Fructose-1-kinase has pleiotropic roles in Escherichia coli.

In Escherichia coli, the master transcription regulator catabolite repressor activator (Cra) regulates >100 genes in central metabolism. Cra binding to DNA is allosterically regulated by binding to fructose-1-phosphate (F-1-P), but the only documented source of F-1-P is from the concurrent import and phosphorylation of exogenous fructose. Thus, many have proposed that fructose-1,6-bisphosphate (F-1,6-BP) is also a physiological regulatory ligand. However, the role of F-1,6-BP has been widely debated. Here, we report that the E. coli enzyme fructose-1-kinase (FruK) can carry out its "reverse" reaction under physiological substrate concentrations to generate F-1-P from F-1,6-BP. We further show that FruK directly binds Cra with nanomolar affinity and forms higher order, heterocomplexes. Growth assays with a ΔfruK strain and fruK complementation show that FruK has a broader role in metabolism than fructose catabolism. Since fruK itself is repressed by Cra, these newly-reported events add layers to the dynamic regulation of E. coli's central metabolism that occur in response to changing nutrients. These findings might have wide-spread relevance to other γ-proteobacteria, which conserve both Cra and FruK.

The Sympathetic Nervous System Is Necessary for Development of CD4+ T-Cell Memory Following Staphylococcus aureus Infection.

Lymph nodes and spleens are innervated by sympathetic nerve fibers that enter alongside arteries. Despite discovery of these nerve fibers nearly 40 years ago, the role of these nerves during response to infection remains poorly defined. We have found that chemical depletion of sympathetic nerve fibers compromises the ability of mice to develop protective immune memory to a Staphylococcus aureus infection. Innate control of the primary infection was not impacted by sympathectomy. Germinal center formation is also compromised in nerve-depleted animals; however, protective antibody responses are still generated. Interestingly, protective CD4+ T-cell memory fails to form in the absence of sympathetic nerves after S aureus infection.

Kinetic Characterization of the Immune Response to Methicillin-Resistant Staphylococcus aureus Subcutaneous Skin Infection.

Staphylococcus aureus is a leading cause of skin and soft tissue infections (SSTIs). Studies examining the immune response to S. aureus have been conducted, yet our understanding of the kinetic response to S. aureus subcutaneous skin infection remains incomplete. In this study, we used C57BL/6J mice and USA300 S. aureus to examine the host-pathogen interface from 8 h postinfection to 15 days postinfection (dpi), with the following outcomes measured: lesion size, bacterial titers, local cytokine and chemokine levels, phenotype of the responding leukocytes, and histopathology and Gram staining of skin tissue. Lesions were largest at 1 dpi, with peak necrotic tissue areas at 3 dpi, and were largely resolved by 15 dpi. During early infection, bacterial titers were high, neutrophils were the most abundant immune cell type, there was a decrease in most leukocyte populations found in uninfected skin, and many different cytokines were produced. Histopathological analysis demonstrated swift and extensive keratinocyte death and robust and persistent neutrophil infiltration. Gram staining revealed subdermal S. aureus colonization and, later, limited migration into upper skin layers. Interleukin-17A/F (IL-17A/F) was detected only starting at 5 dpi and coincided with an immediate decrease in bacterial numbers in the following days. After 9 days, neutrophils were no longer the most abundant immune cell type present as most other leukocyte subsets returned, and surface wounds resolved coincident with declining bacterial titers. Collectively, these data illustrate a dynamic immune response to S. aureus skin infection and suggest a key role for precisely timed IL-17 production for infection clearance and healthy tissue formation.

The Small RNA Teg41 Is a Pleiotropic Regulator of Virulence in Staphylococcus aureus.

