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Background Rapid development of stable, high-level daptomycin-resistance (DAP-R) is frequent among Streptococcus mitis-oralis strains during exposure to DAP in vitro and in vivo. Metabolomic analyses of in vitro-derived daptomycin-resistant (DAP-R) S. mitis-oralis strains (351D10) revealed substantial glycolytic pathway differences vs. its DAP-susceptible (DAP-S) parental strain, 351. To define translatability of such metabolic changes, we assessed combinations of DAP + strategic metabolic inhibitors likely to impact such pathways, including: oxamic acid [OXA], trimetazidine [TMZ) and 6-mercaptopurine [6-MP]. We assessed these combinations vs. our DAP-S/DAP-R isogenic strain pair both in vitro and ex vivo for: bactericidal and synergistic activities; prevention of high-level DAP-R in DAP-S cells; and/or resensitization of DAP-R cells Methods MICs. E test and microbroth dilution In vitro antimicrobial combination assays. Time-kill curves (24 hr) and serial passaging in DAP (10 d). Ex vivo IE model: SEVs (simulated endocardial vegetation’s) were quantitatively cultured at serial time-points post-exposures to DAP +/- inhibitors (0, 4, 8, 24, 32, 48 hr). Results In vitro , combinations of DAP + OXA or DAP + TMZ, as well as OXA or TMZ monotherapy, exerted bactericidal effect against both strains. Moreover, DAP + 6-MP yielded both a bactericidal, as well as synergistic activity against DAP-S 351 (but not against DAP-R 351 D10). None of the combinations prevented the development of DAP-R or resensitized the DAP-R strain to a DAP-S phenotype in vitro. In the ex vivo SEV model, using PK/PD simulations of humanized drug doses, DAP + OXA was the most effective regimen for both the DAP-S 351 and DAP-R 351 D10 strains; both OXA alone and DAP + OXA prevented DAP-R evolution, although not resensitizing the DAP-R strain to a DAP-S phenotype. Conclusion Combinations of DAP plus specific metabolic inhibitors represents a promising approach to enhance killing of S. mitis-oralis strains, as well as to potentially forestall DAP-R emergence. Also, they do provide a solid platform upon which to explore other potential metabolic modifiers for their ability to enhance the efficacy of DAP, based on definable metabolic perturbations in DAP-R S. mitis-oralis. Disclosures All Authors: No reported disclosures.

Carbon catabolite repression (CCR) is a common mechanism pathogenic bacteria use to link central metabolism with virulence factor synthesis. In gram-positive bacteria, catabolite control protein A (CcpA) and the histidine-containing phosphocarrier protein HPr (encoded by ptsH) are the predominant mediators of CCR. In addition to modulating CcpA activity, HPr is essential for glucose import via the phosphotransferase system. While the regulatory functions of CcpA in Staphylococcus aureus are largely known, little is known about the function of HPr in CCR and infectivity. To address this knowledge gap, ptsH mutants were created in S. aureus that either lack the open reading frame or harbor a ptsH variant carrying a thymidine to guanosine mutation at position 136, and the effects of these mutations on growth and metabolism were assessed. Inactivation of ptsH altered bacterial physiology and decreased the ability of S. aureus to form a biofilm and cause infections in mice. These data demonstrate that HPr affects central metabolism and virulence in S. aureus independent of its influence on CcpA regulation.

Staphylococcus aureus is a medically important pathogen with an abundance of virulence factors that are necessary for survival within a host, including the production of cytolytic toxins. The regulation of toxin production is mediated by the Agr quorum sensing system, and a poorly defined post-exponential growth phase signal independent of Agr. As part of a recent genome wide association study (GWAS) to identify novel loci that alter the expression of cytolytic toxins, a polymorphism in the cyoE gene, which encodes a protoheme IX farnesyltransferase, was identified. This enzyme is essential for processing heme into the electron transport chain for use as an electron acceptor. Interestingly, without this enzyme S. aureus were repressed in their ability to secrete cytolytic toxins, and this appears to be mediated through repression of the Agr quorum sensing system. We hypothesize that the loss of electron transport is inducing feedback inhibition of metabolic capabilities that suppress the TCA cycle, and that this coupled with decreased RNAIII transcription prevents synthesis of cytolytic toxins.

BackgroundA major developing problem in the treatment of Staphylococcus aureus infections is the emergence of resistance during treatment with daptomycin. Previous metabolomic analyses of isogenic S. aureus strains prior to and after evolution into a daptomycin non-susceptible (DapNS) state provided important metabolic information about this transition (e.g. perturbations of the tricarboxylic acid cycle).ObjectivesTo assess the significance of these metabolic changes, in vitro susceptibility to daptomycin was determined in daptomycin-susceptible (DapS) and DapNSS. aureus strains cultivated with metabolic inhibitors targeting these changes.MethodsOnly inhibitors that are approved for use in humans were chosen (i.e. fosfomycin, valproate, trimetazidine and 6-mercaptopurine) to assess the importance of metabolic pathways for daptomycin non-susceptibility. The ability of these inhibitors to forestall the emergence of DapNS strains was also assessed.ResultsThe combination of daptomycin and fosfomycin synergistically killed both DapS and DapNS strains in vitro and enhanced the in vivo outcome against a DapNS strain in experimental endocarditis. Interestingly, fosfomycin acts on the peptidoglycan biosynthetic enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA); however, it also had a significant effect on the enzymatic activity of enolase, an essential enzyme in S. aureus. While fosfomycin acted synergistically with daptomycin, it failed to prevent the in vitro evolution of daptomycin non-susceptibility. In contrast, trimetazidine, an anti-angina drug that stimulates glucose oxidation, abolished the ability of DapSS. aureus strains to transition to a DapNS state.ConclusionsThese data reveal that metabolic adaptations associated with DapNS strains can be targeted to prevent the emergence of and/or reverse pre-existing resistance to daptomycin.

Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. The success of S. aureus as a pathogen is due in part to its many virulence determinants and resistance to antimicrobials. In particular, methicillin-resistant S. aureus has emerged as a major cause of infections and led to increased use of the antibiotics vancomycin and daptomycin, which has increased the isolation of vancomycin-intermediate S. aureus and daptomycin-nonsusceptible S. aureus strains. The most common mechanism by which S. aureus acquires intermediate resistance to antibiotics is by adapting its physiology and metabolism to permit growth in the presence of these antibiotics, a process known as adaptive resistance. To better understand the physiological and metabolic changes associated with adaptive resistance, six daptomycin-susceptible and -nonsusceptible isogenic strain pairs were examined for changes in growth, competitive fitness, and metabolic alterations. Interestingly, daptomycin nonsusceptibility coincides with a slightly delayed transition to the postexponential growth phase and alterations in metabolism. Specifically, daptomycin-nonsusceptible strains have decreased tricarboxylic acid cycle activity, which correlates with increased synthesis of pyrimidines and purines and increased carbon flow to pathways associated with wall teichoic acid and peptidoglycan biosynthesis. Importantly, these data provided an opportunity to alter the daptomycin nonsusceptibility phenotype by manipulating bacterial metabolism, a first step in developing compounds that target metabolic pathways that can be used in combination with daptomycin to reduce treatment failures.

