(p)ppGpp/GTP and Malonyl-CoA Modulate Staphylococcus aureus Adaptation to FASII Antibiotics and Provide a Basis for Synergistic Bi-Therapy

Staphylococcus aureus is a major human bacterial pathogen for which new inhibitors are urgently needed. Antibiotic development has centered on the fatty acid synthesis (FASII) pathway, which provides the building blocks for bacterial membrane phospholipids.

B acterial infections that fail to respond to antibiotic treatments are on the rise, especially in the immunocompromised or weakened host, underlining the need for novel antimicrobial strategies (1). The fatty acid synthesis (FASII) enzymes were considered fail-safe targets for eliminating numerous Gram-positive pathogens. Anti-FASII drugs have been a front-line treatment against Mycobacterium tuberculosis, which synthesizes very long-chain fatty acids that cannot be compensated by the host (2). However, Firmicute pathogens, including Staphylococcus aureus and numerous members of the Streptococcaceae, bypass FASII inhibition and satisfy their fatty acid requirements by using host-supplied fatty acids (3)(4)(5). FASII inhibitors, such as triclosan (Tric), MUT056399, fasamycins A and B, amycomicin, and a pipeline FASII antibiotic AFN-1252 (6)(7)(8)(9)(10)(11), would thus have limited use as stand-alone treatments of infections by numerous Gram-positive pathogens (3)(4)(5).
Our recent studies show that S. aureus can adapt to FASII inhibitors by two mechanisms, depending on growth conditions. One involves mutations in a FASII initiation gene, usually fabD. Lower activity of the FabD mutant would increase availability of its substrates, one of which is acyl carrier protein (ACP), for incorporation of exogenous fatty acids (eFA) via the phosphate acyltransferase PlsX (Fig. S1A in the supplemental material) (4,12). The second mode of adaptation occurs without FASII mutations and predominates in serum-supplemented medium. In this case, full adaptation and eFA incorporation in actively growing cells is achieved after a latency phase, whose duration (6 to 12 h) depends on the strain and pregrowth in serum-containing medium. Adaptation is associated with greater intracellular retention of eFA and ACP, both of which contribute to eFA incorporation in membrane phospholipids to compensate FASII inhibition (Fig. S1B) (5).
The factors regulating S. aureus transition from latency to outgrowth upon anti-FASII treatment remain unknown. We hypothesized that initial fatty acid starvation in response to anti-FASII might comprise the signal that delays eFA incorporation in phospholipids and outgrowth. The S. aureus FapR repressor reportedly regulates most FASII genes (except acc, encoding acetyl-CoA carboxylase, and FabZ, b-hydroxyacyl-ACP dehydratase) together with phospholipid synthesis genes plsX and plsC (13,14). Interestingly, malonyl-CoA has a dual function; it is the first dedicated FASII substrate used by FabD (malonyl-CoA transacylase), and it also controls FapR by a feed-forward mechanism (14). FabD uses malonyl-CoA and ACP to synthesize malonyl-ACP (15). Malonyl-CoA binding to FapR reverses FapR repression, leading to upregulation of the FASII and phospholipid synthesis genes (14). Thus, malonyl-CoA is important in both enzymatic and regulatory activities of FASII. In Escherichia coli, expression of the malonyl-CoA synthesis enzyme ACC is regulated by (p)ppGpp, which accumulates in slow growing, nutrient-deficient conditions (16,17); (p)ppGpp also reportedly regulates other FASII and phospholipid synthesis genes (18,19). In Bacillus subtilis, studies of (p)ppGpp null mutants gave evidence for the need to activate the stringent response in order to survive fatty acid starvation; these studies implicated increased GTP in mortality of (p)ppGpp null mutant strains (20). Fatty acid starvation is also associated with cell size via regulation of FASII, although underlying mechanisms remain to be elucidated (21). To our knowledge, no evidence exists for stringent response-mediated FASII regulation in S. aureus.
Here, we first show that stringent response induction exerts control over fatty acid and phospholipid synthesis in S. aureus by modulating FapR repressor activity. FASII antibiotic treatment, like the stringent response, leads to GTP depletion, which is the likely common metabolite linking these two responses. The chain of events revealed here indicate that (p)ppGpp/GTP and malonyl-CoA contribute to adjusting the timing of FASII-antibioticinduced latency transition to outgrowth. Based on our findings, we suggest a bi-therapy approach that combines FASII inhibitors and a (p)ppGpp inducer to prevent S. aureus adaptation.

