Lipidomics of homeoviscous adaptation to low temperatures in Staphylococcus aureus utilizing exogenous straight-chain unsaturated fatty acids.

It is well established that Staphylococcus aureus can incorporate exogenous straight-chain unsaturated fatty acids (SCUFAs) into membrane phospho- and glyco-lipids from various sources in supplemented culture media and when growing in vivo during infection. Given the enhancement of membrane fluidity when oleic acid (C18:1Δ9) is incorporated into lipids, we were prompted to examine the effect of medium supplementation with C18:1Δ9 on growth at low temperatures. C18:1Δ9 supported the growth of a cold-sensitive, branched-chain fatty acid (BCFA)-deficient mutant at 12°C. Interestingly, we found similar results in the BCFA-sufficient parental strain, supported by the fact that the incorporation of C18:1Δ9 into the membrane increased membrane fluidity in both strains. We show that the incorporation of C18:1Δ9 and its elongation product C20:1Δ11 into membrane lipids was required for growth stimulation and relied on a functional FakAB incorporation system. Lipidomics analysis of the phosphatidylglycerol and diglycosyldiacylglycerol lipid classes revealed major impacts of C18:1Δ9 and temperature on lipid species. Growth at 12°C in the presence of C18:1Δ9 also led to increased production of the carotenoid pigment staphyloxanthin. The enhancement of growth by C18:1Δ9 is an example of homeoviscous adaptation to low temperatures utilizing an exogenous fatty acid. This may be significant in the growth of S. aureus at low temperatures in foods that commonly contain C18:1Δ9 and other SCUFAs in various forms. IMPORTANCE: We show that Staphylococcus aureus can use its known ability to incorporate exogenous fatty acids to enhance its growth at low temperatures. Individual species of phosphatidylglycerols and diglycosyldiacylglycerols bearing one or two degrees of unsaturation derived from the incorporation of C18:1Δ9 at 12°C are described for the first time. In addition, enhanced production of the carotenoid staphyloxanthin occurs at low temperatures. The studies describe a biochemical reality underlying membrane biophysics. This is an example of homeoviscous adaptation to low temperatures utilizing exogenous fatty acids over the regulation of the biosynthesis of endogenous fatty acids. The studies have likely relevance to food safety in that unsaturated fatty acids may enhance the growth of S. aureus in the food environment.

Defective pgsA contributes to increased membrane fluidity and cell wall thickening in Staphylococcus aureus with high-level daptomycin resistance.

Daptomycin is a membrane-targeting last-resort antimicrobial therapeutic for the treatment of infections caused by methicillin- and/or vancomycin-resistant Staphylococcus aureus. In the rare event of failed daptomycin therapy, the source of resistance is often attributable to mutations directly within the membrane phospholipid biosynthetic pathway of S. aureus or in the regulatory systems that control cell envelope response and membrane homeostasis. Here we describe the structural changes to the cell envelope in a daptomycin-resistant isolate of S. aureus strain N315 that has acquired mutations in the genes most commonly reported associated with daptomycin resistance: mprF, yycG, and pgsA. In addition to the decreased phosphatidylglycerol (PG) levels that are the hallmark of daptomycin resistance, the mutant with high-level daptomycin resistance had increased branched-chain fatty acids (BCFAs) in its membrane lipids, increased membrane fluidity, and increased cell wall thickness. However, the successful utilization of isotope-labeled straight-chain fatty acids (SCFAs) in lipid synthesis suggested that the aberrant BCFA:SCFA ratio arose from upstream alteration in fatty acid synthesis rather than a structural preference in PgsA. Transcriptomics studies revealed that expression of pyruvate dehydrogenase (pdhB) was suppressed in the daptomycin-resistant isolate, which is known to increase BCFA levels. While complementation with an additional copy of pdhB had no effect, complementation of the pgsA mutation resulted in increased PG formation, reduction in cell wall thickness, restoration of normal BCFA levels, and increased daptomycin susceptibility. Collectively, these results demonstrate that pgsA contributes to daptomycin resistance through its influence on membrane fluidity and cell wall thickness, in addition to phosphatidylglycerol levels. IMPORTANCE: The cationic lipopeptide antimicrobial daptomycin has become an essential tool for combating infections with Staphylococcus aureus that display reduced susceptibility to ß-lactams or vancomycin. Since daptomycin's activity is based on interaction with the negatively charged membrane of S. aureus, routes to daptomycin-resistance occur through mutations in the lipid biosynthetic pathway surrounding phosphatidylglycerols and the regulatory systems that control cell envelope homeostasis. Therefore, there are many avenues to achieve daptomycin resistance and several different, and sometimes contradictory, phenotypes of daptomycin-resistant S. aureus, including both increased and decreased cell wall thickness and membrane fluidity. This study is significant because it demonstrates the unexpected influence of a lipid biosynthesis gene, pgsA, on membrane fluidity and cell wall thickness in S. aureus with high-level daptomycin resistance.