Previously, our group demonstrated a role for the small RNA (sRNA) Teg41 in regulating production of the alpha phenol-soluble modulin toxins (αPSMs) in Staphylococcus aureus. Overexpressing Teg41 increased αPSM production while deleting the 3' end of Teg41 (Teg41Δ3' strain) resulted in a decrease in αPSM production, reduced hemolytic activity of S. aureus culture supernatants, and attenuated virulence in a murine abscess model of infection. In this study, we further explore the attenuation of virulence in the Teg41Δ3' strain. Using both localized and systemic models of infection, we demonstrate that the Teg41Δ3' strain is more severely attenuated than an ΔαPSM mutant, strongly suggesting that Teg41 influences more than the αPSMs. Proteomic and transcriptomic analysis of the wild-type and Teg41Δ3' strains reveals widespread alterations in transcript abundance and protein production in the absence of Teg41, confirming that Teg41 has pleiotropic effects in the cell. We go on to investigate the molecular mechanism underlying Teg41-mediated gene regulation. Surprisingly, results demonstrate that certain Teg41 target genes, including the αPSMs and ßPSMs, are transcriptionally altered in the Teg41Δ3' strain, while other targets, specifically spa (encoding surface protein A), are regulated at the level of transcript stability. Collectively, these data demonstrate that Teg41 is a pleiotropic RNA regulator in S. aureus that influences expression of a variety of genes using multiple different mechanisms.

Fatty acids can inhibit Staphylococcus aureus SaeS activity at the membrane independent of alterations in respiration.

In Staphylococcus aureus, the two-component system SaeRS is responsible for regulating various virulence factors essential for the success of this pathogen. SaeRS can be stimulated by neutrophil-derived products but has also recently been shown to be inactivated by the presence of free fatty acids. A mechanism for how fatty acids negatively impacts SaeRS has not been described. We found that unsaturated fatty acids, as well as fatty acids not commonly found in Staphylococcal membranes, prevent the activation of SaeRS at a lower concentration than their saturated counterparts. These fatty acids can negatively impact SaeRS without altering the respiratory capacity of the bacterium. To uncover a potential mechanism for how fatty acids impact SaeRS function/activity, we utilized a naturally occurring point mutation found in S. aureus as well as chimeric SaeS proteins. Using these tools, we identified that the native transmembrane domains of SaeS dictate the transcriptional response to fatty acids in S. aureus. Our data support a model where free fatty acids alter the activity of the two-component system SaeRS directly through the sensor kinase SaeS and is dependent on the transmembrane domains of the protein.

Exogenous Fatty Acids Remodel Staphylococcus aureus Lipid Composition through Fatty Acid Kinase.

Staphylococcus aureus can utilize exogenous fatty acids for phospholipid synthesis. The fatty acid kinase FakA is essential for this utilization by phosphorylating exogenous fatty acids for incorporation into lipids. How FakA impacts the lipid membrane composition is unknown. In this study, we used mass spectrometry to determine the membrane lipid composition and properties of S. aureus in the absence of fakA We found the fakA mutant to have increased abundance of lipids containing longer acyl chains. Since S. aureus does not synthesize unsaturated fatty acids, we utilized oleic acid (18:1) to track exogenous fatty acid incorporation into lipids. We observed a concentration-dependent incorporation of exogenous fatty acids into the membrane that required FakA. We also tested how FakA and exogenous fatty acids impact membrane-related physiology and identified changes in membrane potential, cellular respiration, and membrane fluidity. To mimic the host environment, we characterized the lipid composition of wild-type and fakA mutant bacteria grown in mouse skin homogenate. We show that wild-type S. aureus can incorporate exogenous unsaturated fatty acids from host tissue, highlighting the importance of FakA in the presence of host skin tissue. In conclusion, FakA is important for maintaining the composition and properties of the phospholipid membrane in the presence of exogenous fatty acids, impacting overall cell physiology.IMPORTANCE Environmental fatty acids can be harvested to supplement endogenous fatty acid synthesis to produce membranes and circumvent fatty acid biosynthesis inhibitors. However, how the inability to use these fatty acids impacts lipids is unclear. Our results reveal lipid composition changes in response to fatty acid addition and when S. aureus is unable to activate fatty acids through FakA. We identify concentration-dependent utilization of oleic acid that, when combined with previous work, provides evidence that fatty acids can serve as a signal to S. aureus Furthermore, using mouse skin homogenates as a surrogate for in vivo conditions, we showed that S. aureus can incorporate host fatty acids. This study highlights how exogenous fatty acids impact bacterial membrane composition and function.

Staphylococcus aureus Fibronectin Binding Protein A Mediates Biofilm Development and Infection.