RESULTS
(p)ppGpp negatively regulates malonyl-CoA levels in S. aureus. We investigated the potential roles of (p)ppGpp and malonyl-CoA in S. aureus response to FASII inhibition. Three S. aureus strains were used in this study, Newman, USA300, and HG1-R (Table S1), which all adapt to anti-FASII with similar kinetics (5, this study). Previous studies reported difficulties in (p)ppGpp measurements in B. subtilis and S. aureus (20,22). Our initial attempts at measuring (p)ppGpp by high-pressure liquid chromatography (HPLC) and the fluorescent dye PyDPA (23) failed to give reliable results (data not shown). We therefore constructed transcriptional fusions to detect conditions when (p)ppGpp-induced genes are activated in vivo (Table S2). The reporter fusion activities responded to mupirocin, which inhibits isoleucyl-tRNA synthetase and triggers (p)ppGpp synthesis (24) (Fig. 1A and data not shown). P ilvD -lacZ (NWMN_1960) and P oppB -lacZ (NWMN_0856) were upregulated, and P cshA -lacZ (NWMN_1985) was downregulated by mupirocin. Nutrient starvation during stationary phase induces the stringent response in E. coli (16). In S. aureus, b-galactosidase (b-gal) activity of the P ilvD -lacZ and P oppB -lacZ sensors were 1.2-and 7-fold higher in stationary phase compared to exponential-phase cells, while P cshA -lacZ activity was ;2-fold lower ( Fig. 1B and data not shown), further validating the in vivo (p)ppGpp sensors.
The stringent response sensors would be expected not to respond to mupirocin in a (p)ppGpp null strain. We compared sensor responses in (p)ppGpp-proficient and deficient strains. These strains derive from HG001 (25) and a (p)ppGpp-null strain (kindly provided by C. Wolz) (26). They were first repaired for a defect in fakB1, which is common to 8325 derivatives (like HG001) and a minority of S. aureus isolates (5). FakB1, a fatty acid kinase subunit, facilitates assimilation of mainly saturated fatty acids (27). Its absence in 8325 derivatives can explain previous reports of S. aureus sensitivity to anti-FASII treatment (11,28), although the majority of S. aureus strains adapt to these antibiotics (4,5). The fakB1-repaired HG001 and HG001 (p)ppGpp0 strains are referred to respectively as HG1-R and ppGpp0. Responses of the P ilvD -lacZ, and P cshA -lacZ reporter fusions were compared in HG1-R and ppGpp0 strains by plate tests (Fig.S2; see Materials and Methods). If the response to mupirocin occurs via its stimulation of (p)ppGpp, then neither induction of ilvD nor suppression of cshA would occur in the ppGpp0 background. Indeed, P ilvD -lacZ (Fig. S2A) and P cshA -lacZ (Fig. S2B) responded to mupirocin as expected in the parental strain, whereas no such responses were observed in the ppGpp0 background. These results also indicate that the stringent response controls these sensors in S. aureus.
We then asked whether (p)ppGpp blocks malonyl-CoA synthesis in S. aureus, as reported in E. coli (29), despite major regulatory differences between these bacteria. Total malonyl-CoA was measured in cells treated or not with mupirocin by enzymelinked immunosorbent assay (ELISA). We also used the in vivo promoter fusion P accBC -lacZ to measure expression of accBC (NWMN_1432 and NWMN_1431), which encode subunits of acetyl-CoA carboxylase (ACC) required for malonyl-CoA synthesis (Table S2). Stringent response induction by mupirocin led to decreases in malonyl-CoA pools (;6-fold) and in P accBC -lacZ b-gal activity (;4-fold) (Fig. 1C). Similarly, stationary-phase cells showed ;2-fold lower malonyl-CoA production and P accBC -lacZ b-gal activity compared to exponential-phase cells (Fig. 1D). Finally, the P accBC -lacZ reporter was inhibited by mupirocin in HG1-R, but not in the ppGpp0 strain (Fig. S2C). These results show that in S. aureus, stringent response induction leads to repression of malonyl-CoA synthesis (13).
