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\begin{document}

\title{In Staphylococcus aureus , the acyl-CoA synthetase MbcS supports
branched-chain fatty acid synthesis from carboxylic acid and aldehyde
precursors}



\author[1]{Shaun R. Brinsmade}%
\author[1]{Marcelle C. dos Santos Ferreira}%
\author[2]{Augustus Pendleton}%
\author[1]{Won-Sik Yeo}%
\author[1]{Fabiana C. Málaga Gadea}%
\author[1]{Danna Camelo}%
\author[1]{Maeve McGuire}%
\affil[1]{Georgetown University}%
\affil[2]{Cornell University Department of Microbiology}%


\vspace{-1em}



  
  \date{May 22, 2024}


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\selectlanguage{english}
\begin{abstract}
In the human pathogen \emph{Staphylococcus aureus}, branched-chain fatty
acids (BCFAs) are the most abundant fatty acids in membrane
phospholipids, and strains deficient for their synthesis experience
BCFAs auxotrophy in laboratory culture and attenuated virulence during
infection. Thus, membrane integrity is essential for \emph{S. aureus}
pathogenesis. Furthermore, the membrane of \emph{S. aureus} is among the
main targets for antibiotic therapy. Therefore, determining the
mechanisms involved in BCFAs synthesis is critical to manage \emph{S.
aureus} infections. Here, we report that overexpression of the bona fide
acyl-CoA synthetase gene \emph{mbcS} (formerly SAUSA300\_2542) restores
BCFAs synthesis in strains lacking the canonical biosynthetic pathway
catalyzed by the branched-chain a-keto acid dehydrogenase (BKDH)
complex. We demonstrate that the acyl-CoA synthetase activity of MbcS
activates branched-chain carboxylic acids, and is required by \emph{S.
aureus} to utilize the isoleucine derivative 2-methylbutyraldehyde to
restore BCFAs synthesis in \emph{S. aureus}. Based on the ability of
some staphylococci to convert branched-chain aldehydes into their
respective branched-chain carboxylic acids and our findings
demonstrating that branched-chain aldehydes are in fact BCFAs
precursors, we propose that MbcS promotes the scavenging of exogenous
branched-chain carboxylic acids (BCCAs) and mediates branched-chain
fatty acids synthesis via a \emph{de novo} alternative pathway.%
\end{abstract}%



\sloppy


\textbf{In \emph{Staphylococcus aureus}, the acyl-CoA synthetase MbcS
supports branched-chain fatty acid synthesis from carboxylic acid and
aldehyde precursors}

Marcelle C. dos Santos Ferreira, Augustus
Pendleton\textsuperscript{\^{}}, Won-Sik Yeo, Fabiana C. M\selectlanguage{ngerman}álaga Gadea,
Danna Camelo, Maeve McGuire and Shaun R. Brinsmade\textsuperscript{\#}

Department of Biology, Georgetown University, Washington, DC, USA

Running title:~MbcS-dependent BCFA synthesis in \emph{Staphylococcus
aureus}

\textsuperscript{\^{}}Present address: Department of Microbiology,
Cornell University, Ithaca, NY, USA

\textsuperscript{\#}Address correspondence to Shaun R.
Brinsmade,\emph{shaun.brinsmade@georgetown.edu}

Key words: \emph{Staphylococcus aureus} , MRSA, branched-chain fatty
acids, BKDH complex, membrane phospholipids, fatty acids metabolism.

\textbf{Summary}

In the human pathogen \emph{Staphylococcus aureus} , branched-chain
fatty acids (BCFAs) are the most abundant fatty acids in membrane
phospholipids, and strains deficient for their synthesis experience
BCFAs auxotrophy in laboratory culture and attenuated virulence during
infection. Thus, membrane integrity is essential for \emph{S.
aureus}pathogenesis. Furthermore, the membrane of \emph{S. aureus} is
among the main targets for antibiotic therapy. Therefore, determining
the mechanisms involved in BCFAs synthesis is critical to manage
\emph{S. aureus} infections. Here, we report that overexpression of the
bona fide acyl-CoA synthetase gene \emph{mbcS} (formerly SAUSA300\_2542)
restores BCFAs synthesis in strains lacking the canonical biosynthetic
pathway catalyzed by the branched-chain \selectlanguage{greek}α-\selectlanguage{english}keto acid dehydrogenase (BKDH)
complex. We demonstrate that the acyl-CoA synthetase activity of MbcS
activates branched-chain carboxylic acids, and is required by \emph{S.
aureus} to utilize the isoleucine derivative 2-methylbutyraldehyde to
restore BCFAs synthesis in \emph{S. aureus} . Based on the ability of
some staphylococci to convert branched-chain aldehydes into their
respective branched-chain carboxylic acids and our findings
demonstrating that branched-chain aldehydes are in fact BCFAs
precursors, we propose that MbcS promotes the scavenging of exogenous
branched-chain carboxylic acids (BCCAs) and mediates branched-chain
fatty acids synthesis via a \emph{de novo} alternative pathway.

\textbf{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\emph{.} , 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
pathogen\emph{Staphylococcus 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). \emph{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 \selectlanguage{greek}α-\selectlanguage{english}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 \selectlanguage{greek}α-\selectlanguage{english}keto acid dehydrogenase complex (BKDH). These acyl-CoAs
are primed by FabH, catalyzing condensation of acyl-CoA with
malonyl-ACP. The resulting \selectlanguage{greek}β-\selectlanguage{english}ketoacyl-ACP is elongated by the type II
fatty acid synthase (FASII) prior to incorporation into
phospholipids\textbf{(Fig 1)}(Parsons \& Rock 2013). BCFAs can be
classified as iso (\emph{i} ) or anteiso (\emph{a} ) depending on the
location of the methyl group in their structure and the fatty acids
\emph{a} 15:0 and \emph{a} 17:0 derived from isoleucine are the most
abundant in the \emph{S. aureus} membrane (Kaneda, 1991).

The BKDH-dependent route of BCFA synthesis has been studied in multiple
Gram-positive bacterial species including \emph{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 \emph{S. aureus} the BKDH complex is
composed of four enzymes: a dehydrogenase (E1\selectlanguage{greek}α\selectlanguage{english}), a decarboxylase (E1\selectlanguage{greek}β\selectlanguage{english}),
a dihydrolipoamide acyltransferase (E2), and a dihydrolipoamide
dehydrogenase (E3), encoded by \emph{bkdA1} , \emph{bkdA2} , \emph{bkdB}
, and \emph{lpdA} , respectively (Singh et al., 2008). We and others
demonstrated that disruption of \emph{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
in\emph{S. aureus} , we wondered whether the canonical BKDH pathway was
the sole source of BCFA synthesis. In the present study, we take
advantage of the \emph{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 for\emph{i} C\textsubscript{4} and \emph{a}
C\textsubscript{5} substrates. We show that the branched-chain aldehyde
2-methylbutyraldehyde can also restore BCFA synthesis in the \emph{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.



\textbf{Results}

\textbf{Mutations in the \emph{mbcS} promoter restore growth of
a\emph{lpdA} mutant during laboratory growth.} A \emph{lpdA} mutant
lacks the dihydrolipoamide dehydrogenase subunit of the BKDH complex and
is deficient for BCFA synthesis (Teoh et al., 2021). As expected, the
strain is not viable in rich, complex medium (tryptic soy broth
{[}TSB{]}) and does not achieve an optical density beyond
\textasciitilde{}0.1 after \textasciitilde{}7-8 h unless supplemented
with a mixture of the branched-chain carboxylic acids (BCCAs;
2-methylbutyric acid {[}\emph{a} C\textsubscript{5}{]}, 3-methylbutyric
acid {[}\emph{i} C\textsubscript{5}{]}, and isobutyric acid {[}\emph{i}
C\textsubscript{4}{]}) or with \emph{a} 15:0 BCFA \textbf{(Fig 2A-C} ,
compare squares to circles\textbf{)} . However, we observed~that~the
\emph{lpdA} mutant formed small colonies upon prolonged incubation on
tryptic soy agar (TSA) in the absence of BCCAs. We reasoned that these
colonies harbor additional mutations that bypass the requirement for the
BKDH complex. To test this, independently isolated mutants were
submitted for whole genome sequencing and the sequences of the apparent
suppressor mutants and the parent strain were compared with BRESEQ
(Barrick et al., 2009). Interestingly, nearly all mutants harbored
either single nucleotide polymorphisms or insertion-deletions in the
promoter region of SAUSA300\_2542\emph{,}which is annotated to encode an
acyl-CoA synthetase \textbf{(Table 1, Fig 3A).} We refer to these
alleles hereafter as \emph{mbcS} alleles to reflect the \emph{m}
ethyl\emph{b} utyri\emph{c} acid supplementation bypassed in these
strains. We first analyzed growth behavior of a representative
\emph{lpdA} mutant with a mutant \emph{mcbS} allele (\emph{i.e., mbcS1}
). This strain achieved a doubling time and cell yield essentially
identical to that of the WT strain, even in the absence of BCFAs or
their carboxylic acid precursors \textbf{(Fig 2A-C,}compare triangles
and squares to circles\textbf{)} .

A recent study reported that TSB and other standard laboratory media
contain trace amounts of branched-chain carboxylic acids (Whaley et al.,
2023) \textbf{.} We wondered whether \emph{mbcS} was required to
scavenge these BCFA precursors and promote BCFA synthesis in the absence
of \emph{lpdA} . To test this, we constructed an \emph{lpdA mbcS} double
mutant using available mutants from the Nebraska Transposon Mutant
Library (Fey et al., 2013) and compared the strain's growth behavior to
the \emph{lpdA} single mutant. Unlike the \emph{lpdA} mutant, whose
growth was restored with branched-chain carboxylic acids, the \emph{lpdA
mbcS} double mutant failed to grow beyond an optical density of
\textasciitilde{}0.1 unless \emph{a} 15:0 was included in the medium
(\textbf{Fig 2} , compare inverted triangles to squares). These data
indicate that the mutations in the \emph{mbcS} promoter suppress the
BCFA auxotrophy, and that \emph{mbcS} is required to restore growth when
BCCAs are provided exogenously in the growth medium.