Implanted medical device-associated infections pose significant health risks, as they are often the result of bacterial biofilm formation. Staphylococcus aureus is a leading cause of biofilm-associated infections which persist due to mechanisms of device surface adhesion, biofilm accumulation, and reprogramming of host innate immune responses. We found that the S. aureus fibronectin binding protein A (FnBPA) is required for normal biofilm development in mammalian serum and that the SaeRS two-component system is required for functional FnBPA activity in serum. Furthermore, serum-developed biofilms deficient in FnBPA were more susceptible to macrophage invasion, and in a model of biofilm-associated implant infection, we found that FnBPA is crucial for the establishment of infection. Together, these findings show that S. aureus FnBPA plays an important role in physical biofilm development and represents a potential therapeutic target for the prevention and treatment of device-associated infections.

Staphylococcus aureus Fatty Acid Kinase FakA Modulates Pathogenesis during Skin Infection via Proteases.

Staphylococcus aureus fatty acid kinase FakA is necessary for the incorporation of exogenous fatty acids into the lipid membrane. We previously demonstrated that the inactivation of fakA leads to decreased α-hemolysin (Hla) production but increased expression of the proteases SspAB and aureolysin in vitro, and that the ΔfakA mutant causes larger lesions than the wild type (WT) during murine skin infection. As expected, necrosis is Hla dependent in the presence or absence of FakA, as both hla and hla ΔfakA mutants are unable to cause necrosis of the skin. At day 4 postinfection, while the ΔfakA mutant maintains larger and more necrotic abscesses, bacterial numbers are similar to those of the WT, indicating the enhanced tissue damage of mice infected with the ΔfakA mutant is not due to an increase in bacterial burden. At this early stage of infection, skin infected with the ΔfakA mutant has decreased levels of proinflammatory cytokines, such as interleukin-17A (IL-17A) and IL-1α, compared to those of WT-infected skin. At a later stage of infection (day 7), abscess resolution and bacterial clearance are hindered in ΔfakA mutant-infected mice. The paradoxical findings of decreased Hla in vitro but increased necrosis in vivo led us to investigate the role of the proteases regulated by FakA. Utilizing Δaur and ΔsspAB mutants in both the WT and fakA mutant backgrounds, we found that the absence of these proteases in a fakA mutant reduced dermonecrosis to levels similar to those of the WT strain. These studies suggest that the overproduction of proteases is one factor contributing to the enhanced pathogenesis of the ΔfakA mutant during skin infection.

CidR and CcpA Synergistically Regulate Staphylococcus aureus cidABC Expression.

The death and lysis of a subpopulation of Staphylococcus aureus cells during biofilm development benefit the whole bacterial population through the release of an important component of the biofilm matrix, extracellular DNA. Previously, we have demonstrated that these processes are affected by the gene products of the cidABC operon, the expression of which is controlled by the LysR-type transcriptional regulator, CidR. In this study, we characterized cis- and trans-acting elements essential for the induction of the cidABC operon. In addition to a CidR-binding site located within the cidABC promoter region, sequence analysis revealed the presence of a putative catabolite responsive element (cre box), suggestive of the involvement of the catabolite control protein A (CcpA) in the regulation of cidABC expression. This was confirmed using electrophoretic mobility shift assays and real-time reverse transcriptase PCR analysis demonstrating the direct positive control of cidABC transcription by the master regulator of carbon metabolism. Furthermore, the importance of CcpA and the identified cre site for the induction of the cidABC operon was demonstrated by examining the expression of P cidABC-lacZ reporter fusions in various mutant strains in which the genes involved in carbon metabolism and carbon catabolite repression were disrupted. Together the results of this study demonstrate the necessity of both transcriptional regulators, CidR and CcpA, for the induction of the cidABC operon and reveal the complexity of molecular interactions controlling its expression.IMPORTANCE This work focuses on the characterization of cis- and trans-acting elements essential for the induction of the cidABC operon in S. aureus The results of this study are the first to demonstrate the synergistic control of cidABC expression by transcriptional regulators CidR and CcpA during carbohydrate metabolism. We established that the full induction of cidABC expression depends on the metabolic state of bacteria and requires both CidR and CcpA. Together, these findings delineate regulatory control of cidABC expression under different metabolic conditions and provide important new insights into our understanding of cell death mechanisms during biofilm development in S. aureus.