The success of Staphylococcus aureus as a pathogen is due in part to its ability to adapt to changing environmental conditions using signal transduction pathways, such as metabolite- responsive regulators and two-component systems. S. aureus has a two-component system encoded by the gene pair sav0224 (hptS) and sav0223 (hptR) that regulate the hexose phosphate transport (uhpT) system in response to extracellular glucose-6-phosphate. Glycolytic intermediates such as glucose-6-phosphate are important carbon sources that also modulate the activity of the global metabolite-responsive transcriptional regulator CcpA. Because uhpT has a putative CcpA binding site in its promoter and it is regulated by HptR, it was hypothesized the regulons of CcpA and HptR might intersect. To determine if the regulatory domains of CcpA and HptRS overlap, ccpA was deleted in strains SA564 and SA564-ΔhptRS and growth, metabolic, proteomic, and transcriptional differences were assessed. As expected, CcpA represses hptS and hptR in a glucose dependent manner; however, upon CcpA derepression, the HptRS system functions as a transcriptional activator of metabolic genes within the CcpA regulon. Importantly, inactivation of ccpA and hptRS altered sensitivity to fosfomycin and ampicillin in the absence of exogenous glucose-6-phosphate, indicating that both CcpA and HptRS modulate antibiotic susceptibility.

Staphylococcus aureus is a major human pathogen whose infections are increasingly difficult to treat due to increased antibiotic resistance, including resistance to vancomycin. Vancomycin-intermediate S. aureus (VISA) strains develop resistance to vancomycin through adaptive changes that are incompletely understood. Central to this adaptation are metabolic changes that permit growth in the presence of vancomycin. To define the metabolic changes associated with adaptive resistance to vancomycin in S. aureus , the metabolomes of a vancomycin-sensitive and VISA strain pair isolated from the same patient shortly after vancomycin therapy began and following vancomycin treatment failure were analyzed. The metabolic adaptations included increases in acetogenesis, carbon flow through the pentose phosphate pathway, wall teichoic acid and peptidoglycan precursor biosynthesis, purine biosynthesis, and decreased tricarboxylic acid (TCA) cycle activity. The significance of these metabolic pathways for vancomycin-intermediate susceptibility was determined by assessing the synergistic potential of human-use-approved inhibitors of these pathways in combination with vancomycin against VISA strains. Importantly, inhibitors of amino sugar and purine biosynthesis acted synergistically with vancomycin to kill a diverse set of VISA strains, suggesting that combinatorial therapy could augment the efficacy of vancomycin even in patients infected with VISA strains.

Many bacteria regulate the expression of virulence factors via carbon catabolite responsive elements. In Gram-positive bacteria, the predominant mediator of carbon catabolite repression is the catabolite control protein A (CcpA). Hyperglycemia is a widespread disorder that predisposes individuals to an array of symptoms and an increased risk of infections. In hyperglycemic individuals, the bacterium Staphylococcus aureus causes serious, life-threatening infections. The importance of CcpA in regulating carbon catabolite repression in S. aureus suggests it may be important for infections in hyperglycemic individuals. To test this suggestion, hyperglycemic non-obese diabetic (NOD; blood glucose level ≥20 mM) mice were challenged with the mouse pathogenic S. aureus strain Newman and the isogenic ccpA deletion mutant (MST14), and the effects on infectivity were determined. Diabetic NOD mice challenged with the ccpA deletion mutant enhanced the symptoms of infection in an acute murine pneumonia model relative to the parental strain. Interestingly, when diabetic NOD mice were used in footpad or catheter infection models, infectivity of the ccpA mutant decreased relative to the parental strain. These differences greatly diminished when normoglycemic NOD mice (blood glucose level ≤ 10 mM) were used. These data suggest that CcpA is important for infectivity of S. aureus in hyperglycemic individuals.

Background Streptococcus mitis-oralis is a leading cause of IE. Treatment of this pathogen is limited by frequent high-level β-lactam resistance and the propensity to develop high-level DAP-R during DAP exposure. The current study elucidated key metabolic perturbations associated with high-level DAP-R in prototype S. mitis-oralis strain 351, following in vitro selection of DAP-R by 10-day serial passage in sub-inhibitory DAP. Furthermore, to test translatability of such metabolic changes (see below), the synergistic activity of combinations of DAP plus a strategic metabolic inhibitor (i.e., fosfomycin) vs. DAP or fosfomycin alone was assessed, using DAP-R S. mitis-oralis strain 351-D10 (MIC >256 µg/mL) in vitro and in an ex vivo IE model. Methods MICs. E test Growth Curve: Optical density (OD600) determined spectrophotometrically at 0–8 hours glyceraldehyde-3-phosphatedehydrogenase (GAPDH) activity. Kit from BioVision®. Metabolomics: one-dimensional 1H NMR-MS and two-dimensional 1H-13C HSQC in vitro time-kill assay: Using sub-MIC/MIC drug concentrations (initial inoculum ~1 × 105 CFU/mL) for 0, 2, 4, 6, and 8 hours. Ex vivo IE model: Simulated endocardial vegetations (SEVs) quantitatively cultured at 0, 4, 8, 24, 32, 48, and 72 hours with DAP or fosfomycin alone or in combination. Results NMR metabolomics analysis identified a number of metabolite differences in the 351 D10 DAP-R vs. 351 DAP-S strain (Figure 1). These data are consistent with a significant reduction in GAPDH activity (a glycolytic enzyme) in 351-D10 vs. 351 strain. Based on these metabolic changes, fosfomycin (a phosphoenolpyruvate analog) was chosen as a strategic metabolic inhibitor to attempt to “resensitize” our DAP-R S. mitis-oralis strain to DAP. The combination of DAP + fosfomycin demonstrated synergistic killing of the DAP-R strain vs. DAP or fosfomycin alone in the in vitro time-killing assays. Moreover, the DAP-R strain was synergistically cleared from SEVs by DAP + fosfomycin in the ex vivo IE model. Conclusion Taken together, these data indicate there are unique metabolome signatures associated with the DAP-R phenotype in S. mitis-oralis. In addition, these data provide support for further studying the use of strategic S. mitis-oralis metabolic inhibitors in additional strain-sets to resensitize DAP-R strains to DAP, using in vitro, ex vivo and in vivo models. Disclosures All authors: No reported disclosures.