FASII-antibiotic-induced latency transiently alters expression of (p)ppGppregulated sensors. We recently showed that host fatty acids can compensate FASII-antibiotic inhibition of S. aureus to promote growth. In low membrane stress conditions, as in serum, adaptation involves a transient latency phase without detection of FASII mutations ( Fig. 2A). Anti-FASII-adapted S. aureus display fatty acid profiles that are fully exogenous (Fig. S1B) (5). As anti-FASII treatment may provoke fatty acid deprivation before eFAs are incorporated, we asked whether the latency preceding FASII bypass corresponds to stringent response induction. Using the stringent response sensors, an ;3.9-fold increase in P ilvD -lacZ and ;7-fold decrease of P cshA -lacZ b-gal activities were observed during the latency phase preceding outgrowth (Fig. 2B), indicating that a factor related to the stringent response is induced in response to anti-FASII treatment. P ilvD -lacZ activity returned to normal levels once bacteria were in the outgrowth phase. P cshA -lacZ b-gal activity was only partially restored during outgrowth, as levels increased by only 2-fold compared to latency. The reason for lower cshA expression is unknown, but it is likely that its expression is subject to other layers of regulation.
FASII antibiotic treatment downregulates accBC and lowers malonyl-CoA pools. Malonyl-CoA, the ACC product, binds FapR and antagonizes repression, and is also a FabD substrate (Fig. 3A). We assessed malonyl-CoA production in nonselective (SerFA) and anti-FASII-treated (SerFA-Tric) latency and outgrowth in cultures of the Newman strain. Pools of malonyl-CoA were measured by ELISA and by P accBC -lacZ expression. Both measurements indicated that malonyl-CoA levels were comparable in SerFA and SerFA-Tric-adapted outgrowth cultures, and were markedly lower during SerFA-Tric latency (Fig. 3B). Taken together, these results show that stringent response induction and anti-FASII-induced latency lead to accBC inhibition, suggesting that a common element links these responses.
We also assessed pools of malonyl-CoA using a FapR activity sensor called FapR-Trap (Fig. S3A, Table S2). FapR-Trap responded as expected: expression was increased in the absence of repressor (DfapR), but decreased in stationary-phase wild-type cells when malonyl-CoA levels were low (Fig. S3B). Interestingly, and in sharp contrast to the above results, malonyl-CoA estimations by FapR-Trap were around 10-fold higher during SerFA-Tric outgrowth compared to nonselective SerFA cultures ( Fig. 3C; compare panel B). These differences (summarized in Table  S3), particularly visible during adaptation outgrowth, indicate that malonyl-CoA distribution in anti-FASII-treated S. aureus favors FapR binding over FabD. They suggest that malonyl-CoA pools and their distribution between FapR and FabD may be central determinants in S. aureus adaptation to FASII antibiotics. . P ilvD -lacZ and P cshA -lacZ expression were evaluated by b-gal assays. Total malonyl-CoA levels were determined by immunoassay (ELISA) and deduced from P accBC -lacZ expression. Genes ilvD and cshA are upregulated and downregulated, respectively, by stringent response induction. Data presented are means 6 standard deviations from triplicate independent experiments. *, P # 0.05 using the Mann-Whitney test.
Reduced FabD competition for malonyl-CoA would increase its availability for FapR (Fig. 3A). We showed previously that fabD mutants may emerge upon FASII-antibiotic selection, but not in serum-supplemented medium as used here (4,5). Indeed, a fabD mutant displayed 5-fold greater FapR-Trap expression than the parental strain in nonselective SerFA (Fig. 3D). However, we ruled out the presence of fabD mutations in our conditions by sequencing the DNA of five independent anti-FASII-adapted cultures (available upon request). These findings could suggest that FabD is intact but disabled for its interactions with malonyl-CoA during S. aureus growth in the presence of anti-FASII. This possibility is currently under study in our laboratory.
GTP depletion is the feature common to the stringent response and FASIIantibiotic-induced latency. We asked whether the stringent response effector (p)ppGpp was directly responsible for the observed phenotypes during anti-FASII treatment, using an S. aureus wild type (WT) strain (HG1-R) and the (p)ppGpp0 isogenic strain (called ppGpp0). AFN-1252 was used as anti-FASII in this strain background due to higher resistance of HG001 derivatives to triclosan. The HG1-R and ppGpp0 strains grew similarly in the presence of anti-FASII treatment, suggesting that the absence of (p)ppGpp did not accelerate anti-FASII adaptation (data not shown). We then compared expression of P ilvD -lacZ and P accBC -lacZ sensors in the WT versus ppGpp0 backgrounds upon anti-FASII treatment (Fig. 4A). Both sensors behaved as described above ( Fig. 2 and 3) in the WT strain. However, these sensors displayed the same responses to anti-FASII treatment in the two strains. Thus, while (p)ppGpp induction inhibits acc and thus lowers malonyl-CoA pools, it is not required for these phenotypes in anti-FASII-treated S. aureus.