Growth of Staphylococcus aureus in the presence of oleic acid shifts the glycolipid fatty acid profile and increases resistance to antimicrobial peptides.

Staphylococcus aureus readily adapts to various environments and quickly develops antibiotic resistance, which has led to an increase in multidrug-resistant infections. Hence, S. aureus presents a significant global health issue and its adaptations to the host environment are crucial for understanding pathogenesis and antibiotic susceptibility. When S. aureus is grown conventionally, its membrane lipids contain a mix of branched-chain and straight-chain saturated fatty acids. However, when unsaturated fatty acids are present in the growth medium, they become a major part of the total fatty acid composition. This study explores the biophysical effects of incorporating straight-chain unsaturated fatty acids into S. aureus membrane lipids. Membrane preparations from cultures supplemented with oleic acid showed more complex differential scanning calorimetry scans than those grown in tryptic soy broth alone. When grown in the presence of oleic acid, the cultures exhibited a transition significantly above the growth temperature, attributed to the presence of glycolipids with long-chain fatty acids causing acyl chain packing frustration within the bilayer. Functional aspects of the membrane were assessed by studying the kinetics of dye release from unilamellar vesicles induced by the antimicrobial peptide mastoparan X. Dye release was slower from liposomes prepared from cells grown in oleic acid-supplemented cultures, suggesting that changes in membrane lipid composition and biophysics protect the cell membrane against peptide-induced lysis. These findings underscore the intricate relationship between the growth environment, membrane lipid composition, and the physical properties of the bacterial membrane, which should be considered when developing new strategies against S. aureus infections.

Lipidomics of homeoviscous adaptation to low temperatures in Staphylococcus aureus utilizing exogenous straight-chain unsaturated fatty acids over biosynthesized endogenous branched-chain fatty acids.

It is well established that Staphylococcus aureus can incorporate exogenous straight-chain unsaturated fatty acids (SCUFAs) into membrane phospho- and glyco-lipids from various sources in supplemented culture media, and when growing in vivo in an infection. Given the enhancement of membrane fluidity when oleic acid (C18:1Δ9) is incorporated into lipids, we were prompted to examine the effect of medium supplementation with C18:1Δ9 on growth at low temperatures. C18:1Δ9 supported the growth of a cold-sensitive, branched-chain fatty acid (BCFA)-deficient mutant at 12°C. Interestingly, we found similar results in the BCFA-sufficient parental strain. We show that incorporation of C18:1Δ9 and its elongation product C20:1Δ9 into membrane lipids was required for growth stimulation and relied on a functional FakAB incorporation system. Lipidomics analysis of the phosphatidylglycerol (PG) and diglycosyldiacylglycerol (DGDG) lipid classes revealed major impacts of C18:1Δ9 and temperature on lipid species. Growth at 12°C in the presence of C18:1Δ9 also led to increased production of the carotenoid pigment staphyloxanthin; however, this was not an obligatory requirement for cold adaptation. Enhancement of growth by C18:1Δ9 is an example of homeoviscous adaptation to low temperatures utilizing an exogenous fatty acid. This may be significant in the growth of S. aureus at low temperatures in foods that commonly contain C18:1Δ9 and other SCUFAs in various forms.

Defective pgsA contributes to increased membrane fluidity and cell wall thickening in S. aureus with high-level daptomycin resistance.

