Conceived and designed the experiments: JGK DW EJ AT MJH. Performed the experiments: JGK DW EJ IMJ MJH. Analyzed the data: JGK DW EJ IMJ JH MJH. Contributed reagents/materials/analysis tools: JL. Wrote the paper: JGK DW HR MJH.
The authors have declared that no competing interests exist.
FFAs of various chain lengths and with different levels of unsaturation are primarily effective against Gram-positive bacteria
A diversity of mechanisms have been proposed to account for resistance to LC-uFFAs in
Previous studies demonstrated that
While the biological effects of free fatty acids as antimicrobial compounds have been catalogued, there remains no unequivocal identification of the targets or mechanisms of action in relation to
The relative resistances of different strains of
A Graph showing percentage survival of wild-type strains of
To facilitate analysis of gene transcription and protein expression, a range of different concentrations of linoleic or oleic acid and the timing of their addition were examined during growth (data not shown). Upon inoculation 0.01 mM linoleic acid was determined to be the maximum concentration, which did not retard the aerobic growth of MRSA252 in BHI broth (
A pronounced differential transcriptional response was observed in MRSA252 cells treated with linoleic acid when it was added to a final concentration of 0.1 mM for 20 min during late-exponential growth (linoleic acid challenge) compared to unexposed control cells; 213 genes were up-regulated (
Group Functions | MRSA252 ORF | MRSA252 Gene | MRSA252 Gene Product | Fold Change Up Regulated | P-value |
Virulence Factors and Regulators | SAR0156 | capsular polysaccharide synthesis enzyme | 2.23 | 4.22E-02 | |
SAR0163 | capsular polysaccharide synthesis enzyme | 2.23 | 3.20E-02 | ||
SAR0164 | capsular polysaccharide synthesis enzyme | 2.83 | 1.79E-02 | ||
SAR0625 | staphylococcal accessory regulator A | 2.14 | 1.50E-02 | ||
SAR0842 | clumping factor | 4.12 | 6.58E-03 | ||
SAR2122 | delta-hemolysin precursor | 3.28 | 1.23E-02 | ||
SAR2295 | putative exported MAP/eap domain protein | 3.21 | 8.77E-04 | ||
SAR2443 | MarR family regulatory protein | 3.15 | 1.76E-03 | ||
RNAIII | RNAIII | RNAIII accessory gene regulator ( |
2.01 | 3.02E-02 | |
Stress Response | SAR0577 | putative proline/betaine transporter | 8.31 | 5.78E-04 | |
SAR0859 | putative organic hydroperoxide resistance protein | 3.82 | 1.23E-02 | ||
SAR0938 | putative ATPase subunit of an ATP-dependent protease | 2.49 | 8.15E-04 | ||
SAR1344 | catalase | 5.71 | 1.86E-03 | ||
SAR1656 | chaperone protein | 2.30 | 4.25E-02 | ||
SAR1657 | chaperone protein | 2.41 | 2.17E-03 | ||
SAR2273 | alkaline shock protein 23 | 2.06 | 3.86E-02 | ||
SAR2276 | glycine betaine transporter 2 | 4.42 | 6.16E-03 | ||
SAR2561 | alkylhydroperoxidase, AhpD family | 6.83 | 8.77E-04 | ||
SAR2628 | putative ATPase subunit of an ATP-dependent protease | 4.06 | 4.79E-03 | ||
Energy Metabolism | SAR0113 | L-lactate permease 1 | 2.15 | 1.07E-03 | |
SAR0188 | putative isochorismatase | 4.91 | 8.03E-04 | ||
SAR0141 | putative phosphopentomutase | 2.45 | 1.31E-02 | ||
SAR0574 | putative hexulose-6-phosphate synthase | 2.36 | 1.80E-03 | ||
SAR0575 | putative 6-phospho-3-hexuloisomerase | 2.16 | 5.11E-03 | ||
SAR0775 | Osmoprotectant ABC transporter | 2.13 | 4.80E-03 | ||
SAR0776 | Osmoprotectant ABC transporter, permease protein | 2.99 | 3.00E-04 | ||
SAR0824 | putative malolactic enzyme | 2.59 | 9.27E-03 | ||
SAR0830 | triosephosphate isomerase | 2.22 | 3.39E-02 | ||
SAR0831 | putative phosphoglycerate mutase | 2.64 | 1.39E-02 | ||
SAR1017 | putative menaquinone biosynthesis bifunctional protein | 2.24 | 1.65E-03 | ||
SAR1018 | putative hydrolase | 2.80 | 1.65E-03 | ||
SAR2386 | putative NAD-dependent dehydrogenase | 3.73 | 3.00E-04 | ||
SAR2506 | putative phosphoglycerate mutase | 2.06 | 7.33E-04 | ||
SAR2684 | fructose-bisphosphate aldolase class I | 2.02 | 5.85E-03 | ||
SAR2687 | putative AMP-binding enzyme | 2.01 | 9.65E-03 | ||
SAR2724 | isochorismatase family protein | 3.00 | 8.30E-04 | ||
DNA Repair and Replication | SAR0363 | putative single-strand DNA-binding protein | 2.26 | 3.29E-03 | |
SAR0744 | putative DNA photolyase | 3.46 | 6.97E-04 | ||
SAR0813 | excinuclease ABC subunit A | 2.45 | 2.06E-03 | ||
SAR0836 | putative ribonuclease R | 3.40 | 3.55E-03 | ||
SAR0837 | putative tmRNA-binding protein | 3.07 | 3.70E-04 | ||
Protein Synthesis | SAR0364 | 30S ribosomal protein S18 | 2.40 | 1.99E-02 | |
SAR0552 | translation elongation factor G | 2.10 | 3.39E-02 | ||
SAR1638 | RNA polymerase sigma factor | 2.86 | 3.70E-04 | ||
SAR2308 | 50S ribosomal protein L17 | 2.60 | 1.99E-02 | ||
SAR2309 | DNA-directed RNA polymerase alpha chain | 2.36 | 3.35E-02 | ||
SAR2310 | 30S ribosomal protein S11 | 2.46 | 3.77E-02 | ||
SAR2311 | 30S ribosomal protein S13 | 2.45 | 3.12E-02 | ||
SAR2313 | translation initiation factor IF-1 | 2.08 | 1.49E-02 | ||
SAR2728 | preprotein translocase SecA subunit-like protein | 3.85 | 6.58E-03 | ||
Peptidoglycan Synthesis | SAR0878 | putative selenocysteine lyase | 2.52 | 3.07E-02 | |
SAR1026 | bifunctional autolysin precursor | 2.65 | 6.16E-03 | ||
SAR1158 | phospho-N-acetylmuramoyl-pentapeptide-transferase | 2.13 | 9.56E-04 | ||
SAR1159 | UDP-N-acetylmuramoylalanine–D-glutamate ligase | 2.39 | 7.64E-03 | ||
SAR1160 | putative cell division protein | 2.10 | 1.31E-02 | ||
SAR1290 | putative exported CHAP domain protein | 3.17 | 6.97E-04 | ||
SAR1430 | putative N-acetylglucosamine transferase | 5.26 | 1.86E-03 | ||
SAR1761 | lysine-specific permease | 2.07 | 3.01E-02 | ||
SAR2109 | putative succinyl-diaminopimelate desuccinylase | 4.89 | 3.00E-03 | ||
SAR2188 | putative carboxyvinyltransferase | 2.94 | 6.54E-03 | ||
SAR2269 | putative alanine racemase | 2.64 | 1.78E-03 | ||
SAR2346 | putative pentaglycine interpeptide biosynthesis protein | 2.49 | 4.22E-03 | ||