(p)ppGpp is known to be intimately linked to GTP, as (p)ppGpp inhibits GTP synthesis (30,31). Lowering GTP levels rescues B. subtilis from ppGpp0 toxicity during lipid starvation (20). We used HPLC to measure GTP levels during anti-FASII adaptation of S. aureus Newman. GTP levels decreased by 4-fold at 3 h post-anti-FASII treatment (Fig. 4B). Consistent with this, the amounts of two GTP synthesis enzymes were decreased during anti-FASII latency of S. aureus USA300, as seen by proteomics (5); HprT (2.35-fold lower [n = 4]; P = 0.014) and GuaA (1.5-fold lower [n = 4]; P = 0.029). These results identify GTP as the metabolite and potential effector common to both the stringent response and anti-FASII-induced latency.
GTP is also a cofactor of the pleiotropic regulator CodY (31). We asked whether CodY is implicated in accBC regulation. P accBC -lacZ expression was visibly lower in a codY insertional mutant compared to expression in the parental WT (USA300) (Fig. 4C). In addition, the anti-FASII latency period was strikingly longer in a codY mutant than in the WT strain (Fig. 4D). This delay is consistent with a role of GTP depletion in delaying anti-FASII latency via CodY. These results lead us to propose that, in S. aureus, the stringent response pathway intersects the initial latency response to FASII inhibitors by the common depletion of GTP, likely via the CodY regulon.
Phospholipid synthesis genes plsX and plsC are differently controlled by FapR. The above results show that malonyl-CoA pools are restored during S. aureus adaptation to FASII antibiotics, and preferentially bind FapR, which alleviates FapR repression (Fig. 3). The S. aureus FapR regulon reportedly includes plsX (NWMN_1139, part of the fapR operon) and plsC (NWMN_1620); however, the S. aureus FapR binding site in the plsC promoter region is highly degenerate (13) (see Fig. 5A), and no proof was given for this interaction. We used promoter reporter fusions P fapR plsX -lacZ and P plsC -lacZ (Table S2) to compare expression in a wild-type strain (HG1-R) and its DfapR derivative. Expression of both reporters was upregulated (each 1.6-fold) in the DfapR strain (Fig. 5B). To determine whether regulation involved direct FapR binding, we performed DNase I footprinting using the plsX and plsC promoters as binding substrates for purified FapR (Fig. 5C). FapR bound efficiently to the plsX promoter region. In contrast, FapR did not bind the plsC upstream region containing the putative binding site. Taken together, these results indicate that in S. aureus, FapR regulates expression of both plsX and plsC, but that its effect on plsC is either indirect or may require other S. aureus factors.  Mupirocin and anti-FASII treatment lead to reduced expression of S. aureus phospholipid synthesis genes plsX and plsC. Repression of accBC FASII by mupirocin would be expected to impact all FapR-regulated genes, including those involved in phospholipid synthesis (Fig. S1A). To test this, we followed P fapRplsX -lacZ and P plsC -lacZ transcriptional fusion expression in the presence of mupirocin (0.1 mg/ml), using P ilvD -lacZ and P accBC -lacZ sensors as references (Table S4). Expression of plsX and plsC sensor fusions were 4-and 3-fold lower, respectively, in mupirocin than in nontreated samples.
Responses of P fapRplsX -lacZ and P plsC -lacZ during anti-FASII-induced latency and outgrowth were then measured. Expression of b-gal from both sensors gradually decreased during latency, followed by abrupt (4-and 2-fold, respectively) increases upon restart of active growth of anti-FASII-adapted cells (Fig. 5D, and data not shown). Expression of P fapRplsX -lacZ reached higher (;3-fold) levels in anti-FASII-adapted outgrowth than in nonselective growth. Anti-FASII treatment thus decreases expression of phospholipid synthesis genes during latency, which recovers upon adaptation.
