Figure 4. Acyl-CoA synthetase activity of MbcS restores 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 fromSaccharomyces cerevisiae (Starai & Escalante-Semerena, 2004); AcsA, acetyl-CoA synthetase from B. subtilis (Gardner et al., 2006); IbuA, isobutyryl-CoA synthetase from R. palustrus (Crosby & Escalante-Semerena, 2014; Crosby et al., 2012); FcsA, fatty acyl-CoA synthetase from R. palustris (Crosby & Escalante-Semerena, 2014). (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 and growth (OD600) was monitored over time. (C, D) WT orlpdA mbcS strains with the empty vector control or the wild-type allele of ibuA from R. palustris(pRpibuA +) (C) with or (D)without 25 ng ml-1 of anhydrotetracycline (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 WTVOC,mbcS VOC andlpdA mbcS pSambcS +. In panel C, asterisks indicate that lpdA mbcS VOC and lpdA mbcS pRpibuA + are statistically different from WTVOC and mbcS VOC. In panel D, asterisks indicate that lpdA mbcS VOC is statistically different from WTVOC,mbcS VOC and lpdA mbcS pRpibuA +.
Figure 5. MbcS is a methylbutyryl-CoA synthetase. (A)The activity of MbcS was tested in vitro with several short, straight and branched carboxylic acids. Acids are indicated as CX , where X denotes the carbon length. Data are plotted as the mean specific activity of the enzyme ± SD of at least three independent trials. (B) Sa MbcS was incubated with 2.5 mM MgATP, 493 μM coenzyme A (CoA) and 73 μM isobutyric acid (IB) to produce isobutyryl-CoA as described in Experimental Procedures . Detection and quantification of CoA and IB-CoA was measured via LC-MS. HK: heat killed enzyme. Traces are representative of three independent trials.
Figure 6. S. aureus utilizes branched-chain aldehydes to produce BCFAs in a MbcS-dependent manner . WT, lpdA mutant,lpdA mbcS1 suppressor mutant, and lpdA mbcS double mutant cells were inoculated into chemically defined medium (CDM) (black) or CDM supplemented with vehicle (DMSO; gray), i C4(2MB; pink), 2-methylbutyraldehyde (2MA; orange), and a 17:0 fatty acid (green). Cell density (OD600) was measured after overnight incubation (16-18 h of growth). Data are plotted as mean ± SD from three biological replicates. **** p<0.0001, ***p<0.001, **p<0.01 two-way ANOVA with Tukey’s multiple comparison test for each genotype; ns, not significant.
Figure 7. Metabolites from S. epidermidis support growth of S. aureus in an MbcS-dependent manner. (A) The lpdAmutant (Left) and the lpdA mbcS double mutant (Right) were inoculated ~5 mm apart from S. epidermidis on TSA plates and incubated for 24 h. Arrow head indicates the point of inoculation. (B) WT, lpdA mutant, and lpdA mbcS double mutant cells were inoculated into chemically defined medium (CDM) (black) or CDM supplemented with a mixture of a C5, i C4, and i C5 (gray), a 17:0 fatty acid (orange), and 10% (green), 1% (pink) or 0.1% (blue), of conditioned CDM from S. epidermidis (i.e., cell-free supernatant). Cell density (OD600) was measured after overnight incubation (16-18 h of growth). Data are plotted as mean ± SD from three biological replicates. **** p<0.0001, *p<0.05, two-way ANOVA with Tukey’s multiple comparison test; ns, not significant.
Figure 8. Working model for the synthesis of BCFAs in S. aureus in a BKDH-independent manner. In a lpdA mutant the BKDH complex is inactive and during laboratory cultivation the synthesis of BCFAs is blocked. S. aureus strains with high MbcS enzyme activity (i.e., overexpression of mbcS ) can synthesize BCFAs independent of the BKDH complex using exogenous or endogenous precursors. We propose α-keto acids are converted into their respective branched-chain aldehydes by an α-keto acid decarboxylase, followed by a reaction catalyzed by an aldehyde dehydrogenase to generate branched-chain carboxylic acids. MbcS-dependent acyl-CoA synthesis feeds FASII to generate BCFAs for incorporation into membrane phospholipids. Whether the two pathways operate in parallel or function under specific conditions is a focus of ongoing research. BrnQ, branched-chain amino acid permease; IlvE, branched-chain amino acid aminotransferase.
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