Interplay of Nitric Oxide Synthase (NOS) and SrrAB in Modulation of Staphylococcus aureus Metabolism and Virulence.

Staphylococcus aureus nitric oxide synthase (saNOS) is a major contributor to virulence, stress resistance, and physiology, yet the specific mechanism(s) by which saNOS intersects with other known regulatory circuits is largely unknown. The SrrAB two-component system, which modulates gene expression in response to the reduced state of respiratory menaquinones, is a positive regulator of nos expression. Several SrrAB-regulated genes were also previously shown to be induced in an aerobically respiring nos mutant, suggesting a potential interplay between saNOS and SrrAB. Therefore, a combination of genetic, molecular, and physiological approaches was employed to characterize a nos srrAB mutant, which had significant reductions in the maximum specific growth rate and oxygen consumption when cultured under conditions promoting aerobic respiration. The nos srrAB mutant secreted elevated lactate levels, correlating with the increased transcription of lactate dehydrogenases. Expression of nitrate and nitrite reductase genes was also significantly enhanced in the nos srrAB double mutant, and its aerobic growth defect could be partially rescued with supplementation with nitrate, nitrite, or ammonia. Furthermore, elevated ornithine and citrulline levels and highly upregulated expression of arginine deiminase genes were observed in the double mutant. These data suggest that a dual deficiency in saNOS and SrrAB limits S. aureus to fermentative metabolism, with a reliance on nitrate assimilation and the urea cycle to help fuel energy production. The nos, srrAB, and nos srrAB mutants showed comparable defects in endothelial intracellular survival, whereas the srrAB and nos srrAB mutants were highly attenuated during murine sepsis, suggesting that SrrAB-mediated metabolic versatility is dominant in vivo.

Contribution of YjbIH to Virulence Factor Expression and Host Colonization in Staphylococcus aureus.

To persist within the host and cause disease, Staphylococcus aureus relies on its ability to precisely fine-tune virulence factor expression in response to rapidly changing environments. During an unbiased transposon mutant screen, we observed that disruption of a two-gene operon, yjbIH, resulted in decreased levels of pigmentation and aureolysin (Aur) activity relative to the wild-type strain. Further analyses revealed that YjbH, a predicted thioredoxin-like oxidoreductase, is predominantly responsible for the observed yjbIH mutant phenotypes, though a minor role exists for the putative truncated hemoglobin YjbI. These differences were due to significantly decreased expression of crtOPQMN and aur Previous studies found that YjbH targets the disulfide- and oxidative stress-responsive regulator Spx for degradation by ClpXP. The absence of yjbH or yjbI resulted in altered sensitivities to nitrosative and oxidative stress and iron deprivation. Additionally, aconitase activity was altered in the yjbH and yjbI mutant strains. Decreased levels of pigmentation and aureolysin (Aur) activity in the yjbH mutant were found to be Spx dependent. Lastly, we used a murine sepsis model to determine the effect of the yjbIH deletion on pathogenesis and found that the mutant was better able to colonize the kidneys and spleens during an acute infection than the wild-type strain. These studies identified changes in pigmentation and protease activity in response to YjbIH and are the first to have shown a role for these proteins during infection.

Inactivation of the exogenous fatty acid utilization pathway leads to increased resistance to unsaturated fatty acids in Staphylococcus aureus.

The human pathogen Staphylococcus aureus produces saturated fatty acids, but can incorporate both exogenous saturated and unsaturated fatty acids into its lipid membrane. S. aureus encounters unsaturated fatty acids in the host skin where they serve as an innate immune defence due to their toxicity. Previously, we identified a fatty acid kinase in S. aureus that is necessary for the utilization of exogenous fatty acids. The goal of this study was to determine the effects of fatty acids on mutants deficient in the exogenous fatty acid utilization machinery. We have demonstrated that mutants lacking a functional fatty acid kinase (fakA) or both fatty acid carrier proteins (fakB1 fakB2) are more resistant to unsaturated fatty acids. Previous studies suggested a role for ammonia-producing enzymes in resistance to unsaturated fatty acids, but these enzymes do not contribute to the resistance of the fakA mutant, despite increased urease transcription and protein activity in the mutant. Additionally, while pigment is altered in mutants unable to use exogenous fatty acids, staphyloxanthin does not contribute to fatty acid resistance of an fakA mutant. Because exposure to unsaturated fatty acids probably initiates a stress response, we investigated the role of the alternative sigma factor σB and determined if it is necessary for the fatty acid resistance observed in the fakA mutant. Collectively, this study demonstrates that the inability to incorporate unsaturated fatty acids leads to increased resistance to those fatty acids, and that resistance requires a σB stress response.

