions between the transposon and the flanking DNA in the wild-type plasmid, in an insertion into a second plasmid, and in two transposon-related deletions. The ends of the transposon consist of an inverted repeat of 40 base pairs flanked by a direct repeat of 5, thus placing the transposon in the same class as Tn3, IS2, Tn501, gamma delta, and bacteriophage Mu. Interestingly, we find that the terminal sequences of the 40 base pairs inverted repeat are very similar to the ends of Tn3, a transposon which one would not have expected to show any relation to Tn551. This result suggests common ancestry for Tn3 and Tn551. The inverted repeat sequence of Tn551 also contains (with one additional inserted base) the internal heptanucleotide sequence which has been found to be common to most of the transposable elements that generate 5-base pair direct repeat sequences.

  178. Murphy E, Novick RP. "Site-specific recombination between plasmids of Staphylococcus aureus," Journal of bacteriology 1980 Jan;141(1):316-26.   (MEDL:6243624 PMID: 6243624 #J0114211)    

    Anomalous recombination between two similar but nonidentical, naturally occurring penicillinase plasmids, pI258 and pI524, leading to duplication and deletion of the beta-lactamase locus, is described. Physical mapping of these plasmids by heteroduplex and restriction analysis revealed that the beta-lactamase loci were homologous and in inverted orientation with respect to one another and that their respective locations were separated by a short region of homology. This intervening region of homology included one copy of a segment that was repeated on pI524 in inverted orientation at a distance of 2.2 kilobase pairs and contained a recognition sequence for a site-specific, rec-independent recombination function that caused reversible inversion of this segment on pI524. It is proposed that site-specific, intermolecular recombination involving this repeated sequence was responsible for the observed results.

  179. NOVICK, RP, KHAN, SA, MURPHY, E, IORDANESCU, S, EDELMAN, I, KROLEWSKI, J, RUSH, M. "HITCHHIKING TRANSPOSONS AND OTHER MOBILE GENETIC ELEMENTS AND SITE-SPECIFIC RECOMBINATION SYSTEMS IN STAPHYLOCOCCUS-AUREUS," Cold Spring Harbor symposia on quantitative biology 1980;45(2):67-76.   (ISI:A1980LV23700012 #J0058643)    

  180. Novick RP. "Plasmids," Scientific american 1980 Dec;243(6):102-4, 106, 110 passim.   (MEDL:6259723 PMID: 6259723 #J0114207)    

  181. Shalita Z, Murphy E, Novick RP. "Penicillinase plasmids of Staphylococcus aureus: structural and evolutionary relationships," Plasmid 1980 May;3(3):291-311.   (MEDL:6100898 PMID: 6100898 #J0114210)    

  182. Campbell A, Berg DE, Botstein D, Lederberg EM, Novick RP, Starlinger P, Szybalski W. "Nomenclature of transposable elements in prokaryotes," Gene 1979 Mar;5(3):197-206.   (MEDL:467979 PMID: 467979 #J0114215)    

    Transposable elements are defined as specific DNA segments that can repeatedly insert into a few or many sites in a genome. They are classified as simple IS elements, more complex Tn transposons and self-replicating episomes. Definitions and nomenclature rules for these three classes of prokaryotic transposable elements are specified.

  183. Campbell A, Starlinger P, Berg DE, Botstein D, Lederberg EM, Novick RP, Szybalski W. "Nomenclature of transposable elements in prokaryotes," Plasmid 1979 Jul;2(3):466-73.   (MEDL:384423 PMID: 384423 #J0114213)    

  184. Murphy E, Novick RP. "Physical mapping of Staphylococcus aureus penicillinase plasmid pI524: characterization of an invertible region," Molecular & general genetics 1979 Aug;175(1):19-30.   (MEDL:316096 PMID: 316096 #J0114212)    

    The staphylococcal penicillinase plasmid pI524 and a series of derivatives have been extensively mapped by restriction endonuclease digestion and by heteroduplex analysis. We report here the identification of a 2.2 kb region that undergoes a reversible, rec-independent inversion. This sequence is bounded by a pair of inverted repeats 650 base pairs in length, and has asymmetrically located recognition sites for at least three restriction endonucleases. A series of deleted derivatives and one naturally occurring, closely related plasmid, were studied. Two of these retain the inversion; the remainder are incapable of inverting and were all found to be locked in the same orientation of the inversion. The invertible sequence is adjacent to the region of the plasmid encoding beta-lactamase (bla); this entire region appears to be transposable and the inversion may be involved in the regulation of beta-lactamase expression or in translocation.