\textbf{Promoter-up mutations in\emph{mbcS} alter the membrane fatty
acid profile in \emph{S. aureus}.}Many of the changes we observed in the
\emph{mbcS} promoter of our mutants either decrease the spacing between
the -35 and -10 boxes from 18 nucleotides to the typical 17 nucleotides
found in \emph{S. aureus} , or refine the -35 and -10 sequences toward a
consensus \selectlanguage{greek}σ\selectlanguage{english}\textsuperscript{A}-dependent promoter \textbf{(Table 1, Fig
3A)}(Helmann, 1995). We hypothesized that these changes would
increase\emph{mbcS} expression. To test~this, we introduced~a
\emph{gfp}reporter plasmid containing either the wild-type (WT) promoter
of\emph{mbcS} or the~promoters isolated from two independently isolated
mutants into wild-type cells and measured promoter activity \textbf{(Fig
3A, B)} . During growth in TSB, \emph{mbcS} expression is relatively low
in WT cells. Mutation of the \emph{mbcS} promoter resulted in a 3- to
4-fold increase in promoter activity (\textbf{Figure 3B)} . We then
wondered whether overexpressing \emph{mbcS} altered the membrane fatty
acid profile, supporting growth in TSB. To address this question, we
grew the WT and our two selected \emph{lpdA} suppressor mutants to
exponential phase and subjected the cells to gas chromatography fatty
acid methyl ester analysis~(GC-FAME). As expected, WT cells have a
relatively high proportion of \emph{a} 15:0 BCFAs in their membranes
with lower levels of \emph{i} 14:0 BCFAs. Interestingly, we measured a
large increase in \emph{i} 14:0 BCFAs and relatively low levels of
\emph{a} 15:0 BCFAs in the suppressor mutants (\textbf{Fig 3C} ). These
data strongly suggest that overexpressing \emph{mbcS} rescues BCFA
auxotrophy by promoting the synthesis of iso but not anteiso BCFAs.

\textbf{MbcS is a branched-chain acyl-CoA synthetase.} SAUSA300\_2542 is
annotated to encode an acyl-CoA synthetase we call MbcS. Three facts
support this annotation. First, MbcS is 56\% similar to
(end-to-end)\emph{S. aureus} acetyl-CoA synthetase (Burckhardt et al.,
2019). Second, MbcS is 43-47\% similar to other bona fide acyl-CoA
synthetases from \emph{Salmonella enterica} and \emph{Rhodopseudomonas
palustris}(Crosby \& Escalante-Semerena, 2014; Crosby et al., 2012;
Starai \& Escalante-Semerena, 2004). Third, close inspection of the
predicted amino acid sequence of MbcS reveals a conserved motif and
catalytic lysine residue among representative members of the AMP-forming
family of acyl-CoA synthetases (\textbf{Fig 4A} ) (Burckhardt et al.,
2019; Crosby \& Escalante-Semerena, 2014; Crosby et al., 2012; Starai et
al., 2002). To begin to test the hypothesis that MbcS has acyl-CoA
synthetase activity, we complemented a \emph{lpdA mbcS} double mutant
with integrative plasmids coding for either the wild-type \emph{S.
aureus mbcS} (\emph{SambcS} \textsuperscript{+}) or a variant
substituting the catalytic lysine residue with alanine (K510A) under the
control of an anhydrotetracycline inducible promoter and the Tet
repressor (TetR) and assessed growth behavior in TSB. When we introduced
the empty vector into the \emph{mbcS lpdA} double mutant, the cells were
unable to achieve an optical density beyond \textasciitilde{}0.1~after
\textasciitilde{}6-8 h \textbf{(Fig 4B} , compare triangles to
circles\textbf{)} . Growth was restored in this mutant when we
introduced a wild-type copy of \emph{mbcS} but not the mutant allele
coding for the catalytically inert MbcS\textsuperscript{K510A} variant,
strongly suggesting that MbcS-dependent acyl-CoA synthetase activity was
necessary and sufficient for growth in TSB \textbf{(Fig 4B} , compare
inverted triangles and diamonds to circles\textbf{)} . Interestingly, we
measured no obvious growth defect in the single \emph{mbcS} mutant. To
test genetically if MbcS indeed supports growth of the \emph{lpdA}mutant
by catalyzing acyl-CoA synthetase activity, we complemented
the\emph{lpdA mbcS} double mutant with an integrative plasmid coding for
the bona fide acyl-CoA synthetase IbuA from \emph{R.
palustris}(\emph{RpibuA\textsuperscript{+}} ), and assessed growth
behavior\textbf{(Fig 4C, D)} . Unlike \emph{SambcS\textsuperscript{+}}
,\emph{RpibuA\textsuperscript{+}} does not complement the \emph{lpdA
mbcS} mutant for growth in TSB in the absence of inducer \textbf{(Fig
4C)} . However, adding 25 ng ml\textsuperscript{-1} of
anhydrotetracycline (aTc) to the growth medium was sufficient to restore
growth of the \emph{lpdA mbcS} strain to a level similar to the WT
strain \textbf{(Fig 4D)} . Taken together, our genetic data indicate
that, in the absence of the BKDH complex, the acyl-CoA synthetase
activity of MbcS bypasses the BCFA auxotrophy of the \emph{lpdA}mutant.

To support the \emph{in vivo} data above, we cloned, overexpressed, and
purified \emph{N-} terminal hexahistidine (His\textsubscript{6})-tagged
MbcS protein (\emph{Sa} MbcS). Following tag removal, we reisolated the
protein to apparent homogeneity and conducted a coupled
spectrophotometric assay with Mg*ATP, coenzyme A (CoA) and a variety of
carboxylic acids as substrates as described in \emph{Experimental
Procedures} .~\emph{Sa} MbcS activated short, straight and
branched-chain monocarboxylic acids and typically preferred branched
substrates over straight substrates with the same carbon length.
\emph{Sa} MbcS had the highest activity with isobutyrate (\emph{i}
C\textsubscript{4})\textbf{(Fig 5A)} . We saw no activity when \emph{a}
15:0 was used as substrate in the reaction (data not shown).

The coupled assay does not provide a direct identification of the
product formed. Therefore, we used LC-MS analysis to authenticate
isobutyryl-CoA (\emph{i} C\textsubscript{4}-CoA) formation from
isobutryate (\emph{i} C\textsubscript{4}). Briefly, we
incubated\emph{Sa} MbcS with Mg*ATP, isobutyrate (IB), and CoA in
buffer. As a negative control, \emph{Sa} MbcS was denatured at 85\selectlanguage{ngerman}°C, and
its activity was tested. Using LC-MS, we successfully separated,
detected, and confirmed the identities of isobutyryl-CoA and CoA in
reaction mixtures containing recombinant \emph{Sa} MbcS. We failed to
detect IB-CoA formation when substrates were incubated with denatured
MbcS, demonstrating product formation was enzyme-dependent \textbf{(Fig
5B)} .

We then determined a limited set of kinetic parameters for \emph{Sa}
MbcS and compared them to bona fide isobutyryl-CoA synthetase from
\emph{R. palustris} (\emph{Rp} IbuA) using the same continuous
spectrophotometric assay. Because our interest lay in the specificity of
the enzyme for substrates relevant to BCFA synthesis, we focused on
carboxylic acid substrates and used a fixed amount of coenzyme A and ATP
co-substrates. Under our \emph{in vitro} conditions, the apparent
affinity of\emph{Sa} MbcS for \emph{i} C\textsubscript{4} and\emph{a}
C\textsubscript{5} were essentially identical (K\textsubscript{m} values
were calculated to be 8.9 ± 2.1 \selectlanguage{greek}μ\selectlanguage{english}M and 5.5 \selectlanguage{ngerman}± 1.0 \selectlanguage{greek}μ\selectlanguage{english}M, respectively).
\emph{Sa} MbcS displayed a significantly lower affinity for \emph{i} C5
(\textgreater{} 500 \selectlanguage{greek}μ\selectlanguage{english}M) \textbf{(Table 2)} . MbcS displayed a measurably
higher affinity for \emph{i} C4 compared to\emph{Rp} IbuA. This seemed
to be offset by the faster rate of reaction catalyzed by \emph{Rp} IbuA
compared to \emph{Sa} MbcS using\emph{i} C\textsubscript{4} as substrate
\textbf{(Table 2} , compare K\textsubscript{cat} values for \emph{Rp}
IbuA and\emph{Sa} MbcS\textbf{)} . This resulted in comparable catalytic
efficiencies for the two enzymes (compare
K\textsubscript{cat}/K\textsubscript{m} values). As expected, the
catalytically inert \emph{Sa} MbcS\textsuperscript{K510A} variant
exhibited no detectable acyl-CoA synthetase activity \textbf{(Table 2)}
. Taken together, these data indicate that \emph{Sa} MbcS is a bona fide
methylbutyryl-CoA synthetase with a preference for\emph{i}
C\textsubscript{4} as substrate and participates in branched-chain fatty
acid synthesis.

