Transcriptional Responses of Pseudomonas aeruginosa to Inhibition of Lipoprotein Transport by a Small Molecule Inhibitor.

Lipoprotein transport from the inner to the outer membrane, carried out by the Lol machinery, is essential for the biogenesis of the Gram-negative cell envelope and, consequently, for bacterial viability. Recently, small molecule inhibitors of the Lol system in Escherichia coli have been identified and shown to inhibit the growth of this organism by interfering with the function of the LolCDE complex. Analysis of the transcriptome of E. coli treated with one such molecule (compound 2) revealed that a number of envelope stress response pathways were induced in response to LolCDE inhibition. However, Pseudomonas aeruginosa is refractory to inhibition by the same small molecule, but we could demonstrate that E. colilolCDE could be substituted for the P. aeruginosa orthologues, where it functions in the correct transport of Pseudomonas lipoproteins, and the cells are inhibited by the more potent compound 2A. In the present study, we took advantage of the functionality of E. coli LolCDE in P. aeruginosa and determined the P. aeruginosa transcriptional response to LolCDE inhibition by compound 2A. We identified key genes that responded to LolCDE inhibition and also demonstrated that the same genes appeared to be affected by genetic depletion of the native P. aeruginosa LolCDE proteins. Several of the major changes were in an upregulated cluster of genes that encode determinants of alginate biosynthesis and transport, and the levels of alginate were found to be increased either by treatment with the small molecule inhibitor or upon depletion of native LolCDE. Finally, we tested several antibiotics with differing mechanisms of action to identify potential specific reporter genes for the further development of compounds that would inhibit the native P. aeruginosa Lol system.IMPORTANCE A key set of lipoprotein transport components, LolCDE, were inhibited by both a small molecule as well as genetic downregulation of their expression. The data show a unique signature in the Pseudomonas aeruginosa transcriptome in response to perturbation of outer membrane biogenesis. In addition, we demonstrate a transcriptional response in key genes with marked specificity compared to several antibiotic classes with different mechanisms of action. As a result of this work, we identified genes that could be of potential use as biomarkers in a cell-based screen for novel antibiotic inhibitors of lipoprotein transport in P. aeruginosa.

Correct Sorting of Lipoproteins into the Inner and Outer Membranes of Pseudomonas aeruginosa by the Escherichia coli LolCDE Transport System.

Biogenesis of the outer membrane of Gram-negative bacteria depends on dedicated macromolecular transport systems. The LolABCDE proteins make up the machinery for lipoprotein trafficking from the inner membrane (IM) across the periplasm to the outer membrane (OM). The Lol apparatus is additionally responsible for differentiating OM lipoproteins from those for the IM. In Enterobacteriaceae, a default sorting mechanism has been proposed whereby an aspartic acid at position +2 of the mature lipoproteins prevents Lol recognition and leads to their IM retention. In other bacteria, the conservation of sequences immediately following the acylated cysteine is variable. Here we show that in Pseudomonas aeruginosa, the three essential Lol proteins (LolCDE) can be replaced with those from Escherichia coli The P. aeruginosa lipoproteins MexA, OprM, PscJ, and FlgH, with different sequences at their N termini, were correctly sorted by either the E. coli or P. aeruginosa LolCDE. We further demonstrate that an inhibitor of E. coli LolCDE is active against P. aeruginosa only when expressing the E. coli orthologues. Our work shows that Lol proteins recognize a wide range of signals, consisting of an acylated cysteine and a specific conformation of the adjacent domain, determining IM retention or transport to the OM.IMPORTANCE Gram-negative bacteria build their outer membranes (OM) from components that are initially located in the inner membrane (IM). A fraction of lipoproteins is transferred to the OM by the transport machinery consisting of LolABCDE proteins. Our work demonstrates that the LolCDE complexes of the transport pathways of Escherichia coli and Pseudomonas aeruginosa are interchangeable, with the E. coli orthologues correctly sorting the P. aeruginosa lipoproteins while retaining their sensitivity to a small-molecule inhibitor. These findings question the nature of IM retention signals, identified in E. coli as aspartate at position +2 of mature lipoproteins. We propose an alternative model for the sorting of IM and OM lipoproteins based on their relative affinities for the IM and the ability of the promiscuous sorting machinery to deliver lipoproteins to their functional sites in the OM.