Mupirocin treatment lowers fatty acid incorporation and is synergistic with anti-FASII treatment to inhibit S. aureus growth. Since mupirocin leads to downregulation of phospholipid synthesis genes, it might consequently affect S. aureus membrane fatty acid composition. To test this, S. aureus strain Newman was grown in SerFA with and without sublethal mupirocin addition (0.05 mg/ml, i.e., 5-fold below the MIC) (32). Incorporated eFA was markedly decreased, from 50% in nontreated to 35% in mupirocin-treated cultures (Fig. 6A). Induction of (p)ppGpp during anti-FASII-induced latency could thus slow or stop eFA incorporation in this transient period.
The above findings led us to hypothesize that FASII inhibitors could be synergistic with a stringent response inducer that prevents compensatory eFA incorporation by repressing the phospholipid synthesis genes plsX and plsC. We first examined anti-FASII adaptation in a strain expressing (p)ppGpp (via relP-expressing plasmid pCG258 in a ppGpp0 strain [26]), (Table S2). While the ppGpp0 control strain (carrying the empty vector pCG248) adapted to anti-FASII after overnight growth, basal RelP expression was sufficient to inhibit anti-FASII adaptation (Fig. 6B). This result shows that (p)ppGpp accumulation synergizes with anti-FASII action to block S. aureus growth. Likewise, addition of a subinhibitory concentration of mupirocin (0.05 mg/ml) and triclosan (0.5 mg/ml) to S. aureus SerFA cultures resulted in extended latency, whereas neither mupirocin nor the anti-FASII treatment separately blocked bacterial growth (Fig. 6C). Similar results were obtained using anti-FASII AFN-1252 (7) and the multidrug-resistant S. aureus (MRSA) strain USA300 FPR3757 (Table S5). Thus, the observed synergistic effect between two flawed antibiotics may offer an effective strategy for development of last-resort treatments against S. aureus infection.

DISCUSSION
This study reveals the nature of cross-control between S. aureus responses to FASII inhibition and to stringent conditions. GTP is depleted in both these conditions, which may explain why the same targets are affected. Our results further show that (p)ppGpp induction lengthens the latency phase preceding adaptation to FASII inhibition. accBC transcription is repressed upon stringent response induction, which sets off a chain of events leading to transient repression of the phospholipid synthesis genes plsX and plsC. These events correlate with limited eFA incorporation and extended latency. During S. aureus adaptation outgrowth, the initial effects of anti-FASII are reversed, allowing eFA incorporation and adaptation to FASII antibiotics. These results suggest a model (Fig. 7) in which (p)ppGpp induction and anti-FASII both initially trigger GTP depletion, resulting in decreased malonyl-CoA pools. The suggested role for CodY in regulating ACC expression remains to be investigated. These events repress phospholipid enzyme synthesis and contribute to anti-FASII latency prior to adaptation outgrowth. Stringent conditions in host niches may be relevant to S. aureus infection (33), and might impact the bacterial response to anti-FASII treatment. While our findings identify a role for (p)ppGpp induction via GTP depletion in anti-FASII adaptation in S. aureus, they do not rule out other roles for these metabolites, or the involvement of other factors in this process.
A new role for malonyl-CoA in anti-FASII adaptation was uncovered in this study, via its increased association with FapR in antibiotic-adapted cultures compared to nonselective cultures. FapR-Trap showed ;10-fold greater expression in anti-FASII-adapted cultures than in nonselective cultures, while total malonyl-CoA pools were the same in both conditions (Fig. 3). Increased malonyl-CoA interaction with FapR, i.e., FapR derepression, during anti-FASII adaptation is consistent with increased plsX and plsC expression (Fig. 5). Malonyl-CoA rerouting in anti-FASII treatment may be explained by FabD inactivation in anti-FASII adaptation conditions, e.g., by an intermediate metabolite, as suggested in E. coli (34). Along this line, a recent study proposed that acyl-ACP accumulation could inhibit FabD (35). Interestingly, acyl-ACP accumulates in a fabD mutant during anti-FASII adaptation (4). Alternative possibilities may be considered, such as (i) post-translational FabD modification (36) or (ii) FabD reversal upon FASII inhibition due to a pile-up of its endproduct, malonyl-ACP. We are currently investigating these hypotheses. These findings indicate limits to the reliability of FapR operon-based sensors to estimate malonyl-CoA pools, for which readouts vary according to growth conditions. This may be important to consider in bioengineering applications that rely on FapR operon-like sensors to optimize malonyl-CoA production (37).
