Results
Mutations in the mbcS promoter restore growth of alpdA mutant during laboratory growth. A 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
~0.1 after ~7-8 h unless supplemented
with a mixture of the branched-chain carboxylic acids (BCCAs;
2-methylbutyric acid [a C5], 3-methylbutyric
acid [i C5], and isobutyric acid
[i C4]) or with a 15:0 BCFA (Fig
2A-C , compare squares to circles) . However, we
observed that the 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,which is annotated to encode an acyl-CoA synthetase (Table 1,
Fig 3A). We refer to these alleles hereafter as mbcS alleles to
reflect the m ethylb utyric acid supplementation
bypassed in these strains. We first analyzed growth behavior of a
representative lpdA mutant with a mutant mcbS allele
(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 (Fig 2A-C,compare triangles and squares to circles) .
A recent study reported that TSB and other standard laboratory media
contain trace amounts of branched-chain carboxylic acids (Whaley et al.,
2023) . We wondered whether mbcS was required to
scavenge these BCFA precursors and promote BCFA synthesis in the absence
of lpdA . To test this, we constructed an 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 lpdA single mutant. Unlike the lpdA mutant, whose
growth was restored with branched-chain carboxylic acids, the lpdA
mbcS double mutant failed to grow beyond an optical density of
~0.1 unless a 15:0 was included in the medium
(Fig 2 , compare inverted triangles to squares). These data
indicate that the mutations in the mbcS promoter suppress the
BCFA auxotrophy, and that mbcS is required to restore growth when
BCCAs are provided exogenously in the growth medium.
Promoter-up mutations inmbcS alter the membrane fatty acid profile in S. aureus.Many of the changes we observed in the 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 S. aureus ,
or refine the -35 and -10 sequences toward a consensus
σA-dependent promoter (Table 1, Fig 3A)(Helmann, 1995). We hypothesized that these changes would increasembcS expression. To test this, we introduced a gfpreporter plasmid containing either the wild-type (WT) promoter ofmbcS or the promoters isolated from two independently isolated
mutants into wild-type cells and measured promoter activity (Fig
3A, B) . During growth in TSB, mbcS expression is relatively low
in WT cells. Mutation of the mbcS promoter resulted in a 3- to
4-fold increase in promoter activity (Figure 3B) . We then
wondered whether overexpressing mbcS altered the membrane fatty
acid profile, supporting growth in TSB. To address this question, we
grew the WT and our two selected 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 a 15:0 BCFAs in their membranes with
lower levels of i 14:0 BCFAs. Interestingly, we measured a large
increase in i 14:0 BCFAs and relatively low levels of a 15:0
BCFAs in the suppressor mutants (Fig 3C ). These data strongly
suggest that overexpressing mbcS rescues BCFA auxotrophy by
promoting the synthesis of iso but not anteiso BCFAs.
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)S. aureus acetyl-CoA synthetase (Burckhardt et al., 2019).
Second, MbcS is 43-47% similar to other bona fide acyl-CoA synthetases
from Salmonella enterica and 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 (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 lpdA mbcS double mutant with
integrative plasmids coding for either the wild-type S. aureus
mbcS (SambcS +) 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 mbcS lpdA double mutant, the cells were unable to
achieve an optical density beyond ~0.1 after
~6-8 h (Fig 4B , compare triangles to
circles) . Growth was restored in this mutant when we introduced
a wild-type copy of mbcS but not the mutant allele coding for the
catalytically inert MbcSK510A variant, strongly
suggesting that MbcS-dependent acyl-CoA synthetase activity was
necessary and sufficient for growth in TSB (Fig 4B , compare
inverted triangles and diamonds to circles) . Interestingly, we
measured no obvious growth defect in the single mbcS mutant. To
test genetically if MbcS indeed supports growth of the lpdAmutant by catalyzing acyl-CoA synthetase activity, we complemented thelpdA mbcS double mutant with an integrative plasmid coding for
the bona fide acyl-CoA synthetase IbuA from R. palustris(RpibuA+ ), and assessed growth behavior(Fig 4C, D) . Unlike SambcS+ ,RpibuA+ does not complement the lpdA
mbcS mutant for growth in TSB in the absence of inducer (Fig
4C) . However, adding 25 ng ml-1 of
anhydrotetracycline (aTc) to the growth medium was sufficient to restore
growth of the lpdA mbcS strain to a level similar to the WT
strain (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 lpdAmutant.
To support the in vivo data above, we cloned, overexpressed, and
purified N- terminal hexahistidine (His6)-tagged
MbcS protein (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 Experimental
Procedures . Sa MbcS activated short, straight and branched-chain
monocarboxylic acids and typically preferred branched substrates over
straight substrates with the same carbon length. Sa MbcS had the
highest activity with isobutyrate (i C4)(Fig 5A) . We saw no activity when 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 (i C4-CoA) formation from
isobutryate (i C4). Briefly, we incubatedSa MbcS with Mg*ATP, isobutyrate (IB), and CoA in buffer. As a
negative control, Sa MbcS was denatured at 85°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 Sa MbcS. We failed to detect IB-CoA
formation when substrates were incubated with denatured MbcS,
demonstrating product formation was enzyme-dependent (Fig 5B) .
