Introduction
Bacterial cytoplasmic membranes serve as the interface between the cell and the extracellular environment. They are essential for cell integrity in the face of environmental stress (Denichet al., 2003; Sun et al., 2012; Wang et al. , 2014). They also serve as the signaling platform for coordinating cellular responses to achieve cell homeostasis (Gohrbandt et al., 2022; Jung et al., 2018; Schneider et al., 2015). In many Gram-positive bacteria, branched-chain fatty acids (BCFAs) are the predominant fatty acids for membrane phospholipids under normal growth conditions (de Mendoza et al., 1993; Kaneda, 1991; Singh et al., 2008; Sun et al., 2012). In the human opportunistic pathogenStaphylococcus aureus , BCFA content is as high as 70% (Whittaker et al., 2005), with the balance mostly straight chain fatty acids and lower levels of cardiolipin and carotenoids (Schleifer & Kropenstedt, 1990; Sen et al., 2016). S. aureus cannot synthesize unsaturated fatty acids or desaturate saturated fatty acids. Thus BCFAs fluidize staphylococcal membranes to avoid membrane phase separation, protein segregation, and cell death (Gohrbandt et al., 2022). While the organism can incorporate host unsaturated fatty acids to support growth, it is unclear if this salvaging is sufficient to maintain full virulence during infection, since specific BCFAs promote the production of secreted and surface-associated factors via major regulators of virulence (Pendleton & Yeo et al., 2022).
The synthesis of BCFAs starts with the branched-chain amino acids isoleucine, leucine, and valine. Following transamination by IlvE, the respective α-keto acids undergo oxidative decarboxylation to branched-chain carboxylic acids and subsequent activation to their acyl-CoA derivatives. This series of reactions is catalyzed by the branched-chain α-keto acid dehydrogenase complex (BKDH). These acyl-CoAs are primed by FabH, catalyzing condensation of acyl-CoA with malonyl-ACP. The resulting β-ketoacyl-ACP is elongated by the type II fatty acid synthase (FASII) prior to incorporation into phospholipids(Fig 1)(Parsons & Rock 2013). BCFAs can be classified as iso (i ) or anteiso (a ) depending on the location of the methyl group in their structure and the fatty acids a 15:0 and a 17:0 derived from isoleucine are the most abundant in the S. aureus membrane (Kaneda, 1991).
The BKDH-dependent route of BCFA synthesis has been studied in multiple Gram-positive bacterial species including S. aureus, and is essential for growth in laboratory culture (Brinsmade & Sonenshein, 2011; Gohrbandt et al., 2022; Pendleton & Yeo et al., 2022; Singh et al., 2008; Ward et al., 1999). In S. aureus the BKDH complex is composed of four enzymes: a dehydrogenase (E1α), a decarboxylase (E1β), a dihydrolipoamide acyltransferase (E2), and a dihydrolipoamide dehydrogenase (E3), encoded by bkdA1 , bkdA2 , bkdB , and lpdA , respectively (Singh et al., 2008). We and others demonstrated that disruption of lpdA results in a significant decrease in the percentage of membrane BCFAs and attenuates virulence due to a loss of virulence regulator activity, indicating that a functional BKDH complex is indispensable for BCFA synthesis and full virulence gene expression (Pendleton & Yeo et al., 2022; Singh et al., 2008). Given their importance in bacterial physiology and virulence inS. aureus , we wondered whether the canonical BKDH pathway was the sole source of BCFA synthesis. In the present study, we take advantage of the lpdA mutant and looked for mutants that bypassed BCFA auxotrophy. We report that suppressor mutations that increase promoter activity of a putative acyl-CoA synthetase restore BCFA synthesis during laboratory growth in rich, complex medium. We present genetic and biochemical evidence that the gene product we now call MbcS is a member of the AMP-forming acyl-CoA synthetases and catalyzes the activation of branched-chain carboxylic acids to their acyl-CoAs with a preference fori C4 and a C5 substrates. We show that the branched-chain aldehyde 2-methylbutyraldehyde can also restore BCFA synthesis in the lpdA mutant, revealing the existence of an alternative route to BCFA synthesis. Our data have implications for colonization of human skin as well as interspecies interactions during skin and soft tissue infection.