\emph{mbcS} \textbf{supports the utilization of 2-methylbutyraldehyde as
a precursor for BCFA synthesis.} The BKDH complex catalyzes an oxidative
decarboxylation reaction, converting the branched-chain \selectlanguage{greek}α-\selectlanguage{english}keto acids to
their cognate BCCAs. These BCCAs are activated to acyl-CoAs that feed
fatty acid synthesis (Frank et al., 2021; Sen et al., 2015; Singh et
al., 2008; Ward et al., 1999). Blocking BKDH function revealed a second
enzyme that performs this activation step. \emph{S. aureus} produces
aldehydes during isoleucine, leucine, and valine catabolism in
peptide-rich environments (Bos et al., 2013; Filipiak et al., 2012).
These aldehydes are conceivably oxidized to carboxylic acids and
activated by MbcS. Indeed, staphylococci used in the food industry, such
as \emph{S. carnosus} , oxidize branched-chain aldehydes to their
respective BCCAs to synthesize BCFAs (Beck, 2005; Beck et al., 2002). To
test if this is also true for \emph{S. aureus} , we grew cells in
chemically defined medium (CDM) (Sheldon et al., 2014) supplemented with
the isoleucine-derived aldehyde 2-methylbutyraldehyde (2MA), the BCCA
2-methylbutyric acid (\emph{a} C\textsubscript{5}), and the BCFA\emph{a}
17:0. As expected, the \emph{lpdA} single mutant failed to achieve a
final optical density beyond \textasciitilde{}0.1 in the absence of
supplementation. Both the \emph{lpdA} mutant and the\emph{lpdA mbcS1}
suppressor mutant grew to similar levels in medium containing the
aldehyde and the carboxylic acid (albeit not to WT levels for the
\emph{lpdA mbcS1} strain). BCCA- and aldehyde-dependent growth was
dependent on \emph{mbcS} \textbf{(Fig 6} , compare pink and orange bars,
\emph{lpdA::kan\textsuperscript{+} mbcS::} \selectlanguage{greek}φ\selectlanguage{english}N\selectlanguage{greek}Σ
\selectlanguage{english}vs.\emph{lpdA::kan\textsuperscript{+}} \textbf{)} . Indeed, the double
mutant only grew in the presence of \emph{a} 17:0 BCFA, indicating that
lack of viability was due to insufficient intracellular branched-chain
acyl-CoA precursors.

\emph{S. aureus} heavily suppresses branched-chain amino acid (BCAA)
synthesis during rapid growth (Kaiser et al., 2018; Waters et al.,
2016). Rather, BCAAs are preferentially imported during growth as either
free amino acids or as peptides and play critical roles for cell
physiology. As abundant amino acids found in proteins, BCAAs can be used
directly for protein synthesis and drive folding via the hydrophobic
effect (Brosnan \& Brosnan, 2006; Dill 1990). BCAAs are also readily
interconverted to their \selectlanguage{greek}α-\selectlanguage{english}keto acids that serve as precursors for
pantothenate and coenzyme A, and for the BCFAs. Thus, the metabolism of
BCAAs controls membrane phospholipid composition (Frank et al., 2021;
Richardson et al., 2015). \emph{Lactococcus lactis} strains are capable
of decarboxylating BCAAs, particularly leucine, to their corresponding
aldehyde 3-methylbutanal. These aldehydes are important for flavor
formation in certain cheeses by \emph{Lactococcus} spp\emph{.} (Rijnen
et al., 2003; Smit et al., 2005; Smit et al., 2004). We wondered
whether\emph{S. aureus} might also be capable of generating BCFAs
\emph{de novo} through a pathway parallel to that formed by the BKDH
complex. To test this, we compared the growth behavior of the
\emph{lpdA} mutant to the WT parent strain, with and without \emph{mbcS}
overexpressed. Unlike the \emph{lpdA} mutant, the \emph{lpdA mbcS1}
mutant grew, albeit poorly, in CDM medium without BCFA or precursor
supplementation. Again, this growth was abrogated in the \emph{lpdA
mbcS} double mutant\textbf{(Fig 6} , grey and black bars, mutant strains
compared to WT\textbf{)} .

\textbf{Metabolites from \emph{S. epidermidis} restore growth
of\emph{lpdA} mutant in an MbcS-dependent manner.} The human skin is an
important habitat for microbial colonization. Metagenomic studies show
that different species of bacteria, fungi and viruses compose the skin
microbiota (Byrd et al., 2018). Coagulase negative staphylococci (CoNS)
such as \emph{S. epidermidis} , \emph{S. hominis} , \emph{S.
haemolyticus} , and \emph{S. lugdunensis} are the most abundant skin
colonizers and serve as a barrier to protect against local and systemic
infections caused by pathogenic species like \emph{S. aureus} (Brown \&
Horswill, 2020; Byrd et al., 2018; Zipperer et al., 2016). Indeed, skin
commensals successfully compete with \emph{S. aureus} when resources
become limited in the skin environment. For instance, \emph{S.
lugdunensis} synthesizes the antimicrobial peptide lugdunin that
effectively inhibits the growth of \emph{S. aureus} \emph{in vitro} and
interferes with nasal colonization in a mouse model (Zipperer et al.,
2016).

Microbial cells within natural communities also can exchange metabolites
with one another, working together to distribute labor before
interactions potentially become antagonistic (Pande et al., 2014).
Considering the interactions between \emph{S. aureus} and staphylococci
in the skin, we wondered if \emph{S. epidermidis} could feed our\emph{S.
aureus} \emph{lpdA} mutant given its propensity to make volatile
compounds derived from the branched-chain amino acids. To address this,
we streaked the \emph{lpdA} single mutant on medium lacking
branched-chain fatty acids next to \emph{S. epidermidis.} If\emph{S.
epidermidis} can cross-feed \emph{S. aureus,} we expect to observe
growth adjacent to S. \emph{epidermidis,} with the zone supporting
growth proportional to the amount of the nutrient being secreted and
diffusing through the medium. Interestingly, the\emph{lpdA} mutant grew
only in close proximity to \emph{S. epidermidis,} and this growth was
abrogated when we streaked the\emph{lpdA mbcS} double mutant next to
\emph{S. epidermidis}(\textbf{Fig 7A} ). We then quantified the effect
using \emph{S. epidermidis} conditioned medium. As expected, the
\emph{lpdA} single mutant was chemically complemented with a mix of
branched-chain carboxylic acids or \emph{a} 17:0 BCFAs \textbf{(Fig 7B}
, compare grey, orange bars to black bars, respectively\textbf{)} . We
then determined that 10\% conditioned medium from \emph{S. epidermidis}
restored near WT growth. The effect was lost when the cells were
supplemented with decreasing concentrations of conditioned medium
(\textbf{Fig 7B} , compare green, pink, and blue bars)\emph{.} Only
\emph{a} 17:0 supplementation restored growth of the \emph{lpdA mbcS}
double mutant\textbf{(Fig 7B)} . Taken together, these data indicate
\emph{S. aureus}can salvage isoleucine-derived, exogenous branched-chain
aldehydes in addition to branched-chain carboxylic acids in a
\emph{mbcS-} dependent manner for BCFA synthesis. Our data also strongly
suggest that \emph{S. epidermidis} can support \emph{S. aureus} growth
by providing these same essential precursors for BCFA synthesis.



\textbf{Discussion}

Branched-chain fatty acids, specifically \emph{a} 15:0 and \emph{a}
17:0, are the predominant fatty acids in Gram-positive bacterial
membranes. They are essential for growth and virulence in pathogenic
species including \emph{S. aureus} (Annous et al., 1997; Beck 2005;
Pendleton \& Yeo et al., 2022; Singh et al., 2008; Whaley et al., 2023).
Indeed,\emph{a} 15:0 fatty acid is essential for full activity of the
major\emph{S. aureus} virulence two-component regulatory system SaeRS,
and\emph{lpdA} 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 \emph{mbcS} ). These mutations result in overexpression
of\emph{mbcS} and resolve the BCFA auxotrophy \textbf{(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 \textbf{(Table 2, Figs 4-7)} .

Our data are in strong agreement with a recent report by Whaley \emph{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 \textbf{(Figs 4-5)} (Whaley et al., 2023). Our suppressor
screen also points to MbcS, and kinetic analysis of the enzyme indicates
a high (\emph{i.e.,} low micromolar) affinity for\emph{i}
C\textsubscript{4} and \emph{a} C\textsubscript{5} substrates. This is
consistent with the finding that TSB medium contains a trace amount of
these compounds. Interestingly, unlike in TSB, the \emph{lpdA}single
mutant does not exhibit auxotrophy for BCFAs when grown in lysogeny
broth, prompting Whaley \emph{et al.} to hunt for \emph{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 \emph{i} 14:0 BCFA into membranes derived from\emph{i}
C\textsubscript{4}. This makes physiological sense - while FabH prefers
\emph{a} C\textsubscript{5}-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 \emph{et al.} study where they report a
\textasciitilde{}5-fold increase in affinity for\emph{a}
C\textsubscript{5} over \emph{i} C\textsubscript{4}, we did not measure
a significant difference in MbcS affinity for\emph{i} C\textsubscript{4}
or \emph{a} C\textsubscript{5}. 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 K\textsubscript{m} values below 5
\selectlanguage{greek}µ\selectlanguage{english}M.