The tricarboxylic acid cycle (TCA cycle) is a central metabolic pathway that provides energy, reducing potential, and biosynthetic intermediates. In Staphylococcus aureus, TCA cycle activity is controlled by several regulators (e.g. CcpA, CodY, and RpiRc) in response to the availability of sugars, amino acids, and environmental stress. Developing a bioinformatic search for additional carbon catabolite-responsive regulators in S. aureus, we identified a LysR-type regulator, catabolite control protein E (CcpE), with homology to the Bacillus subtilis CcpC regulator. Inactivation of ccpE in S. aureus strain Newman revealed that CcpE is a positive transcriptional effector of the first two enzymes of the TCA cycle, aconitase (citB) and to a lesser extent citrate synthase (citZ). Consistent with the transcriptional data, aconitase activity dramatically decreased in the ccpE mutant relative to the wild-type strain. The effect of ccpE inactivation on citB transcription and the lesser effect on citZ transcription were also reflected in electrophoretic mobility shift assays where CcpE bound to the citB promoter but not the citZ promoter. Metabolomic studies showed that inactivation of ccpE resulted in increased intracellular concentrations of acetate, citrate, lactate, and alanine, consistent with a redirection of carbon away from the TCA cycle. Taken together, our data suggest that CcpE is a major direct positive regulator of the TCA cycle gene citB.

Streptomyces aureofaciens is a Gram-positive actinomycete that produces the antibiotics tetracycline and chlortetracycline. Here, we report the assembly and initial annotation of the draft genome sequence of S. aureofaciens ATCC strain 10762.

In Staphylococcus aureus , metabolism is intimately linked with virulence determinant biosynthesis, and several metabolite-responsive regulators have been reported to mediate this linkage. S. aureus possesses at least three members of the RpiR family of transcriptional regulators. Of the three RpiR homologs, RpiRc is a potential regulator of the pentose phosphate pathway, which also regulates RNAIII levels. RNAIII is the regulatory RNA of the agr quorum-sensing system that controls virulence determinant synthesis. The effect of RpiRc on RNAIII likely involves other regulators, as the regulators that bind the RNAIII promoter have been intensely studied. To determine which regulators might bridge the gap between RpiRc and RNAIII, sarA , sigB , mgrA , and acnA mutations were introduced into an rpiRc mutant background, and the effects on RNAIII were determined. Additionally, phenotypic and genotypic differences were examined in the single and double mutant strains, and the virulence of select strains was examined using two different murine infection models. The data suggest that RpiRc affects RNAIII transcription and the synthesis of virulence determinants in concert with σ B , SarA, and the bacterial metabolic status to negatively affect virulence.

Inactivation of the Staphylococcus aureus tricarboxylic acid (TCA) cycle delays the resolution of cutaneous ulcers in a mouse soft tissue infection model. In this study, it was observed that cutaneous lesions in mice infected with wild-type or isogenic aconitase mutant S. aureus strains contained comparable inflammatory infiltrates, suggesting the delayed resolution was independent of the recruitment of immune cells. These observations led us to hypothesize that staphylococcal metabolism can modulate the host immune response. Using an in vitro model system involving RAW 264.7 cells, the authors observed that cells cultured with S. aureus aconitase mutant strains produced significantly lower amounts of nitric oxide (NO(•)) and an inducible nitric oxide synthase as compared to those cells exposed to wild-type bacteria. Despite the decrease in NO(•) synthesis, the expression of antigen-presentation and costimulatory molecules was similar in cells cultured with wild-type and those cultured with aconitase mutant bacteria. The data suggest that staphylococci can evade innate immune responses and potentially enhance their ability to survive in infected hosts by altering their metabolism. This may also explain the occurrence of TCA cycle mutants in clinical S. aureus isolates.

CodY is a global regulatory protein that was first discovered in Bacillus subtilis , where it couples gene expression to changes in the pools of critical metabolites through its activation by GTP and branched-chain amino acids. Homologs of CodY can be found encoded in the genomes of nearly all low-G+C gram-positive bacteria, including Staphylococcus aureus . The introduction of a codY -null mutation into two S. aureus clinical isolates, SA564 and UAMS-1, through allelic replacement, resulted in the overexpression of several virulence genes. The mutant strains had higher levels of hemolytic activity toward rabbit erythrocytes in their culture fluid, produced more polysaccharide intercellular adhesin (PIA), and formed more robust biofilms than did their isogenic parent strains. These phenotypes were associated with derepressed levels of RNA for the hemolytic alpha-toxin ( hla ), the accessory gene regulator ( agr ) ( RNAII and RNAIII/hld ), and the operon responsible for the production of PIA ( icaADBC ). These data suggest that CodY represses, either directly or indirectly, the synthesis of a number of virulence factors of S. aureus .

The most common mechanism by which Staphylococcus aureus gains resistance to vancomycin is by adapting its physiology and metabolism to permit growth in the presence of vancomycin. Several studies have examined the adaptive changes occurring during the transition to vancomycin-intermediate resistance, leading to a model of vancomycin resistance in which decreased cell wall turnover and autolysis result in increased cell wall thickness and resistance to vancomycin. In the present study, we identified metabolic changes common to vancomycin-intermediate S. aureus (VISA) strains by assessing the metabolic and growth characteristics of two VISA strains (vancomycin MICs of 8 μg/ml) and two isogenic derivative strains with vancomycin MICs of 32 μg/ml. Interestingly, we observed the parental strains had impaired catabolism of nonpreferred carbon sources (i.e., acetate), and this impairment became more pronounced as vancomycin resistance increased. To determine if acetate catabolism impairment is common to VISA strains, we assessed the ability of VISA and vancomycin-sensitive S. aureus (VSSA) clinical isolates to catabolize acetate. As expected, a significantly greater percentage of VISA strains (71%) had impaired acetate catabolism relative to VSSA (8%). This is an important observation because staphylococcal acetate catabolism is implicated in growth yield and antibiotic tolerance and in regulating cell death and polysaccharide intercellular adhesin synthesis.

