Discussion
Branched-chain fatty acids, specifically a 15:0 and a 17:0,
are the predominant fatty acids in Gram-positive bacterial membranes.
They are essential for growth and virulence in pathogenic species
including S. aureus (Annous et al., 1997; Beck 2005; Pendleton &
Yeo et al., 2022; Singh et al., 2008; Whaley et al., 2023). Indeed,a 15:0 fatty acid is essential for full activity of the majorS. aureus virulence two-component regulatory system SaeRS, andlpdA mutant cells are attenuated for virulence (Pendleton & Yeo
et al., 2022). Given their critical roles, it is difficult to imagine
that there is no redundancy in the pathways that lead to their
synthesis. On the other hand, disrupting the BKDH complex results in
BCFA auxotrophy. In the present study we took advantage of this
phenotype and looked for mutants that might reveal an alternative route
to BCFA synthesis. Extragenic suppressor mutants isolated in TSB medium
map to the regulatory region of a putative acyl-CoA synthetase gene (now
referred to as mbcS ). These mutations result in overexpression ofmbcS and resolve the BCFA auxotrophy (Table 1, Figs
2-4) . Using genetic and biochemical approaches, we demonstrate herein
that MbcS is an acyl-CoA synthetase with specificity for short, branched
carboxylic acid substrates, and MbcS activity is required for salvaging
exogenous carboxylic acids and aldehydes for BCFA synthesis when the
BKDH complex is inactivated (Table 2, Figs 4-7) .
Our data are in strong agreement with a recent report by Whaley et
al. , demonstrating MbcS is an AMP-forming acyl-CoA synthetase that
selectivity catalyzes the activation of isobutyrate and
2-methylbutyrate. In that manuscript, they clearly show extracellular
carboxylic acids are converted to their CoA derivatives and flow into
the FASII elongation cycle, and argue that MbcS serves in a salvaging
capacity (Figs 4-5) (Whaley et al., 2023). Our suppressor
screen also points to MbcS, and kinetic analysis of the enzyme indicates
a high (i.e., low micromolar) affinity fori C4 and a C5 substrates.
This is consistent with the finding that TSB medium contains a trace
amount of these compounds. Interestingly, unlike in TSB, the lpdAsingle mutant does not exhibit auxotrophy for BCFAs when grown in
lysogeny broth, prompting Whaley et al. to hunt for mbcS.This is likely due to the relative levels of carboxylic acids in these
two media. Indeed, this accounts for the distinct membrane fatty acid
profiles seen for WT cells (Sen et al., 2016; Whaley et al., 2023).
Moreover, BCFA auxotrophy is resolved in our suppressor mutants by
incorporating i 14:0 BCFA into membranes derived fromi C4. This makes physiological sense - while FabH
prefers a C5-CoA to initiate fatty acid synthesis,
incorporating iso even fatty acids into the bilayer reflects not only
the affinity of MbcS for substrate, but also the availability of the
acyl-CoA precursor pool (Whaley et al., 2023). We note that in contrast
to the Whaley et al. study where they report a
~5-fold increase in affinity fora C5 over i C4, we did not
measure a significant difference in MbcS affinity fori C4 or a C5. The
discrepancy may be explained by the assay used to determine kinetic
constants. Our coupled assay may simply be not sensitive enough to
resolve the Km values below 5 µM.
Our data validate and expand the idea that S. aureus salvages
BCFA precursors by showing that branched-chain aldehydes are catabolized(Figs 6-7) . At the same time, it is intriguing that strains
lacking a functional BKDH complex with heightened MbcS enzyme activity
are able to grow, albeit poorly, in unsupplemented, defined medium. We
posit this growth is the result of a cryptic alternative de novosynthesis pathway. Previous studies investigating BCFA synthesis in
related Gram-positive bacteria inform our working model for an
alternative route to BCFAs synthesis (Fig 8) . For instance, the
Gram-positive bacterium Lactococcus lactis , which is largely used
for the fermentation of dairy products, harbors an α-keto acid
decarboxylase KdcA that converts the branched-chain α-keto acids into
their respective branched-chain aldehydes, and these are recognized to
be important for flavor formation (Smit et al., 2005). In USA300
strains, SAUSA300_0190 is annotated to encode an indole-pyruvate
decarboxylase (IpdC). IpdC is 64% similar to L. lactis KdcA that
has highest activity for the α-keto acid of valine (Smit et al., 2005),
consistent with our GC-FAME data showing incorporation of valine
derived, i1 4:0 BCFA in the lpdA mbcS1 and lpdA
mbcS2 strains (Fig 3) . We propose that IpdC is a
branched-chain α-keto acid decarboxylase. Three additional facts support
this hypothesis: i) IpdCs are known to have broad specificity (Parsons
et al., 2015); ii) ipdC expression is repressed by S.