SAR2394 | putative protein associated with cell-envelope regulation | 2.34 | 2.55E-03 | ||
SAR2420 | arginase family protein | 2.83 | 4.71E-03 | ||
SAR2521 | putative membrane GtrA-like protein | 3.11 | 5.78E-04 | ||
Fatty Acid Metabolism | SAR1438 | conserved hypothetical protein | 2.64 | 4.94E-03 | |
SAR2187 | putative hydroxymyristoyl-(acyl carrier protein) dehydratase | 2.41 | 4.22E-02 | ||
Carotenoid Biosynthesis | SAR0596 | mevalonate kinase | 2.32 | 3.00E-04 | |
SAR0597 | mevalonate diphosphate decarboxylase | 3.35 | 9.23E-04 | ||
SAR0598 | phosphomevalonate kinase | 3.18 | 5.09E-04 | ||
SAR2642 | squalene synthase | 4.95 | 8.03E-04 | ||
SAR2643 | squalene desaturase | 7.18 | 2.38E-02 | ||
SAR2645 | putative glycosyl transferase | 6.07 | 3.00E-03 | ||
SAR2646 | putative phytoene dehydrogenase related protein | 6.28 | 1.73E-03 | ||
SAR2647 | putative membrane protein | 4.47 | 1.73E-03 | ||
Antibiotic Resistance | SAR0139 | putative tetracycline resistance protein | 4.06 | 1.59E-03 | |
SAR1622 | metallo-beta-lactamase superfamily protein | 2.08 | 3.93E-03 | ||
SAR1785 | metallo-beta-lactamase superfamily protein | 3.05 | 1.08E-03 | ||
SAR1831 | beta-lactamase precursor | 2.02 | 2.72E-02 | ||
SAR2505 | putative transport system protein | 3.93 | 7.74E-03 | ||
SAR2558 | conserved hypothetical beta-lactamase-like protein | 8.72 | 3.70E-04 | ||
SAR2632 | Putative MMPL efflux pump | 2.03 | 4.58E-02 | ||
SAR2655 | putative glyoxalase | 5.15 | 1.11E-03 | ||
SAR2668 | hypothetical aminoglycoside phosphotransferase protein | 4.35 | 6.30E-03 | ||
Miscellaneous | SAR1738 | transposase B 2 | 2.14 | 1.25E-03 | |
SAR2725 | putative surface anchored protein | 16.80 | 4.68E-05 | ||
Metabolism | SAR0108 | putative peptidase | 2.98 | 5.22E-03 | |
SAR0109 | putative transporter protein | 2.37 | 1.52E-02 | ||
SAR0170 | putative cation efflux system protein | 2.50 | 1.77E-03 | ||
SAR0306 | ABC transporter ATP-binding protein | 6.10 | 1.68E-03 | ||
SAR0324 | putative lipoate-protein ligase A | 2.09 | 4.31E-03 | ||
SAR0325 | putative reductase | 4.80 | 8.17E-04 | ||
SAR0556 | ThiJ/PfpI family protein | 7.20 | 7.59E-04 | ||
SAR0589 | putative amino acid permease | 4.19 | 3.75E-03 | ||
SAR0600 | pyridine nucleotide-disulphide oxidoreductase protein | 2.26 | 2.77E-04 | ||
SAR0624 | putative esterase | 6.49 | 7.59E-04 | ||
SAR0729 | putative acetyltransferase | 2.92 | 3.23E-03 | ||
SAR0732 | putative acetyltransferase | 2.34 | 3.00E-04 | ||
SAR0756 | aldo/keto reductase family protein | 2.96 | 6.16E-04 | ||
SAR0757 | putative glucosyl transferase | 3.49 | 7.59E-04 | ||
SAR0764 | putative 6-pyruvoyl tetrahydropterin synthase | 3.70 | 8.03E-04 | ||
SAR0841 | putative acetyltransferase | 5.22 | 3.29E-03 | ||
SAR0883 | putative dioxygenase | 5.40 | 1.75E-03 | ||
SAR0903 | putative pyridine nucleotide-disulphide oxidoreductase | 2.82 | 3.00E-04 | ||
SAR0953 | transport system extracellular binding lipoprotein | 2.18 | 6.05E-03 | ||
SAR1076 | Spermidine/putrescine-binding protein homolog. | 4.46 | 8.77E-04 | ||
SAR1247 | putative tRNA pseudouridine synthase B | 2.31 | 2.10E-03 | ||
SAR1340 | homoserine kinase | 2.20 | 3.71E-02 | ||
SAR1431 | putative acetyltransferase | 4.58 | 1.80E-03 | ||
SAR1439 | dihydrofolate reductase type I | 2.13 | 6.34E-03 | ||
SAR1440 | thymidylate synthase | 5.32 | 1.58E-03 | ||
SAR1585 | maltose operon transcriptional repressor | 2.21 | 2.20E-02 | ||
SAR1655 | putative methyltransferase | 2.25 | 6.08E-03 | ||
SAR2210 | aldehyde dehydrogenase family protein | 5.48 | 1.51E-03 | ||
SAR2352 | putative molybdenum cofactor biosynthesis protein A | 2.07 | 1.86E-03 | ||
SAR2385 | putative Na+/H+ antiporter | 2.35 | 1.34E-02 | ||
SAR2395 | inositol monophosphatase family protein | 2.90 | 7.59E-04 | ||
SAR2413 | putative short chain dehydrogenase | 4.63 | 3.66E-03 | ||
SAR2460 | putative acetyltransferase (GNAT) family protein | 2.26 | 6.97E-04 | ||
SAR2485 | nitrate reductase beta chain | 2.16 | 1.02E-02 | ||
SAR2541 | putative carboxylesterase | 2.45 | 1.79E-02 | ||
SAR2544 | ABC transporter ATP-binding protein | 6.01 | 1.68E-03 | ||
SAR2559 | putative short chain dehydrogenase | 6.85 | 6.16E-04 | ||
SAR2659 | putative short chain dehydrogenase | 2.65 | 1.76E-03 | ||
SAR2661 | putative hydrolase | 8.11 | 8.27E-04 | ||
SAR2754 | putative histidine biosynthesis bifunctional protein | 2.09 | 2.19E-02 | ||
SAR2778 | putative nickel transport protein | 2.51 | 3.59E-03 | ||
Hypothetical Genes | SAR0111 | putative myosin-crossreactive antigen | 5.96 | 2.43E-03 | |
SAR0112 | putative membrane protein | 3.57 | 4.80E-04 | ||
SAR0171 | hypothetical protein | 2.64 | 3.01E-02 | ||
SAR0269 | LacI family regulatory protein | 2.57 | 3.59E-03 | ||
SAR0299 | hypothetical protein | 2.05 | 4.99E-02 | ||
SAR0305 | putative membrane protein | 3.89 | 6.02E-03 | ||
SAR0390 | putative lipoprotein | 3.97 | 1.68E-03 | ||
SAR0392 | putative membrane protein | 2.54 | 1.20E-02 | ||
SAR0405 | hypothetical protein | 2.76 | 1.07E-02 | ||
SAR0444 | putative lipoprotein | 2.31 | 2.16E-03 | ||
SAR0498 | putative regulatory protein | 3.65 | 1.07E-03 | ||
SAR0499 | stage V sporulation protein G | 2.83 | 1.11E-02 | ||
SAR0601 | putative DNA-binding protein | 2.15 | 4.48E-04 | ||
SAR0670 | putative sensor histidine kinase protein | 2.01 | 4.80E-04 | ||
SAR0721 | multicopper oxidase protein | 2.29 | 4.39E-03 | ||
SAR0733 | conserved hypothetical protein | 3.04 | 1.99E-03 | ||
SAR0734 | conserved hypothetical protein | 2.23 | 1.59E-03 | ||
SAR0821 | conserved hypothetical protein | 3.19 | 6.54E-03 | ||
SAR0825 | conserved hypothetical protein | 5.06 | 1.86E-03 | ||
SAR0840 | putative membrane protein | 5.25 | 2.63E-03 | ||
SAR0849 | hypothetical protein | 2.81 | 6.05E-03 | ||
SAR0850 | hypothetical protein | 2.94 | 6.81E-04 | ||
SAR0854 | hypothetical protein | 4.07 | 1.56E-02 | ||
SAR0855 | hypothetical protein | 2.53 | 1.94E-03 | ||
