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.