Daptomycin is a membrane-targeting last-resort antimicrobial therapeutic for the treatment of infections caused by methicillin- and/or vancomycin-resistant Staphylococcus aureus. In the rare event of failed daptomycin therapy, the source of resistance is often attributable to mutations directly within the membrane phospholipid biosynthetic pathway of S. aureus or in the regulatory systems that control cell envelope response and membrane homeostasis. Here we describe the structural changes to the cell envelope in a daptomycin-resistant isolate of S. aureus strain N315 that has acquired mutations in the genes most commonly reported associated with daptomycin-resistance: mprF, yycG, and pgsA. In addition to the decreased phosphatidylglycerol (PG) levels that are the hallmark of daptomycin-resistance, the mutant with high-level daptomycin resistance had increased branched-chain fatty acids (BCFAs) in its membrane lipids, increased membrane fluidity, and increased cell wall thickness. However, the successful utilization of isotope-labeled straight-chain fatty acids (SCFAs) in lipid synthesis suggested that the aberrant BCFA:SCFA ratio arose from upstream alteration in fatty acid synthesis rather than a structural preference in PgsA. RT-qPCR studies revealed that expression of pyruvate dehydrogenase (pdhB) was suppressed in the daptomycin-resistant isolate, which is known to increase BCFA levels. While complementation with an additional copy of pdhB had no effect, complementation of the pgsA mutation resulted in increased PG formation, reduction in cell wall thickness, restoration of normal BCFA levels, and increased daptomycin susceptibility. Collectively, these results demonstrate that pgsA contributes to daptomycin resistance through its influence on membrane fluidity and cell wall thickness, in addition to phosphatidylglycerol levels.

Transcriptomic and Metabolomic Analysis of a Fusidic Acid-Selected fusA Mutant of Staphylococcus aureus.

Physiological experimentation, transcriptomics, and metabolomics were engaged to compare a fusidic acid-resistant Staphylococcus aureus mutant SH10001st-2 to its parent strain SH1000. SH10001st-2 harbored a mutation (H457Y) in the gene fusA which encodes the fusidic acid target, elongation factor G, as well as mutations in a putative phage gene of unknown function. SH10001st-2 grew slower than SH1000 at three temperatures and had reduced coagulase activity, two indicators of the fitness penalty reported for fusA-mediated fusidic acid- resistance in the absence of compensatory mutations. Despite the difference in growth rates, the levels of O2 consumption and CO2 production were comparable. Transcriptomic profiling revealed 326 genes were upregulated and 287 were downregulated in SH10001st-2 compared to SH1000. Cell envelope and transport and binding protein genes were the predominant functional categories of both upregulated and downregulated genes in SH10001st-2. Genes of virulence regulators, notably the agr and kdp systems, were highly upregulated as were genes encoding capsule production. Contrary to what is expected of mid-exponential phase cells, genes encoding secreted virulence factors were generally upregulated while those for adhesion-associated virulence factors were downregulated in SH10001st-2. Metabolomic analysis showed an overall increase in metabolite pools in SH10001st-2 compared to SH1000, mostly for amino acids and sugars. Slowed growth and metabolite accumulation may be byproducts of fusA mutation-mediated protein synthesis impairment, but the overall results indicate that SH10001st-2 is compensating for the H457Y fitness penalty by repurposing its virulence machinery, in conjunction with increasing metabolite uptake capacity, in order to increase nutrient acquisition.

Fluorescence Polarization (FP) Assay for Measuring Staphylococcus aureus Membrane Fluidity.

Fluorescence polarization is a method to determine membrane fluidity using a hydrophobic fluorescent dye that intercalates into the fatty acid bilayer. A spectrofluorometer is used to polarize UV light as a vertical excitation beam which passes through the dye-labeled membrane where the dye fluoresces. The beams perpendicular and horizontal to the excitation light are then collected and analyzed. Membrane structural properties are largely due to the packing of the fatty acids in the lipid bilayer that determines the membrane biophysical parameters. Staphylococcus aureus contains straight-chain (SCFAs) and branched-chain (BCFAs) fatty acids in the membrane and alters the proportion of membrane fluidizing BCFAs and stabilizing SCFAs as a response to a variety of stresses. Herein, we describe a method for determination of membrane fluidity in S. aureus using diphenylhexatriene, one of the most used fluorescent dyes for this purpose.

Apt (Adenine Phosphoribosyltransferase) Mutation in Laboratory-Selected Vancomycin-Intermediate Staphylococcus aureus.