SAR0867 | hypothetical protein | 2.54 | 4.55E-03 | ||
SAR0877 | conserved hypothetical protein | 2.34 | 3.01E-02 | ||
SAR0879 | NifU-like protein | 2.06 | 3.39E-02 | ||
SAR0880 | conserved hypothetical protein | 2.14 | 3.47E-03 | ||
SAR0882 | putative membrane protein | 4.05 | 4.12E-03 | ||
SAR0931 | putative membrane protein | 7.87 | 4.28E-04 | ||
SAR1055 | hypothetical protein | 4.50 | 3.08E-03 | ||
SAR1077 | putative membrane protein | 2.54 | 5.69E-03 | ||
SAR1227 | conserved hypothetical protein | 2.11 | 1.71E-02 | ||
SAR1258 | putative DNA-binding protein | 2.12 | 3.07E-04 | ||
SAR1289 | putative exported protein | 3.49 | 1.65E-03 | ||
SAR1306 | hypothetical protein | 2.20 | 5.66E-03 | ||
SAR1429 | putative membrane protein | 5.74 | 1.84E-03 | ||
SAR1528 | hypothetical phage protein | 6.04 | 4.99E-02 | ||
SAR1623 | conserved hypothetical protein | 2.29 | 1.39E-02 | ||
SAR1669 | conserved hypothetical protein | 2.01 | 1.03E-02 | ||
SAR1670 | conserved hypothetical protein | 2.53 | 5.36E-03 | ||
SAR1671 | probable nicotinate-nucleotide adenylyltransferase | 2.03 | 8.07E-03 | ||
SAR1816 | putative membrane protein | 2.82 | 6.16E-04 | ||
SAR1854 | hypothetical protein | 4.98 | 1.27E-03 | ||
SAR1965 | ThiJ/PfpI family protein | 2.25 | 4.83E-02 | ||
SAR1970 | conserved hypothetical protein | 2.17 | 4.68E-02 | ||
SAR1972 | putative exported protein | 5.71 | 6.59E-03 | ||
SAR2010 | hypothetical protein | 3.49 | 6.58E-03 | ||
SAR2047 | hypothetical phage protein | 2.12 | 1.16E-02 | ||
SAR2085 | hypothetical phage RecT family protein | 2.18 | 9.84E-04 | ||
SAR2088 | hypothetical phage protein | 2.62 | 2.28E-02 | ||
SAR2094 | hypothetical phage protein | 2.69 | 3.06E-03 | ||
SAR2095 | hypothetical phage protein | 4.03 | 5.44E-03 | ||
SAR2098 | hypothetical phage protein | 2.02 | 2.87E-03 | ||
SAR2189 | putative membrane protein | 2.94 | 6.07E-03 | ||
SAR2232 | conserved hypothetical protein | 8.26 | 2.06E-03 | ||
SAR2245 | putative transcriptional antiterminator | 6.03 | 1.76E-03 | ||
SAR2270 | hypothetical IucA/IucC family protein | 3.36 | 3.80E-03 | ||
SAR2274 | putative membrane protein | 4.59 | 2.04E-03 | ||
SAR2275 | putative membrane protein | 3.98 | 6.16E-04 | ||
SAR2347 | putative membrane protein | 2.21 | 6.54E-03 | ||
SAR2392 | conserved hypothetical protein | 3.03 | 5.20E-03 | ||
SAR2393 | putative molydopterin dinucleotide binding domain protein | 3.26 | 2.50E-03 | ||
SAR2444 | putative membrane protein | 4.38 | 2.77E-04 | ||
SAR2469 | putative pyridoxamine 5′-phosphate oxidase | 4.72 | 1.11E-03 | ||
SAR2496 | putative solute binding lipoprotein | 2.60 | 3.66E-03 | ||
SAR2525 | hypothetical protein | 5.28 | 2.33E-05 | ||
SAR2532 | CapD domain protein | 2.48 | 4.16E-03 | ||
SAR2542 | putative transport protein | 2.01 | 5.64E-03 | ||
SAR2543 | putative membrane protein | 6.37 | 6.16E-04 | ||
SAR2568 | hypothetical protein | 4.66 | 1.65E-03 | ||
SAR2656 | conserved hypothetical protein | 3.35 | 6.16E-04 | ||
SAR2657 | hypothetical protein | 2.40 | 8.80E-04 | ||
SAR2658 | TetR family regulatory protein | 2.22 | 4.39E-04 | ||
SAR2660 | conserved hypothetical protein | 7.26 | 6.16E-04 | ||
SAR2665 | conserved hypothetical protein | 2.19 | 4.06E-03 | ||
SAR2666 | hypothetical protein | 2.75 | 1.87E-03 | ||
SAR2667 | hypothetical protein | 2.19 | 1.37E-02 | ||
SAR2688 | hypothetical protein | 7.55 | 3.70E-04 | ||
SAR2689 | hypothetical protein | 2.53 | 1.84E-02 | ||
SAR2726 | conserved hypothetical protein | 5.07 | 1.80E-03 | ||
SAR2727 | glycosyl transferase, group 1 family protein | 4.11 | 6.70E-03 | ||
SAR2739 | conserved hypothetical protein | 4.21 | 2.06E-03 | ||
SAR2740 | conserved hypothetical protein | 2.05 | 3.09E-02 | ||
SAR2777 | putative DNA-binding protein | 2.40 | 1.90E-03 | ||
SAR2780 | putative membrane protein | 7.38 | 4.80E-04 |
Group Functions | MRSA 252 ORF | MRSA252 Gene | MRSA252 Gene Product | Fold Change Down Regulated | P-value |
Virulence Factors and Regulators | SAR0105 | 1-phosphatidylinositol phosphodiesterase | 3.85 | 4.39E-04 | |
SAR1574 | iron uptake regulatory protein | 2.08 | 2.46E-02 | ||
SAR1984 | ferritin | 2.56 | 4.39E-03 | ||
SAR2001 | staphopain protease | 2.44 | 3.00E-04 | ||
SAR2474 | MarR family virulence regulator | 2.22 | 2.18E-02 | ||
SAR2155 | putative sigma factor sigB regulation protein | 2.56 | 1.58E-03 | ||
SAR2715 | arginine repressor family protein | 2.27 | 4.27E-03 | ||
SAR2716 | zinc metalloproteinase aureolysin precursor | 2.17 | 1.76E-03 | ||
Energy Metabolism | SAR0234 | L-lactate dehydrogenase 1 | 2.94 | 2.99E-05 | |
SAR0235 | putative PTS system, IIBC component | 2.22 | 1.30E-03 | ||
SAR0242 | putative galactitol PTS component | 2.13 | 1.76E-02 | ||
SAR0263 | putative PTS system protein | 2.50 | 3.44E-02 | ||
SAR0355 | Cys/Met metabolism PLP-dependent enzyme | 2.04 | 2.74E-02 | ||
SAR0522 | putative pyridoxal 5-phosphate biosynthesis protein | 3.03 | 3.00E-04 | ||
SAR0523 | SNO glutamine amidotransferase family protein | 2.70 | 3.70E-04 | ||
SAR0752 | putative phosphofructokinase | 2.38 | 3.27E-02 | ||
SAR0753 | fructose-specific PTS system component | 2.50 | 3.78E-02 | ||
SAR0766 | glutamine amidotransferase class-I protein | 2.04 | 6.16E-04 | ||
SAR1088 | putative pyruvate carboxylase | 2.5 | 8.78E-04 | ||
SAR1450 | putative threonine dehydratase | 2.22 | 5.47E-03 | ||
SAR1451 | alanine dehydrogenase 2 | 3.03 | 1.15E-03 | ||
SAR1777 | 6-phosphofructokinase | 2.86 | 2.87E-03 | ||
SAR1789 | acetate kinase | 2.33 | 9.23E-03 | ||
SAR2143 | ketol-acid reductoisomerase | 2.22 | 2.80E-02 | ||
SAR2213 | putative tagatose-bisphosphate aldolase | 3.13 | 6.59E-03 | ||
SAR2262 | putative uridylyltransferase | 2.27 | 8.35E-03 | ||
SAR2579 | putative uridylyltransferase | 2.63 | 6.84E-03 | ||
SAR2720 | putative PTS system component | 4.17 | 3.54E-03 | ||
SAR2721 | mannose-6-phosphate isomerase | 3.57 | 2.76E-03 | ||