Transcriptional Responses of Escherichia coli to a Small-Molecule Inhibitor of LolCDE, an Essential Component of the Lipoprotein Transport Pathway.

In Gram-negative bacteria, a dedicated machinery consisting of LolABCDE components targets lipoproteins to the outer membrane. We used a previously identified small-molecule inhibitor of the LolCDE complex of Escherichia coli to assess the global transcriptional consequences of interference with lipoprotein transport. Exposure of E. coli to the LolCDE inhibitor at concentrations leading to minimal and significant growth inhibition, followed by transcriptome sequencing, identified a small group of genes whose transcript levels were decreased and a larger group whose mRNA levels increased 10- to 100-fold compared to those of untreated cells. The majority of the genes whose mRNA concentrations were reduced were part of the flagellar assembly pathway, which contains an essential lipoprotein component. Most of the genes whose transcript levels were elevated encode proteins involved in selected cell stress pathways. Many of these genes are involved with envelope stress responses induced by the mislocalization of outer membrane lipoproteins. Although several of the genes whose RNAs were induced have previously been shown to be associated with the general perturbation of the cell envelope by antibiotics, a small subset was affected only by LolCDE inhibition. Findings from this work suggest that the efficiency of the Lol system function may be coupled to a specific monitoring system, which could be exploited in the development of reporter constructs suitable for use for screening for additional inhibitors of lipoprotein trafficking. IMPORTANCE: Inhibition of the lipoprotein transport pathway leads to E. coli death and subsequent lysis. Early significant changes in the levels of RNA for a subset of genes identified to be associated with some periplasmic and envelope stress responses were observed. Together these findings suggest that disruption of this key pathway can have a severe impact on balanced outer membrane synthesis sufficient to affect viability.

Novel antibacterial targets and compounds revealed by a high-throughput cell wall reporter assay.

UNLABELLED: A high-throughput phenotypic screen based on a Citrobacter freundii AmpC reporter expressed in Escherichia coli was executed to discover novel inhibitors of bacterial cell wall synthesis, an attractive, well-validated target for antibiotic intervention. Here we describe the discovery and characterization of sulfonyl piperazine and pyrazole compounds, each with novel mechanisms of action. E. coli mutants resistant to these compounds display no cross-resistance to antibiotics of other classes. Resistance to the sulfonyl piperazine maps to LpxH, which catalyzes the fourth step in the synthesis of lipid A, the outer membrane anchor of lipopolysaccharide (LPS). To our knowledge, this compound is the first reported inhibitor of LpxH. Resistance to the pyrazole compound mapped to mutations in either LolC or LolE, components of the essential LolCDE transporter complex, which is required for trafficking of lipoproteins to the outer membrane. Biochemical experiments with E. coli spheroplasts showed that the pyrazole compound is capable of inhibiting the release of lipoproteins from the inner membrane. Both of these compounds have significant promise as chemical probes to further interrogate the potential of these novel cell wall components for antimicrobial therapy. IMPORTANCE: The prevalence of antibacterial resistance, particularly among Gram-negative organisms, signals a need for novel antibacterial agents. A phenotypic screen using AmpC as a sensor for compounds that inhibit processes involved in Gram-negative envelope biogenesis led to the identification of two novel inhibitors with unique mechanisms of action targeting Escherichia coli outer membrane biogenesis. One compound inhibits the transport system for lipoprotein transport to the outer membrane, while the other compound inhibits synthesis of lipopolysaccharide. These results indicate that it is still possible to uncover new compounds with intrinsic antibacterial activity that inhibit novel targets related to the cell envelope, suggesting that the Gram-negative cell envelope still has untapped potential for therapeutic intervention.

Target-based resistance in Pseudomonas aeruginosa and Escherichia coli to NBTI 5463, a novel bacterial type II topoisomerase inhibitor.

