Repression of branched-chain amino acid synthesis in <i>Staphylococcus aureus</i> is mediated by isoleucine via CodY, and by a leucine-rich attenuator peptide

<div><p><i>Staphylococcus aureus</i> 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 <i>S</i>. <i>aureus</i> physiology necessitates that it either synthesize them or scavenge them from the environment. Indeed <i>S</i>. <i>aureus</i> 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 <i>S</i>. <i>aureus</i> 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 <i>trans-</i>acting mechanism involving isoleucine-dependent repression by the global transcriptional regulator CodY and a <i>cis</i>-acting leucine-responsive attenuator, uncovering how <i>S</i>. <i>aureus</i> 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 <i>S</i>. <i>aureus</i>, 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 <i>S</i>. <i>aureus</i> environmental adaptation and virulence via CodY.</p></div

Mutations identified in CodY.

<p>Mutations identified in CodY.</p

Organization of the <i>ilv</i>-<i>leu</i> operon in <i>S</i>. <i>aureus</i>.

<p>Top diagram is a schematic of the BCAA biosynthetic pathway in <i>S</i>. <i>aureus</i>. Bottom diagram depicts the genomic context of the BCAA biosynthetic genes in the USA300 FPR3757 genome. Regulatory features and their coordinates relative to the transcription start site are depicted, including the canonical CodY binding motifs [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007159#pgen.1007159.ref057" target="_blank">57</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007159#pgen.1007159.ref097" target="_blank">97</a>] and the ribosome binding site (RBS).</p

Ile is the predominant BCAA to regulate CodY activity on the <i>ilvD</i> promoter.

<p>WT <i>S</i>. <i>aureus</i> containing the <i>lux</i> reporter vector with either A) the partial <i>ilvD</i> promoter region (pGY<i>ilvD</i>^P::<i>lux</i>) or B) the complete <i>ilvD</i> promoter region (pGY<i>ilvD</i>^C::<i>lux</i>) was pre-grown in complete CDM to mid-exponential phase and then sub-cultured into either complete CDM or CDM with limiting concentrations of BCAAs, as indicated. Concentrations of Ile, Leu, and Val in complete CDM are 228 μM, 684 μM, 684 μM, respectively. Concentrations of Ile, Leu, and Val in limited media are 23 μM, 68 μM, and 68 μM, respectively. Luminescence values were read when cells reached mid-exponential phase and were normalized to the OD_600nm. Data are the mean of three biological replicates +/- SD. <i>S</i>. <i>aureus</i> strains with mutations in either <i>codY</i> (<i>codY</i>::ϕNΣ) or BCAA transporters and containing either C) the partial <i>ilvD</i> promoter region (pGY<i>ilvD</i>^P::<i>lux</i>) or D) the complete <i>ilvD</i> promoter region (pGY<i>ilvD</i>^C::<i>lux</i>) were pre-grown in complete CDM to mid-exponential phase and then sub-cultured into complete CDM. Luminescence values were read when cells reached mid-exponential phase and were normalized to the OD_600nm. Data are the mean of three biological replicates +/- SD. Data were analyzed by one-way ANOVA with Dunnet’s multiple comparisons test. *** <i>P</i> < 0.001.</p

The absence of exogenous valine selects for mutations that inactivate CodY.

<p>A) Schematic representation of the mutations identified in CodY. B) Mutations identified are indicated on the CodY structure (PDB ID:5EY0) in yellow, except for the Ser180 to Pro mutation, which is indicated in green. CodY ligands, Ile and GTP, are coloured based on atomic composition. C) Strains were pre-grown in TSB to mid-exponential phase, then sub-cultured into TSB for 16 hr. Supernatants were collected and proteins were precipitated using TCA. Protein samples were normalized to the equivalent of 5 ODs and run on a 12% SDS-PAGE gel. D) Strains with unique mutations in <i>codY</i> (Val^Sup-2 carries an identical mutation to Val^Sup-4) were pre-grown in complete CDM to mid-exponential phase, then sub-cultured into either complete CDM or CDM with Val omitted. OD_600nm was read after 16 hr of growth. USA300 with a transposon insertion in <i>codY</i> (<i>codY</i>::ϕNΣ) was used for comparison. Val^Sup is abbreviated to Val^S. Data are the mean +/- SD of three biological replicates.</p

Mutations identified in the 5’ UTR of <i>ilvD</i>.

<p>Mutations identified in the 5’ UTR of <i>ilvD</i>.</p

Isoleucine limitation induces <i>nuc</i> expression.

<p>Strains were pre-grown in complete CDM to exponential phase and then sub-cultured into either complete CDM or CDM lacking Ile, as indicated. Fluorescence values were read when cells achieved mid-exponential phase and were normalized to OD_600nm. Data are the mean of three biological replicates +/- SEM.</p

Omission of Ile from CDM restores growth in media lacking Leu, but not Val.

<p>A-C) WT USA300 was pre-grown in complete CDM to mid-exponential phase and then sub-cultured into either complete CDM or CDM with amino acids omitted, as indicated. Data are the mean +/- SD of three biological replicates.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Mutations in the <i>ilvD</i> promoter result in an increase in promoter activity and <i>ilv-leu</i> operon expression.

<p>A) Position map of the mutations identified in the <i>ilvD</i> promoter relative to the transcriptional start site, the canonical CodY binding motifs, and the region used for cloning in the promoter:reporter construct. B) WT USA300 with the pGY::<i>lux</i> plasmid containing either the WT <i>ilvD</i> promoter, or the mutant <i>ilvD</i> promoter was grown in complete CDM to mid-exponential phase and then sub-cultured into complete CDM. Luminescence was read at mid-exponential phase and normalized to the OD_600nm. Data are the mean +/- SD of three biological replicates. Data were analyzed by one-way ANOVA with Dunnet’s multiple comparison test. *** <i>P</i> < 0.001. C, D) Strains were grown in complete CDM to mid-exponential phase and then sub-cultured into complete CDM. Cells were harvested at mid-exponential phase and RNA was isolated. Expression of <i>ilvD</i> and <i>ilvC</i> was normalized to expression of <i>rpoB</i>. Val^Sup is abbreviated to Val^S. Data are the mean +/- SD of three biological replicates. Data were analyzed by an Student’s unpaired <i>t</i>-test. *** <i>P</i> < 0.001, ** <i>P</i> < 0.01, * <i>P</i> < 0.05.</p