  185. Novick RP, Edelman I, Schwesinger MD, Gruss AD, Swanson EC, Pattee PA. "Genetic translocation in Staphylococcus aureus," Proceedings of the National Academy of Sciences of the United States of America 1979 Jan;76(1):400-4.   (MEDL:284355 PMID: 284355 #J0114219)    

    A 5.2-kilobase pair transposon, Tn551, has been found in Staphylococcus aureus, a Gram-positive bacterium. Initially detected on plasmid pI258, it undergoes rec-independent transposition to multiple chromosomal and plasmid sites, sometimes causing insertional inactivation. Unlike most other transposons, Tn551 undergoes apparently precise excision as a rule. The initial observation of Tn551 transition involved UV inactivation of the carrier plasmid; this would appear to be a general means of detecting transposable elements.

  186. Novick RP. "The molecular basis of the plasmid state," Contributions to microbiology & immunology 1979;6:1-15.   (MEDL:535397 PMID: 535397 #J0114217)    

  187. Novick RP, Murphy E, Gryczan TJ, Baron E, Edelman I. "Penicillinase plasmids of Staphylococcus aureus: restriction-deletion maps," Plasmid 1979 Jan;2(1):109-29.   (MEDL:314115 PMID: 314115 #J0114218)    

  188. Novick RP, Edelman I, Latta PD, Swanson EC, Pattee PA. "Translocatable elements in Staphylococcus aureus," Contributions to microbiology & immunology 1979;6:41-55.   (MEDL:535401 PMID: 535401 #J0114216)    

    The properties of the first translocatable element in Gram-positive bacteria, a 5.2 kb segment encoding erythromycin resistance in S. aureus, are described. This element translocates from plasmid to multiple chromosomal sites and from chromosome to multiple plasmid sites, sometimes causing insertional inactivation and deletion. The genetic control of translocation and its role in natural plasmid evolution are discussed and preliminary evidence for translocation of penicillin and chloramphenicol resistance is presented. In the latter case, translocation involves in intact plasmid.

  189. Phillips S, Novick RP. "Tn554--a site-specific repressor-controlled transposon in Staphylococcus aureus," Nature 1979 Mar 29;278(5703):476-8.   (MEDL:156306 PMID: 156306 #J0114214)    

  190. Della Latta P, Bouanchaud D, Novick RP. "Partition kinetics and thermosensitive replication of pT169, a naturally occurring multicopy tetracycline resistance plasmid of Staphylococcus aureus," Plasmid 1978 Jun;1(3):366-75.   (MEDL:748952 PMID: 748952 #J0114221)    

  191. Novick RP, Hoppensteadt FC. "On plasmid incompatibility," Plasmid 1978 Sep;1(4):421-34.   (MEDL:372974 PMID: 372974 #J0114220)    

  192. Novick RP. "The mechanism of translocation in bacteria," Brookhaven Symposia on Biology 1977 May 12-20;(29):272-6.   (MEDL:754864 PMID: 754864 #J0114223)    

  193. Pattee PA, Thompson NE, Haubrich D, Novick RP. "Chromosomal map locations of integrated plasmids and related elements in Staphylococcus aureus," Plasmid 1977 Nov;1(1):38-51.   (MEDL:618184 PMID: 618184 #J0114222)    

