Figure - available from: Molecular Microbiology
This content is subject to copyright. Terms and conditions apply.
![Acyl‐CoA synthetase activity of MbcS restores the growth of a S. aureus lpdA mbcS strain in rich, complex medium lacking BCFAs. (a) Clustal Omega (Sievers et al., 2011) was used to align MbcS with amino acid sequences of previously characterized acyl‐CoA synthetases. Amino acids that constitute a conserved motif in the C‐terminal catalytic domain are shown. Amino acids strictly conserved in the selected proteins are shaded; the conserved lysine residue required for the catalytic activity of acyl‐CoA synthetases is highlighted in yellow. Acs, acetyl‐CoA synthetase from Salmonella enterica (Starai & Escalante‐Semerena, 2004); Acs2, acetyl‐CoA synthetase from Saccharomyces cerevisiae (Starai & Escalante‐Semerena, 2004); AcsA, acetyl‐CoA synthetase from B. subtilis (Gardner et al., 2006); IbuA, isobutyryl‐CoA synthetase from R. palustrus (Crosby et al., 2012; Crosby & Escalante‐Semerena, 2014); FcsA, fatty acyl‐CoA synthetase from R. palustris (Crosby et al., 2012). (b) WT, mbcS, or lpdA mbcS strains containing either the empty integration vector pCT3 (vector‐only control [VOC]), pCT3 containing the WT allele of S. aureus mbcS under the control of the anhydrotetracycline‐inducible tet promoter (pSambcS⁺), or the allele of mbcS that codes for a lysine‐to‐alanine substitution at the residue 510 [pSambcS(K510A)] were grown in TSB without anhydrotetracycline (‐aTc) and growth (OD600) was monitored over time. (c, d) WT or lpdA mbcS strains with the empty vector control or the wild‐type allele of ibuA from R. palustris (pRpibuA⁺) (c) without or (d) with 25 ng mL⁻¹ of aTc as gratuitous inducer were grown in TSB and OD600 was monitored over time. Data are plotted as mean ± SD from three biological replicates. ****p < 0.0001, two‐way ANOVA with Tukey's multiple comparison test. ns, not significant. In panel b, asterisks indicate that lpdA mbcS + VOC and lpdA mbcS + pSambcS(K510A) are statistically different from WT + VOC, mbcS + VOC and lpdA mbcS + pSambcS⁺. In panel c, asterisks indicate that lpdA mbcS + VOC and lpdA mbcS + pRpibuA⁺ are statistically different from WT + VOC. In panel d, asterisks indicate that lpdA mbcS + VOC is statistically different from WT + VOC, and lpdA mbcS + pRpibuA⁺.](publication/378267595/figure/fig4/AS:11431281243951875@1715780954172/Acyl-CoA-synthetase-activity-of-MbcS-restores-the-growth-of-a-S-aureus-lpdA-mbcS-strain.png)
Acyl‐CoA synthetase activity of MbcS restores the growth of a S. aureus lpdA mbcS strain in rich, complex medium lacking BCFAs. (a) Clustal Omega (Sievers et al., 2011) was used to align MbcS with amino acid sequences of previously characterized acyl‐CoA synthetases. Amino acids that constitute a conserved motif in the C‐terminal catalytic domain are shown. Amino acids strictly conserved in the selected proteins are shaded; the conserved lysine residue required for the catalytic activity of acyl‐CoA synthetases is highlighted in yellow. Acs, acetyl‐CoA synthetase from Salmonella enterica (Starai & Escalante‐Semerena, 2004); Acs2, acetyl‐CoA synthetase from Saccharomyces cerevisiae (Starai & Escalante‐Semerena, 2004); AcsA, acetyl‐CoA synthetase from B. subtilis (Gardner et al., 2006); IbuA, isobutyryl‐CoA synthetase from R. palustrus (Crosby et al., 2012; Crosby & Escalante‐Semerena, 2014); FcsA, fatty acyl‐CoA synthetase from R. palustris (Crosby et al., 2012). (b) WT, mbcS, or lpdA mbcS strains containing either the empty integration vector pCT3 (vector‐only control [VOC]), pCT3 containing the WT allele of S. aureus mbcS under the control of the anhydrotetracycline‐inducible tet promoter (pSambcS⁺), or the allele of mbcS that codes for a lysine‐to‐alanine substitution at the residue 510 [pSambcS(K510A)] were grown in TSB without anhydrotetracycline (‐aTc) and growth (OD600) was monitored over time. (c, d) WT or lpdA mbcS strains with the empty vector control or the wild‐type allele of ibuA from R. palustris (pRpibuA⁺) (c) without or (d) with 25 ng mL⁻¹ of aTc as gratuitous inducer were grown in TSB and OD600 was monitored over time. Data are plotted as mean ± SD from three biological replicates. ****p < 0.0001, two‐way ANOVA with Tukey's multiple comparison test. ns, not significant. In panel b, asterisks indicate that lpdA mbcS + VOC and lpdA mbcS + pSambcS(K510A) are statistically different from WT + VOC, mbcS + VOC and lpdA mbcS + pSambcS⁺. In panel c, asterisks indicate that lpdA mbcS + VOC and lpdA mbcS + pRpibuA⁺ are statistically different from WT + VOC. In panel d, asterisks indicate that lpdA mbcS + VOC is statistically different from WT + VOC, and lpdA mbcS + pRpibuA⁺.
Source publication
In the human pathogen Staphylococcus aureus, branched‐chain fatty acids (BCFAs) are the most abundant fatty acids in membrane phospholipids. Strains deficient for BCFAs synthesis experience auxotrophy in laboratory culture and attenuated virulence during infection. Furthermore, the membrane of S. aureus is among the main targets for antibiotic ther...