Our data validate and expand the idea that \emph{S. aureus} salvages
BCFA precursors by showing that branched-chain aldehydes are
catabolized\textbf{(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
\emph{de novo}synthesis pathway.~Previous studies investigating BCFA
synthesis in related Gram-positive bacteria inform our working model for
an alternative route to BCFAs synthesis \textbf{(Fig 8)} . For instance,
the Gram-positive bacterium \emph{Lactococcus lactis} , which is largely
used for the fermentation of dairy products, harbors an \selectlanguage{greek}α-\selectlanguage{english}keto acid
decarboxylase KdcA that converts the branched-chain \selectlanguage{greek}α-\selectlanguage{english}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 \emph{L. lactis} KdcA that
has highest activity for the \selectlanguage{greek}α-\selectlanguage{english}keto acid of valine (Smit et al., 2005),
consistent with our GC-FAME data showing incorporation of valine
derived, \emph{i1} 4:0 BCFA in the \emph{lpdA mbcS1} and \emph{lpdA
mbcS2~} strains \textbf{(Fig 3)} . We propose that IpdC is a
branched-chain \selectlanguage{greek}α-\selectlanguage{english}keto acid decarboxylase. Three additional facts support
this hypothesis: i) IpdCs are known to have broad specificity (Parsons
et al., 2015); ii) \emph{ipdC} expression is repressed by \emph{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) \emph{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). \emph{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 \emph{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
like\emph{Cutibacterium acnes} and \emph{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 \textbf{(Fig 7)} , suggesting these compounds
are salvaged by \emph{S. aureus} using this pathway during infection.
Taken together, data from our lab and other labs indicate that \emph{S.
aureus} oxidizes the branched-chain aldehyde to the carboxylic acid.
The\emph{S. 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 \selectlanguage{greek}α-\selectlanguage{english}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 \selectlanguage{greek}α-\selectlanguage{english}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
of\emph{mbcS} (either by mutation of the native promoter or by
artificial overexpression using an inducible promoter) restores growth
in the BKDH-deficient strain \textbf{(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 \emph{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 \emph{N-} acetyltransferase AcuA was recently shown to
acetylate acetyl-CoA synthetase in \emph{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.

\textbf{Experimental Procedures}

\emph{Bacterial strains, growth media, and culture conditions}

Strains used in the present study are listed in \textbf{Table S1} . To
routinely cultivate \emph{S. aureus} strains, tryptic soy broth (TSB)
containing 0.25\% (wt/vol) dextrose (BD Biosciences) or complete
chemically defined medium (CDM, pH 6.5) were used (Sheldon et al.,
2014). Briefly, CDM medium was formulated with alanine (672 \selectlanguage{greek}μ\selectlanguage{english}M),
arginine (287 \selectlanguage{greek}μ\selectlanguage{english}M), aspartic acid (684 \selectlanguage{greek}μ\selectlanguage{english}M), cysteine (166 \selectlanguage{greek}μ\selectlanguage{english}M), glutamic
acid (680 \selectlanguage{greek}μ\selectlanguage{english}M), glycine (670 \selectlanguage{greek}μ\selectlanguage{english}M), histidine (129 \selectlanguage{greek}μ\selectlanguage{english}M), isoleucine (228
\selectlanguage{greek}μ\selectlanguage{english}M), leucine (684 \selectlanguage{greek}μ\selectlanguage{english}M), lysine (342 \selectlanguage{greek}μ\selectlanguage{english}M), methionine (20 \selectlanguage{greek}μ\selectlanguage{english}M),
phenylalanine (240 \selectlanguage{greek}μ\selectlanguage{english}M), proline (690 \selectlanguage{greek}μ\selectlanguage{english}M), serine (285 \selectlanguage{greek}μ\selectlanguage{english}M), threonine
(260 \selectlanguage{greek}μ\selectlanguage{english}M), tryptophan (50 \selectlanguage{greek}μ\selectlanguage{english}M), tyrosine (275 \selectlanguage{greek}μ\selectlanguage{english}M), valine (684 \selectlanguage{greek}μ\selectlanguage{english}M),
thiamine (56 \selectlanguage{greek}μ\selectlanguage{english}M), nicotinic acid (10 \selectlanguage{greek}μ\selectlanguage{english}M), biotin (0.04 \selectlanguage{greek}μ\selectlanguage{english}M), pantothenic
acid (2.3 \selectlanguage{greek}μ\selectlanguage{english}M), MgCl\textsubscript{2} (1,000 \selectlanguage{greek}μ\selectlanguage{english}M),
CaCl\textsubscript{2}(100 \selectlanguage{greek}μ\selectlanguage{english}M), monopotassium phosphate (40,000 \selectlanguage{greek}μ\selectlanguage{english}M),
dipotassium phosphate (14,700 \selectlanguage{greek}μ\selectlanguage{english}M), sodium citrate dehydrate (1,400 \selectlanguage{greek}μ\selectlanguage{english}M),
magnesium sulfate (400 \selectlanguage{greek}μ\selectlanguage{english}M), ammonium sulfate (7,600 \selectlanguage{greek}μ\selectlanguage{english}M), and glucose
(27,753 \selectlanguage{greek}μ\selectlanguage{english}M). Blood agar plates were used to propagate the \emph{lpdA
mbcS} double mutant and its derivatives to mitigate the severe growth
defect on TSB.\emph{Escherichia coli} strains were grown in lysogeny
broth (LB) medium without glucose (10 g L\textsuperscript{-1} tryptone,
5 g L\textsuperscript{-1} yeast extract, and 10 g
L\textsuperscript{-1}sodium chloride) (Bertani, 1951). When necessary,
media were solidified~with~agar~at~1.5\%~{[}w/v{]} and supplemented~
with~ antibiotics~ at~ the~ following~ concentrations~ to maintain
selection: ampicillin (Ap) 100 \selectlanguage{greek}μ\selectlanguage{english}g ml\textsuperscript{-1},
chloramphenicol (Cm) 10 \selectlanguage{greek}μ\selectlanguage{english}g ml\textsuperscript{-1}, erythromycin (Em) 10
\selectlanguage{greek}μ\selectlanguage{english}g ml\textsuperscript{-1}, kanamycin (Km) 100 \selectlanguage{greek}μ\selectlanguage{english}g ml\textsuperscript{-1}
or tetracycline~ (Tc) 1.5 \selectlanguage{greek}μ\selectlanguage{english}g ml\textsuperscript{-1}. When required,
media were supplemented with the short branched-chain carboxylic
acids\emph{i} C\textsubscript{4}, \emph{i} C\textsubscript{5},
and\emph{a} C\textsubscript{5} (Sigma-Aldrich), with the branched-chain
fatty acids \emph{a} 15:0 (Avanti Polar Lipids) and \emph{a} 17:0
(Sigma-Aldrich), or with the branched-chain aldehyde
2-methylbutyraldehyde (Sigma-Aldrich) to a final concentration of 0.5
mM. For genetic complementation, TSB was supplemented with 50 ng
ml\textsuperscript{-1} anhydrotetracycline (aTc). Unless otherwise
noted, all strains were grown at 37\textsuperscript{o}C. A double-back
dilution scheme was used to grow cells for growth curves and GC-FAME
analysis to ensure steady-state growth. Briefly, for growth curve
experiments, overnight cultures were used to inoculate precultures in
disposable 16x125 mm borosilicate glass tubes (Fisher Scientific) to an
optical density at 600 nm (OD\textsubscript{600}) of 0.05 and incubated
with rotation. Cell growth was monitored by measuring
OD\textsubscript{600} using an Amersham Ultraspec 2100 Pro UV-visible
spectrophotometer. After streaking from frozen stocks, overnight
cultures were inoculated from single colonies and were used to initiate
pre-cultures that were grown to exponential phase to an
OD\textsubscript{600} of 1.0 and diluted into fresh TSB to an
OD\textsubscript{600} of 0.05 and continued to grow. Exponential-phase
samples were collected at an OD\textsubscript{600} of 0.5\selectlanguage{ngerman}±0.2. Samples
were inoculated into a fresh medium in a 96-well plate to an
OD\textsubscript{600} of 0.05 and cell density was monitored over time
in a computer-controlled Synergy H1~plate reader (BioTek/Agilent)
running Gen5 software ver3.14. Cells for GC-FAME analysis were grown
following the same scheme as above but in 125-mL DeLong shake flasks
with 12.5 mL TSB (10:1 flask/medium ratio) and incubated in a gyratory
water bath shaking. Exponential-phase samples were collected at an
OD\textsubscript{600} of 0.5 ± 0.1. Cells were grown to stationary phase
in disposable borosilicate glass tubes for reporter and chemical
complementation assays.

\emph{Genetic techniques}

Oligonucleotides used~in~this study~were synthesized by Integrated DNA~
Technologies~(IDT; Coralville,~ IA) and are listed in \textbf{Table
S2.}Genomic DNAs (gDNAs) were sequencing using Illumina whole genome
sequencing (SeqCenter; Pittsburgh, PA) and results were analyzed using
BRESEQ software (Barrick et al., 2009). Plasmids used in this study are
listed in \textbf{Table S3} and were constructed using Gibson assembly
as described previously (Gibson et al., 2009)~and verified by Sanger
sequencing (Azenta Life Sciences) or whole plasmid sequencing
(Plasmidsaurus; Eugene, OR). \emph{E. coli} NEB 5\selectlanguage{greek}α \selectlanguage{english}(NEB) was used as a
host for plasmid constructions, which were then transferred into\emph{S.
aureus} strain RN4220 by electroporation as previously described (Schenk
\&~Laddaga, 1992). Plasmids~and~marked~ mutations~ were~ moved~ between~
\emph{S.~ aureus} strains via \selectlanguage{greek}Φ\selectlanguage{english}85-mediated transduction (Novick, 1991).

\emph{Construction of the lpdA::kan\textsuperscript{+}} strain\emph{:}To
construct the \emph{lpdA::kan\textsuperscript{+}} strain (SRB3000), we
moved the transposon insertion containing the
Km\textsuperscript{R}marker from the \emph{S. aureus} SH1000 strain
VKS102 (Singh et al., 2008) into the \emph{S. aureus} USA300 LAC SRB2421
(\emph{lpdA} ::\selectlanguage{greek}φ\selectlanguage{english}N\selectlanguage{greek}Σ\selectlanguage{english}) via \selectlanguage{greek}Φ\selectlanguage{english}85-mediated transduction (Novick,
1991).~Recombinants were selected on TSA Km medium and scored for Em
susceptibility. We then confirmed the allele by PCR.