Previous studies identified S. aureus plsC as containing a FapR-binding site (13). This is disproven here, as FapR failed to bind the published plsC consensus site, which lacks a consensus palindromic sequence (Fig. 5C). Nevertheless, plsC expression is increased in a DfapR mutant, indicating that FapR-mediated control is indirect.
The need for antimicrobial alternatives is urgent and, besides the discovery of new molecules or targets, the development of efficient combinations based on existing but  individually ineffective drugs remains to be explored. Our clarification of the link between the stringent response and anti-FASII adaptation opens perspectives for combinatorial antibiotic strategies, using FASII inhibitory and subinhibitory concentrations of stringent response inducers that delay or prevent anti-FASII adaptation of multidrug resistant pathogens like S. aureus. Mupirocin, which is usually used topically, was recently proposed as a potentially active systemic antibiotic when presented in liposomes (38). The proof-of-concept demonstrated here using anti-FASII antibiotics and mupirocin suggests a useful bi-therapy approach for reducing S. aureus survival during infection. Table S1 in the supplemental material. Brain heart infusion (BHI) and Luria-Bertani (LB) media were used, respectively, for S. aureus and E.coli growth. S. aureus precultures were routinely prepared in BHI medium. Three fatty acids (C14:0, myristic acid; C16:0, palmitic acid; and C18:1, oleic acid) (Larodan Fine Chemicals, Stockholm, Sweden) were prepared as 100 mM stocks in dimethyl sulfoxide (DMSO) and used at final equimolar concentrations of 0.17 mM each in experiments (referred to as eFA). Ser-FA (BHI containing eFA 1 10% newborn calf serum) (Sigma-Aldrich, St. Louis, MO) and SerFA-Tric (SerFA plus triclosan, 0.5 mg/ml), or SerFA-AFN (SerFA plus AFN-1252, 0.5 mg/ml) were the modified media used as indicated. Antibiotics kanamycin (50 mg/ml) and erythromycin (5 mg/ml) were used in E. coli and S. aureus, respectively, to select pTCV-lac-based reporter fusion plasmids (39). Antibiotic adaptation experiments with plasmid-carrying strains were done in SerFA-Tric containing 2 mg/ml erythromycin; note that the latency period was extended by about 4 to 6 h under this condition. Mupirocin (Clinisciences, Nanterre, France), a functional analogue of isoleucyl-AMP and a stringent response inducer of (p)ppGpp (40), was prepared in DMSO and used at 0.1 mg/ml (32,41) to validate (p)ppGpp sensors, or at 0.05 mg/ml when used in combination with anti-FASII antibiotics. Equal volumes of DMSO were added to control samples when mupirocin was used.

Strains and media. Strains are listed in
Growth experiments in anti-FASII conditions. Three S. aureus strains or their derivatives were used to follow anti-FASII adaptation: Newman, USA300, and HG1-R. The latter strain corresponds to HG001 that was repaired for a fakB1 defect present in the 8325 lineage, and in a minority of S. aureus strains; fakB1 encodes a fatty acid kinase subunit for saturated fatty acid phosphorylation, which enables the use of eFA during anti-FASII adaptation. The above strains showed comparable responses to conditions tested in this work. In experiments using triclosan as the anti-FASII antibiotic, cells were precultured in BHI and then diluted to absorbance at 600 nm (A 600 ) of 0.01 in SerFA-Tric. Growth was followed by A 600  (20,31). We showed that inhibition of FASII also leads to GTP depletion, pointing to an intersecting link between these pathways. Both conditions activate stringent response sensors (P ilvD -lacZ, P oppB -lacZ, and P cshA -lacZ) and lower malonyl-CoA (mal-CoA) pools, such that FapR (for which mal-CoA acts as anti-repressor) exerts repression (13). As a consequence, genes under FapR control, including plsX and plsC, remain repressed, blocking phospholipid synthesis. Both stringent response induction and FASII inhibition during the latency phase lead to membrane synthesis arrest. (Right) Upon FASII adaptation, GTP levels are restored. accBC expression is restored to normal, and mal-CoA levels are increased, leading to FapR derepression. In consequence, PlsX and PlsC are both increased, such that phospholipid synthesis resumes. Red and green arrows and circled metabolites correspond to functions analyzed in this study. * refers to activities based on previous studies. readings as indicated. Nonselective exponential-and stationary-phase cultures were harvested at A 600 = 1 to 4, and ;10, respectively. If AFN-1252 was used as the anti-FASII antibiotic, the procedure was the same except that precultures were done in SerFA. Both triclosan and AFN-1252 specifically inhibit FabI, a FASII enzyme (7,42). Triclosan causes nonspecific membrane damage at higher concentrations (42) and was therefore not used in studies with the HG1-R strain, which showed higher resistance to this drug.