Active Bax and Bak are functional holins.

The mechanism of Bax/Bak-dependent mitochondrial outer membrane permeabilization (MOMP), a central apoptotic event primarily controlled by the Bcl-2 family proteins, remains not well understood. Here, we express active Bax/Bak in bacteria, the putative origin of mitochondria, and examine their functional similarities to the λ bacteriophage (λ) holin. As critical effectors for bacterial lysis, holin oligomers form membrane lesions, through which endolysin, a muralytic enzyme, escapes the cytoplasm to attack the cell wall at the end of the infection cycle. We found that active Bax/Bak, but not any other Bcl-2 family protein, displays holin behavior, causing bacterial lysis by releasing endolysin in an oligomerization-dependent manner. Strikingly, replacing the holin gene with active alleles of Bax/Bak results in plaque-forming phages. Furthermore, we provide evidence that active Bax produces large membrane holes, the size of which is controlled by structural elements of Bax. Notably, lysis by active Bax is inhibited by Bcl-xL, and the lysis activity of the wild-type Bax is stimulated by a BH3-only protein. Together, these results mechanistically link MOMP to holin-mediated hole formation in the bacterial plasma membrane.

Redirection of Metabolism in Response to Fatty Acid Kinase in Staphylococcus aureus.

Staphylococcus aureus is capable of phosphorylating exogenous fatty acids for incorporation into the bacterium's membrane via the fatty acid kinase, FakA. Additionally, FakA plays a significant role in virulence factor regulation and skin infections. We previously showed that a fakA mutant displays altered growth kinetics in vitro, observed during the late-exponential phase of growth. Here, we demonstrate that the absence of FakA leads to key metabolic changes. First, the fakA mutant has an altered acetate metabolism, with acetate being consumed at an increased rate than in the wild-type strain. Moreover, the growth benefit was diminished with inactivation of the acetate-generating enzyme AckA. Using a mass spectrometry-based approach, we identified altered concentrations of tricarboxylic acid (TCA) cycle intermediates and both intracellular and extracellular amino acids. Together, these data demonstrate a change in carbohydrate carbon utilization and altered amino acid metabolism in the fakA mutant. Energy status analysis revealed the mutant had a similar ADP/ATP ratio to that of the wild type, but a reduced adenylate energy charge. The inactivation of fakA changed the NAD+/NADH and NADP+/NADPH ratios, indicating a more oxidized cellular environment. Evidence points to the global metabolic regulatory proteins CcpA and CodY being important contributors to the altered growth in a fakA mutant. Indeed, it was found that directing amino acids from the urea cycle into the TCA cycle via glutamate dehydrogenase was an essential component of S. aureus growth after glucose depletion. Together, these data identify a previously unidentified role of FakA in the global physiology of S. aureus, linking external fatty acid utilization and central metabolism.IMPORTANCE The fatty acid kinase, FakA, of Staphylococcus aureus plays several important roles in the cell. FakA is important for the activation of the SaeRS two-component system and secreted virulence factors like α-hemolysin. However, the contribution of FakA to cellular metabolism has not been explored. Here, we highlight the metabolic consequence of removal of FakA from the cell. The absence of FakA leads to altered acetate metabolism and altered redox balance, as well as a change in intracellular amino acids. Additionally, the use of environmental amino acid sources is affected by FakA. Together, these results demonstrate for the first time that FakA provides a link between the pathways for exogenous fatty acid use, virulence factor regulation, and other metabolic processes.

VfrB Is a Key Activator of the Staphylococcus aureus SaeRS Two-Component System.