  194. Novick RP, Schwesinger M. "Indepencence of plasmid incompatibility and replication control functions in Staphylococcus aureus," Nature 1976 Aug 12;262(5569):623-6.   (MEDL:958433 PMID: 958433 #J0114224)    

  195. Novick RP, Clowes RC, Cohen SN, Curtiss R 3rd, Datta N, Falkow S. "Uniform nomenclature for bacterial plasmids: a proposal," Bacteriological reviews 1976 Mar;40(1):168-89.   (MEDL:1267736 PMID: 1267736 #J0114225)    

  196. Novick R, Zouzias D, Rush M. "Nucleic acid hybridization analysis of an integrated plasmid in Staphylococcus aureus," Journal of bacteriology 1975 Dec;124(3):1424-8..   (MEDL:76069107 PMID: 1194240 #J0018832)    

    A series of studies were performed on a Staphylococcus aureus strain thought to contain a pencillinase plasmid integrated into the host chromosome. Reassociation kinetics analysis of whole-cell deoxyribonucleic acid (DNA) in the presence of pure radioactive plasmid DNA revealed that plasmid-specific sequences were present at about 1 copy per chromosome equivalent as compared to 3.6 copies for the same plasmid in its autonomous state. Consistent with this observation was the finding that penicillinase activity was lower for the former strain than for the latter. It was shown further that the plasmid-specific sequences cosedimented on neutral sucrose gradients with fragments of whole-cell DNA many times larger than the plasmid. These two findings were taken as strongly confirmatory of the integrated state. Analysis of whole-cell ribonucleic acid for the presence of plasmid-specific messengers revealed that these were present in approximately the amounts expected on the basis of the DNA study.

  197. Ruby C, Novick RP. "Plasmid interactions in Staphylococcus aureus: nonadditivity of compatible plasmid DNA pools," Proceedings of the National Academy of Sciences of the United States of America 1975 Dec;72(12):5031-5.   (MEDL:1061089 PMID: 1061089 #J0114226)    

    Six different Staphylococcus aureus plasmids have been examined for compatibility relationships and their intracellular DNA pools have been measured singly and in various combinations. All six were mutually compatible, but contrary to expectation, their intracellular DNA pools were not additive; instead, there appeared to be a maximum level of extrachromosomal DNA that could be supported by the cell, and the plasmids studied approached this level individually as well as in varous combinations. One exception was encountered: a plasmid encoding kanamycin/neomycin resistance was present in a small but constant number of copies regardless of the presence of other plasmids.

  198. Rush, M, Novick, R, Delap, R. "DETECTION AND QUANTITATION OF STAPHYLOCOCCUS-AUREUS PENICILLINASE PLASMID DEOXYRIBONUCLEIC-ACID BY REASSOCIATION KINETICS," Journal of bacteriology 1975;124(3):1417-1423.   (ISI:A1975AZ80200053 #J0035546)    

  199. Schwesinger MD, Novick RP. "Prophage-dependent plasmid integration in Staphylococcus aureus," Journal of bacteriology 1975 Aug;123(2):724-38.   (MEDL:125745 PMID: 125745 #J0114227)    

    A study has been done of reversion to thermostability of thermosensitive, replication-defective (TSR) mutant penicillinase plasmids. All three of the expected classes of reversions were encountered: back mutation, suppression, and integration. The latter class was examined in some detail and it was found that the presence of the phi 11 phophage enhance the frequency of reversion by integration some 103-fold. Prophage-dependent integration resulted in inactivation of plasmid-linked arsenate and arsenite resistance; these revertant strains gave rise to high frequency tranducing lysates where the plasmid was restored upon transduction to its original TSR state including recovery of these resistances. The integrated plasmid-prophage complexes were stable at high temperatures (43 C) but slow growing and unstable at low (32 C); loss of either plasmid or prophage restored normal growth and stability. Sometimes restoration of the plasmid to its autonomous TSR state was observed and molecular studies showed that in most cases the plasmid was essentially the same size as before integration. In some cases an excision complex was recovered that was more than twice the size of the plasmid and could have been a plasmid-phage co-integrate. Integration also took place in the absence of the l 11 prophage. These integrations retained all plasmid-linked resistances, were stable at all temperatures, and gave rise to low frequency transducing lysates in which the integrated state was retained upon transduction. On the basis of these results it is suggested that the prophage promotes integration at or near its attachment site.