We describe the genome sequence of a macrolide-resistant strain (MGAS10394) of serotype M6 group A Streptococcus (GAS). The genome is 1,900,156 bp in length, and 8 prophage-like elements or remnants compose 12.4% of the chromosome. A 8.3-kb prophage remnant encodes the SpeA4 variant of streptococcal pyrogenic exotoxin A. The genome of strain MGAS10394 contains a chimeric genetic element composed of prophage genes and a transposon encoding the mefA gene conferring macrolide resistance. This chimeric element also has a gene encoding a novel surface-exposed protein (designated "R6 protein"), with an LPKTG cell-anchor motif located at the carboxyterminus. Surface expression of this protein was confirmed by flow cytometry. Humans with GAS pharyngitis caused by serotype M6 strains had antibody against the R6 protein present in convalescent, but not acute, serum samples. Our studies add to the theme that GAS prophage-encoded extracellular proteins contribute to host-pathogen interactions in a strain-specific fashion.

In bacteria, translation initiates with formyl-methionine; however, the N-terminal formyl group is usually removed by peptide deformylase, an enzymatic activity requiring iron. Staphylococcus aureus δ-toxin is a 26-amino-acid polypeptide secreted predominantly with a formylated N-terminal methionine, which led us to investigate regulation of δ-toxin deformylation. We observed that during exponential and early postexponential growth, δ-toxin accumulated in the culture medium in formylated and deformylated forms. In contrast, only formylated δ-toxin accumulated after the early postexponential phase. The transition from producing both species of δ-toxin to producing only formyl-methionine-containing δ-toxin coincided with increased tricarboxylic acid (TCA) cycle activity. The TCA cycle contains several iron-requiring enzymes, which led us to hypothesize that TCA cycle induction depletes the iron in the culture medium, thereby inhibiting peptide deformylase activity. As expected, S. aureus depletes the iron in the culture medium between the postexponential and stationary phases of growth. Inhibition of δ-toxin deformylation was relieved by TCA cycle inactivation or by addition of supplemental iron to the culture medium. Of interest, peptides containing formyl-methionine are potent chemoattractants for neutrophils, suggesting that δ-toxin deformylation may have functional consequences. We found neutrophil chemotactic activity only with formylated δ-toxin. The S. aureus TCA cycle is derepressed upon depletion of rapidly catabolizable carbon sources; this coincides with the transition to producing only formylated δ-toxin and results in an increased inflammatory response. The proinflammatory response should increase host cell damage and result in the release of nutrients. Taken together, these results establish that there is an important linkage between bacterial metabolism and pathogenesis.

Pathogenic spirochetes belonging to the genus Borrelia are defined in part by their obligatory biological transmission by blood-feeding arthropods (6). These bacteria cause a variety of diseases in humans and other animals, are transmitted by numerous species of ticks or the human body louse, and are widely distributed around much of the world (12). Most of the species fall into one of two major groups related either to the relapsing-fever or Lyme disease spirochetes (28, 31). Relapsing-fever spirochetes cause a recurrent, febrile illness associated with pronounced bacteremias and are transmitted by fast-feeding argasid ticks of the genus Ornithodoros (1, 9). Lyme disease spirochetes cause a wide range of symptoms and are transmitted by the slow-feeding ixodid ticks of the genus Ixodes (19). A striking difference between the two groups of spirochetes is the cell density attained by the different groups in the peripheral blood. Relapsing-fever spirochetes and the related agent of fowl spirochetosis, Borrelia anserina, can achieve cell densities of 108 or more bacteria per ml of blood, making them easily detectable by light microscopy (Fig. ​(Fig.1).1). Lyme disease spirochetes also infect the peripheral blood and may be cultured from this tissue (3, 24, 43, 49); however, no reports demonstrate that these spirochetes attain cell densities high enough to allow their direct microscopic detection. This discrepancy in the levels of spirochetemia between relapsing-fever and Lyme disease spirochetes suggests a fundamental difference in the way these two groups of bacteria exploit or survive in the blood. FIG. 1. Spirochetemia of B. anserina with a very high cell density in the blood of a 12-day-old chicken 6 days after i.p. inoculation. The spirochetes far outnumber the nucleated red blood cells. Scale bar, 25 μm. During the last 6 years, we have investigated a highly immunogenic protein in relapsing-fever spirochetes that stimulates a strong antibody response in patients having had tick-borne or louse-borne relapsing fever (27, 34). The bacterial gene encoding this protein, glycerophosphodiester phosphodiesterase (GlpQ), was first identified in Escherichia coli and was shown to hydrolyze deacylated phospholipids to an alcohol and glycerol-3-phosphate (G3P) (20). Our initial work also demonstrated that people who have had Lyme disease do not react serologically to this protein, and we were unable to find evidence by immunoblotting, PCR, or Southern blotting for its presence in the Lyme disease spirochete, Borrelia burgdorferi (34). The lack of glpQ in B. burgdorferi was confirmed when no ortholog to this gene was found in its genome (8, 13). The presence or absence of a glpQ ortholog in spirochetes does not establish their ability to hydrolyze phospholipids. Therefore, in the present work we investigated numerous species of Borrelia for GlpQ activity and examined the glp locus at the DNA level. Our hypothesis is that GlpQ permits only the relapsing-fever group of spirochetes to acquire G3P from phospholipids. This metabolic advantage may contribute, in part, to their ability to achieve higher cell densities in the blood than do the Lyme disease spirochetes. The work we describe here is the first step toward testing this hypothesis.

Human polymorphonuclear leukocytes (PMNs) are an essential part of innate immunity and contribute significantly to inflammation. Although much is nderstood about the inflammatory response, the molecular basis for termination of inflammation in humans is largely undefined. We used human oligonucleotide microarrays to identify genes differentially regulated during the onset of apoptosis occurring after PMN phagocytosis. Genes encoding proteins that regulate cell metabolism and vesicle trafficking comprised 198 (98 genes induced, 100 genes repressed) of 867 differentially expressed genes. We discovered that complex cellular pathways involving glutathione and thioredoxin detoxification systems, heme catabolism, ubiquitin-proteasome degradation, purine nucleotide metabolism, and nuclear import were regulated at the level of gene expression during the initial stages of PMN apoptosis. Eleven genes encoding key regulators of glycolysis, the hexose monophosphate shunt, the glycerol-phosphate shuttle, and oxidative phosphorylation were induced. Increased levels of cellular reduced glutathione and γ-glutamyltransferase and glycolytic activity confirmed that several of these metabolic pathways were up-regulated. In contrast, seven genes encoding critical enzymes involved in fatty acid β-oxidation, which can generate toxic lipid peroxides, were down-regulated. Our results indicate that energy metabolism and oxidative stress-response pathways are gene-regulated during PMN apoptosis. We propose that changes in PMN gene expression leading to programmed cell death are part of an apoptosis-differentiation program, a final stage of transcriptionally regulated PMN maturation that is accelerated significantly by phagocytosis. These findings provide new insight into the molecular events that contribute to the resolution of inflammation in humans.