In previous studies, we identified the fatty acid kinase virulence factor regulator B (VfrB) as a potent regulator of α-hemolysin and other virulence factors in Staphylococcus aureus In this study, we demonstrated that VfrB is a positive activator of the SaeRS two-component regulatory system. Analysis of vfrB, saeR, and saeS mutant strains revealed that VfrB functions in the same pathway as SaeRS. At the transcriptional level, the promoter activities of SaeRS class I (coa) and class II (hla) target genes were downregulated during the exponential growth phase in the vfrB mutant, compared to the wild-type strain. In addition, saePQRS expression was decreased in the vfrB mutant strain, demonstrating a need for this protein in the autoregulation of SaeRS. The requirement for VfrB-mediated activation was circumvented when SaeS was constitutively active due to an SaeS (L18P) substitution. Furthermore, activation of SaeS via human neutrophil peptide 1 (HNP-1) overcame the dependence on VfrB for transcription from class I Sae promoters. Consistent with the role of VfrB in fatty acid metabolism, hla expression was decreased in the vfrB mutant with the addition of exogenous myristic acid. Lastly, we determined that aspartic acid residues D38 and D40, which are predicted to be key to VfrB enzymatic activity, were required for VfrB-mediated α-hemolysin production. Collectively, this study implicates VfrB as a novel accessory protein needed for the activation of SaeRS in S. aureusIMPORTANCE The SaeRS two-component system is a key regulator of virulence determinant production in Staphylococcus aureus Although the regulon of this two-component system is well characterized, the activation mechanisms, including the specific signaling molecules, remain elusive. Elucidating the complex regulatory circuit of SaeRS regulation is important for understanding how the system contributes to disease causation by this pathogen. To this end, we have identified the fatty acid kinase VfrB as a positive regulatory modulator of SaeRS-mediated transcription of virulence factors in S. aureus In addition to describing a new regulatory aspect of SaeRS, this study establishes a link between fatty acid kinase activity and virulence factor regulation.

The LysR-type transcriptional regulator, CidR, regulates stationary phase cell death in Staphylococcus aureus.

The Staphylococcus aureus LysR-type transcriptional regulator, CidR, activates the expression of two operons including cidABC and alsSD that display pro- and anti-death functions, respectively. Although several investigations have focused on the functions of different genes associated with these operons, the collective role of the CidR regulon in staphylococcal physiology is not clearly understood. Here we reveal that the primary role of this regulon is to limit acetate-dependent potentiation of cell death in staphylococcal populations. Although both CidB and CidC promote acetate generation and cell death, the CidR-dependent co-activation of CidA and AlsSD counters the effects of CidBC by redirecting intracellular carbon flux towards acetoin formation. From a mechanistic standpoint, we demonstrate that CidB is necessary for full activation of CidC, whereas CidA limits the abundance of CidC in the cell.

Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus.

Extracellular fatty acid incorporation into the phospholipids of Staphylococcus aureus occurs via fatty acid phosphorylation. We show that fatty acid kinase (Fak) is composed of two dissociable protein subunits encoded by separate genes. FakA provides the ATP binding domain and interacts with two distinct FakB proteins to produce acyl-phosphate. The FakBs are fatty acid binding proteins that exchange bound fatty acid/acyl-phosphate with fatty acid/acyl-phosphate presented in detergent micelles or liposomes. The ΔfakA and ΔfakB1 ΔfakB2 strains were unable to incorporate extracellular fatty acids into phospholipid. FakB1 selectively bound saturated fatty acids whereas FakB2 preferred unsaturated fatty acids. Affymetrix array showed a global perturbation in the expression of virulence genes in the ΔfakA strain. The severe deficiency in α-hemolysin protein secretion in ΔfakA and ΔfakB1 ΔfakB2 mutants coupled with quantitative mRNA measurements showed that fatty acid kinase activity was required to support virulence factor transcription. These data reveal the function of two conserved gene families, their essential role in the incorporation of host fatty acids by Gram-positive pathogens, and connects fatty acid kinase to the regulation of virulence factor transcription in S. aureus.

SrrAB Modulates Staphylococcus aureus Cell Death through Regulation of cidABC Transcription.