Construction of fakB1-repaired strains. The fakB1 gene of S. aureus HG001, as the entire 8325 lineage, displays a 483-bp deletion removing 56% of the 867-bp functional gene. To repair this deletion, a 1,939-bp DNA fragment containing a functional fakB1 was amplified by PCR from S. aureus JE2 with primers fakB1_fp and fakB1_rp (Table S6). This fragment was cloned using the Gibson assembly protocol in the thermosensitive vector pG1 (43) amplified with the primers pG1_GibBam and pG1_GibEco (Table  S6). The resulting vector pG1XfakB1 was introduced by electroporation into S. aureus HG001, HG001DfapR, and HG001ppGpp0 strains to generate corresponding fakB1-repaired strains (Table S1). Electroporation of S. aureus strains and allelic exchange were performed as described previously (44). The expected fakB1 repair in these strains was confirmed by PCR and sequence analysis.
Reporter fusions. Promoter regions of ilvD, oppB, cshA, as well as fapR, plsC, and accBC, were cloned in pTCV-lac or pAW8 plasmids (Table S2) using the appropriate primers (Table S6). PCR-amplified DNA fragments and plasmid were treated with restriction enzymes EcoRI and BamHI and ligated products were transformed into DH5a, Top10, or IM08B E. coli cells. The obtained constructs were confirmed by DNA sequencing. Plasmids obtained from IM08B were used directly to transform the S. aureus Newman strain; clones obtained in DH5a or Top10 were first established in the S. aureus strain RN4220. A standard electroporation protocol was used to transform DNA in S. aureus (45).
FapR activity sensor. To estimate malonyl-CoA pools bound to FapR, we designed a transcriptional fusion with promoter and operator sequences containing a consensus FapR-binding site and called it FapR-Trap (Fig. S2, Table S2). The construction was based on similar previous studies to estimate malonyl-CoA pools (37,47).
b-Galactosidase assays. Fresh cultures were prepared at A 600 = 0.1 from overnight BHI cultures and b-galactosidase (b-gal) activities were measured at the indicated A 600 or time of sampling. When mupirocin was used, cultures were treated or not at A 600 = 0.1 after growth from an initial A 600 = 0.01 and processed 1 h later. All samples of a set were stored at 220°C prior to measurements, which were performed for all samples of a same set. b-Gal activities were measured as described previously (48), except that samples derived from SerFA-containing medium were incubated with lysostaphin (0.1 mg/ml; AMBI Products, Tarrytown, NY) for 30 min at room temperature prior to processing with b-Glo reagents (Promega Co., Madison, WI). The values of b-gal (mean 6 standard deviation) were determined from three independently performed experiments.
Malonyl-CoA measurement by ELISA. Bacterial cultures were prepared as described above, and samples were processed at the indicated A 600 /time interval according to our test conditions. For each sample, the equivalent of A 600 = 30 was centrifuged at 8,000 rpm at room temperature for 5 min. Pelleted cells were immediately frozen in liquid nitrogen and transferred to -80°C overnight. Ice-cold phosphate-buffered saline (PBS) was used to resuspend cells at 4°C, which were then sonicated in FastPrep (MP Biomedical, Solon, OH). Supernatants were collected by centrifuging the cell slurry at 13,000 rpm at 4°C for 5 min, and stored at -80°C until use. ELISAs for total malonyl-CoA measurements were performed as per the manufacturer's instructions (CUSABIO Life Sciences, College Park, MD). Malonyl-CoA standards were run along with test samples. Each experiment was performed on three independent cultures. Mean values 6 standard deviation are presented.
For malonyl-CoA measurements under stringent response conditions (Fig. 1C, left), the Newman strain was first grown in BHI to an A 600 of 0.5 from an initial inoculum of 0.01. Cultures were treated or not with 0.1 mg/ml mupirocin for 30 min (A 600 = ;1 for both samples). ELISAs were performed as described above.