Carbon metabolism and virulence determinant production are often linked in pathogenic bacteria, and several regulatory elements have been reported to mediate this linkage in Staphylococcus aureus. Previously, we described a novel protein, catabolite control protein E (CcpE) that functions as a regulator of the tricarboxylic acid cycle. Here we demonstrate that CcpE also regulates virulence determinant biosynthesis and pathogenesis. Specifically, deletion of ccpE in S. aureus strain Newman revealed that CcpE affects transcription of virulence factors such as capA, the first gene in the capsule biosynthetic operon; hla, encoding α-toxin; and psmα, encoding the phenol-soluble modulin cluster α. Electrophoretic mobility shift assays demonstrated that CcpE binds to the hla promoter. Mice challenged with S. aureus strain Newman or its isogenic ΔccpE derivative revealed increased disease severity in the ΔccpE mutant using two animal models; an acute lung infection model and a skin infection model. Complementation of the mutant with the ccpE wild-type allele restored all phenotypes, demonstrating that CcpE is negative regulator of virulence in S. aureus.

Staphylococcus aureus is a prominent nosocomial pathogen and a major cause of biomaterial-associated infections. The success of S. aureus as a pathogen is due in part to its ability to adapt to stressful environments. As an example, the transition from residing in the nares to residing in the blood or deeper tissues is accompanied by changes in the availability of nutrients and elements such as oxygen and iron. As such, nutrients, oxygen, and iron are important determinants of virulence factor synthesis in S. aureus . In addition to influencing virulence factor synthesis, oxygen and iron are critical cofactors in enzymatic and electron transfer reactions; thus, a change in iron or oxygen availability alters the bacterial metabolome. Changes in metabolism create intracellular signals that alter the activity of metabolite-responsive regulators such as CodY, RpiRc, and CcpA. To assess the extent of metabolomic changes associated with oxygen and iron limitation, S. aureus cells were cultivated in iron-limited medium and/or with decreasing aeration, and the metabolomes were examined by nuclear magnetic resonance (NMR) spectroscopy. As expected, oxygen and iron limitation dramatically decreased tricarboxylic acid (TCA) cycle activity, creating a metabolic block and significantly altering the metabolome. These changes were most prominent during post-exponential-phase growth, when TCA cycle activity was maximal. Importantly, many of the effects of iron limitation were obscured by aeration limitation. Aeration limitation not only obscured the metabolic effects of iron limitation but also overrode the transcription of iron-regulated genes. Finally, in contrast to previous speculation, we confirmed that acidification of the culture medium occurs independent of the availability of iron.

Glutamate enhances the yield of exotoxin A (ETA), which is induced by iron limitation, from Pseudomonas aeruginosa . We tested the possibility that glutamate affects growth during iron restriction. We confirmed that iron limitation caused early entry into stationary phase but had no effect on the exponential growth rate. We showed that glutamate, as well as citrate and isocitrate, partially overcame this growth limitation. Glutamate had no effect on toxA (ETA-encoding) transcription, which implies that glutamate primarily increases the number of toxin-producing cells. In contrast, citrate and isocitrate diminished toxA transcription. Since glutamate, citrate, and isocitrate stimulated growth, we suspected a block in the citric acid cycle. Iron limitation reduced the activity of the iron-containing aconitase 12-fold but had no effect on isocitrate dehydrogenase activity, which was assayed as a control. There is a reciprocal relationship between aconitase activity and ETA synthesis, and this correlation does not appear to be coincidental because aconitase-specific effectors affect ETA synthesis. We tested whether a metabolic block is sufficient to induce ETA synthesis, but an aconitase-specific inhibitor diminished ETA production, which argues against this possibility. Finally, we present preliminary evidence that iron limitation may reversibly and posttranslationally inactivate aconitase in vivo. In summary, the environmental factors that stimulate ETA synthesis are related: glutamate bypasses an iron limitation-dependent metabolic block that causes entry into stationary phase. We speculate that one or more of the aconitases in P. aeruginosa may contribute to the control of virulence factor synthesis.

The "omics" era began with transcriptomics and this progressed into proteomics. While useful, these approaches provide only circumstantial information about carbon flow, metabolic status, redox poise, etc. To more directly address these metabolic concerns, researchers have turned to the emerging field of metabolomics. In our laboratories, we frequently use NMR metabolomics to acquire a snapshot of bacterial metabolomes during stressful or transition events. Irrespective of the "omics" method of choice, the experimental outcome depends on the proper cultivation and preparation of bacterial samples. In addition, the integration of these large datasets requires that these cultivation conditions be clearly defined.