  200. Sheehy RJ, Novick RP. "Studies on plasmid replication. V Replicative intermediates," Journal of molecular biology 1975 Apr 5;93(2):237-53.   (MEDL:1152052 PMID: 1152052 #J0114228)    

  201. Novick RP. "Studies on plasmid replication. III. Isolation and characterization of replication-defective mutants," Molecular & general genetics 1974;135(2):131-47.   (MEDL:4457754 PMID: 4457754 #J0114231)    

    Some 85 Staphylococcus aureus mutants phenotypically thermosensitive for penicillinase plasmid segregation (Seg-) have been isolated and characterized. Some of the mutations were plasmid-linked and those studied in detail were found to be defective in plasmid replication, most problbly at the initiation stage. Analysis of the segregation behavior of these mutants suggested a figure of 2.7 for the average number of plasmid copies per cell in a random culture. Other mutations were host chromosome-linked and these could be divided into at least three classes on the basis of their ability to maintain plasmids of the two different incompatibility sets: some were defective for type I plasmids, some for type II, and some for both types. One host mutant, defective in segregation of type I but not type II plasmids, was defective in polymerization of both.

  202. Wyman L, Goering RV, Novick RP. "Genetic control of chromosomal and plasmid recombination in Staphylococcus aureus," Genetics 1974 Apr;76(4):681-702.   (MEDL:4275652 PMID: 4275652 #J0114229)    

  203. Wyman L, Novick RP. "Studies on plasmid replication. IV. Complementation of replication-defective mutants by an incompatibility-deficient plasmid," Molecular & general genetics 1974;135(2):149-61.   (MEDL:4457755 PMID: 4457755 #J0114230)    

    This paper described a complementation test system for replication-defective S. aureus pencillinase plasmids in which the incompatibility barrier has been overcome by the isolation of an incompatibility-defective (Inc-) plasmid. This plasmid appears to be stably and irreversibly integrated into the host chromosome as attempts to restore it to its original independent state have been unsuccessful. The Inc- plasmid was able to complement the thermosensitive replication defects of Seg- plasmids belonging to the same original incompatibility class but was unable to complement onels belonging to a different incompatibility class. Positive and negative phenotypic complementation tests were confirmed at the molecular level by isotopec labeling of plasmid-specific DNA molecules.

  204. Lindberg M, Novick RP. "Plasmid-specific transformation in Staphylococcus aureus," Journal of bacteriology 1973 Jul;115(1):139-45.   (MEDL:4717511 PMID: 4717511 #J0114233)    

  205. Novick RP, Smith K, Sheehy RJ, Murphy E. "A catenated intermediate in plasmid replication," Biochemical & biophysical research communications 1973 Oct 15;54(4):1460-9.   (MEDL:4754722 PMID: 4754722 #J0114232)    

  206. Zouzias D, Rush M, Murphy E, Novick R. "Studies on plasmid replication. II. In vivo transcription and its control in penicillinase plasmids from Staphylococcus aureus," Journal of molecular biology 1973 Sep 25;79(3):471-80..   (MEDL:74043802 PMID: 4758063 #J0018833)    

  207. Novick RP, Brodsky R. "Studies on plasmid replication. I. Plasmid incompatibility and establishment in Staphylococcus aureus," Journal of molecular biology 1972 Jul 21;68(2):285-302.   (MEDL:4262657 PMID: 4262657 #J0114235)    

  208. Smith K, Novick RP. "Genetic studies on plasmid-linked cadmium resistance in Staphylococcus aureus," Journal of bacteriology 1972 Nov;112(2):761-72.   (MEDL:4343823 PMID: 4343823 #J0114234)    