Purification of S. aureus FapR. TheS. aureus fapR gene was amplified by PCR with FapRORFfp and FapRORFrp primers (Table S6) and cloned into pET-21b to produce a recombinant FapR carrying an Nterminal His tag and tobacco etch virus (TEV) site expressed in E. coli BL21/pDIA17 cells (13,49). Bacterial cultures were grown at 37°C in LB containing ampicillin (100 mg/ml) and chloramphenicol (10 mg/ml) until A 600 = 0.6; expression was then induced following addition of IPTG (isopropyl-b-D-thiogalactopyranoside; 0.5 mM) at 20°C for 17 h. Bacteria were harvested by centrifugation (5 g wet weight), washed twice in PBS, and resuspended in 30 ml of buffer A (50 mM Tris-HCl [pH 7.5], 300 mM NaCl, 1 mM dithiothreitol [DTT]), benzonase nuclease (Sigma-Aldrich, St. Louis, MO), and a protease inhibitor cocktail (Roche, Basel, Switzerland). Bacteria were lysed by passage through a CF cell-disrupter (Constant Systems Ltd., Cambridge, United Kingdom) at 4°C. The lysed culture was centrifuged at 46,000 Â g for 1 h and the supernatant was loaded onto a 1-ml Protino Ni-NTA column (Macherey-Nagel, Diiren, Germany). The protein was eluted with buffer A 1 300 mM imidazole and protein-containing fractions were pooled and dialyzed overnight in buffer A with TEV protease (1/10 wt/wt ratio) at 4°C (produced by the Pasteur Institute Production and Purification of Recombinant Proteins Technological Platform). The His-tag-free protein was loaded onto a 1-ml Ni-NTA column and collected. FapR was further purified using a HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare, Madison, WI) equilibrated with 20 mM Tris (pH 7.5), 50 mM NaCl. The purified protein was concentrated and stored at 280°C.
Determination of S. aureus fatty acid profiles. Fatty acid profiles were performed as described previously (4). Newman strain precultures prepared from two independent colonies were diluted to A 600 = 0.1 in SerFA and grown 3 h with and without mupirocin (0.05 mg/ml). A 600 values of SerFA samples were ;2.5 and treated samples were ;1.0. Percentages of eFA are shown (mean 6 standard deviation).
GTP determinations. All extraction steps were performed on ice. Cellular pellets were deproteinized with an equal volume of 6% perchloric acid (PCA), vortex mixed for 20 s, ice bathed for 10 min, and vortex mixed again for 20 s. Acid cell extracts were centrifuged at 13,000 rpm for 10 min at 4°C. The resulting supernatants were supplemented with an equal volume of bi-distilled water, vortex mixed for 60 s, and neutralized by addition of 2 M Na 2 CO 3 . Extracts were injected onto a C 18 Supelco 5 mm (250 Â 4.6 mm) column (Sigma-Aldrich, St. Louis, MO) at 45°C. The mobile phase was delivered at a flow rate of 1 ml/min using the following stepwise gradient elution program: A to B (60:40) at 0 min!(40:60) at 30 min!(40:60) at 60 min. Buffer A contained 10 mM tetrabutylammonium hydroxide, 10 mM KH 2 PO 4 , and 0.25% MeOH, and was adjusted to pH 6.9 with 1 M HCl. Buffer B consisted of 5.6 mM tetrabutylammonium hydroxide, 50 mM KH 2 PO 4 , and 30% MeOH, and was neutralized to pH 7.0 with 1 M NaOH. Detection was done with a diode array detector (PDA). The LC solution workstation chromatography manager was used to pilot the HPLC instrument and to process the data. Products were monitored spectrophotometrically at 254 nm and quantified by integration of the peak absorbance area, employing a calibration curve established with various known nucleosides. Finally, a correction coefficient was applied to correct raw data for minor differences in the total number of cells determined in each culture condition (by A 600 measurements).
Statistical analyses. Graphs and statistical analyses were prepared using GraphPad Prism software. Means and standard deviations are presented for sensor fusions, ELISA readouts, fatty acid profile comparisons, and GTP measurements. Statistical significance was determined by unpaired, nonparametric Mann-Whitney tests, as recommended for small sample sizes (here biological triplicates) and by a nonparametric, unpaired Kruskal-Wallis test for three-way comparisons.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.