Cell Wall Synthesis | SAR0228 | putative glutamine amidotransferase class-I | 2.13 | 2.08E-03 | |
SAR0257 | autolysin sensor kinase protein | 3.33 | 3.00E-03 | ||
SAR0258 | autolysin response regulator protein | 3.57 | 9.15E-03 | ||
SAR0259 | holin-like protein | 2.22 | 1.20E-03 | ||
SAR0646 | teichoic acid biosynthesis protein | 2.78 | 6.80E-03 | ||
SAR0648 | teichoic acid ABC transporter permease protein | 2.38 | 1.32E-02 | ||
SAR0649 | teichoic acid biosynthesis protein | 2.50 | 4.41E-04 | ||
SAR1143 | putative carbamate kinase | 2.27 | 6.79E-03 | ||
SAR1752 | glutamyl-tRNA reductase | 2.27 | 3.93E-02 | ||
SAR1807 | putative transglycosylase | 2.04 | 8.78E-04 | ||
SAR2472 | putative proton/sodium-glutamate symport protein | 2.04 | 2.97E-02 | ||
SAR2621 | holin-like protein | 2.27 | 1.18E-02 | ||
SAR2669 | putative dihydroorotate dehydrogenase | 2.86 | 3.75E-03 | ||
Fatty Acid Metabolism | SAR0225 | putative acyl-CoA dehydrogenase | 2.17 | 3.27E-02 | |
SAR0227 | putative acetyl-CoA transferase | 2.13 | 4.95E-02 | ||
SAR0803 | conserved hypothetical protein | 3.23 | 1.02E-02 | ||
Carotenoid Biosynthesis | SAR1278 | putative isopentenylpyrophosphate transferase | 2.00 | 9.23E-03 | |
SAR1479 | putative heptaprenyl diphosphate synthase | 2.78 | 1.62E-02 | ||
SAR1480 | heptaprenylnaphthoquinone methyltransferase | 2.27 | 3.54E-02 | ||
SAR1481 | putative hexaprenyl diphosphate synthase | 3.13 | 1.79E-02 | ||
DNA Repair and Replication | SAR0001 | chromosomal replication initiator protein DnaA | 2.04 | 2.17E-03 | |
SAR0004 | DNA replication and repair protein RecF | 2.08 | 8.51E-03 | ||
SAR0028 | replication protein (pseudogene) | 4.35 | 1.48E-02 | ||
SAR0485 | putative DNA polymerase III, delta' subunit | 3.03 | 1.26E-02 | ||
SAR0711 | putative replication initiation protein | 2.50 | 3.43E-02 | ||
SAR2429 | putative 3-methylpurine glycosylase | 2.22 | 1.87E-03 | ||
Metabolism | SAR0246 | conserved hypothetical protein | 2.00 | 2.27E-03 | |
SAR0261 | putative nitric oxide reductase | 2.22 | 6.16E-04 | ||
SAR0302 | putative formate/nitrite transporter | 2.38 | 8.03E-03 | ||
SAR0524 | nucleoside permease | 2.94 | 3.96E-03 | ||
SAR0562 | putative deoxyadenosine kinase protein | 2.17 | 2.64E-02 | ||
SAR0563 | putative deaminase | 2.50 | 3.75E-03 | ||
SAR0569 | putative glycosyl transferase | 2.13 | 4.40E-03 | ||
SAR0642 | ABC transporter permease protein | 2.56 | 9.65E-03 | ||
SAR0643 | ABC transporter ATP-binding protein | 3.70 | 9.25E-03 | ||
SAR0655 | putative Na+ dependent nucleoside transporter | 2.17 | 2.25E-03 | ||
SAR0743 | putative sodium:sulfate symporter protein | 2.22 | 4.39E-04 | ||
SAR0847 | thermonuclease precursor | 3.33 | 3.70E-04 | ||
SAR0916 | putative peptidyl-prolyl cis-trans isomerase | 2.13 | 6.54E-03 | ||
SAR1008 | putative glycosyl transferases | 4.00 | 1.65E-03 | ||
SAR1014 | acetyltransferase (GNAT) family protein | 2.27 | 6.54E-03 | ||
SAR1090 | putative protoheme IX farnesyltransferase | 2.04 | 2.66E-02 | ||
SAR1185 | putative guanylate kinase | 2.78 | 6.16E-03 | ||
SAR1449 | amino acid permease | 2.50 | 2.65E-03 | ||
SAR1478 | putative nucleoside diphosphate kinase | 2.38 | 3.03E-02 | ||
SAR1598 | arginine repressor | 2.50 | 3.23E-03 | ||
SAR1627 | 5-formyltetrahydrofolate cyclo-ligase family protein | 2.78 | 2.27E-03 | ||
SAR1707 | putative ATPase | 2.13 | 1.36E-02 | ||
SAR1714 | GTP pyrophosphokinase | 2.27 | 3.96E-03 | ||
SAR1717 | putative protein-export membrane protein | 2.27 | 6.05E-03 | ||
SAR1804 | putative acyltransferase | 2.44 | 2.99E-02 | ||
SAR2129 | sucrose operon repressor | 2.56 | 1.38E-02 | ||
SAR2130 | ammonium transporter family protein | 2.04 | 1.65E-03 | ||
SAR2340 | acetyltransferase (GNAT) family protein | 3.03 | 8.77E-04 | ||
SAR2363 | putative molybdate-binding lipoprotein precursor | 2.08 | 2.97E-02 | ||
SAR2432 | CorA-like Mg2+ transporter protein | 2.44 | 5.61E-03 | ||
SAR2493 | putative formate/nitrite transporter | 2.22 | 8.71E-03 | ||
SAR2594 | ABC transporter ATP-binding protein | 2.38 | 1.65E-03 | ||
SAR2789 | putative subtilase family protease | 2.04 | 2.27E-03 | ||
Hypothetical Genes | SAR0013 | putative membrane protein | 2.17 | 1.81E-02 | |
SAR0024 | conserved hypothetical protein | 3.03 | 2.27E-03 | ||
SAR0030 | hypothetical protein | 2.38 | 6.16E-03 | ||
SAR0048 | putative membrane protein | 2.08 | 1.08E-02 | ||
SAR0061 | putative membrane protein | 2.08 | 4.25E-02 | ||
SAR0063 | hypothetical protein | 2.56 | 1.02E-02 | ||
SAR0075 | hypothetical protein | 2.04 | 6.16E-04 | ||
SAR0078 | hypothetical protein | 2.08 | 9.65E-03 | ||
SAR0097 | putative DNA-binding protein | 2.17 | 2.99E-03 | ||
SAR0145 | putative lipoprotein | 2.13 | 1.56E-02 | ||
SAR0197 | hypothetical protein | 286 | 2.14E-02 | ||
SAR0216 | putative lipoprotein | 2.04 | 6.16E-04 | ||
SAR0338 | putative membrane protein | 2.86 | 2.40E-03 | ||
SAR0383 | abortive infection bacteriophage resistance related | 4.76 | 1.99E-02 | ||
SAR0618 | putative iron compound-binding protein | 2.27 | 4.08E-02 | ||
SAR0673 | conserved hypothetical protein | 2.70 | 4.55E-03 | ||
SAR0694 | putative bacteriocin | 2.38 | 3.75E-03 | ||
SAR0695 | putative bacteriocin-immunity membrane protein | 2.22 | 2.08E-03 | ||
SAR0718 | putative membrane protein | 3.33 | 9.93E-04 | ||
SAR0761 | putative lipoprotein | 2.86 | 3.00E-04 | ||
SAR0793 | hypothetical protein | 2.56 | 1.58E-02 | ||
SAR0846 | secreted von Willebrand factor-binding homolog | 2.17 | 1.94E-02 | ||
SAR0890 | conserved hypothetical protein | 2.56 | 8.06E-04 | ||
SAR0893 | putative membrane protein | 2.13 | 4.74E-02 | ||
SAR0898 | conserved hypothetical protein | 2.70 | 1.59E-02 | ||
SAR0899 | conserved hypothetical protein | 2.33 | 4.94E-03 | ||
SAR0915 | kinase-associated protein B | 2.44 | 8.06E-04 | ||
SAR0970 | protozoan/cyanobacterial globin family protein | 2.38 | 1.11E-02 | ||
SAR0971 | conserved hypothetical protein | 2.78 | 1.87E-03 | ||