Comparative genomic sequencing of laboratory-derived vancomycin-intermediate Staphylococcusaureus (VISA) (MM66-3 and MM66-4) revealed unique mutations in both MM66-3 (in apt and ssaA6), and MM66-4 (in apt and walK), compared to hetero-VISA parent strain MM66. Transcriptional profiling revealed that both MM66 VISA shared 79 upregulated genes and eight downregulated genes. Of these, 30.4% of the upregulated genes were associated with the cell envelope, whereas 75% of the downregulated genes were associated with virulence. In concordance with mutations and transcriptome alterations, both VISA strains demonstrated reduced autolysis, reduced growth in the presence of salt and reduced virulence factor activity. In addition to mutations in genes linked to cell wall metabolism (ssaA6 and walK), the same mutation in apt which encodes adenine phosphoribosyltransferase, was confirmed in both MM66 VISA. Apt plays a role in both adenine metabolism and accumulation and both MM66 VISA grew better than MM66 in the presence of adenine or 2-fluoroadenine indicating a reduction in the accumulation of these growth inhibiting compounds in the VISA strains. MM66 apt mutants isolated via 2-fluoroadenine selection also demonstrated reduced susceptibility to the cell wall lytic dye Congo red and vancomycin. Finding that apt mutations contribute to reduced vancomycin susceptibility once again suggests a role for altered purine metabolism in a VISA mechanism.

Revealing Fatty Acid Heterogeneity in Staphylococcal Lipids with Isotope Labeling and RPLC-IM-MS.

Up to 80% of the fatty acids in Staphylococcus aureus membrane lipids are branched, rather than straight-chain, fatty acids. The branched fatty acids (BCFAs) may have either an even or odd number of carbons, and the branch position may be at the penultimate carbon (iso) or the antepenultimate (anteiso) carbon of the tail. This results in two sets of isomeric fatty acid species with the same number of carbons that cannot be resolved by mass spectrometry. The isomer/isobar challenge is further complicated when the mixture of BCFAs and straight-chain fatty acids (SCFAs) are esterified into diacylated lipids such as the phosphatidylglycerol (PG) species of the S. aureus membrane. No conventional chromatographic method has been able to resolve diacylated lipids containing mixtures of SCFAs, anteiso-odd, iso-odd, and iso-even BCFAs. A major hurdle to method development in this area is the lack of relevant analytical standards for lipids containing BCFA isomers. The diversity of the S. aureus lipidome and its naturally high levels of BCFAs present an opportunity to explore the potential of resolving diacylated lipids containing BCFAs and SFCAs. Using our knowledge of lipid and fatty acid biosynthesis in S. aureus, we have used a stable-isotope-labeling strategy to develop and validate a 30 min C18 reversed-phase liquid chromatography method combined with traveling-wave ion mobility-mass spectrometry to provide resolution of diacylated lipids based on the number of BCFAs that they contain.

Lipidomic and Ultrastructural Characterization of the Cell Envelope of Staphylococcus aureus Grown in the Presence of Human Serum.

Staphylococcus aureus can incorporate exogenous straight-chain unsaturated and saturated fatty acids (SCUFAs and SCFAs, respectively) to replace some of the normally biosynthesized branched-chain fatty acids and SCFAs. In this study, the impact of human serum on the S. aureus lipidome and cell envelope structure was comprehensively characterized. When S. aureus was grown in the presence of 20% human serum, typical human serum lipids, such as cholesterol, sphingomyelin, phosphatidylethanolamines, and phosphatidylcholines, were present in the total lipid extracts. Mass spectrometry showed that SCUFAs were incorporated into all major S. aureus lipid classes, i.e., phosphatidylglycerols, lysyl-phosphatidylglycerols, cardiolipins, and diglucosyldiacylglycerols. Heat-killed S. aureus retained fewer serum lipids and failed to incorporate SCUFAs, suggesting that association and incorporation of serum lipids with S. aureus require a living or nondenatured cell. Cytoplasmic membranes isolated from lysostaphin-produced protoplasts of serum-grown cells retained serum lipids, but washing cells with Triton X-100 removed most of them. Furthermore, electron microscopy studies showed that serum-grown cells had thicker cell envelopes and associated material on the surface, which was partially removed by Triton X-100 washing. To investigate which serum lipids were preferentially hydrolyzed by S. aureus lipases for incorporation, we incubated individual serum lipid classes with S. aureus and found that cholesteryl esters (CEs) and triglycerides (TGs) are the major donors of the incorporated fatty acids. Further experiments using purified Geh lipase confirmed that CEs and TGs were the substrates of this enzyme. Thus, growth in the presence of serum altered the nature of the cell surface with implications for interactions with the host.IMPORTANCE Comprehensive lipidomics of S. aureus grown in the presence of human serum suggests that human serum lipids can associate with the cell envelope without being truly integrated into the lipid membrane. However, fatty acids derived from human serum lipids, including unsaturated fatty acids, can be incorporated into lipid classes that can be biosynthesized by S. aureus itself. Cholesteryl esters and triglycerides are found to be the major source of incorporated fatty acids upon hydrolysis by lipases. These findings have significant implications for the nature of the S. aureus cell surface when grown in vivo Changes in phospholipid and glycolipid abundances and fatty acid composition could affect membrane biophysics and function and the activity of membrane-targeting antimicrobials. Finally, the association of serum lipids with the cell envelope has implications for the physicochemical nature of the cell surface and its interaction with host defense systems.