In a previous report (T. J. Dougherty, A. Nayar, J. V. Newman, S. Hopkins, G. G. Stone, M. Johnstone, A. B. Shapiro, M. Cronin, F. Reck, and D. E. Ehmann, Antimicrob Agents Chemother 58:2657-2664, 2014), a novel bacterial type II topoisomerase inhibitor, NBTI 5463, with activity against Gram-negative pathogens was described. First-step resistance mutations in Pseudomonas aeruginosa arose exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux pump system. The present report describes further resistance studies with NBTI 5463 in both Pseudomonas aeruginosa and Escherichia coli. Second-step mutations in P. aeruginosa arose at aspartate 82 of the gyrase A subunit and led to 4- to 8-fold increases in the MIC over those seen in the parental strain with a first-step nfxB efflux mutation. A third-step mutant showed additional GyrA changes, with no changes in topoisomerase IV. Despite repeated efforts, resistance mutations could not be selected in E. coli. Genetic introduction of the Asp82 mutations observed in P. aeruginosa did not significantly increase the NBTI MIC in E. coli. However, with the aspartate 82 mutation present, it was possible to select second-step mutations in topoisomerase IV that did lead to MIC increases of 16- and 128-fold. As with the gyrase aspartate 82 mutation, the mutations in topoisomerase IV did not by themselves raise the NBTI MIC in E. coli. Only the presence of mutations in both targets of E. coli led to an increase in NBTI MIC values. This represents a demonstration of the value of balanced dual-target activity in mitigating resistance development.

Optimization of physicochemical properties and safety profile of novel bacterial topoisomerase type II inhibitors (NBTIs) with activity against Pseudomonas aeruginosa.

Type II bacterial topoisomerases are well validated targets for antimicrobial chemotherapy. Novel bacterial type II topoisomerase inhibitors (NBTIs) of these targets are of interest for the development of new antibacterial agents that are not impacted by target-mediated cross-resistance with fluoroquinolones. We now disclose the optimization of a class of NBTIs towards Gram-negative pathogens, especially against drug-resistant Pseudomonas aeruginosa. Physicochemical properties (pKa and logD) were optimized for activity against P. aeruginosa and for reduced inhibition of the hERG channel. The optimized analogs 9g and 9i displayed potent antibacterial activity against P. aeruginosa, and a significantly improved hERG profile over previously reported analogs. Compound 9g showed an improved QT profile in in vivo models and lower clearance in rat over earlier compounds. The compounds show promise for the development of new antimicrobial agents against drug-resistant Pseudomonas aeruginosa.

NBTI 5463 is a novel bacterial type II topoisomerase inhibitor with activity against gram-negative bacteria and in vivo efficacy.

The need for new antibiotics that address serious Gram-negative infections is well recognized. Our efforts with a series of novel bacterial type II topoisomerase inhibitors (NBTIs) led to the discovery of NBTI 5463, an agent with improved activity over other NBTIs against Gram-negative bacteria, in particular against Pseudomonas aeruginosa (F. Reck, D. E. Ehmann, T. J. Dougherty, J. V. Newman, S. Hopkins, G. Stone, N. Agrawal, P. Ciaccio, J. McNulty, H. Barthlow, J. O'Donnell, K. Goteti, J. Breen, J. Comita-Prevoir, M. Cornebise, M. Cronin, C. J. Eyermann, B. Geng, G. R. Carr, L. Pandarinathan, X. Tang, A. Cottone, L. Zhao, N. Bezdenejnih-Snyder, submitted for publication). In the present work, NBTI 5463 demonstrated promising activity against a broad range of Gram-negative pathogens. In contrast to fluoroquinolones, the compound did not form a double-strand DNA cleavable complex with Escherichia coli DNA gyrase and DNA, but it was a potent inhibitor of both DNA gyrase and E. coli topoisomerase IV catalytic activities. In studies with P. aeruginosa, NBTI 5463 was bactericidal. Resistant mutants arose at a low rate, and the mutations were found exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux system. Levofloxacin-selected resistance mutations in GyrA did not result in decreased susceptibility to NBTI 5463. Animal infection studies demonstrated that NBTI 5463 was efficacious in mouse models of lung, thigh, and ascending urinary tract infections.

Photodynamic therapy (PDT) using HPPH for the treatment of precancerous lesions associated with Barrett's esophagus.