The genus Corynebacterium consists of nearly seventy species and is closely related to the genera Mycobacterium, Nocardia, and Rhodococcus. The characteristic traits of Corynebacteria include cells that are shaped like straight rods with clubbed ends, as well as an extra cell wall layer consisting of mycolic acids covalently bound to the peptidoglycan layer which adds an additional layer of protection against antibiotics. Corynebacteria are very well studied and outstandingly important for the large scale biotechnological production of amino acids and nucleotides.[1] Moreover, Corynebacteria produce several pathogens that affect humans and livestock. Toxins produced by C. diphtheriae and C. ulcerans cause diphtheria, a highly contagious respiratory infection in humans,[2] or diphtheria-like symptoms,[2–3] respectively. C. pseudotuberculosis causes “cheesy gland” disease in goats and sheep resulting in significant economic losses.[2, 4] Moreover, while rare, many other Corynebacteria species have also been shown to cause infections. Therefore, elucidating the functional roles of uncharacterized proteins from Corynebacteria is of high biomedical and economic importance. Protein CG2496 from Corynebacterium glutamicum (UniProtKB ID: {"type":"entrez-protein","attrs":{"text":"Q6M3G5","term_id":"75368461","term_text":"Q6M3G5"}}Q6M3G5, {"type":"entrez-protein","attrs":{"text":"Q8NNC9","term_id":"81760150","term_text":"Q8NNC9"}}Q8NNC9; Gene ID: CG2496, Cgl2275) is predicted to be an integral membrane protein comprised of 684 amino acids, where the N-terminal and C-terminal polypeptide segments relative to a single transmembrane helix are extracellular and cytoplasmic, respectively. In the NCBI RefSeq and UniProtKB[5] databases, CG2496 is annotated as a chromosome segregation ATPase, but there is currently no experimental evidence for this particular annotation. Homologous proteins are found in genomes of 43 other species of Corynebacteria. A significant portion of the N-terminal domain of CG2496 (residues 63–171) belongs to the TPM domain (named after proteins TLP18.3, Psb32 and MOLO-1) family (Pfam[6] accession: PF04536), which currently contains 3,085 protein sequences from 1,821 species, including bacteria, plants, protozoa and lower metazoa, such as nematodes and lancelets. Two TPM domain-containing proteins, TLP18.3 from A. thaliana and Psb32(Sll1390) from Synechocystis sp., were shown to be involved in the photosystem II (PSII) repair cycle.[7] Phosphatase activity was reported for TLP18.3,[7b] however, the measured enzymatic activity levels were very low. In C. elegans, the TPM domain protein MOLO-1 acts as a modulator of the levamisole-sensitive acetylcholine receptor (L-AChR), but the function of TPM domain proteins in other organisms is still unknown. The Northeast Structural Genomics Consortium (NESG; http://www.nesg.org) recently determined the solution NMR structure[8] of the TPM domain of CG2496 comprising residues 41-180 (PDB ID: 2KPT; NESG target ID: CgR26A) which revealed a distinct architecture and provided the first structural representative for PF04536. Here we describe the identification of low molecular weight compounds binding to the extracellular N-terminal domain of protein CG2496 from C. glutamicum using an NMR-based screening approach (FAST-NMR).[9] We expect that the newly identified compounds will also support future functional characterization of protein CG2496. Furthermore, assuming that protein CG2496 plays an important role for proliferation of C. glutamicum, we investigated to which extent one of the compounds, that is, methiothepin, inhibits cell growth. 1D 1H NMR screening of the FAST-NMR library of 460 low molecular weight compounds resulted in 13 initial hits which showed broadening of 1H NMR peaks in the presence of CG2496(41-180) (Table 1). Specific interaction with CG2496(41-180) was revealed by assessing chemical shift perturbations in 2D [15N,1H]-HSQC spectra recorded for CG2496(41-180) in the presence of each of the 13 compounds. Additionally, 5 more compounds from the library, which did not show line-broadening in the 1D 1H NMR screen, were evaluated using the 2D [15N,1H]-HSQC screen. These compounds were selected to test hypotheses of CG2496(41-180) function. Of the 18 total compounds screened by 2D [15N,1H]-HSQC, 10 induced minor perturbations of a small number (

A recent controversial hypothesis suggested that the bactericidal action of antibiotics is due to the generation of endogenous reactive oxygen species (ROS), a process requiring the citric acid cycle (tricarboxylic acid [TCA] cycle). To test this hypothesis, we assessed the ability of oxacillin to induce ROS production and cell death in Staphylococcus epidermidis strain 1457 and an isogenic citric acid cycle mutant. Our results confirm a contributory role for TCA-dependent ROS in enhancing susceptibility of S. epidermidis toward β-lactam antibiotics and also revealed a propensity for clinical isolates to accumulate TCA cycle dysfunctions presumably as a way to tolerate these antibiotics. The increased protection from β-lactam antibiotics could result from pleiotropic effects of a dysfunctional TCA cycle, including increased resistance to oxidative stress, reduced susceptibility to autolysis, and a more positively charged cell surface., IMPORTANCE Staphylococcus epidermidis, a normal inhabitant of the human skin microflora, is the most common cause of indwelling medical device infections. In the present study, we analyzed 126 clinical S. epidermidis isolates and discovered that tricarboxylic acid (TCA) cycle dysfunctions are relatively common in the clinical environment. We determined that a dysfunctional TCA cycle enables S. epidermidis to resist oxidative stress and alter its cell surface properties, making it less susceptible to β-lactam antibiotics.

Background The Per-Arnt-Sim (PAS) domain represents a ubiquitous structural fold that is involved in bacterial sensing and adaptation systems, including several virulence related functions. Although PAS domains and the subclass of PhoQ-DcuS-CitA (PDC) domains have a common structure, there is limited amino acid sequence similarity. To gain greater insight into the evolution of PDC/PAS domains present in the bacterial kingdom and staphylococci in specific, the PDC/PAS domains from the genomic sequences of 48 bacteria, representing 5 phyla, were identified using the sensitive search method based on HMM-to-HMM comparisons (HHblits). Results A total of 1,007 PAS domains and 686 PDC domains distributed over 1,174 proteins were identified. For 28 Gram-positive bacteria, the distribution, organization, and molecular evolution of PDC/PAS domains were analyzed in greater detail, with a special emphasis on the genus Staphylococcus. Compared to other bacteria the staphylococci have relatively fewer proteins (6–9) containing PDC/PAS domains. As a general rule, the staphylococcal genomes examined in this study contain a core group of seven PDC/PAS domain-containing proteins consisting of WalK, SrrB, PhoR, ArlS, HssS, NreB, and GdpP. The exceptions to this rule are: 1) S. saprophyticus lacks the core NreB protein; 2) S. carnosus has two additional PAS domain containing proteins; 3) S. epidermidis, S. aureus, and S. pseudintermedius have an additional protein with two PDC domains that is predicted to code for a sensor histidine kinase; 4) S. lugdunensis has an additional PDC containing protein predicted to be a sensor histidine kinase. Conclusions This comprehensive analysis demonstrates that variation in PDC/PAS domains among bacteria has limited correlations to the genome size or pathogenicity; however, our analysis established that bacteria having a motile phase in their life cycle have significantly more PDC/PAS-containing proteins. In addition, our analysis revealed a tremendous amount of variation in the number of PDC/PAS-containing proteins within genera. This variation extended to the Staphylococcus genus, which had between 6 and 9 PDC/PAS proteins and some of these appear to be previously undescribed signaling proteins. This latter point is important because most staphylococcal proteins that contain PDC/PAS domains regulate virulence factor synthesis or antibiotic resistance.

Over the past decade, metabolomics has emerged as an important technique for systems biology. Measuring all the metabolites in a biological system provides an invaluable source of information to explore various cellular processes, and to investigate the impact of environmental factors and genetic modifications. Nuclear magnetic resonance (NMR) spectroscopy is an important method routinely employed in metabolomics. NMR provides comprehensive structural and quantitative information useful for metabolomics fingerprinting, chemometric analysis, metabolite identification and metabolic pathway construction. A successful metabolomics study relies on proper experimental protocols for the collection, handling, processing and analysis of metabolomics data. Critically, these protocols should eliminate or avoid biologically-irrelevant changes to the metabolome. We provide a comprehensive description of our NMR-based metabolomics procedures optimized for the analysis of bacterial metabolomes. The technical details described within this manuscript should provide a useful guide to reliably apply our NMR-based metabolomics methodology to systems biology studies.