Plasticity of Coagulase-Negative Staphylococcal Membrane Fatty Acid Composition and Implications for Responses to Antimicrobial Agents.

Staphylococcus aureus demonstrates considerable membrane lipid plasticity in response to different growth environments, which is of potential relevance to response and resistance to various antimicrobial agents. This information is not available for various species of coagulase-negative staphylococci, which are common skin inhabitants, can be significant human pathogens, and are resistant to multiple antibiotics. We determined the total fatty acid compositions of Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, and Staphylococcus aureus for comparison purposes. Different proportions of branched-chain and straight-chain fatty acids were observed amongst the different species. However, growth in cation-supplemented Mueller-Hinton broth significantly increased the proportion of branched-chain fatty acids, and membrane fluidities as measured by fluorescence anisotropy. Cation-supplemented Mueller-Hinton broth is used for routine determination of antimicrobial susceptibilities. Growth in serum led to significant increases in straight-chain unsaturated fatty acids in the total fatty acid profiles, and decreases in branched-chain fatty acids. This indicates preformed fatty acids can replace biosynthesized fatty acids in the glycerolipids of coagulase-negative staphylococci, and indicates that bacterial fatty acid biosynthesis system II may not be a good target for antimicrobial agents in these organisms. Even though the different species are expected to be exposed to skin antimicrobial fatty acids, they were susceptible to the major skin antimicrobial fatty acid sapienic acid (C16:1Δ6). Certain species were not susceptible to linoleic acid (C18:2Δ9,12), but no obvious relationship to fatty acid composition could be discerned.

Transcriptional profiling and metabolomic analysis of Staphylococcus aureus grown on autoclaved chicken breast.

Although Staphylococcus aureus is a major cause of food poisoning, little is known about its response to growth on food. Utilizing a transcriptional profiling and metabolomics approach, we compared S. aureus grown on autoclaved chicken breast (ACB) to Luria broth agar. ACB cultures demonstrated increased expression of genes associated with protein synthesis, cofactors, secondary metabolites, nitrogen and nucleotide metabolism, amino acid transport, and reduced expression of general stress, lipid metabolism, and virulence genes. The ACB culture also displayed characteristics of catabolite de-repression and anaerobic growth, and increased expression of arginine biosynthesis genes (argFGH) and an arginine/ornithine antiporter gene (arcD). S. aureus synthesizes arginine from proline and the ACB culture exhibited increased expression of proline transport genes (opuBA, opuBB and putP) and increased proline accumulation. Amino acid and sugar content in the ACB grown culture increased, and this was attributed to the consumption of ACB, transport of amino acids, and gluconeogenesis. Genes involved with biotin biosynthesis and uptake were upregulated and biotin is required for amino acid catabolism. Genes encoding urease and urease activity were upregulated in ACB cultures, while urea levels were reduced. This research provides fundamental information on the response of S. aureus growing on chicken meat that could find application in future attempts to reduce the growth of S. aureus in food.

Interrelationships between Fatty Acid Composition, Staphyloxanthin Content, Fluidity, and Carbon Flow in the Staphylococcus aureus Membrane.