BACKGROUND AND OBJECTIVES: Photodynamic therapy (PDT) with porfimer sodium, FDA approved to treat premalignant lesions in Barrett's esophagus, causes photosensitivity for 6-8 weeks. HPPH (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a) shows minimal photosensitization of short duration and promising efficacy in preclinical studies. Here we explore toxicity and optimal drug and light dose with endoscopic HPPH-PDT. We also want to know the efficacy of one time treatment with HPPH-PDT. STUDY DESIGN/MATERIALS AND METHODS: Two nonrandomized dose escalation studies were performed (18 patients each) with biopsy-proven high grade dysplasia or early intramucosal adenocarcinoma of esophagus. HPPH doses ranged from 3 to 6 mg/m2 . At 24 or 48 hours after HPPH administration the lesions received one endoscopic exposure to 150, 175, or 200 J/cm of 665 nm light. RESULTS: Most patients experienced mild to moderate chest pain requiring symptomatic treatment only. Six patients experienced grade 3 and 4 adverse events (16.6%). Three esophageal strictures were treated with dilatation. No clear pattern of dose dependence of toxicities emerged. In the drug dose ranging study (light dose of 150 J/cm at 48 hours), 3 and 4 mg/m2 of HPPH emerged as most effective. In the light dose ranging study (3 or 4 mg/m2 HPPH, light at 24 hours), complete response rates (disappearance of high grade dysplasia and early carcinoma) of 72% were achieved at 1 year, with all patients treated with 3 mg/m2 HPPH plus 175 J/cm and 4 mg/m2 HPPH plus 150 J/cm showing complete responses at 1 year. CONCLUSIONS: HPPH-PDT for precancerous lesions in Barrett's esophagus appears to be safe and showing promising efficacy. Further clinical studies are required to establish the use of HPPH-PDT.

Introduction.

In this book you will find chapters describing the most up-to-date information concerning the important clinical applications and methodology of photodynamic therapy. This introduction will provide some background on general mechanisms, cellular targets, and note a few significant new findings relevant to photodynamic action in clinical practice. A short description of the development of photodynamic therapy at Roswell Park Cancer Institute is included.

Discovery of azetidinyl ketolides for the treatment of susceptible and multidrug resistant community-acquired respiratory tract infections.

Respiratory tract bacterial strains are becoming increasingly resistant to currently marketed macrolide antibiotics. The current alternative telithromycin (1) from the newer ketolide class of macrolides addresses resistance but is hampered by serious safety concerns, hepatotoxicity in particular. We have discovered a novel series of azetidinyl ketolides that focus on mitigation of hepatotoxicity by minimizing hepatic turnover and time-dependent inactivation of CYP3A isoforms in the liver without compromising the potency and efficacy of 1.

Purification of the large ribosomal subunit via its association with the small subunit.

We have developed an affinity purification of the large ribosomal subunit from Deinococcus radiodurans that exploits its association with FLAG-tagged 30S subunits. Thus, capture is indirect so that no modification of the 50S is required and elution is achieved under mild conditions (low magnesium) that disrupt the association, avoiding the addition of competitor ligands or coelution of common contaminants. Efficient purification of highly pure 50S is achieved, and the chromatography simultaneously sorts the 50S into three classes according to their association status (unassociated, loosely associated, or tightly associated), improving homogeneity.

Comparative genome analysis of high-level penicillin resistance in Streptococcus pneumoniae.

Streptococcus pneumoniae strains with very high levels of penicillin resistance (minimum inhibitory concentration [MIC] >or=8 microg/ml) emerged in the 1990 s. Previous studies have traced the changes in penicillin binding proteins (PBP) that result in decreased penicillin susceptibility, and the role of several PBP genes in high-level resistance. In the present study, we investigated the changes that occurred at the two highest levels of penicillin resistance using NimbleGen's Comparative Genome Sequencing (CGS) technology. DNA from a highly resistant (Pen MIC 16 microg/ml) pneumococcus was used to serially transform the R6 strain to high-level resistance. Four distinct levels of penicillin resistance above the susceptible R6 strain (MIC 0.016 microg/ml) were identified. Using CGS technology, the entire genome sequences of the two highest levels of resistant transformants were examined for changes associated with the resistance phenotypes. At the third level of resistance, changes in PBPs 1a, 2b, and 2x were found, very similar to previous reports. At the fourth resistance level, two additional changes were observed in the R6 transformants. More changes were observed in PBP2x, as well as in peptidoglycan GlcNAc deacetylase (pdgA), which had a missense mutation in the coding region. Genetic transformation with polymerase chain reaction (PCR) products generated from the high-level resistant parent containing either the additional PBP2x or mutant pdgA gene did not increase the MIC of the third-level transformant. Only when both PCR products were simultaneously transformed into the third-level transformant did colonies emerge that were at the highest level of resistance (16-32 microg/ml), equivalent to the highly resistant parent strain. This is the first instance of the involvement of a variant pdgA gene in penicillin resistance. It is also clear from these experiments and the literature that there are multiple paths to the pneumococcus achieving high-level penicillin resistance.

