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  1. 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.

  2. Geisinger, E, George, EA, Muir, TW, Novick, RP. "Identification of ligand specificity determinants in AgrC, the Staphylococcus aureus quorum-sensing receptor," Journal of biological chemistry 2008 APR 4;283(14):8930-8938.   (ISI:000254465800021 #J0135144)    

    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.

  3. George, EA, Novick, RP, Muir, TW. "Cyclic peptide inhibitors of staphylococcal virulence prepared by Fmoc-based thiolactone peptide synthesis," Journal of the American Chemical Society 2008 APR 9;130(14):4914-4924.   (ISI:000254643900047 #J0135415)    

    Virulence factor production in Staphylococcus aureus is largely under the control of the accessory gene regulator (ago 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.

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

  5. Novick, R. "MR," Scientist 2008 FEB;22(2):14-15.   (ISI:000252530200004 #J0133561)    

  6. Tormo, MA, Ferrer, MD, Maiques, E, Ubeda, C, Selva, L, Lasa, I, Calvete, JJ, Novick, RP, Penades, JR. "Staphylococcus aureus pathogenicity island DNA is packaged in particles composed of phage proteins," Journal of bacteriology 2008 APR;190(7):2434-2440.   (ISI:000254323300021 #J0135145)    

    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.

  7. Ubeda C., Maiques E., Barry P., Matthews A., Tormo M.A., Lasa I., Novick R.P., Penades J.R.. "SaPI mutations affecting replication and transfer and enabling autonomous replication in the absence of helper phage," Molecular microbiology 2008;67(3):493-503.   (EMBASE:2008012964 #J0132958)    

    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. copyright 2007 The Authors.

  8. 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. Epub 2007 Jul 2.   (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.

  9. 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.

  10. Maiques, E, Ubeda, C, Tormo, MA, Ferrer, MD, Lasa, I, Novick, RP, Penades, JR. "Role of staphylococcal phage and SaPI integrase in intra- and interspecies SaPI transfer," Journal of bacteriology 2007 AUG;189(15):5608-5616.   (ISI:000248358100023 #J0128697)    

    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.

  11. 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)    

  12. 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..

  13. 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.

  14. 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. Epub 2007 Aug 10.   (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.

  15. Ubeda, C, Maiques, E, Tormo, MA, Campoy, S, Lasa, I, Barbe, J, Novick, RP, Penades, JR. "SaPI operon I is required fpr SaPI packaging and is controlled by LexA," Molecular microbiology 2007 JUL;65(1):41-50.   (ISI:000247871600005 #J0129180)    

    Transfer of Staphylococcus aureus pathogenicity islands (SaPls) 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 SaPlbovl 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 SaP1 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 SaPlbovl 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  21. 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)    

  22. 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.

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

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

  25. 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.

  26. 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.

  27. 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.

  28. 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. Epub 2005 Jan 21.   (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.

  29. 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.

  30. 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.

  31. 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.

  32. 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. Epub 2003 Nov 30.   (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.

  33. 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.

  34. 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. Epub 2004 Mar 29.   (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.

  35. 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. Epub 2004 Nov 04.   (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.

  36. 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.

  37. 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.

  38. 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.

  39. 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. Epub 2003 Mar 6.   (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.

  40. 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.

  41. 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.

  42. 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. Epub 2001 Dec 3.   (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.

  43. 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.

  44. 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.

  45. 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.

  46. 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. Epub 2002 Jul 16.   (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.

  47. 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.

  48. 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.

  49. 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.

  50. 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.

  51. 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.

  52. 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.

  53. 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.

  54. 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!   

  55. 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.

  56. 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.

  57. 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.

  58. 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)    

  59. 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.

  60. 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.

  61. 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.

  62. 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.

  63. 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.

  64. 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.

  65. 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.

  66. 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.

  67. 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.

  68. 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.

  69. 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.

  70. 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.

  71. 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)