  209. Novick RP, Bouanchaud D. "The problems of drug-resistant pathogenic bacteria. Extrachromosomal nature of drug resistance in Staphylococcus aureus," Annals of the New York Academy of Sciences 1971 Jun 11;182:279-94.   (MEDL:5285292 PMID: 5285292 #J0114236)    

  210. Novick RP. "Extrachromosomal inheritance in bacteria," Bacteriological reviews 1969 Jun;33(2):210-63.   (MEDL:4896350 PMID: 4896350 #J0114237)    

  211. Novick RP. "Staphylococcal plasmids," Journal of general microbiology 1969 Mar;55(3):3.   (MEDL:5783890 PMID: 5783890 #J0114239)    

  212. Peyru G, Wexler LF, Novick RP. "Naturally occurring penicillinase plasmids in Staphylococcus aureus," Journal of bacteriology 1969 Apr;98(1):215-21.   (MEDL:5781577 PMID: 5781577 #J0114238)    

  213. Rush MG, Gordon CN, Novick RP, Warner RC. "Penicillinase plasmid DNA from Staphylococcus aureus," Proceedings of the National Academy of Sciences of the United States of America 1969 Aug;63(4):1304-10..   (MEDL:70050806 PMID: 5260933 #J0019222)    

  214. Novick RP, Roth C. "Plasmid-linked resistance to inorganic salts in Staphylococcus aureus," Journal of bacteriology 1968 Apr;95(4):1335-42.   (MEDL:5646621 PMID: 5646621 #J0114241)    

  215. Peyru G, Novick RP. "Streptomycin suppression of plasmid-linked mutations in Staphylococcus aureus," Journal of bacteriology 1968 Nov;96(5):1863-6.   (MEDL:5726314 PMID: 5726314 #J0114240)    

  216. Novick RP, Morse SI. "In vivo transmission of drug resistance factors between strains of Staphylococcus aureus," Journal of experimental medicine 1967 Jan 1;125(1):45-59.   (MEDL:6016896 PMID: 6016896 #J0114242)    

  217. Novick RP. "Penicillinase plasmids of Staphylococcus aureus," Federation Proceedings (Federation of American Societies for Experimental Biology) 1967 Jan-Feb;26(1):29-38.   (MEDL:4225241 PMID: 4225241 #J0114243)    

  218. Novick RP. "The genetic determinant of staphylococcal penicillinase," Annals of the New York Academy of Sciences 1965 Jul 23;128(1):165-82.   (MEDL:5323638 PMID: 5323638 #J0114245)    

  219. NOVICK RP, RICHMOND MH. "NATURE AND INTERACTIONS OF THE GENETIC ELEMENTS GOVERNING PENICILLINASE SYNTHESIS IN STAPHYLOCOCCUS AUREUS," Journal of bacteriology 1965 Aug;90:467-80.   (MEDL:14329463 PMID: 14329463 #J0114244)    

  220. NOVICK RP. "ANALYSIS BY TRANSDUCTION OF MUTATIONS AFFECTING PENICILLINASE FORMATION IN STAPHYLOCOCCUS AUREUS," Journal of general microbiology 1963 Oct;33:121-36.   (MEDL:14072829 PMID: 14072829 #J0114246)    

  221. NOVICK RP. "Micro-iodometric assay for penicillinase," Biochemical journal 1962 May;83:236-40.   (MEDL:14480578 PMID: 14480578 #J0114248)    

  222. NOVICK RP. "Staphylococcal penicillinase and the new penicillins," Biochemical journal 1962 May;83:229-35.   (MEDL:14480579 PMID: 14480579 #J0114247)    

  223. NOVICK RP, MAAS WK. "Control by endogenously synthesized arginine of the formation of ornithine transcarbamylase in Escherichia coli," Journal of bacteriology 1961 Feb;81:236-40.   (MEDL:13729753 PMID: 13729753 #J0114249)    





Ehrman Medical Library Faculty Bibliography Search printed 11/23/2009 10:15


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