Photodynamic therapy for the treatment of periocular squamous cell carcinoma in horses: a pilot study.

OBJECTIVE: Local photodynamic therapy (PDT) is a novel cancer therapy in veterinary ophthalmology. A prospective pilot study seeking to demonstrate proof of principle and safety for the treatment of equine periocular squamous cell carcinoma (PSCC) was therefore conducted. We hypothesized that surgical excision with adjunctive local PDT is an effective and safe treatment for equine PSCC. PROCEDURES: Nine horses (10 eyes) with PSCC were treated with surgical resection, local infiltration of resulting wound beds with 2-[1-hexyloxyethyl]-2-devinylpyropheophorbide-a (HPPH) and irradiation with 665-nm wavelength diode laser. Regular follow-up ophthalmic examinations were performed. RESULTS: Surgical resection and PDT yielded disease-free intervals of 25-68 months in our study horses as of January, 2008. These results were obtained following a single treatment in seven horses and two treatments in one horse. In one horse, carcinoma in situ developed 2.5 months after partial surgical excision and PDT, requiring local excision under standing sedation. CONCLUSIONS: Preliminary results suggest that surgical resection and adjunctive local PDT is a safe and effective novel treatment for PSCC in horses. More research is needed before PDT for the treatment of equine PSCC can be adequately compared with other current modalities. Important to future investigations regarding PDT, tumor recurrence rate, length of hospitalization, number of treatment episodes required to effect tumor remission, and total treatment costs should be examined in a controlled manner. Our present results and experiences suggest that this treatment may be useful in the treatment of equine PSCC.

A method to assay penicillin-binding proteins.

Key enzymes that assemble the bacterial cell wall are also the target of the Beta-lactam class of antibiotics. The covalent binding of labeled penicillin to these proteins has been used in numerous studies in drug discovery, antibiotic mechanisms of action and resistance, and cell wall physiology. Methods to label and measure penicillin binding proteins in two prototypical organisms, a Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus), are described. The methods discussed include identifying penicillin-binding proteins in both intact cells (in vivo measurements) and isolated cell membranes.

Identification of an inhibitor of the MurC enzyme, which catalyzes an essential step in the peptidoglycan precursor synthesis pathway.

The pathway for synthesis of the peptidoglycan precursor UDP-N-acetylmuramyl pentapeptide is essential in Gram-positive and Gram-negative bacteria. This pathway has been exploited in the recent past to identify potential new antibiotics as inhibitors of one or more of the Mur enzymes. In the present study, a high-throughput screen was employed to identify potential inhibitors of the Escherichia coli MurC (UDP-N-acetylmuramic acid:L-alanine ligase), the first of four paralogous amino acid-adding enzymes. Inhibition of ATP consumed during the MurC reaction, using an adaptation of a kinase assay format, identified a number of potential inhibitory chemotypes. After nonspecific inhibition testing and chemical attractiveness were assessed, C-1 emerged as a compound for further characterization. The inhibition of MurC by this compound was confirmed in both a kinetic-coupled enzyme assay and a direct nuclear magnetic resonance product detection assay. C-1 was found to be a low micromolar inhibitor of the E. coli MurC reaction, with preferential inhibition by one of two enantiomeric forms. Experiments indicated that it was a competitive inhibitor of ATP binding to the MurC enzyme. Further work with MurC enzymes from several bacterial sources revealed that while the compound was equally effective at inhibiting MurC from genera (Proteus mirabilis and Klebsiella pneumoniae) closely related to E. coli, MurC enzymes from more distant Gram-negative species such as Haemophilus influenzae, Acinetobacter baylyi, and Pseudomonas aeruginosa were not inhibited.

Nucleotide sequence changes between Streptococcus pneumoniae R6 and D39 strains determined by an oligonucleotide hybridization DNA sequencing technology.