Fatty acids play a major role in determining membrane biophysical properties. Staphylococcus aureus produces branched-chain fatty acids (BCFAs) and straight-chain saturated fatty acids (SCSFAs), and can directly incorporate exogenous SCSFAs and straight-chain unsaturated fatty acids (SCUFAs). Many S. aureus strains produce the triterpenoid pigment staphyloxanthin, and the balance of BCFAs, SCSFAs and staphyloxanthin determines membrane fluidity. Here, we investigated the relationship of fatty acid and carotenoid production in S. aureus using a pigmented strain (Pig1), its carotenoid-deficient mutant (Pig1ΔcrtM) and the naturally non-pigmented Staphylococcus argenteus that lacks carotenoid biosynthesis genes and is closely related to S. aureus. Fatty acid compositions in all strains were similar under a given culture condition indicating that staphyloxanthin does not influence fatty acid composition. Strain Pig1 had decreased membrane fluidity as measured by fluorescence anisotropy compared to the other strains under all conditions indicating that staphyloxanthin helps maintain membrane rigidity. We could find no evidence for correlation of expression of crtM and fatty acid biosynthesis genes. Supplementation of medium with glucose increased SCSFA production and decreased BCFA and staphyloxanthin production, whereas acetate-supplementation also decreased BCFAs but increased staphyloxanthin production. We believe that staphyloxanthin levels are influenced more through metabolic regulation than responding to fatty acids incorporated into the membrane.

Exposure of Staphylococcus aureus to Targocil Blocks Translocation of the Major Autolysin Atl across the Membrane, Resulting in a Significant Decrease in Autolysis.

Peptidoglycan (PG) and wall teichoic acid (WTA) are the major staphylococcal cell wall components, and WTA biosynthesis has recently been explored for drug development. Targocil is a novel agent that targets the TarG subunit of the WTA translocase (TarGH) that transports WTA across the membrane to the wall. Previously we showed that targocil treatment of a methicillin-susceptible Staphylococcus aureus strain led to a rapid shut down of cellular autolysis. Targocil II, which targets the TarH subunit of TarGH, also resulted in a drastic decrease in autolysis. Here, we address the mechanism of targocil-mediated decreased autolysis. The mechanism is WTA dependent since targocil treatment decreased autolysis in methicillin-resistant strains but not in a WTA-deficient mutant. Similar to cellular autolysis, autolysin-retaining crude cell walls isolated from targocil-treated cells had vastly decreased autolytic activity compared to those from untreated cells. Purified cell walls from control and targocil-treated cells, which lack autolytic activity, were similarly susceptible to lysozyme and lysostaphin and had similar O-acetyl contents, indicating that targocil treatment did not grossly alter PG structure and chemistry. Purified cell walls from targocil-treated cells were highly susceptible to autolysin extracts, supporting the notion that targocil treatment led to decreased autolysin in the crude cell walls. Quantitative real-time PCR analysis revealed that the decrease in autolysis in the targocil-exposed cells was not due to transcriptional repression of the autolysin genes atl, lytM, lytN, and sle1 Zymographic analysis of peptidoglycan hydrolase profiles showed a deficiency of cell surface autolysins in targocil-treated cells but higher activity in cell membrane fractions. Here, we propose that the untranslocated WTA molecules in the targocil-exposed cells sequester Atl at the membrane, resulting in significantly decreased autolysis.

Roles of pyruvate dehydrogenase and branched-chain α-keto acid dehydrogenase in branched-chain membrane fatty acid levels and associated functions in Staphylococcus aureus.

PURPOSE: Membrane fluidity to a large extent is governed by the presence of branched-chain fatty acids (BCFAs). Branched-chain α-keto acid dehydrogenase (BKD) is the key enzyme in BCFA synthesis. A Staphylococcus aureus BKD-deficient strain still produced substantial levels of BCFAs. Pyruvate dehydrogenase (PDH) with structural similarity to BKD has been speculated to contribute to BCFAs in S. aureus. METHODOLOGY: This study was carried out using BKD-, PDH- and BKD : PDH-deficient derivatives of methicillin-resistant S. aureus strain JE2. Differences in growth kinetics were evaluated spectrophotometrically, membrane BCFAs using gas chromatography and membrane fluidity by fluorescence polarization. Carotenoid levels were estimated by measuring A465 of methanol extracts from 48 h cultures. MIC values were determined by broth microdilution.Results/Key findings. BCFAs made up 50 % of membrane fatty acids in wild-type but only 31 % in the BKD-deficient mutant. BCFA level was ~80 % in the PDH-deficient strain and 38 % in the BKD : PDH-deficient strain. BKD-deficient mutant showed decreased membrane fluidity, the PDH-deficient mutant showed increased membrane fluidity. The BKD- and PDH-deficient strains grew slower and the BKD : PDH-deficient strain grew slowest at 37 °C. However at 20 °C, the BKD- and BKD : PDH-deficient strains grew only a little followed by autolysis of these cells. The BKD-deficient strain produced higher levels of staphyloxanthin. The PDH-deficient and BKD : PDH-deficient strains produced very little staphyloxanthin. The BKD-deficient strain showed increased susceptibility to daptomycin. CONCLUSION: The BCFA composition of the cell membrane in S. aureus seems to significantly impact cell growth, membrane fluidity and resistance to daptomycin.