Similar publications
Background
Acidobacteriota are abundant in soil, peatlands and sediments, but there are so far only a few studies on those in freshwater environments. Genome streamlined bacteria have reduced genomes as an evolutionary process to adapt to oligotrophic environments such as oceans and lakes. UBA12189, an Acidobacteriota genus under the family Holopha...
Citations
... BCFA activation of SaeS may be direct or indirect, altering protein-protein or protein-lipid interactions at the SaeS signaling complex. Green box, potential activator protein; the solid line denotes the BKDH-dependent BCFA synthesis pathway; the dotted arrow denotes a second, BKDH-independent and MbcS-dependent route to BCFA synthesis, as described previously (61). the latter, and powerful new lipidomics techniques would reveal the former (81,82). ...
... pOS-saeP1-gfp (a gift from Dr. Taeok Bae, Indiana University, 6,644 bp) harboring the saeP1 promoter region fused to GFP was linearized and amplified using primers oDD171 and oDD172. The tetracycline resistance gene (tetM, 2,265 bp) was amplified from S. aureus integration vector pWY53 [pCT3; (61)] using primers oDD173 and oDD174. Each primer was designed to add 25 bp overlapping for later Gibson assembly. ...
Staphylococcus aureus is a Gram-positive, opportunistic human pathogen that is a leading cause of skin and soft tissue infections and invasive disease worldwide. Virulence in this bacterium is tightly controlled by a network of regulatory factors. One such factor is the global regulatory protein CodY. CodY links branched-chain amino acid sufficiency to the production of surface-associated and secreted factors that facilitate immune evasion and subversion. Our previous work revealed that CodY regulates virulence factor gene expression indirectly in part by controlling the activity of the SaeRS two-component system (TCS). While this is correlated with an increase in membrane anteiso-15:0 and −17:0 branched-chain fatty acids (BCFAs) derived from isoleucine, the true mechanism of control has remained elusive. Herein, we report that CodY-dependent regulation of SaeS sensor kinase activity requires BCFA synthesis. During periods of nutrient sufficiency, BCFA synthesis and Sae TCS activity are kept relatively low by CodY-dependent repression of the ilv-leu operon and the isoleucine-specific permease gene brnQ2. In a codY null mutant, which simulates extreme nutrient limitation, de-repression of ilv-leu and brnQ2 directs the synthesis of enzymes in redundant de novo and import pathways to upregulate production of BCFA precursors. Overexpression of brnQ2, independent of CodY, is sufficient to increase membrane anteiso BCFAs, Sae-dependent promoter activity, and SaeR ~P levels. Our results further clarify the molecular mechanisms by which CodY controls virulence in S. aureus.
IMPORTANCE
Expression of bacterial virulence genes often correlates with the exhaustion of nutrients, but how the signaling of nutrient availability and the resulting physiological responses are coordinated is unclear. In S. aureus, CodY controls the activity of two major regulators of virulence—the Agr and Sae two-component systems (TCSs)—by unknown mechanisms. This work identifies a mechanism by which CodY controls the activity of the sensor kinase SaeS by modulating the levels of anteiso branched-chain amino acids that are incorporated into the membrane. Understanding the mechanism adds to our understanding of how bacterial physiology and metabolism are linked to virulence and underscores the role virulence in maintaining homeostasis. Understanding the mechanism also opens potential avenues for targeted therapeutic strategies against S. aureus infections.
Staphylococcus aureus is a Gram-positive, opportunistic human pathogen that is a leading cause of skin and soft tissue infections and invasive disease worldwide. Virulence in this bacterium is tightly controlled by a network of regulatory factors. One such factor is the global regulatory protein CodY. CodY links branched-chain amino acid sufficiency to the production of surface-associated and secreted factors that facilitate immune evasion and subversion. Our previous work revealed that CodY regulates virulence factor gene expression indirectly in part by controlling the activity of the SaeRS two-component system. While this is correlated with an increase in membrane anteiso-15:0 and −17:0 branched-chain fatty acids (BCFAs) derived from isoleucine, the true mechanism of control has remained elusive. Herein, we report that CodY-dependent regulation of SaeS sensor kinase activity requires BCFA synthesis. During periods of nutrient sufficiency, BCFA synthesis and Sae TCS activity is heavily suppressed by CodY-dependent repression of the ilv-leu operon and the isoleucine-specific permease gene brnQ2. In a codY null mutant, which simulates extreme nutrient limitation, de-repression of ilv-leu and brnQ2 directs the synthesis of enzymes in redundant de novo and import pathways to catalyze the production of BCFA precursors. Overexpression of brnQ2 independent of CodY is sufficient to increase membrane anteiso BCFAs, Sae-dependent promoter activity, and SaeR∼P levels. Our results further clarify the molecular mechanism by which CodY controls virulence in S. aureus .
IMPORTANCE
Expression of bacterial virulence genes often correlates with the exhaustion of nutrients, but how the signaling of nutrient availability and the resulting physiological responses are coordinated is unclear. In S. aureus, CodY controls the activity of two major regulators of virulence – the Agr and Sae two-component systems – by unknown mechanisms. This work identifies a mechanism by which CodY controls the activity of the sensor kinase SaeS by modulating the flux of anteiso branched-chain amino acids to the membrane. Understanding the mechanism adds to our understanding of how bacterial physiology and metabolism are linked to virulence and underscores the homeostatic nature of virulence. Understanding the mechanism also opens potential avenues for targeted therapeutic strategies against S. aureus infections.