The Gram-positive pathogen Streptococcus pneumoniae, which can be responsible for serious cases of pneumonia and meningitis, has been intensely studied for almost 100 years. Many of the key experiments have been performed in two strains; the non-pathogenic S. pneumoniae R6 and its pathogenic progenitor, S. pneumoniae D39. Whereas the genomic sequence of the R6 strain has been published, there is relatively little genomic information available on D39. Since R6 was derived from D39, we wished to explore the utility of a new technology, Comparative Genome Sequencing, which uses a set of custom oligonucleotide arrays to compare DNA sequences between similar strains. We report here the nucleotide polymorphisms identified between the R6 strain and D39 based on an R6 sequencing array. During the process, we were also able to confirm all of the high confidence changes reported by the oligonucleotide array chip by sequencing the region in the genome around the changes identified with the genome hybridization chip. We also discuss the potential impact of some of the amino acid changes found between these two widely used strains of pneumococci.

Purpurinimide carbohydrate conjugates: effect of the position of the carbohydrate moiety in photosensitizing efficacy.

A lactose moiety was regioselectively introduced at various positions of N-hexyl-mesopurpurinimide (a class of chlorin containing a fused six-membered imide ring system, lambda(max): 700 nm) to investigate the effect of its presence and position on photosensitizing efficacy. The resulting novel structures produced a significant difference in in vitro and in vivo efficacy. Among the positional isomers in which the lactose moiety was introduced at positions 3, 8, and 12, the 3-lactose purpurin-18-N-hexylimide produced the best efficacy. Compared to these analogues, the lactose moiety joined with an amide bond at position 17(2), and with an N-benzyl group bearing a -C[triple bond]C- linkage at position 13(2) showed reduced in vitro/in vivo photosensitivity. A noticeable difference between lactose conjugates in cell uptake (RIF tumor cells) was observed at 3 and 24 h postincubation. Replacing the lactose (Galbeta1 --> 4Glc) with beta-galactose and glucose moieties at position 3 of purpurinimide produced an increase in both cell uptake and in in vitro efficacy, but with reduced in vivo efficacy. Sites of intracellular localization differed among photosensitizers with and without carbohydrate moieties. Molecular modeling shows favorable interactions of 3- and 12-lactose-purpurinimide analogues with both galectin-1 and galectin-3, but clear contributions were not found for the conjugate containing lactose moiety at position 8. In a comparative ELISA study of the lactose conjugates with free lactose, all carbohydrate-purpurinimides showed binding to both galectins with a significant variation between the batches of galectins.

Cyclopentanone ring-cleaved pleuromutilin derivatives.

Ring-cleaved pleuromutilin derivatives comprised of a [5.3.1] bicyclic core structure have been synthesized and evaluated in vitro as antibacterial agents. Four of the compounds described were found to have MICs

Extensive and genome-wide changes in the transcription profile of Staphylococcus aureus induced by modulating the transcription of the cell wall synthesis gene murF.

A murF conditional mutant was used to evaluate the effect of suboptimal transcription of this gene on the transcriptome of the methicillin-resistant Staphylococcus aureus strain COL. The mutant was grown in the presence of optimal and suboptimal concentrations of the inducer, and the relative levels of transcription of genes were evaluated genome wide with an Affymetrix DNA microarray that included all open reading frames (ORFs) as well as intergenic sequences derived from four sequenced S. aureus strains. Using a sensitivity threshold value of 1.5, suboptimal expression of murF altered the transcription of a surprisingly large number of genes, i.e., 668 out of the 2,740 ORFs (close to one-fourth of all ORFs), of the genome of S. aureus strain COL. The genes with altered transcription were distributed evenly around the S. aureus chromosome, and groups of genes involved with distinct metabolic functions responded in unique and operon-specific manners to modulation in murF transcription. For instance, all genes belonging to the isd operon and all but 2 of the 35 genes of prophage L54a were down-regulated, whereas all but one of the 21 members of the vraSR regulon and most of the 79 virulence-related genes (those for fibronectin binding proteins A and B, clumping factor A, gamma hemolysin, enterotoxin B, etc.) were up-regulated in cells with suboptimal expression of murF. Most importantly, the majority of these altered gene expression profiles were reversible by resupplying the optimal concentration of IPTG (isopropyl-beta-D-thiogalactopyranoside) to the culture. The observations suggest the coordinate regulation of a large sector of the S. aureus transcriptome in response to a disturbance in cell wall synthesis.