UNLABELLED: The death and lysis of a subpopulation in Staphylococcus aureus biofilm cells are thought to benefit the surviving population by releasing extracellular DNA, a critical component of the biofilm extracellular matrix. Although the means by which S. aureus controls cell death and lysis is not understood, studies implicate the role of the cidABC and lrgAB operons in this process. Recently, disruption of the srrAB regulatory locus was found to cause increased cell death during biofilm development, likely as a result of the sensitivity of this mutant to hypoxic growth. In the current study, we extended these findings by demonstrating that cell death in the ΔsrrAB mutant is dependent on expression of the cidABC operon. The effect of cidABC expression resulted in the generation of increased reactive oxygen species (ROS) accumulation and was independent of acetate production. Interestingly, consistently with previous studies, cidC-encoded pyruvate oxidase was found to be important for the generation of acetic acid, which initiates the cell death process. However, these studies also revealed for the first time an important role of the cidB gene in cell death, as disruption of cidB in the ΔsrrAB mutant background decreased ROS generation and cell death in a cidC-independent manner. The cidB mutation also caused decreased sensitivity to hydrogen peroxide, which suggests a complex role for this system in ROS metabolism. Overall, the results of this study provide further insight into the function of the cidABC operon in cell death and reveal its contribution to the oxidative stress response. IMPORTANCE: The manuscript focuses on cell death mechanisms in Staphylococcus aureus and provides important new insights into the genes involved in this ill-defined process. By exploring the cause of increased stationary-phase death in an S. aureus ΔsrrAB regulatory mutant, we found that the decreased viability of this mutant was a consequence of the overexpression of the cidABC operon, previously shown to be a key mediator of cell death. These investigations highlight the role of the cidB gene in the death process and the accumulation of reactive oxygen species. Overall, the results of this study are the first to demonstrate a positive role for CidB in cell death and to provide an important paradigm for understanding this process in all bacteria.

Bright luminescence of Vibrio fischeri aconitase mutants reveals a connection between citrate and the Gac/Csr regulatory system.

The Gac/Csr regulatory system is conserved throughout the γ-proteobacteria and controls key pathways in central carbon metabolism, quorum sensing, biofilm formation and virulence in important plant and animal pathogens. Here we show that elevated intracellular citrate levels in a Vibrio fischeri aconitase mutant correlate with activation of the Gac/Csr cascade and induction of bright luminescence. Spontaneous or directed mutations in the gene that encodes citrate synthase reversed the bright luminescence of aconitase mutants, eliminated their citrate accumulation and reversed their elevated expression of CsrB. Our data elucidate a correlative link between central metabolic and regulatory pathways, and they suggest that the Gac system senses a blockage at the aconitase step of the tricarboxylic acid cycle, either through elevated citrate levels or a secondary metabolic effect of citrate accumulation, and responds by modulating carbon flow and various functions associated with host colonization, including bioluminescence.

Identification of the amino acids essential for LytSR-mediated signal transduction in Staphylococcus aureus and their roles in biofilm-specific gene expression.

Recent studies have demonstrated that expression of the Staphylococcus aureus lrgAB operon is specifically localized within tower structures during biofilm development. To gain a better understanding of the mechanisms underlying this spatial control of lrgAB expression, we carried out a detailed analysis of the LytSR two-component system. Specifically, a conserved aspartic acid (Asp53) of the LytR response regulator was shown to be the target of phosphorylation, which resulted in enhanced binding to the lrgAB promoter and activation of transcription. In addition, we identified His390 of the LytS histidine kinase as the site of autophosphorylation and Asn394 as a critical amino acid involved in phosphatase activity. Interestingly, LytS-independent activation of LytR was observed during planktonic growth, with acetyl phosphate acting as a phosphodonor to LytR. In contrast, mutations disrupting the function of LytS prevented tower-specific lrgAB expression, providing insight into the physiologic environment within these structures. In addition, overactivation of LytR led to increased lrgAB promoter activity during planktonic and biofilm growth and a change in biofilm morphology. Overall, the results of this study are the first to define the LytSR signal transduction pathway, as well as determine the metabolic context within biofilm tower structures that triggers these signaling events.