aureus CodY, which controls BCFAs synthesis by regulating the genes
that direct the synthesis of the short BCFAs precursors (Waters et al.,
2016); and iii) S. aureus synthesizes tryptophan but, unlike
other organisms, does not catabolize the amino acid (Proctor &
Kloos,1973; Spaepen et al., 2007; ). AlsSD (acetolactate
synthase/decarboxylase) and CidC (pyruvate oxidase) also have
significant similarity to KdcA. However, how these enzymes would fit
into the model is unclear.
As described above, other staphylococcal species are commonly used in
the food industry due to their ability to form aldehydes, which are
important for flavor formation (Beck, 2005; Beck et al., 2002). S.
aureus itself was described to synthesize nine different aldehydes
among them the branched chain 2-methylbutyraldehyde, 3-methylbutanal,
and 3-methylpropanal (Bos et al., 2013; Filipiak et al., 2012). Mass
spectrometry data from S. xylosus showed these cells can convert
3-methylbutanal to its respective carboxylic acid, the 3-methylbutanoic
acid (Beck et al., 2002). In addition, human commensals likeCutibacterium acnes and S. epidermidis also produce these
compounds on skin (Bos et al., 2013; Duffy & Morrin, 2019; Tait et al.,
2014; Verhulst 2011) and salvage them in laboratory culture in an
MbcS-dependent manner (Fig 7) , suggesting these compounds are
salvaged by S. aureus using this pathway during infection. Taken
together, data from our lab and other labs indicate that S.
aureus oxidizes the branched-chain aldehyde to the carboxylic acid. TheS. aureus genome encodes several potential aldehyde
dehydrogenases (Alds); some of which have been characterized and are
known to have broad specificity (Imber et al., 2018). Thus, we
posit that one or more of these Alds converts the aldehyde derivatives
of the branched-chain α-keto acids to their cognate carboxylic acids.
MbcS then would serve to activate the carboxylic acid to its acyl-CoA to
feed fatty acid synthesis. The α-keto acid decarboxylase and the
aldehyde dehydrogenase(s) involved in this proposed alternative pathway
are still to be found but this is currently an active focus of our
laboratory.
Why has this pathway remained hidden until now? Overexpression ofmbcS (either by mutation of the native promoter or by artificial
overexpression using an inducible promoter) restores growth in the
BKDH-deficient strain (Figs 2-4) . This suggests that MbcS
activity in laboratory cultures is normally too low to support growth.
Interestingly, MbcS is a member of the AMP-forming family of acyl-CoA
synthetases. These enzymes (including Rp IbuA used in this study)
are known to be regulated by reversible lysine modification of a
conserved lysine residue by acetylation (Crosby & Escalante, 2014;
Crosby et al., 2010; Gardner et al., 2006). Acetylation down-regulates
enzyme activity (Gardner et al., 2006; Starai et al., 2002). It is
conceivable that MbcS is acetylated under our laboratory conditions, and
overproducing MbcS increases enzyme activity because the fraction of
MbcS that escapes the acetylation machinery is increased. Indeed, the
GCN5-related N- acetyltransferase AcuA was recently shown to
acetylate acetyl-CoA synthetase in S. aureus (Burckhardt et al.,
2019). The role of AcuA in bacterial physiology is unknown. Whether MbcS
is acetylated and is a substrate for AcuA is not known. We are actively
pursuing these questions in our laboratory.