Bacterial cultivation requires consideration of three things: The bacterial strain, cultivation medium, and cultivation conditions. Most microbiologists dutifully report their choice of strains and cultivation media in manuscripts; however, these same microbiologists often overlook reporting cultivation conditions. Without this information, it is difficult to determine if cultures were grown aerobically, microaerobically, or anaerobically. To cultivate bacteria aerobically, it is necessary to understand that oxygen does not readily diffuse into culture media; it needs help to get in. Microbiologists can do this by altering the flask-to-medium ratio, rpm of agitation, and/or the concentration of atmospheric oxygen, or by using baffled flasks.

Staphylococci are a versatile genus of bacteria that are capable of causing acute and chronic infections in diverse host species. The success of staphylococci as pathogens is due in part to their ability to mitigate endogenous and exogenous oxidative and nitrosative stress. Endogenous oxidative stress is a consequence of life in an aerobic environment; whereas, exogenous oxidative and nitrosative stress are often due to the bacteria’s interaction with host immune systems. To overcome the deleterious effects of oxidative and nitrosative stress, staphylococci have evolved protection, detoxification, and repair mechanisms that are controlled by a network of regulators. In this review, we summarize the cellular targets of oxidative stress, the mechanisms by which staphylococci sense oxidative stress and damage, oxidative stress protection and repair mechanisms, and regulation of the oxidative stress response. When possible, special attention is given to how the oxidative stress defense mechanisms help staphylococci control oxidative stress in the host.

Staphylococcus epidermidis is an opportunistic bacterium whose infections often involve the formation of a biofilm on implanted biomaterials. In S. epidermidis, the exopolysaccharide facilitating bacterial adherence in a biofilm is polysaccharide intercellular adhesin (PIA), whose synthesis requires the enzymes encoded within the intercellular adhesin operon (icaADBC). In vitro, the formation of S. epidermidis biofilms is enhanced by conditions that repress tricarboxylic acid (TCA) cycle activity, such as growth in a medium containing glucose. In many Gram-positive bacteria, repression of TCA cycle genes in response to glucose is accomplished by catabolite control protein A (CcpA). CcpA is a member of the GalR–LacI repressor family that mediates carbon catabolite repression, leading us to hypothesize that catabolite control of S. epidermidis biofilm formation is indirectly regulated by CcpA-dependent repression of the TCA cycle. To test this hypothesis, ccpA deletion mutants were constructed in strain 1457 and 1457-acnA and the effects on TCA cycle activity, biofilm formation and virulence were assessed. As anticipated, deletion of ccpA derepressed TCA cycle activity and inhibited biofilm formation; however, ccpA deletion had only a modest effect on icaADBC transcription. Surprisingly, deletion of ccpA in strain 1457-acnA, a strain whose TCA cycle is inactive and where icaADBC transcription is derepressed, strongly inhibited icaADBC transcription. These observations demonstrate that CcpA is a positive effector of biofilm formation and icaADBC transcription and a repressor of TCA cycle activity.

Staphylococcus aureus is a medically important pathogen that synthesizes a wide range of virulence determinants. The synthesis of many staphylococcal virulence determinants is regulated in part by stress-induced changes in the activity of the tricarboxylic acid (TCA) cycle. One metabolic change associated with TCA cycle stress is an increased concentration of ribose, leading us to hypothesize that a pentose phosphate pathway (PPP)-responsive regulator mediates some of the TCA cycle-dependent regulatory effects. Using bioinformatics, we identified three potential ribose-responsive regulators that belong to the RpiR family of transcriptional regulators. To determine whether these RpiR homologues affect PPP activity and virulence determinant synthesis, the rpiR homologues were inactivated, and the effects on PPP activity and virulence factor synthesis were assessed. Two of the three homologues (RpiRB and RpiRC) positively influence the transcription of the PPP genes rpiA and zwf , while the third homologue (RpiRA) is slightly antagonistic to the other homologues. In addition, inactivation of RpiRC altered the temporal transcription of RNAIII, the effector molecule of the agr quorum-sensing system. These data confirm the close linkage of central metabolism and virulence determinant synthesis, and they establish a metabolic override for quorum-sensing-dependent regulation of RNAIII transcription.

We previously hypothesized that Staphylococcus epidermidis senses a diverse set of environmental and nutritional factors associated with biofilm formation through a modulation in the activity of the tricarboxylic acid (TCA) cycle. Herein, we report our further investigation of the impact of additional environmental stress factors on TCA cycle activity and provide a detailed description of our NMR methodology. S. epidermidis wild-type strain 1457 was treated with stressors that are associated with biofilm formation, a sublethal dose of tetracycline, 5% NaCl, 2% glucose, and autoinducer-2 (AI-2). As controls and to integrate our current data with our previous study, 4% ethanol stress and iron-limitation were also used. Consistent with our prior observations, the effect of many environmental stress factors on the S. epidermidis metabolome was essentially identical to the effect of TCA cycle inactivation in the aconitase mutant strain 1457-acnA::tetM. A detailed quantitative analysis of metabolite concentration changes using 2D (1)H-(13)C HSQC and (1)H-(1)H TOCSY spectra identified a network of 37 metabolites uniformly affected by the stressors and TCA cycle inactivation. We postulate that the TCA cycle acts as the central pathway in a metabolic signaling network.

Staphylococcus epidermidis is a skin-resident bacterium and a major cause of biomaterial-associated infections. The transition from residing on the skin to residing on an implanted biomaterial is accompanied by regulatory changes that facilitate bacterial survival in the new environment. These regulatory changes are dependent upon the ability of bacteria to "sense" environmental changes. In S. epidermidis, disparate environmental signals can affect synthesis of the biofilm matrix polysaccharide intercellular adhesin (PIA). Previously, we demonstrated that PIA biosynthesis is regulated by tricarboxylic acid (TCA) cycle activity. The observations that very different environmental signals result in a common phenotype (i.e. increased PIA synthesis) and that TCA cycle activity regulates PIA biosynthesis led us to hypothesize that S. epidermidis is "sensing" disparate environmental signals through the modulation of TCA cycle activity. In this study, we used NMR metabolomics to demonstrate that divergent environmental signals are transduced into common metabolomic changes that are "sensed" by metabolite-responsive regulators, such as CcpA, to affect PIA biosynthesis. These data clarify one mechanism by which very different environmental signals cause common phenotypic changes. In addition, due to the frequency of the TCA cycle in diverse genera of bacteria and the intrinsic properties of TCA cycle enzymes, it is likely the TCA cycle acts as a signal transduction pathway in many bacteria.