SAR0979 | putative membrane protein | 2.50 | 2.25E-03 | ||
SAR0981 | putative esterase | 2.44 | 1.55E-03 | ||
SAR0982 | putative restriction-modification system protein | 2.44 | 1.37E-03 | ||
SAR0983 | putative restriction-modification system protein | 2.56 | 2.42E-03 | ||
SAR0985 | putative 2′,5′ RNA ligase family | 2.13 | 2.32E-02 | ||
SAR0987 | putative monogalactosyldiacylglycerol synthase | 2.56 | 6.54E-03 | ||
SAR1066 | putative lipoprotein | 2.50 | 4.74E-02 | ||
SAR1085 | conserved hypothetical protein | 2.33 | 3.44E-02 | ||
SAR1086 | conserved hypothetical protein | 3.45 | 6.54E-03 | ||
SAR1095 | conserved hypothetical protein | 2.86 | 2.16E-02 | ||
SAR1114 | putative cell division protein ZapA | 2.38 | 3.96E-03 | ||
SAR1148 | putative DNA-binding protein | 2.38 | 2.66E-02 | ||
SAR1154 | MraZ protein | 2.50 | 3.00E-03 | ||
SAR1312 | hypothetical protein | 3.85 | 3.27E-02 | ||
SAR1315 | hypothetical protein | 2.38 | 2.99E-03 | ||
SAR1316 | hypothetical protein | 2.27 | 1.79E-02 | ||
SAR1320 | hypothetical protein | 4.00 | 1.46E-02 | ||
SAR1335 | putative exported protein | 2.27 | 7.38E-03 | ||
SAR1389 | conserved hypothetical protein (pseudogene) | 2.33 | 5.63E-03 | ||
SAR1448 | major facilitator superfamily transporter protein | 2.04 | 4.57E-03 | ||
SAR1556 | putative phage regulatory protein | 2.08 | 5.64E-03 | ||
SAR1558 | putative phage lipoprotein | 2.44 | 1.24E-03 | ||
SAR1559 | hypothetical phage protein | 2.33 | 4.80E-04 | ||
SAR1560 | hypothetical phage protein | 2.04 | 1.72E-02 | ||
SAR1561 | putative phage membrane protein | 2.13 | 1.10E-03 | ||
SAR1581 | conserved hypothetical protein | 2.86 | 1.81E-02 | ||
SAR1592 | conserved hypothetical protein | 2.27 | 1.16E-02 | ||
SAR1699 | conserved hypothetical protein | 2.00 | 3.92E-03 | ||
SAR1706 | putative transcriptional regulator | 3.45 | 2.16E-02 | ||
SAR1708 | conserved hypothetical protein | 2.04 | 3.14E-03 | ||
SAR1770 | putative membrane protein | 2.13 | 2.99E-03 | ||
SAR1834 | putative leucyl-tRNA synthetase | 2.17 | 3.65E-02 | ||
SAR1885 | hypothetical protein | 2.63 | 6.97E-04 | ||
SAR1897 | hypothetical protein | 3.03 | 2.86E-02 | ||
SAR1935 | probable phosphoesterase | 2.78 | 2.37E-03 | ||
SAR1938 | putative DNA-binding protein | 2.38 | 2.80E-02 | ||
SAR2020 | putative membrane protein | 2.44 | 6.39E-03 | ||
SAR2035 | putative exported protein | 2.86 | 1.61E-02 | ||
SAR2113 | hypothetical protein | 2.86 | 4.64E-02 | ||
SAR2114 | hypothetical protein | 2.56 | 3.43E-02 | ||
SAR2115 | hypothetical protein | 2.86 | 4.22E-02 | ||
SAR2118 | putative membrane protein | 2.00 | 3.28E-03 | ||
SAR2119 | membrane anchored protein | 2.44 | 1.08E-03 | ||
SAR2156 | pemK-like protein | 3.03 | 5.61E-03 | ||
SAR2219 | hypothetical protein | 2.78 | 6.21E-03 | ||
SAR2261 | putative membrane protein | 2.08 | 6.02E-04 | ||
SAR2263 | putative membrane protein | 2.17 | 2.89E-03 | ||
SAR2299 | hypothetical protein | 2.04 | 3.47E-03 | ||
SAR2369 | putative acyl-CoA dehydrogenase | 2.86 | 1.94E-03 | ||
SAR2425 | putative membrane protein | 2.13 | 5.63E-03 | ||
SAR2428 | putative membrane protein | 2.00 | 3.23E-03 | ||
SAR2435 | putative acyl hydrolase | 2.50 | 3.02E-02 | ||
SAR2439 | tetR family regulatory protein | 2.22 | 3.23E-03 | ||
SAR2473 | putative exported protein | 3.85 | 3.97E-03 | ||
SAR2500 | putative lipoprotein | 2.86 | 8.48E-04 | ||
SAR2546 | putative lipoprotein | 3.13 | 2.76E-02 | ||
SAR2595 | putative membrane protein | 2.78 | 1.65E-03 | ||
SAR2718 | putative exported protein | 2.04 | 4.80E-04 | ||
SAR2719 | transcriptional regulator | 3.13 | 3.07E-04 | ||
SAR2792 | putative membrane protein | 3.85 | 2.78E-03 | ||
SAR2793 | putative membrane protein | 3.70 | 8.17E-04 |
Group Functions | MRSA252 ORF | MRSA252 Gene | MRSA252 Gene Product | Fold Change Up Regulated | P-value |
Virulence Factors and Regulators | SAR0279 | virulence factor esxA | 5.93 | 7.03E-08 | |
SAR0280 | putative membrane protein | 4.32 | 4.53E-06 | ||
SAR0281 | putative membrane protein | 2.70 | 1.66E-03 | ||
SAR0282 | conserved hypothetical protein | 2.65 | 3.35E-03 | ||
SAR0284 | putative membrane protein | 2.56 | 1.25E-03 | ||
SAR0284v | putative membrane protein | 2.49 | 4.23E-03 | ||
SAR2123 | putative autoinducer processing protein | 9.36 | 1.74E-05 | ||
SAR2125 | autoinducer sensor protein | 5.39 | 4.33E-05 | ||
SAR2126 | autoinducer sensor protein response regulator protein | 2.25 | 1.41E-03 | ||
agrIII | Class III accessory gene regulator ( |
8.71 | 4.16E-06 | ||
RNAIII | RNAIII | RNAIII accessory gene regulator ( |
10.20 | 1.21E-05 | |
Metabolism | SAR0150 | putative aldehyde-alcohol dehydrogenase | 2.25 | 1.67E-02 | |
SAR0190 | glucose-specific PTS transporter protein, IIABC component | 2.05 | 3.76E-02 | ||
SAR0829 | phosphoglycerate kinase | 2.76 | 2.16E-03 | ||
SAR0830 | triosephosphate isomerase | 2.75 | 1.69E-03 | ||
SAR0831 | putative phosphoglycerate mutase | 2.83 | 2.22E-03 | ||
SAR0832 | putative enolase | 2.15 | 5.88E-03 | ||
SAR2296 | putative acetolactate decarboxylase | 2.43 | 3.32E-03 | ||
SAR2297 | putative acetolactate synthase | 2.17 | 1.41E-03 | ||
SAR2618 | PTS system, glucose-specific IIABC component | 2.78 | 1.41E-02 | ||
SAR2711 | carbamate kinase | 2.40 | 3.41E-02 | ||
SAR2712 | arginine/ornithine antiporter | 2.21 | 1.88E-02 | ||
SAR2713 | putative ornithine carbamoyltransferase | 2.31 | 1.88E-02 | ||
SAR2714 | arginine deiminase | 2.89 | 1.41E-02 | ||
Hypothetical Genes | SAR0111 | putative myosin-crossreactive antigen | 2.44 | 6.52E-05 | |
SAR0277 | putative exported protein | 3.76 | 4.22E-05 | ||
SAR0278 | putative CHAP domain protein | 2.89 | 1.22E-04 | ||
SAR0299 | possible pseudogene | 2.95 | 3.30E-03 | ||
SAR0301 | putative membrane protein | 3.44 | 1.93E-03 | ||
SAR0385 | similar to putative pathogenicity island gene |
4.09 | 1.93E-03 | ||
SAR0839 | putative lipoprotein | 3.36 | 5.41E-05 | ||
SAR1564 | hypothetical protein | 2.09 | 5.32E-04 | ||