Ethanol-induced stress response of Staphylococcus aureus.

Transcriptional profiles of 2 unrelated clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates were analyzed following 10% (v/v) ethanol challenge (15 min), which arrested growth but did not reduce viability. Ethanol-induced stress (EIS) resulted in differential gene expression of 1091 genes, 600 common to both strains, of which 291 were upregulated. With the exception of the downregulation of genes involved with osmotic stress functions, EIS resulted in the upregulation of genes that contribute to stress response networks, notably those altered by oxidative stress, protein quality control in general, and heat shock in particular. In addition, genes involved with transcription, translation, and nucleotide biosynthesis were downregulated. relP, which encodes a small alarmone synthetase (RelP), was highly upregulated in both MRSA strains following ethanol challenge, and relP inactivation experiments indicated that this gene contributed to EIS growth arrest. A number of persistence-associated genes were also upregulated during EIS, including those that encode toxin-antitoxin systems. Overall, transcriptional profiling indicated that the MRSA investigated responded to EIS by entering a state of dormancy and by altering the expression of elements from cross protective stress response systems in an effort to protect preexisting proteins.

Utilization of multiple substrates by butyrate kinase from Listeria monocytogenes.

Listeria monocytogenes, the causative agent of listeriosis, can build up to dangerous levels in refrigerated foods potentially leading to expensive product recalls. An important aspect of the bacterium's growth at low temperatures is its ability to increase the branched-chain fatty acid anteiso C15:0 content of its membrane at lower growth temperatures, which imparts greater membrane fluidity. Mutants in the branched-chain α-keto dehydrogenase (bkd) complex are deficient in branched-chain fatty acids (BCFAs,) but these can be restored by feeding C4 and C5 branched-chain carboxylic acids (BCCAs). This suggests the presence of an alternate pathway for production of acyl CoA precursors for fatty acid biosynthesis. We hypothesize that the alternate pathway is composed of butyrate kinase (buk) and phosphotransbutyrylase (ptb) encoded in the bkd complex which produce acyl CoA products by their sequential action through the metabolism of carboxylic acids. We determined the steady state kinetics of recombinant His-tagged Buk using 11 different straight-chain and BCCA substrates in the acyl phosphate forming direction. Buk demonstrated highest catalytic efficiency with pentanoate as the substrate. Low product formation observed with acetate (C2) and hexanoate (C6) as the substrates indicates that Buk is not involved in either acetate metabolism or long chain carboxylic acid activation. We were also able to show that Buk catalysis occurs through a ternary complex intermediate. Additionally, Buk demonstrates a strong preference for BCCAs at low temperatures. These results indicate that Buk may be involved in the activation and assimilation of exogenous carboxylic acids for membrane fatty acid biosynthesis.

Growth-Environment Dependent Modulation of Staphylococcus aureus Branched-Chain to Straight-Chain Fatty Acid Ratio and Incorporation of Unsaturated Fatty Acids.