More than 200 direct CodY target genes in Staphylococcus aureus were identified by genome-wide analysis of in vitro DNA binding. This analysis, which was confirmed for some genes by DNase I footprinting assays, revealed that CodY is a direct regulator of numerous transcription units associated with amino acid biosynthesis, transport of macromolecules, and virulence. The virulence genes regulated by CodY fell into three groups. One group was dependent on the Agr system for its expression; these genes were indirectly regulated by CodY through its repression of the agr locus. A second group was regulated directly by CodY. The third group, which includes genes for alpha-toxin and capsule synthesis, was regulated by CodY in two ways, i.e., by direct repression and by repression of the agr locus. Since S. aureus CodY was activated in vitro by the branched chain amino acids and GTP, CodY appears to link changes in intracellular metabolite pools with the induction of numerous adaptive responses, including virulence.

Staphylococcus aureus capsule synthesis requires the precursor N -acetyl-glucosamine; however, capsule is synthesized during post-exponential growth when the availability of N -acetyl-glucosamine is limited. Capsule biosynthesis also requires aerobic respiration, leading us to hypothesize that capsule synthesis requires tricarboxylic acid cycle intermediates. Consistent with this hypothesis, S. aureus tricarboxylic acid cycle mutants fail to make capsule.

a more certain classification of staphylococci than pigment production, wherein a positive coagulase test, which confirmed the identity of S. aureus, correlated much better with pathogenicity. Another common human pathogen is S. saprophyticus, which produces urinary tract infections in young women [2]. Staphylococcus haemolyticus is somewhat less common, but is important because it can be highly antibiotic resistant even to glycopeptides and linezolid [3]. Many other coagulase-negative strains (41 species identified at present) such as S. schleiferi and S. lugdunesis have been described, and they can produce a variety of nosocomial infections (reviewed by von Eiff et al. [4,5]).

Staphylococci are the leading causes of endovascular infections worldwide. Commonly, these infections involve the formation of biofilms on the surface of biomaterials. Biofilms are a complex aggregation of bacteria commonly encapsulated by an adhesive exopolysaccharide matrix. In staphylococci, this exopolysaccharide matrix is composed of polysaccharide intercellular adhesin (PIA). PIA is synthesized when the tricarboxylic acid (TCA) cycle is repressed. The inverse correlation between PIA synthesis and TCA cycle activity led us to hypothesize that increasing TCA cycle activity would decrease PIA synthesis and biofilm formation and reduce virulence in a rabbit catheter-induced model of biofilm infection. TCA cycle activity can be induced by preventing staphylococci from exogenously acquiring a TCA cycle-derived amino acid necessary for growth. To determine if TCA cycle induction would decrease PIA synthesis in Staphylococcus aureus , the glutamine permease gene ( glnP ) was inactivated and TCA cycle activity, PIA accumulation, biofilm forming ability, and virulence in an experimental catheter-induced endovascular biofilm (endocarditis) model were determined. Inactivation of this major glutamine transporter increased TCA cycle activity, transiently decreased PIA synthesis, and significantly reduced in vivo virulence in the endocarditis model in terms of achievable bacterial densities in biofilm-associated cardiac vegetations, kidneys, and spleen. These data confirm the close linkage of TCA cycle activity and virulence factor production and establish that this metabolic linkage can be manipulated to alter infectious outcomes.

SUMMARY Bacteria live in environments that are subject to rapid changes in the availability of the nutrients that are necessary to provide energy and biosynthetic intermediates for the synthesis of macromolecules. Consequently, bacterial survival depends on the ability of bacteria to regulate the expression of genes coding for enzymes required for growth in the altered environment. In pathogenic bacteria, adaptation to an altered environment often includes activating the transcription of virulence genes; hence, many virulence genes are regulated by environmental and nutritional signals. Consistent with this observation, the regulation of most, if not all, virulence determinants in staphylococci is mediated by environmental and nutritional signals. Some of these external signals can be directly transduced into a regulatory response by two-component regulators such as SrrAB; however, other external signals require transduction into intracellular signals. Many of the external environmental and nutritional signals that regulate virulence determinant expression can also alter bacterial metabolic status (e.g., iron limitation). Altering the metabolic status results in the transduction of external signals into intracellular metabolic signals that can be “sensed” by regulatory proteins (e.g., CodY, Rex, and GlnR). This review uses information derived primarily using Bacillus subtilis and Escherichia coli to articulate how gram-positive pathogens, with emphasis on Staphylococcus aureus and Staphylococcus epidermidis , regulate virulence determinant expression in response to a changing environment.

Staphylococcus aureus Clp ATPases (molecular chaperones) alter normal physiological functions including an aconitase-mediated effect on post-stationary growth, acetate catabolism, and entry into death phase (Chatterjee et al., J. Bacteriol. 2005, 187, 4488-4496). In the present study, the global function of ClpC in physiology, metabolism, and late-stationary phase survival was examined using DNA microarrays and 2-D PAGE followed by MALDI-TOF MS. The results suggest that ClpC is involved in regulating the expression of genes and/or proteins of gluconeogenesis, the pentose-phosphate pathway, pyruvate metabolism, the electron transport chain, nucleotide metabolism, oxidative stress, metal ion homeostasis, stringent response, and programmed cell death. Thus, one major function of ClpC is balancing late growth phase carbon metabolism. Furthermore, these changes in carbon metabolism result in alterations of the intracellular concentration of free NADH, the amount of cell-associated iron, and fatty acid metabolism. This study provides strong evidence for ClpC as a critical factor in staphylococcal energy metabolism, stress regulation, and late-stationary phase survival; therefore, these data provide important insight into the adaptation of S. aureus toward a persister state in chronic infections.

Staphylococcus epidermidis is a major nosocomial pathogen primarily infecting immunocompromised individuals or those with implanted biomaterials (e.g., catheters). Biomaterial-associated infections often involve the formation of a biofilm on the surface of the medical device. In S. epidermidis , polysaccharide intercellular adhesin (PIA) is an important mediator of biofilm formation and pathogenesis. Synthesis of PIA is regulated by at least three DNA binding proteins (IcaR, SarA, and σ B ) and several environmental and nutritional conditions. Previously, we observed the environmental conditions that increased PIA synthesis decreased tricarboxylic acid (TCA) cycle activity. In this study, S. epidermidis TCA cycle mutants were constructed, and the function of central metabolism in PIA biosynthesis was examined. TCA cycle inactivation altered the metabolic status of S. epidermidis , resulting in a massive derepression of PIA biosynthetic genes and a redirection of carbon from growth into PIA biosynthesis. These data demonstrate that the bacterial metabolic status is a critical regulatory determinant of PIA synthesis. In addition, these data lead us to propose that the TCA cycle acts as a signal transduction pathway to translate external environmental cues into intracellular metabolic signals that modulate the activity of transcriptional regulators.