SAR1565 | putative lipoprotein | 2.38 | 3.02E-03 | ||
SAR2426 | putative membrane protein | 2.09 | 2.11E-03 | ||
SAR2427 | ABC transporter ATP-binding protein | 2.14 | 4.82E-03 | ||
SAR2428 | putative membrane protein | 3.73 | 1.21E-05 | ||
SAR2569 | hypothetical protein | 6.01 | 4.75E-02 |
Group Functions | MRSA252 ORF | MRSA252 Gene | MRSA252 Gene Product | Fold Change Down Regulated | P-value |
Stess Response | SAR0525 | stress regulatory protein | 3.57 | 6.52E-05 | |
SAR0526 | uvrB/uvrC domain protein | 4.35 | 4.16E-06 | ||
SAR0528 | putative stress response-related Clp ATPase | 4.17 | 5.47E-05 | ||
SAR0823 | putative ATP-dependent Clp protease proteolytic subunit | 2.04 | 3.82E-04 | ||
SAR0938 | putative ATPase subunit of an ATP-dependent protease | 9.09 | 5.72E-06 | ||
SAR1657 | chaperone protein | 2.94 | 5.41E-05 | ||
SAR1658 | GrpE protein | 3.57 | 7.03E-08 | ||
SAR2116 | 60 kDa chaperonin | 2.44 | 1.05E-03 | ||
SAR2117 | 10 kDa chaperonin | 2.78 | 1.92E-04 | ||
Metabolism | SAR0189 | putative thiamine pyrophosphate enzyme | 2.94 | 1.51E-04 | |
SAR0208 | putative sugar transport system permease | 2.94 | 2.68E-02 | ||
SAR0209 | putative oxidoreductase | 4.75 | 1.21E-02 | ||
SAR0210 | putative oxidoreductase | 9.09 | 3.75E-03 | ||
SAR0527 | putative phosphotransferase | 4.55 | 7.03E-08 | ||
SAR0752 | putative phosphofructokinase | 2.44 | 3.20E-02 | ||
SAR0753 | fructose-specific PTS system component | 3.45 | 1.21E-02 | ||
SAR1274 | putative glycerol uptake facilitator protein | 3.70 | 3.36E-03 | ||
SAR1275 | glycerol kinase | 4.17 | 6.42E-04 | ||
SAR1276 | glycerol-3-phosphate dehydrogenase | 7.69 | 4.83E-06 | ||
SAR2244 | mannitol-specific PTS system component | 2.08 | 4.75E-02 | ||
SAR2445 | Heme-regulated transporter ATPase | 2.94 | 3.11E-04 | ||
SAR2594 | ABC transporter ATP-binding protein | 2.33 | 3.32E-03 | ||
Hypothetical Genes | SAR0100 | putative membrane protein | 2.56 | 2.28E-02 | |
SAR0211 | conserved hypothetical protein | 11.11 | 3.02E-03 | ||
SAR0584 | predicted role in ipenimen resistance | 2.27 | 3.15E-02 | ||
SAR0750 | conserved hypothetical protein | 2.22 | 1.32E-02 | ||
SAR0939 | LysR family regulatory protein | 2.94 | 5.81E-05 | ||
SAR2595 | putative membrane protein | 2.04 | 7.18E-03 |
Group Functions | MRSA252 ORF | MRSA252 Gene | MRSA252 Gene Product | Fold Change Up Regulated | P-value |
Virulence Factors and Regulators | SAR0279 | virulence factor EsxA | 3.20 | 1.51E-05 | |
SAR0280 | putative membrane protein | 2.67 | 1.07E-04 | ||
SAR2122 | delta-hemolysin precursor | 6.02 | 5.53E-04 | ||
SAR2123 | putative autoinducer processing protein | 6.54 | 3.55E-07 | ||
SAR2125 | autoinducer sensor protein | 3.77 | 9.89E-05 | ||
SAR2126 | autoinducer sensor protein response regulator protein | 2.01 | 2.21E-04 | ||
agrIII | Class III accessory gene regulator ( |
6.30 | 1.12E-07 | ||
RNAIII | RNAIII accessory gene regulator ( |
7.02 | 3.21E-05 | ||
Metabolism | SAR0753 | fructose-specific PTS system component | 2.07 | 1.34E-02 | |
SAR2296 | conserved hypothetical protein | 2.05 | 4.08E-03 | ||
SAR2297 | putative acetolactate synthase | 2.49 | 1.19E-03 | ||
SAR2711 | carbamate kinase | 4.09 | 6.41E-03 | ||
SAR2712 | arginine/ornithine antiporter | 3.55 | 7.34E-04 | ||
SAR2713 | putative ornithine carbamoyltransferase | 3.41 | 1.97E-03 | ||
SAR2714 | arginine deiminase | 4.03 | 3.28E-03 | ||
Hypothetical Genes | SAR0277 | putative exported protein | 2.00 | 7.31E-03 | |
SAR0301 | putative membrane protein | 2.15 | 2.54E-02 | ||
SAR0385 | putative membrane protein | 2.88 | 8.02E-03 | ||
SAR0839 | putative lipoprotein | 2.07 | 1.97E-03 | ||
SAR1448 | major facilitator superfamily | 2.03 | 1.32E-02 | ||
SAR2710 | putative regulatory protein | 2.62 | 6.93E-05 |
Group Functions | MRSA252 ORF | MRSA252 Gene | MRSA252 Gene Product | Fold Change Down Regulated | P-value |
Stress Response | SAR0525 | stress regulatory protein | 3.23 | 3.26E-05 | |
SAR0526 | uvrB/uvrC domain protein | 4.17 | 3.26E-05 | ||
SAR0527 | putative phosphotransferase | 3.85 | 2.20E-07 | ||
SAR0528 | putative stress response-related Clp ATPase | 3.23 | 1.01E-04 | ||
SAR0938 | putative ATPase subunit of an ATP-dependent protease | 8.33 | 1.30E-06 | ||
SAR1119 | putative excinuclease ABC subunit C | 3.70 | 7.00E-03 | ||
SAR1657 | chaperone protein | 2.63 | 1.01E-04 | ||
SAR1658 | GrpE protein | 2.86 | 4.78E-06 | ||
SAR2116 | 60 kDa chaperonin | 2.38 | 1.82E-03 | ||
SAR2117 | 10 kDa chaperonin | 2.44 | 2.21E-04 | ||
Metabolism | SAR0120 | putative ornithine cyclodeaminase | 2.38 | 4.46E-02 | |
SAR0354 | putative homocysteine S-methyltransferase | 2.13 | 1.60E-02 | ||
SAR0452 | putative NADH-Ubiquinone protein | 2.00 | 1.32E-02 | ||
SAR1274 | putative glycerol uptake facilitator protein | 4.35 | 9.16E-03 | ||
SAR1275 | glycerol kinase | 3.57 | 1.59E-02 | ||
SAR1276 | aerobic glycerol-3-phosphate dehydrogenase | 4.76 | 3.26E-05 | ||
SAR1849 | proline dehydrogenase | 3.23 | 8.02E-03 | ||
SAR2445 | Heme-regulated transporter ATPase | 2.94 | 1.01E-04 | ||
SAR2446 | Heme-regulated transporter permease | 2.22 | 3.98E-02 | ||
SAR2582 | putative gluconate permease | 5.88 | 4.53E-03 | ||
SAR2583 | putative gluconokinase | 4.55 | 3.60E-02 | ||
Hypothetical Genes | SAR0939 | LysR family regulatory protein | 2.86 | 2.76E-04 | |
SAR2581 | hypothetical protein | 4.55 | 3.99E-02 |
The sudden imposition of linoleic acid during exponential growth at OD600 = 3 (linoleic acid challenge) resulted in large-scale transcriptional reprogramming of genes in four major discernible categories, including: virulence, energy metabolism, stress resistance and cell wall synthesis. In contrast, the presence of linoleic at 0.01 mM, a non-growth limiting concentration (linoleic acid growth exposure), resulted in changes in transcription of fewer genes in the same categories, with the exception of cell wall synthesis.
