Staphylococcus aureus requires branched-chain amino acids (BCAAs; isoleucine, leucine, valine) for protein synthesis, branched-chain fatty acid synthesis, and environmental adaptation by responding to their availability via the global transcriptional regulator CodY. The importance of BCAAs for S. aureus physiology necessitates that it either synthesize them or scavenge them from the environment. Indeed S. aureus uses specialized transporters to scavenge BCAAs, however, its ability to synthesize them has remained conflicted by reports that it is auxotrophic for leucine and valine despite carrying an intact BCAA biosynthetic operon. In revisiting these findings, we have observed that S. aureus can engage in leucine and valine synthesis, but the level of BCAA synthesis is dependent on the BCAA it is deprived of, leading us to hypothesize that each BCAA differentially regulates the biosynthetic operon. Here we show that two mechanisms of transcriptional repression regulate the level of endogenous BCAA biosynthesis in response to specific BCAA availability. We identify a trans-acting mechanism involving isoleucine-dependent repression by the global transcriptional regulator CodY and a cis-acting leucine-responsive attenuator, uncovering how S. aureus regulates endogenous biosynthesis in response to exogenous BCAA availability. Moreover, given that isoleucine can dominate CodY-dependent regulation of BCAA biosynthesis, and that CodY is a global regulator of metabolism and virulence in S. aureus, we extend the importance of isoleucine availability for CodY-dependent regulation of other metabolic and virulence genes. These data resolve the previous conflicting observations regarding BCAA biosynthesis, and reveal the environmental signals that not only induce BCAA biosynthesis, but that could also have broader consequences on S. aureus environmental adaptation and virulence via CodY.
In Staphylococcus aureus, the transcription factor CodY modulates the expression of hundreds of genes, including most virulence factors, in response to the availability of key nutrients like GTP and branched‐chain amino acids. Despite numerous studies examining how CodY controls gene expression directly or indirectly, virtually nothing is known about the extent to which CodY exerts its effect through small regulatory RNAs (sRNAs). Herein, we report the first set of sRNAs under the control of CodY. We reveal that staphylococcal sRNA RsaD is overexpressed >20‐fold in a CodY‐deficient strain in three S. aureus clinical isolates and in S. epidermidis. We validated the CodY‐dependent regulation of rsaD and demonstrated that CodY directly represses rsaD expression by binding the promoter. Using a combination of molecular techniques, we show that RsaD posttranscriptionally regulates alsS (acetolactate synthase) mRNA and enzyme levels. We further show that RsaD redirects carbon overflow metabolism, contributing to stationary phase cell death during exposure to weak acid stress. Taken together, our data delineate a role for CodY in controlling sRNA expression in a major human pathogen and indicate that RsaD may integrate nutrient depletion and other signals to mount a response to physiological stress experienced by S. aureus in diverse environments.
Organisms growing aerobically generate reactive oxygen-containing molecules, such as hydrogen peroxide (H 2 O 2 ). These reactive oxygen molecules damage enzymes and DNA and may even cause cell death. In response, Bacillus subtilis produces at least nine potential peroxide-scavenging enzymes, two of which appear to be the primary enzymes responsible for detoxifying peroxides during vegetative growth: a catalase (encoded by katA) and an alkylhydroperoxide reductase (Ahp, encoded by ahpC). AhpC uses two redox-active cysteine residues to reduce peroxides to nontoxic molecules. A specialized thioredoxin-like protein, AhpF, is then required to restore oxidized AhpC back to its reduced state. Curiously, B. subtilis has two genes encoding Ahp: ahpC and ahpA. Although AhpC is well characterized, very little is known about AhpA. In fact, numerous bacterial species have multiple ahp genes; however, these additional Ahp proteins are generally uncharacterized. We seek to understand the role of AhpA in the bacterium's defense against toxic peroxide molecules in relation to the roles previously assigned to AhpC and catalase. Our results demonstrate that AhpA has catalytic activity similar to that of the primary enzyme, AhpC. Furthermore, our results suggest that a unique thioredoxin redox protein, AhpT, may reduce AhpA upon its oxidation by peroxides. However, unlike AhpC, which is expressed well during vegetative growth, our results suggest that AhpA is expressed primarily during postexponential growth. IMPORTANCEB. subtilis appears to produce nine enzymes designed to protect cells against peroxides; two belong to the Ahp class of peroxidases. These studies provide an initial characterization of one of these Ahp homologs and demonstrate that the two Ahp enzymes are not simply replicates of each other, suggesting that they instead are expressed at different times during growth of the cells. These results highlight the need to further study the Ahp homologs to better understand how they differ from one another and to identify their function, if any, in protection against oxidative stress. Through these studies, we may better understand why bacteria have multiple enzymes designed to scavenge peroxides and thus have a more accurate understanding of oxidative stress resistance.
In , the global transcriptional regulator CodY modulates the expression of hundreds of genes in response to the availability of GTP and the branched-chain amino acids isoleucine, leucine, and valine (ILV). CodY DNA-binding activity is high when GTP and ILV are abundant. When GTP and ILV are limited, CodY's affinity for DNA drops, altering expression of CodY regulated targets. In this work, we investigated the impact of guanine nucleotides on physiology and CodY activity by constructing a null mutant (Δ). biosynthesis of guanine monophosphate is abolished due to the mutation; thus, the mutant cells require exogenous guanosine for growth. We also found that CodY activity was reduced when we knocked out , activating the Agr two-component system and increasing secreted protease activity. Notably, in a rich, complex medium, we detected an increase in alternative sigma factor B activity in the Δ mutant, which results in a 5-fold increase in production of the antioxidant pigment staphyloxanthin. Under biologically relevant flow conditions, Δ cells failed to form robust biofilms when limited for guanine or guanosine. RNA-seq analysis of transcriptome during growth in guanosine-limited chemostats revealed substantial CodY-dependent and -independent alteration of gene expression profiles. Importantly, these changes increase production of proteases and δ-toxin, suggesting that exhibits a more invasive lifestyle when limited for guanosine. Further, gene-products upregulated under GN limitation, including those necessary for lipoic acid biosynthesis and sugar transport, may prove to be useful drug targets for treating Gram-positive infections. infections impose a serious economic burden on healthcare facilities and patients because of the emergence of strains resistant to last-line antibiotics. Understanding the physiological processes governing fitness and virulence of in response to environmental cues is critical for developing efficient diagnostics and treatments. purine biosynthesis is essential for both fitness and virulence in , since inhibiting production cripples's ability to cause infection. Here, we corroborate these findings and show that blocking guanine nucleotide synthesis severely affects fitness by altering metabolic and virulence gene expression. Characterizing pathways and gene products upregulated in response to guanine limitation can aid in the development of novel adjuvant strategies to combat infections.
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