\emph{Plasmids construction}

\emph{(i) Construction of gfp reporter plasmids}

To create the P\emph{\textsubscript{mbcS}-gfp} reporter fusion, a 236-bp
fragment upstream of the translation initiation codon of SAUSA300\_2542
(\emph{i.e.} , \emph{mbcS} ) was amplified from \emph{S. aureus} LAC
with Q5 DNA polymerase (NEB) using oAP41 and oAP42 as primers. In
parallel, the promoterless \emph{gfp} reporter plasmid pMRSI (Waters et
al., 2016) was amplified and linearized using oAP39 and oAP40 as
primers. PCR products were treated with DpnI (NEB), subjected to Gibson
Assembly, and inserted into \emph{E. coli} NEB 5\selectlanguage{greek}α \selectlanguage{english}(NEB). The resulting
plasmid was named pAP4 (\emph{mbcSp\textsuperscript{+}-gfp} ). Plasmids
pAP10 (\emph{mbcS1p-gfp} ), and pAP11 (\emph{mbcS2p-gfp} ), containing
the same\emph{mbcS} regulatory region but with point mutations
identified during suppressor analysis, were constructed in a similar way
by cloning the fragments into pMRS1.

\emph{~(ii) Construction of new vectors}

pWY51 (pCT1). A 821-bp fragment containing the \emph{tetR} gene encoding
the TetR repressor and the \emph{tetR} promoter region
(\emph{tetR\textsuperscript{+}} -P\emph{\textsubscript{tet}} ) was
PCR-amplified from pYJ335 (Ji et al.,1999) using primers oWY466 and
oWY467. pCL55 (Lee et al., 1991) was used as the backbone plasmid and
was amplified by PCR using primers oWY468 and oWY469. The DNA fragments
were assembled by the Gibson method (Gibson et al., 2009), inserting
the\emph{tetR\textsuperscript{+}} -P\emph{\textsubscript{tet}} cassette
at the multicloning site of pCL55, and the DNA mixture was transformed
into \emph{E. coli} DH5\selectlanguage{greek}α.

\selectlanguage{english}pWY52 (pCT2), and pWY53 (pCT3). A 2225-bp fragment containing
the\emph{tetM\textsuperscript{.}} gene from pTET (Bose et al., 2013) was
amplified by PCR using primers oWY486 and oWY487. pCL55 or pWY51 as a
backbone plasmid was amplified by PCR using primers oWY488 and oWY489
.The DNA fragments were assembled by the Gibson method and the DNA
mixture was transformed into \emph{E. coli} DH5\selectlanguage{greek}α, \selectlanguage{english}resulting in pWY52
(pCT2) or pWY53 (pCT3) that confer tetracycline resistance instead of
chloramphenicol resistance.

\emph{(iii) Construction of complementation plasmids}

\begin{enumerate}
\tightlist
\item
  pWY54 (P\emph{\textsubscript{tet}-mbcS\textsuperscript{+}} ). A
  1648-bp fragment containing the \emph{mbcS} Shine Delgarno sequence
  and coding sequence was PCR-amplified from the USA300 LAC* chromosome
  using primer pair oWY470/oWY471. This also appends the sequence
  (DYKDDDDK) for a C-terminal FLAG epitope tag (Hopp et al., 1988).
  pWY53 as a backbone plasmid was amplified with primer pair
  oWY472/oWY473. The DNA fragments were assembled by the Gibson method
  and the DNA mixture was transformed into \emph{E. coli} DH5\selectlanguage{greek}α,
  \selectlanguage{english}resulting in pWY54. This places the production of \emph{Sa} MbcS-FLAG
  under the control of the inducible P\textsubscript{tet} promoter by
  anhydrotetracycline addition.
\item
  pWY55 (P\emph{\textsubscript{tet}-mbcS} {[}K510A{]}). The 1523-bp
  fragment containing the \emph{mbcS} Shine Delgarno sequence (25bp) and
  coding sequence (1498bp) was PCR-amplified from the USA300 LAC*
  chromosome using the primer pair oWY470 and oWY500. A point mutation
  in the active site (K510A) of \emph{mbcS} was generated by a gblock
  (125bp, oWY499). As before, pWY53 as a backbone plasmid was amplified
  with primer pair oWY469 and oWY473. The DNA fragments were assembled
  by the Gibson method and the DNA mixture was transformed into \emph{E.
  coli} DH5\selectlanguage{greek}α, \selectlanguage{english}resulting in pWY55. This results in the production
  of\emph{Sa} MbcS\textsuperscript{K510A}-FLAG under the control of the
  inducible P\textsubscript{tet} promoter by anhydrotetracycline
  addition.
\item
  pWY75 (P\emph{\textsubscript{tet}-RpibuA\textsuperscript{+}} ). A
  1716-bp fragment containing the coding sequence of Rp\emph{ibuA}
  \textsuperscript{+} and a FLAG tag at the C-terminus from\emph{R.
  palustris} was optimized and synthesized by GenScript and the RBS and
  ORF from \emph{SambcS} were utilized. This fragment was inserted into
  plasmid pWY53 and verified by GenScript. The resulting plasmid was
  transformed into \emph{E. coli} DH5\selectlanguage{greek}α, \selectlanguage{english}resulting in pWY75. The plasmid
  directs the synthesis of \emph{Rp} IbuA-FLAG under the control of the
  inducible P\textsubscript{tet} promoter by anhydrotetracycline
  addition.
\end{enumerate}

\emph{(iv) Construction of plasmids used for protein overproductio} n

pFM01. A 1636-bp fragment containing the\emph{SambcS\textsuperscript{+}}
coding sequence was amplified from\emph{S. aureus} LAC using oWY543 and
oWY544. In parallel, pTEV5 (Rocco et al., 2008) was amplified from
plasmid pRpIBUA1 using oWY545 and oWY546. Both DNA fragments were
assembled via the Gibson method (Gibson et al., 2009) and the product
was transformed into \emph{E. coli} DH5\selectlanguage{greek}α, \selectlanguage{english}resulting in pFM01.

pFM03. The \emph{SambcS} allele coding for the\emph{Sa}
MbcS\textsuperscript{K510A} variant protein was amplified from pWY55
using oWY543 and oWY544. In parallel, pTEV5 was amplified using oWY545
and oWY546. The DNA fragments were assembled via the Gibson method
(Gibson et al., 2009) and the mixture was transformed into \emph{E.
coli} DH5\selectlanguage{greek}α, \selectlanguage{english}resulting in pFM03.

\emph{Identification of suppressor mutants~}

To screen for spontaneous mutations, the \emph{lpdA} mutant was streaked
from frozen stocks on TSA plate supplemented with a mixture of the
branched-chain carboxylic acids \emph{a} C5, \emph{i} C5, and \emph{i}
C4. Independent colonies were then grown overnight in borosilicate glass
tubes containing TSB supplemented with the mixture of the branched-chain
carboxylic acids. A double-back dilution was performed in unsupplemented
medium, and once cells reached an OD\textsubscript{600} of 0.1 (growth
arrest) they were serial diluted, plated on TSA without supplementation,
and incubated at 37\textsuperscript{o}C. Colonies (one per liquid
culture) were picked, restreaked, and growth in the absence of
carboxylic acids was confirmed. Following confirmation, gDNAs were
purified and submitted for whole genome sequencing as a fee for service
at SeqCenter. Analysis of spontaneous mutations was performed using
BRESEQ software (Barrick et al., 2009).

\emph{Analysis of fatty acid composition~}

\emph{S. aureus} strains were grown to exponential phase
(OD\textsubscript{600} \textasciitilde{} 0.5), at which time 10-50 ml of
each culture (\textasciitilde{}100 mg of wet cells) were pelleted,
washed twice with phosphate-buffered saline (PBS), and stored at -80\selectlanguage{ngerman}°C.
Fatty acids were saponified and methylated, and then analyzed by gas
chromatographic analysis of fatty acid methyl esters (GC-FAME) as a fee
for service at the Center for Microbial Identification and Taxonomy
(Norman, OK).

\emph{GFP reporter assays~}

Single colonies of cells carrying the indicated reporter fusions were
inoculated into sterile borosilicate glass tubes 16x125 mm (Fisher
Scientific) containing 2 ml of TSB with appropriate~antibiotic to
maintain selection. Cells were grown to a stationary phase~overnight.
Pellets were collected and washed twice with PBS and resuspended in the
same buffer. 100 \selectlanguage{greek}μ\selectlanguage{english}L of the cell suspension was transferred into a flat
bottom black 96-well plate (Corning). Fluorescence was measured using a
Synergy H1 plate reader (BioTek/Agilent) tuning the monochromator to 485
nm and 535 nm (excitation/emission, respectively). Relative fluorescence
units (RFUs) were calculated by subtracting the fluorescence of plain
PBS and dividing by OD\textsubscript{600} to correct for cell density.

\emph{Protein Purification}

\emph{rTEV.} The plasmid encoding rTEV protease was transformed
into\emph{E. coli} BL21 Codon Plus and purified as described (Blommel \&
Fox, 2007).