A distinctive feature of linoleic acid addition to cells of MRSA252 under both challenge and growth exposure conditions was observed to be the 10- and 2-fold up-regulation of the virulence regulator RNAIII, respectively (
Many genes involved in sugar metabolism showed altered levels of regulation. In particular, several genes in the fructose and mannose metabolism pathways were down-regulated. These include
In addition to increased transcription of genes encoding glycolytic enzymes, the cells exposed to a linoleic acid challenge alter metabolism to maintain levels of pyruvate. The up-regulation of
There was up-regulation of many genes involved in cellular stress responses, including the CtsR regulon genes
Genes concerned with cell wall biosynthesis were observed to be modulated in linoleic acid challenge conditions but not in the growth exposure conditions. The genes
The fatty acid biosynthesis enzyme FabI was previously reported to be inhibited by linoleic acid and was therefore proposed to be a key target for its antibacterial activity
Confirmation of the microarray data was performed using qRT-PCR to test selected transcriptional changes of known genes from different functional subsets. To this end, the expression level of genes involved in staphyloxanthin synthesis (
ORF | Gene | Linoleic Challenge | Linoleic Growth | Oleic Growth | Linoleic Growth | Oleic Growth |
OD600 = 3 | OD600 = 3 | OD600 = 8 | OD600 = 8 | |||
SAR0114 | 1.46 (0.37) | 1.01 (0.07) | −1.08 (0.11) | −3.03 (0.14) | −2.92 (0.24) | |
SAR0223 | 3.10 (0.08) | nd | nd | nd | nd | |
SAR0225 | 3.16 (0.61) | −1.34 (0.03) | −1.88 (0.02) | −1.06 (0.03) | −1.01 (0.02) | |
SAR0258 | −5.03 (0.01) | nd | nd | nd | nd | |
SAR0625 | 3.84 (0.81) | 1.10 (0.11) | −1.12 (0.07) | 1.52 (0.55) | 2.05 (1.12) | |
SAR2621 | −1.93 (0.02) | 1.52 (0.03) | 1.41 (0.03) | −2.75 (0.01) | −1.39 (0.02) | |
SAR0938 | 3.90 (0.08) | −8.55 (0.01) | −10.31 (0.01) | −3.16 (0.01) | −3.00 (0.01) | |
SAR0978 | 1.25 (0.02) | nd | nd | nd | nd | |
SAR1136 | −1.60 (0.20) | 1.19 (0.19) | −1.71 (0.09) | 6.38 (2.91) | 7.88 (5.16) | |
SAR1344 | 7.27 (0.18) | nd | nd | nd | nd | |
SAR1430 | 7.59 (0.22) | nd | nd | nd | nd | |
SAR2187 | 3.37 (0.64) | −1.10 (0.02) | 1.40 (0.02) | 1.10 (0.04) | 1.20 (0.06) | |
SAR2643 | 3.72 (0.08) | nd | nd | nd | nd | |
SAR2714 | 1.61 (0.03) | 1.86 (0.04) | 2.17 (0.04) | −2.19 (0.02) | −1.50 (0.01) | |
SAR2725 | 31.86 (0.69) | nd | nd | nd | nd | |
RNAIII | 7.86 (0.15) | 56.14 (1.40) | 34.28 (0.74) | 156.12 (6.95) | 153.30 (2.81) |
The values correspond to the fold change for each gene tested under the relevant fatty acid treatment conditions when compared to the untreated control. The standard deviation for each measurement is in parentheses. nd, not detemined. ORF indicates the gene locus in MRSA252 (
The transcription of a subset of genes was examined by qRT-PCR during mid-exponential growth phase and late exponential-phase (OD600 = 8) (
Several genes showed fluctuations in relative transcript levels during the growth cycle. For example,
The observation of increased expression of RNAIII,
ORF | Gene | Linoleic Challenge | Linoleic Growth | Oleic Growth |
OD600 = 3 | OD600 = 3 | |||
SAR0114 | 2.19 (0.09) | −1.94 (0.02) | 1.66 (0.04) | |
SAR0258 | −2.31 (0.02) | nd | nd | |
SAR0625 | 1.26 (0.05) | −3.79 (0.02) | −3.45 (0.03) | |
SAR0938 | 1.95 (0.08) | −2.11 (0.02) | −2.58 (0.03) | |
SAR0978 | −1.20 (0.04) | nd | nd | |
SAR1136 | −3.60 (0.01) | −2.11 (0.02) | −2.58 (0.03) | |
SAR1430 | 1.84 (0.11) | nd | nd | |
SAR2643 | 1.32 (0.05) | nd | nd | |
SAR2714 | nd | 2.19 (0.10) | 4.39 (0.18) | |
SAR2725 | 1.49 (0.06) | nd | nd | |
RNAIII | −1.79 (0.03) | −3.29 (0.01) | −1.95 (0.01) |
The values correspond to the fold change for each gene tested under the relevant fatty acid treatment conditions when compared to the untreated control. The standard deviation for each measurement is in parentheses. nd, not detemined. ORF indicates the gene locus in MRSA252 (
The proteome of MRSA252 was analysed by 2D-PAGE to identify protein expression changes in exponentially growing cells that were exposed to linoleic acid under the challenge conditions used for the microarray experiments. This analysis was performed to determine whether the large-scale transcriptional modulation described above was translated into a correspondingly large-scale proteomic shift. Under these conditions, 58 proteins were significantly (P≤0.05) up-regulated ≥2-fold and 15 proteins were significantly (P≤0.05) down-regulated ≥2-fold. MALDI-MS was used to identify the most intense protein spots on the gel corresponding to proteins that were modulated by linoleic acid, and the identities of 38 up-regulated and 5 down-regulated proteins were unambiguously determined (
Group Functions | MRSA252 ORF | MRSA252 Gene | MRSA252 Gene Product | Fold Change Up Regulated | P-value |
Virulence Factors and Regulators | SAR2745 | Capsular polysaccharide biosynthesis protein | 3.36 | 2.27E-04 | |
Energy Metabolism | SAR0140 | deoxyribose-phosphate aldolase | 4.34 | 2.49E-03 | |
SAR0217 | formate acetyltransferase | 2.30 | 1.50E-03 | ||
SAR0394 | phosphoglycerate mutase family protein | 2.94 | 1.67E-02 | ||
SAR0828 | glyceraldehyde 3-phosphate dehydrogenase 1 | 2.07 | 3.53E-03 | ||
SAR0924 | glucose-6-phosphate isomerase | 4.18 | 7.43E-03 | ||
SAR1451 | alanine dehydrogenase 2 | 2.13 | 1.32E-03 | ||
SAR1789 | acetate kinase | 2.81 | 6.97E-04 | ||
SAR2506 | putative phosphoglycerate mutase | 2.15 | 3.52E-02 | ||
SAR2685 | malate:quinone oxidoreductase | 3.39 | 8.08E-03 | ||