We then determined a limited set of kinetic parameters for Sa MbcS
and compared them to bona fide isobutyryl-CoA synthetase from R.
palustris (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 in vitro conditions, the apparent affinity ofSa MbcS for i C4 anda C5 were essentially identical
(Km values were calculated to be 8.9 ± 2.1 μM and 5.5 ±
1.0 μM, respectively). Sa MbcS displayed a significantly lower
affinity for i C5 (> 500 μM) (Table 2) . MbcS
displayed a measurably higher affinity for i C4 compared toRp IbuA. This seemed to be offset by the faster rate of reaction
catalyzed by Rp IbuA compared to Sa MbcS usingi C4 as substrate (Table 2 , compare
Kcat values for Rp IbuA andSa MbcS) . This resulted in comparable catalytic
efficiencies for the two enzymes (compare
Kcat/Km values). As expected, the
catalytically inert Sa MbcSK510A variant
exhibited no detectable acyl-CoA synthetase activity (Table 2) .
Taken together, these data indicate that Sa MbcS is a bona fide
methylbutyryl-CoA synthetase with a preference fori C4 as substrate and participates in
branched-chain fatty acid synthesis.
mbcS supports the utilization of 2-methylbutyraldehyde as
a precursor for BCFA synthesis. The BKDH complex catalyzes an oxidative
decarboxylation reaction, converting the branched-chain α-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. 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 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 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 (a C5), and the BCFAa 17:0. As expected, the lpdA single mutant failed to
achieve a final optical density beyond ~0.1 in the
absence of supplementation. Both the lpdA mutant and thelpdA mbcS1 suppressor mutant grew to similar levels in medium
containing the aldehyde and the carboxylic acid (albeit not to WT levels
for the lpdA mbcS1 strain). BCCA- and aldehyde-dependent growth
was dependent on mbcS (Fig 6 , compare pink and orange
bars, lpdA::kan+ mbcS:: φNΣ vs.lpdA::kan+ ) . Indeed, the double mutant
only grew in the presence of a 17:0 BCFA, indicating that lack of
viability was due to insufficient intracellular branched-chain acyl-CoA
precursors.
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 α-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). 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 Lactococcus spp. (Rijnen
et al., 2003; Smit et al., 2005; Smit et al., 2004). We wondered whetherS. aureus might also be capable of generating BCFAs de
novo through a pathway parallel to that formed by the BKDH complex. To
test this, we compared the growth behavior of the lpdA mutant to
the WT parent strain, with and without mbcS overexpressed. Unlike
the lpdA mutant, the lpdA mbcS1 mutant grew, albeit
poorly, in CDM medium without BCFA or precursor supplementation. Again,
this growth was abrogated in the lpdA mbcS double mutant(Fig 6 , grey and black bars, mutant strains compared to
WT) .
Metabolites from S. epidermidis restore growth oflpdA 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 S. epidermidis , S. hominis , S.
haemolyticus , and 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 S. aureus (Brown &
Horswill, 2020; Byrd et al., 2018; Zipperer et al., 2016). Indeed, skin
commensals successfully compete with S. aureus when resources
become limited in the skin environment. For instance, S.
lugdunensis synthesizes the antimicrobial peptide lugdunin that
effectively inhibits the growth of S. aureus 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 S. aureus and staphylococci
in the skin, we wondered if S. epidermidis could feed ourS. aureus lpdA mutant given its propensity to make
volatile compounds derived from the branched-chain amino acids. To
address this, we streaked the lpdA single mutant on medium
lacking branched-chain fatty acids next to S. epidermidis. IfS. epidermidis can cross-feed S. aureus, we expect to
observe growth adjacent to S. epidermidis, with the zone
supporting growth proportional to the amount of the nutrient being
secreted and diffusing through the medium. Interestingly, thelpdA mutant grew only in close proximity to S.
epidermidis, and this growth was abrogated when we streaked thelpdA mbcS double mutant next to S. epidermidis(Fig 7A ). We then quantified the effect using S.
epidermidis conditioned medium. As expected, the lpdA single
mutant was chemically complemented with a mix of branched-chain
carboxylic acids or a 17:0 BCFAs (Fig 7B , compare grey,
orange bars to black bars, respectively) . We then determined
that 10% conditioned medium from S. epidermidis restored near WT
growth. The effect was lost when the cells were supplemented with
decreasing concentrations of conditioned medium (Fig 7B ,
compare green, pink, and blue bars). Only a 17:0
supplementation restored growth of the lpdA mbcS double mutant(Fig 7B) . Taken together, these data indicate S. aureuscan salvage isoleucine-derived, exogenous branched-chain aldehydes in
addition to branched-chain carboxylic acids in a mbcS- dependent
manner for BCFA synthesis. Our data also strongly suggest that S.
epidermidis can support S. aureus growth by providing these same
essential precursors for BCFA synthesis.