\emph{Acyl-CoA synthetases} . Plasmids containing the coding sequences
for \emph{Sa} MbcS\textsuperscript{WT},\emph{Sa}
MbcS\textsuperscript{K510A}, and \emph{Rp} IbuA, all fused to cleavable
\emph{N} -terminal hexahistidine (His\textsubscript{6}) tags, were
overproduced and purified using a two-step procedure as described
(Crosby et al., 2012). Briefly, the plasmids were introduced
into\emph{E. coli} BL21 C41\selectlanguage{greek}λ \selectlanguage{english}(DE3) cells. The resulting strains were
grown in LB medium supplemented with 100 mg L\textsuperscript{-1} Ap at
37\selectlanguage{ngerman}°C first overnight in borosilicate tubes. The overnights were then
subcultured at a 1:20 ratio with shaking until early stationary phase
(OD\textsubscript{600} \textasciitilde{}2). A subculture was prepared by
diluting the initial culture at a 1:100 ratio into 500 mL of LB medium
supplemented with 100 mg L\textsuperscript{-1} Ap, and cells were grown
with shaking until reaching an OD\textsubscript{600} of 0.6. Expression
was induced using 0.5 mM isopropyl \selectlanguage{greek}β-\selectlanguage{english}D-1-thiogalactopyranoside (IPTG),
followed by overnight incubation at 30\selectlanguage{ngerman}°C. Cells were harvested by
centrifugation at 8000 x g for 12 minutes, and the resulting pellets
were stored at -80°C. Pellets were resuspended in 30 mL of buffer 1 (50
mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole). Cells were lysed
using a Digital Sonifier (Branson) for 2- pulses separated by 5-s pauses
on wet ice (25\% amplitude) for 1.5-2 minutes. Cellular debris was
removed by centrifugation at 38000 x g for 30 minutes at 4°C, and the
soluble fraction was filtered using a 0.45 \selectlanguage{greek}μ\selectlanguage{english}m filter. The protein was
purified by two-step Ni\textsuperscript{2+} affinity chromatography via
a 4 mL bed volume of nickel-nitrilotriacetic acid resin (Thermo
Scientific) pre-equilibrated with buffer 1 for 10 column volumes. The
lysate was loaded on the column, and the column was washed with wash
buffer 2 (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole)
containing 1 mg ml\textsuperscript{-1} lysozyme (Thermo Scientific) and
0.2mM phenylmethanesulfonyl fluoride (PMSF) (Thermo Scientific). for a
total of seven column volumes. Proteins were eluted with buffer 3 (50 mM
Tris-HCl pH 8.0, 300 mM NaCl, 250 mM imidazole) over six column volumes.
Eluted protein fractions were analyzed using SDS-PAGE, and those
fractions containing the target protein were pooled. The
His\textsubscript{6} tag was cleaved with rTEV protease using a 1:100
{[}v/v{]} rTEV to protein ratio overnight on at 4\selectlanguage{ngerman}°C ice. Following the
cleavage, proteins were dialyzed sequentially against buffer 4 (50 mM
Tris-HCl pH 8.0, 300 mM NaCl, 0.5 mM EDTA), dialysis buffer 5 (50mM
Tris-HCl pH 8.0, 300 mM NaCl), and finally against buffer 1. The
proteins were reapplied to a pre-equilibrated Ni\textsuperscript{2+}-NTA
column to capture the His\textsubscript{6}tag and rTEV. Untagged target
proteins were isolated to apparent homogeneity from the flow-through and
dialyzed against buffer 6 (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.5 mM
EDTA, 20\% {[}v/v{]} glycerol) and then buffer 7 (50 mM Tris-HCl pH 7.5,
100 mM NaCl, 20\% glycerol). Proteins were concentrated, drop-frozen in
liquid nitrogen, and stored at -80°C until used. Protein purity was
evaluated using SDS-PAGE.

\emph{In vitro acyl-CoA synthetase activity assay~}

The activities of MbcS and IbuA were evaluated using a coupled and
continuous spectrophotometric assay based on NADH consumption, as
described (Crosby et al., 2012). Briefly, 100 \selectlanguage{greek}μ\selectlanguage{english}L reactions were composed
of 50 mM HEPES pH 7.5, 1 mM tris (2-carboxyethyl) phosphine (TCEP), 2.5
mM ATP, 5 mM MgCl\textsubscript{2}, 0.5 mM coenzyme A, 3 mM
phosphoenolpyruvate, 0.1 mM NADH, 1 unit of pyruvate kinase, 5 units of
myokinase, 1.5 units of lactate dehydrogenase, and 60 nM MbcS
(calculated for monomeric enzyme) (or 30 nM IbuA). The reactions were
initiated by adding 2 \selectlanguage{greek}μ\selectlanguage{english}L of the indicated carboxylic acid substrate to
the 98 \selectlanguage{greek}μ\selectlanguage{english}L reaction mixture. The changes in the absorbance, measured at
340 nM, were recorded over time at 37\selectlanguage{ngerman}°C degrees in a computer-controlled
Synergy H1 plate reader running Gen5 software ver3.14 (Agilent/BioTek).
Each experiment was performed in triplicate. All reagents and enzymes
were purchased from Sigma, except for lactate dehydrogenase, which was
purchased from Worthington Biochemical Corporation.~The specific
activity of MbcS for different carboxylic acids as substrates was
calculated using the molar extinction coefficient of NADH (6220
M\textsuperscript{-1} cm\textsuperscript{-1}) and taking into account
that two moles of NADH are oxidized for every mole of AMP being produced
(Garrity et al., 2007). Specific activity was reported as \selectlanguage{greek}μ\selectlanguage{english}mol of AMP
per minute per milligram of MbcS.

\emph{Liquid Chromatography-Mass Spectrometry (LC-MS)}

\emph{Reagents and chemicals}

The following reagents were purchased: water, acetonitrile, isopropanol,
and methanol from Optima grade (Fisher Scientific); high purity formic
acid (Thermo-Scientific); 3-HPMA-d6 and Isobutyril-Coenzyme A were
purchased from Cayman chemicals.

\emph{Preparation of standards and internal standard stock solutions}

CoA was resuspended in HEPES buffer pH 7.5 to generate a 3.83 mg
ml\textsuperscript{-1} stock solution (stock A). A 1 mg
mL\textsuperscript{-1} solution (stock B) of Isobutyryl-Coenzyme A
(IB-CoA) was prepared by dissolving IB-CoA in HEPES buffer pH 7.5. The
stock A and B were diluted in extraction buffer (3-HPMA-d6 was dissolved
in methanol to achieve a 1 mg mL\textsuperscript{-1} stock solution, and
subsequently diluted to a final concentration 500 ng
mL\textsuperscript{-1} of IS) to generate stock C (20 \selectlanguage{greek}µ\selectlanguage{english}g
ml\textsuperscript{-1} of CoA and 10 \selectlanguage{greek}µ\selectlanguage{english}g ml\textsuperscript{-1} IB-CoA).
Stock C was then serially diluted to generate calibration curve
standards ranging from 20 ng ml\textsuperscript{-1} to 10 \selectlanguage{greek}µ\selectlanguage{english}g
ml\textsuperscript{-1} (IB-CoA) and 40 ng ml\textsuperscript{-1} to 20
\selectlanguage{greek}µ\selectlanguage{english}g ml\textsuperscript{-1} (CoA).

\emph{Sample preparation}

MbcS acyl-CoA activity was assessed via LC-MS. The reaction mixture
consisted of 50 mM HEPES pH 7.5, 2.5 mM Mg-ATP, 493 \selectlanguage{greek}μ\selectlanguage{english}M CoA, 73 \selectlanguage{greek}μ\selectlanguage{english}M
isobutyric acid, and 60 nM MbcS. MbcS was denatured through incubation
at 85\selectlanguage{ngerman}°C for 10 minutes and used as a negative control. The reactions
were initiated by adding isobutyric acid and incubated at 37°C for 2
hours, followed by placing the samples on ice. For IB-CoA measurements,
to the 40 \selectlanguage{greek}µ\selectlanguage{english}l of samples was added 360 \selectlanguage{greek}µ\selectlanguage{english}l of extraction buffer and
vortexed. For CoA measurements, the samples prepared for IB-CoA
measurements were diluted 10-fold using extraction buffer. The samples
were transferred to the MS vials for data acquisition.

\emph{LC-MS methods}

This method was designed to measure CoA and Isobutyryl-CoA by UPLC-MS.
The samples were resolved on a HSS T3, 1.8 \selectlanguage{greek}µ\selectlanguage{english}m, 2.1 x 100 mm column
online with a triple quadrupole mass spectrometer (Xevo-TQ-S, Waters
Corporation) operating in the multiple reaction monitoring (MRM) mode.
The LC gradient method started with 98\% of mobile phase A (10 mM
ammonium formate in water) that involved a gradient change from 2\% B
(ACN:water {[}95:5{]} with 10 mM ammonium formate) to 98\% phase B in
2.5 minutes after an initial lag phase of 0.5 minutes with flow rate 0.6
ml/min. The column was maintained at 25 \selectlanguage{ngerman}°C and injection volume was kept
at 5 \selectlanguage{greek}µ\selectlanguage{english}L. The autosampler was maintained at 5 \selectlanguage{ngerman}°C. The column eluent was
introduced directly into the TQS mass spectrometer by electrospray
operating in positive mode at a capillary voltage of 3.0 kV and a
sampling cone voltage of 83 V. The desolvation gas flow was set to 1000
l/h and the desolvation temperature was set to 500 °C. The cone gas flow
was 150 l/h and the source temperature was set to 150 °C.