The fatty acid composition of membrane glycerolipids is a major determinant of Staphylococcus aureus membrane biophysical properties that impacts key factors in cell physiology including susceptibility to membrane active antimicrobials, pathogenesis, and response to environmental stress. The fatty acids of S. aureus are considered to be a mixture of branched-chain fatty acids (BCFAs), which increase membrane fluidity, and straight-chain fatty acids (SCFAs) that decrease it. The balance of BCFAs and SCFAs in USA300 strain JE2 and strain SH1000 was affected considerably by differences in the conventional laboratory medium in which the strains were grown with media such as Mueller-Hinton broth and Luria broth resulting in high BCFAs and low SCFAs, whereas growth in Tryptic Soy Broth and Brain-Heart Infusion broth led to reduction in BCFAs and an increase in SCFAs. Straight-chain unsaturated fatty acids (SCUFAs) were not detected. However, when S. aureus was grown ex vivo in serum, the fatty acid composition was radically different with SCUFAs, which increase membrane fluidity, making up a substantial proportion of the total (<25%) with SCFAs (>37%) and BCFAs (>36%) making up the rest. Staphyloxanthin, an additional major membrane lipid component unique to S. aureus, tended to be greater in content in cells with high BCFAs or SCUFAs. Cells with high staphyloxanthin content had a lower membrane fluidity that was attributed to increased production of staphyloxanthin. S. aureus saves energy and carbon by utilizing host fatty acids for part of its total fatty acids when growing in serum, which may impact biophysical properties and pathogenesis given the role of SCUFAs in virulence. The nutritional environment in which S. aureus is grown in vitro or in vivo in an infection is likely to be a major determinant of membrane fatty acid composition.

Insights into the Mechanism of Homeoviscous Adaptation to Low Temperature in Branched-Chain Fatty Acid-Containing Bacteria through Modeling FabH Kinetics from the Foodborne Pathogen Listeria monocytogenes.

The psychrotolerant foodborne pathogen Listeria monocytogenes withstands the stress of low temperatures and can proliferate in refrigerated food. Bacteria adapt to growth at low temperatures by increasing the production of fatty acids that increase membrane fluidity. The mechanism of homeoviscous increases in unsaturated fatty acid amounts in bacteria that predominantly contain straight-chain fatty acids is relatively well understood. By contrast the analogous mechanism in branched-chain fatty acid-containing bacteria, such as L. monocytogenes, is poorly understood. L. monocytogenes grows at low temperatures by altering its membrane composition to increase membrane fluidity, primarily by decreasing the length of fatty acid chains and increasing the anteiso to iso fatty acid ratio. FabH, the initiator of fatty acid biosynthesis, has been identified as the primary determinant of membrane fatty acid composition, but the extent of this effect has not been quantified. In this study, previously determined FabH steady-state parameters and substrate concentrations were used to calculate expected fatty acid compositions at 30°C and 10°C. FabH substrates 2-methylbutyryl-CoA, isobutyryl-CoA, and isovaleryl-CoA produce the primary fatty acids in L. monocytogenes, i.e., anteiso-odd, iso-even, and iso-odd fatty acids, respectively. In vivo concentrations of CoA derivatives were measured, but not all were resolved completely. In this case, estimates were calculated from overall fatty acid composition and FabH steady-state parameters. These relative substrate concentrations were used to calculate the expected fatty acid compositions at 10°C. Our model predicted a higher level of anteiso lipids at 10°C than was observed, indicative of an additional step beyond FabH influencing fatty acid composition at low temperatures. The potential for control of low temperature growth by feeding compounds that result in the production of butyryl-CoA, the precursor of SCFAs that rigidify the membrane and are incompatible with growth at low temperatures, is recognized.

The role of two branched-chain amino acid transporters in Staphylococcus aureus growth, membrane fatty acid composition and virulence.

The branched-chain amino acids (BCAAs) are vital to both growth and virulence of the human pathogen Staphylococcus aureus. In addition to supporting protein synthesis, the BCAAs serve as precursors for branched-chain fatty acids (BCFAs), which are predominant membrane fatty acids, and, in association with the global regulatory protein CodY, the BCAAs are key co-regulators of virulence factors. Despite these critical functions, S. aureus represses Leu and Val synthesis, instead preferring to acquire them from the extracellular milieu. We previously identified BrnQ1 as a BCAA transporter, yet a brnQ1 mutant remained capable of BCAA acquisition. Here, we describe BcaP as an additional BCAA transporter, and determine that it plays a secondary role to BrnQ1 during S. aureus growth in a chemically defined medium. Furthermore, membrane fatty acid composition analysis revealed that BrnQ1, and not BcaP, is required for transporting Leu and Val to be used for iso-BCFA synthesis. Despite a predominant role for BrnQ1 in vitro, both BrnQ1 and BcaP are required for S. aureus fitness in vivo in a hematogenous spread infection model and a nasal colonisation model. These data demonstrate the importance of BrnQ1 and BcaP for growth, environmental adaptation and virulence of S. aureus.