The sample cone voltage and collision energies were optimized for the
analyte to obtain maximum ion intensity for parent and daughter ions
using ``IntelliStart'' feature of MassLynx software (Waters
Corporation). The instrument parameters were optimized to gain maximum
specificity and sensitivity of ionization for the parent and daughter
ions. Signal intensities from the MRM Q1\textgreater{}Q3 ion pairs for
the CoA (768.1\textgreater{}261.1), IB-CoA (838.1\textgreater{}331.1)
and 3-HPMA-d6 (228\textgreater{}169) were ranked to ensure selection of
the most intense precursor and fragment ion pair for MRM-based
quantitation. This approach resulted in selection of cone voltages and
collision energies that maximized the generation of each fragment ion
species. The details for the calibration curves for standards are
included in Supplementary Materials \textbf{(Table S4)} . CoA was
detectable as low as 5.0 ng ml\textsuperscript{-1} (limit of detection
{[}LOD{]}) and showed 250 ng ml\textsuperscript{-1} as the lowest limit
of quantification (LLOQ). While IB-CoA showed the LOD and LLOQ as 25 ng
ml\textsuperscript{-1} and 50 ng ml\textsuperscript{-1}, respectively.
The quantification range (QR) for CoA and IB-CoA were 250 ng
ml\textsuperscript{-1} to 20 \selectlanguage{greek}µ\selectlanguage{english}g ml\textsuperscript{-1} and 50 ng
ml\textsuperscript{-1} to 10 \selectlanguage{greek}µ\selectlanguage{english}g ml\textsuperscript{-1}, respectively. As
ascertained by blanks run on either side of sample sets, no
sample-to-sample carryover was observed. Each experiment was conducted
in triplicate. Blanks (solvent alone) were injected on either side of
test samples to assess sample carryover. Data was processed using Target
Lynx 4.1. The relative quantification values of analytes were determined
by calculating the ratio of peak areas of transitions of samples
normalized to the peak area of the internal standard.

\emph{Kinetic analysis}

Reactions were performed using the acyl-CoA synthetase activity assay
described above. Pseudo-first order kinetic parameters were calculated
using Prism version 9.0 (GraphPad Software), fitting the data to
Equation 1,

\emph{V\textsubscript{o}} = \emph{V} x {[}S{]} /
(\emph{K}\textsubscript{m} + {[}S{]}) (Equation 1)

where \emph{V\textsubscript{o}} is initial velocity, {[}S{]} is
substrate concentration, V is maximum velocity, and K\textsubscript{M}is
the Michaelis-Menten constant (Segel, 1975).

\emph{Cross-feeding assay}

Single colonies of \emph{S. epidermidis} 1457 were inoculated into
sterile, 16x125 mm borosilicate glass tubes containing 2 ml of CDM.
Cells were grown to stationary phase~overnight. The liquid culture was
centrifuged at 5450 x g for 15 minutes and the supernatant was
filter-sterilized using 0.45 \selectlanguage{greek}µ\selectlanguage{english}m membranes (Avantor Sciences) to prepare
conditioned medium. A single colony each of \emph{S. aureus}
WT,\emph{lpdA} single mutant, and \emph{lpdA} \emph{mbcS} double mutant
cells was inoculated to OD\textsubscript{600} 0.05 in CDM, CDM
supplemented with either 0.5 mM short branched-chain carboxylic acids
or\emph{a} 17:0 branched-chain fatty acid (Sigma-Aldrich), or decreasing
concentrations of conditioned medium as indicated in Fig 7. Cells were
grown to stationary phase~overnight and OD\textsubscript{600} was
measured using Amersham Ultraspec 2100 Pro UV-visible spectrophotometer.

\emph{Statistical analyses~}

Prism version 9.0 (GraphPad Software) was used to perform all
statistical analyses. Statistical significance was calculated using
measurements from at least three biological replicates and one-way or
two-way analysis of variance (ANOVA); for time course experiments,
two-way ANOVA with repeated measures was used to assess differences and,
if significant, analyses were followed by Tukey's honestly significant
difference (Tukey's HSD) tests for pairwise comparisons. Statistical
significance was assumed at a P value of \textless{}0.05.

\textbf{Acknowledgements}

We thank Jorge C. Escalante-Semerena and Vineet K. Singh for the
generous gift of strains and plasmids, Dr. Paul Lawson and the Center
for Microbial Identification and Taxonomy for help with GC-FAME
analysis, and Drs. Sunil Bansal and Amrita Cheema in the Georgetown
University Metabolomics and Proteomics Shared Resource for help with
LC-MS analysis, which is partially supported by NIH/NCI/CCSG grant
P30-CA051008. We also thank members of the Georgetown University
Microbial Interest Group as well as Brinsmade lab members for their
helpful comments and discussions.

This work was supported in part by NIH grant R01 AI137403 to SRB. The
funders had no role in study design, data collection and interpretation,
or the decision to submit the work for publication.

\textbf{Author Contributions}

SRB conceived the study; MCDSF and SRB conceptualized the research goals
and aims; MCDSF, AP, WY, FCMG, DC, and MM performed the investigations
and analyzed the data, SRB, MCDSF, WY, FCMG, DC, and MM prepared the
original manuscript draft; SRB and MCDSF prepared the final manuscript.

\textbf{Figure legends and tables}

\textbf{Table 1.} Extragenic suppressor mutations map to the promoter
region of \emph{mbcS} .\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\emph{mbcS} \textbf{Allele} & \textbf{Location of Mutation} &
\textbf{Mutation Effect}\tabularnewline
\emph{mbcS1} & Deletion at 105/C-\textgreater{}T at 109 & Shortens
spacing between -10 and -35 to 17 bp/Unknown Effect\tabularnewline
\emph{mbcS2} & C-\textgreater{} T at 126 in -10 Box & Unknown
Effect\tabularnewline
\emph{mbcS4} & Deletion at 102 in -35 Box & Shortens spacing between -10
and -35 to 17 bp\tabularnewline
\emph{mbcS5} & A-\textgreater{}G at 109 & Unknown Effect\tabularnewline
\emph{mbcS8} (x2)\textsuperscript{a} & Deletion at 108 & Shortens
spacing between -10 and -35 to 17 bp\tabularnewline
\emph{mbcS11} & Deletion at 120 & Shortens spacing between -10 and -35
to 17 bp\tabularnewline
\emph{mbcS19} & Deletion at 121 & Shortens spacing between -10 and -35
to 17 bp\tabularnewline
\emph{mbcS21} & A-\textgreater{} C at 104 in -35 Box & Unknown
Effect\tabularnewline
\emph{mbcS22} & Deletion at 105 & Shortens spacing between -10 and -35
to 17 bp\tabularnewline
\emph{mbcS25} & G-\textgreater{} A at 126 in the -10 Box & Creates
perfect -10 Box (TATAA)\tabularnewline
\emph{mbcS30} & A-\textgreater{} C at 103 in -35 Box & Unknown
Effect\tabularnewline
\emph{mbcS31} & AG-\textgreater{}TC at 117 and 118 & Unknown
Effect\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{a}Independently isolated twice.

\textbf{Table 2.} Comparison of kinetic parameters for \emph{Sa} MbcS
and\emph{Rp} IbuA.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\textbf{Protein}\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\textbf{Substrate}\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\textbf{K\textsubscript{m}} (\selectlanguage{greek}μ\selectlanguage{english}M)\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\textbf{V\textsubscript{max}\emph{\textsuperscript{a}}}\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\textbf{K\textsubscript{cat}} (s\textsuperscript{-1})\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\textbf{K\textsubscript{cat}/ K\textsubscript{m}} (s\textsuperscript{-1}
\selectlanguage{greek}μ\selectlanguage{english}M \textsuperscript{-1})\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Sa}MbcS\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
isobutyrate (\emph{i}C4)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
8.9 \selectlanguage{ngerman}± 2.1\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.6 \selectlanguage{ngerman}± 0.3\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.43 \selectlanguage{ngerman}± 0.09\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.049\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2-methylbutyrate (\emph{a}C5)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
5.5 \selectlanguage{ngerman}± 1.0\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.8 \selectlanguage{ngerman}± 0.2\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.23 \selectlanguage{ngerman}± 0.05\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.042\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
isovalerate (\emph{i}C5)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textgreater{} 500\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\emph{\textsuperscript{b}}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\emph{\textsuperscript{b}}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\emph{\textsuperscript{b}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Sa}MbcS\textsuperscript{K510A}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2-methylbutyrate (\emph{a}C5)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\emph{\textsuperscript{b}}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\emph{\textsuperscript{b}}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\emph{\textsuperscript{b}}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
ND\emph{\textsuperscript{b}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Rp}IbuA\textsuperscript{WT}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
isobutyrate (\emph{i}C4)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
72.3 \selectlanguage{ngerman}± 26.9\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
5.4 \selectlanguage{ngerman}± 1.6\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
3.01 \selectlanguage{ngerman}± 0.87\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.042\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{All values are mean} \selectlanguage{ngerman}± \emph{standard deviation}
\emph{\textsuperscript{a}}\selectlanguage{greek}μ\selectlanguage{english}M of AMP produced min\textsuperscript{-1}
\emph{\textsuperscript{b}}Not determined; enzyme is catalytically
inactive.\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{All values are mean} \selectlanguage{ngerman}± \emph{standard deviation}
\emph{\textsuperscript{a}}\selectlanguage{greek}μ\selectlanguage{english}M of AMP produced min\textsuperscript{-1}
\emph{\textsuperscript{b}}Not determined; enzyme is catalytically
inactive.\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Figure 1.} \textbf{Canonical pathway for the synthesis of
branched-chain fatty acids (BCFA) via the BKDH complex.} The
branched-chain amino acids isoleucine (Ile), leucine (Leu), and valine
(Val) are converted into their respective \selectlanguage{greek}α-\selectlanguage{english}ketoacids by the
transaminase IlvE; \selectlanguage{greek}α-\selectlanguage{english}KMV (\selectlanguage{greek}α-\selectlanguage{english}keto-\selectlanguage{greek}β-\selectlanguage{english}methylvalerate), \selectlanguage{greek}α-\selectlanguage{english}KIC
(\selectlanguage{greek}α-\selectlanguage{english}ketoisocaproate), and \selectlanguage{greek}α-\selectlanguage{english}KIV (\selectlanguage{greek}α-\selectlanguage{english}ketoisovalerate) undergo oxidative
decarboxylation catalyzed by the \selectlanguage{greek}α-\selectlanguage{english}keto acid dehydrogenase (BKDH)
complex and the respective acyl-CoA primers, once condensed with malonyl
CoA via 3-ketoacyl-ACP synthase III (FabH), are elongated into their
respective BCFAs through the type II fatty acid synthase (FASII).

\textbf{Figure 2. The putative acyl-CoA synthetase gene
\emph{mbcS}contributes to BCFAs synthesis in the absence of an active
BKDH complex} . Wild-type (WT), \emph{lpdA} mutant, \emph{lpdA}
suppressor mutant with a modified \emph{mbcS} promoter (\emph{lpdA
mbcS1)} and\emph{lpdA mbcS} double mutant cells were grown in
\textbf{(A)} TSB,\textbf{(B)} TSB supplemented with a mixture of 0.5 mM
BCCAs \emph{i} C4,\emph{i} C5, and \emph{a} C5 or \textbf{(C)} TSB
supplemented with 0.5 mM\emph{a} 15:0 fatty acid, and growth behavior
was monitored over time as an increase in optical density at 600 nm
(OD\textsubscript{600}). Data are plotted as mean \selectlanguage{ngerman}± SD of three
biological replicates. ****p\textless{}0.0001, using two-way ANOVA with
Tukey's multiple comparison test at 5-8 h.~In panel A, asterisks
indicate that\emph{lpdA} single mutant and \emph{lpdA mbcS} double
mutant are significantly different compared to WT and \emph{lpdA mbcS1}
. In panels B and C, asterisks indicate that \emph{lpdA mbcS} double
mutant is significantly different compared to WT, \emph{lpdA} single
mutant, and\emph{lpdA mbcS1} .

\textbf{Figure 3. Mutations in the \emph{mbcS} promoter increase its
activity and result in synthesis of \emph{i}14:0 BCFAs.}
\textbf{(A)}Selected alleles identified during suppressor analysis are
compared to the WT allele of the \emph{mbcS} promoter region. Changes
are highlighted in magenta, and -10 and -35 boxs are indicated by the
gray boxes. \textbf{(B)} WT cells harboring plasmids with either WT or
mutant\emph{mbcS} promoter regions\emph{~} fused translationally to
\emph{gfp}(P\emph{\textsubscript{mbcS}-gfp} ) were grown to stationary
phase in TSB, at which time cells were pelleted and washed with PBS.
Promoter activity was then measured (relative fluorescence units
{[}RFUs{]}; GFP/OD\textsubscript{600}). \emph{****} p\textless{}0.000,
one-way ANOVA with Tukey's multiple comparison test. \textbf{(C)}
Suppressor mutants with the indicated \emph{mbcS} alleles were grown to
exponential phase in TSB. Cells were washed with PBS, and membrane fatty
acid content was analyzed by GC-FAME. Data are presented as mean ± SD
from three biological replicates. \emph{****} p\textless{}0.0001,
***p\textless{}0.001, two-way ANOVA with Tukey's multiple comparison
test; ns, not significant.

\textbf{Figure 4. Acyl-CoA synthetase activity of MbcS restores growth
of a \emph{S. aureus lpdA mbcS} strain in rich, complex medium lacking
BCFAs.}

\textbf{(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 \emph{Salmonella
enterica}(Starai \& Escalante-Semerena, 2004); Acs2, acetyl-CoA
synthetase from\emph{Saccharomyces cerevisiae} (Starai \&
Escalante-Semerena, 2004); AcsA, acetyl-CoA synthetase from \emph{B.
subtilis} (Gardner et al., 2006); IbuA, isobutyryl-CoA synthetase from
\emph{R. palustrus} (Crosby \& Escalante-Semerena, 2014; Crosby et al.,
2012); FcsA, fatty acyl-CoA synthetase from \emph{R. palustris} (Crosby
\& Escalante-Semerena, 2014). \textbf{(B)} WT, \emph{mbcS,} or
\emph{lpdA mbcS} strains containing either the empty integration vector
pCT3 (vector-only control {[}VOC{]}), pCT3 containing the WT allele of
\emph{S. aureus mbcS} under the control of the anhydrotetracycline
inducible \emph{tet} promoter (p\emph{SambcS} \textsuperscript{+}), or
the allele of \emph{mbcS} that codes for a lysine-to-alanine
substitution at the residue 510 {[}p\emph{SambcS} (K510A){]} were grown
in TSB and growth (OD\textsubscript{600}) was monitored over time.
\textbf{(C, D)} WT or\emph{lpdA mbcS} strains with the empty vector
control or the wild-type allele of \emph{ibuA} from \emph{R.
palustris}(p\emph{RpibuA} \textsuperscript{+}) \textbf{(C)} with or
\textbf{(D)}without 25 ng ml\textsuperscript{-1} of anhydrotetracycline
(aTc) as gratuitous inducer were grown in TSB and OD\textsubscript{600}
was monitored over time. Data are plotted as mean ± SD from three
biological replicates. \emph{****} p\textless{}0.0001, two-way ANOVA
with Tukey's multiple comparison test. ns, not significant. In panel B,
asterisks indicate that \emph{lpdA mbcS} \textsuperscript{VOC} and
\emph{lpdA mbcS} \textsuperscript{p\emph{SambcS} (K510A)} are
statistically different from WT\textsuperscript{VOC},\emph{mbcS}
\textsuperscript{VOC} and\emph{lpdA mbcS}
\textsuperscript{pSa\emph{mbcS} +}. In panel C, asterisks indicate that
\emph{lpdA mbcS} \textsuperscript{VOC} and \emph{lpdA mbcS}
\textsuperscript{pRp\emph{ibuA} +} are statistically different from
WT\textsuperscript{VOC} and \emph{mbcS} \textsuperscript{VOC}. In panel
D, asterisks indicate that \emph{lpdA mbcS} \textsuperscript{VOC} is
statistically different from WT\textsuperscript{VOC},\emph{mbcS}
\textsuperscript{VOC} and \emph{lpdA mbcS}
\textsuperscript{pRp\emph{ibuA} +}.

\textbf{Figure 5. MbcS is a methylbutyryl-CoA synthetase\emph{.} (A)}The
activity of MbcS was tested \emph{in vitro} with several short, straight
and branched carboxylic acids. Acids are indicated as
C\emph{\textsubscript{X}} , where \emph{X} denotes the carbon length.
Data are plotted as the mean specific activity of the enzyme ± SD of at
least three independent trials. \textbf{(B)} \emph{Sa} MbcS was
incubated with 2.5 mM MgATP, 493 \selectlanguage{greek}μ\selectlanguage{english}M coenzyme A (CoA) and 73 \selectlanguage{greek}μ\selectlanguage{english}M
isobutyric acid (IB) to produce isobutyryl-CoA as described in
\emph{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.

\textbf{Figure 6. \emph{S. aureus} utilizes branched-chain aldehydes to
produce BCFAs in a MbcS-dependent manner} . WT, \emph{lpdA}
mutant,\emph{lpdA mbcS1} suppressor mutant, and \emph{lpdA mbcS} double
mutant cells were inoculated into chemically defined medium (CDM)
(black) or CDM supplemented with vehicle (DMSO; gray), \emph{i}
C\textsubscript{4}(2MB; pink), 2-methylbutyraldehyde (2MA; orange), and
\emph{a} 17:0 fatty acid (green). Cell density (OD\textsubscript{600})
was measured after overnight incubation (16-18 h of growth). Data are
plotted as mean \selectlanguage{ngerman}± SD from three biological replicates. \emph{****}
p\textless{}0.0001, ***p\textless{}0.001, **p\textless{}0.01 two-way
ANOVA with Tukey's multiple comparison test for each genotype; ns, not
significant.

\textbf{Figure 7. Metabolites from \emph{S. epidermidis} support growth
of \emph{S. aureus} in an MbcS-dependent manner.} (A) The
\emph{lpdA}mutant (Left) and the \emph{lpdA mbcS} double mutant (Right)
were inoculated \textasciitilde{}5 mm apart from \emph{S. epidermidis}
on TSA plates and incubated for 24 h. Arrow head indicates the point of
inoculation. (B) WT, \emph{lpdA} mutant, and \emph{lpdA mbcS} double
mutant cells were inoculated into chemically defined medium (CDM)
(black) or CDM supplemented with a mixture of \emph{a} C5, \emph{i} C4,
and \emph{i} C5 (gray), \emph{a} 17:0 fatty acid (orange), and 10\%
(green), 1\% (pink) or 0.1\% (blue), of conditioned CDM from \emph{S.
epidermidis} (i.e., cell-free supernatant). Cell density
(OD\textsubscript{600}) was measured after overnight incubation (16-18 h
of growth). Data are plotted as mean ± SD from three biological
replicates. \emph{****} p\textless{}0.0001, *p\textless{}0.05, two-way
ANOVA with Tukey's multiple comparison test; ns, not significant.

\textbf{Figure 8. Working model for the synthesis of BCFAs in \emph{S.
aureus} in a BKDH-independent manner.} In a \emph{lpdA} mutant the BKDH
complex is inactive and during laboratory cultivation the synthesis of
BCFAs is blocked. \emph{S. aureus} strains with high MbcS enzyme
activity (\emph{i.e.,} overexpression of \emph{mbcS} ) can synthesize
BCFAs independent of the BKDH complex using exogenous or endogenous
precursors. We propose \selectlanguage{greek}α-\selectlanguage{english}keto acids are converted into their respective
branched-chain aldehydes by an \selectlanguage{greek}α-\selectlanguage{english}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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