DNA Repair and Replication | SAR1639 | DNA primase | 2.66 | 2.61E-03 | |
SAR1996 | DNA Ligase | 2.09 | 6.11E-04 | ||
Protein Synthesis | SAR0552 | elongation factor G | 4.47 | 2.51E-04 | |
SAR0552 | elongation factor G | 2.10 | 2.09E-02 | ||
SAR1216 | putative tRNA (guanine-7-)-methyltransferase | 2.33 | 4.84E-04 | ||
SAR1720 | S-adenosylmethionine:tRNA ribosyltransferase-isomerase | 2.11 | 1.68E-02 | ||
SAR2309 | RNA polymerase alpha subunit | 2.61 | 2.40E-03 | ||
Peptidoglycan Synthesis | SAR0470 | family regulatory protein | 2.20 | 1.07E-02 | |
SAR1762 | threonyl-tRNA synthetase | 2.77 | 9.07E-04 | ||
SAR1991 | aspartyl/glutamyl-tRNA amidotransferase subunit B | 2.25 | 3.52E-02 | ||
SAR2201 | serine hydroxymethyltransferase | 3.55 | 8.43E-03 | ||
SAR2201 | serine hydroxymethyltransferase | 2.15 | 3.74E-03 | ||
Carotenoid Biosynthesis | SAR1378 | prephenate dehydrogenase | 2.37 | 8.08E-03 | |
Miscellaneous | SAR0218 | putative pyruvate formate-lyase activating enzyme | 2.60 | 3.46E-02 | |
SAR0403 | putative DNA binding protein | 2.72 | 3.48E-02 | ||
SAR2007 | putative oxygenase/mitric oxide synthase | 2.04 | 7.13E-03 | ||
SAR2007 | putative nitric oxide synthase | 2.66 | 2.38E-02 | ||
Metabolism | SAR0150 | putative aldehyde-alcohol dehydrogenase | 3.48 | 5.07E-03 | |
SAR0246 | 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase | 2.43 | 4.84E-04 | ||
SAR0246 | 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase | 2.21 | 8.75E-03 | ||
SAR0564 | putative haloacid dehalogenase-like hydrolase | 2.22 | 1.01E-02 | ||
SAR1070 | dihydrolipoamide dehydrogenase | 2.22 | 2.62E-02 | ||
SAR2353 | molybdopterin-guanine dinucleotide biosynthesis protein | 3.12 | 8.32E-04 | ||
SAR2513 | 6-carboxyhexanoate–CoA ligase | 4.84 | 4.13E-05 | ||
SAR2641 | putative aminotransferase | 2.07 | 1.54E-02 | ||
Hypothetical Proteins | SAR0985 | putative RNA ligase protein | 2.31 | 2.12E-02 | |
SAR2064 | hypothetical phage protein | 2.06 | 1.41E-03 | ||
SAR2369 | Acyl-CoA dehydrogenase-related protein | 3.36 | 4.69E-03 |
Group Functions | MRSA252 ORF | MRSA252 Gene | MRSA252 Gene Product | Fold Change Down Regulated | P-value |
Protein Synthesis | SAR0927 | signal peptidase Ib | 5.88 | 9.88E-04 | |
SAR1755 | trigger factor | 2.56 | 1.93E-02 | ||
SAR2179 | putative membrane protein | 2.17 | 2.05E-02 | ||
Peptidoglycan Synthesis | SAR1284 | glutamine synthetase | 2.33 | 5.00E-02 | |
Metabolism | SAR0814 | kinase/phosphorylase | 3.03 | 6.66E-03 |
In addition to linoleic acid, the effect of the skin-associated LC-uFFA hexadecenoic acid [C16:1 (n-6)] on the cellular proteome was studied to determine whether there was a common response to LC-uFFAs on
Group Functions | MRSA252 ORF | MRSA252 Gene | MRSA252 Gene Product | Fold Change Up regulated | P-value |
Stress Response | SAR2116 | chaperonin | 2.03 | 3.49E-02 | |
SAR2273 | alkaline shock protein 23 | 2.89 | 8.90E-03 | ||
SAR2461 | pyridine nucleotide-disulphide oxidoreductase family protein | 2.05 | 3.99E-02 | ||
SAR2461 | pyridine nucleotide-disulphide oxidoreductase family protein | 2.00 | 2.13E-03 | ||
SAR2461 | pyridine nucleotide-disulphide oxidoreductase family protein | 2.59 | 2.49E-02 | ||
Energy Metabolism | SAR0140 | deoxyribose-phosphate aldolase | 4.21 | 4.86E-03 | |
SAR0394 | phosphoglycerate mutase family protein | 4.79 | 1.06E-03 | ||
SAR0828 | glyceraldehyde 3-phosphate dehydrogenase 1 | 2.86 | 2.51E-03 | ||
SAR0830 | triosephosphate isomerase | 3.40 | 4.42E-03 | ||
SAR0832 | enolase | 3.20 | 1.83E-02 | ||
SAR0832 | enolase | 3.09 | 4.60E-03 | ||
SAR0832 | enolase | 2.07 | 5.15E-03 | ||
SAR0924 | glucose-6-phosphate isomerase | 2.31 | 2.73E-02 | ||
SAR1068 | putative pyruvate dehydrogenase E1 component | 2.72 | 2.29E-04 | ||
SAR1068 | putative pyruvate dehydrogenase E1 component | 2.72 | 2.29E-04 | ||
SAR1121 | putative succinate dehydrogenase flavoprotein | 2.24 | 1.36E-02 | ||
SAR1451 | alanine dehydrogenase 2 | 2.21 | 2.30E-03 | ||
SAR2605 | D-lactate dehydrogenase | 3.11 | 8.87E-03 | ||
SAR2685 | malate:quinone oxidoreductase | 4.79 | 1.06E-03 | ||
SAR2685 | malate:quinone oxidoreductase | 3.13 | 2.18E-03 | ||
DNA Repair and Replication | SAR1997 | ATP-dependent DNA helicase | 2.23 | 3.49E-02 | |
Protein Synthesis | SAR0552 | translation elongation factor G | 3.56 | 3.84E-02 | |
SAR0553 | translation elongation factor Tu | 2.65 | 3.41E-02 | ||
SAR0553 | elongation factor Tu | 4.13 | 7.56E-03 | ||
SAR1216 | putative tRNA (guanine-7-)-methyltransferase | 2.80 | 1.69E-03 | ||
SAR1485 | putative 30S ribosomal protein S1 | 2.91 | 4.91E-02 | ||
SAR1719 | queuine tRNA-ribosyltransferase | 2.01 | 4.96E-02 | ||
SAR1720 | S-adenosylmethionine:tRNA ribosyltransferase-isomerase | 2.49 | 1.44E-02 | ||
SAR2309 | DNA-directed RNA polymerase subunit alpha | 2.44 | 9.73E-03 | ||
Peptidoglycan Synthesis | SAR1048 | putative phosphoribosylamine–glycine ligase | 3.98 | 2.08E-02 | |
SAR1762 | threonyl-tRNA synthetase | 2.51 | 1.51E-02 | ||
SAR2212 | UDP-N-acetylglucosamine 1-carboxyvinyltransferase | 2.13 | 1.36E-02 | ||
Cell Division | SAR1795 | putative septation ring formation regulator | 3.11 | 3.71E-03 | |
SAR1795 | putative septation ring formation regulator | 2.56 | 5.16E-03 | ||
Miscellaneous | SAR0218 | putative pyruvate formate-lyase activating enzyme | 2.39 | 4.03E-03 | |
SAR0403 | putative DNA-binding protein | 2.84 | 8.32E-03 | ||
SAR2007 | putative oxygenase | 2.85 | 2.92E-02 | ||
Metabolism | SAR0351 | acetyl-CoA acetyltransferase | 2.85 | 2.59E-04 | |
SAR0351 | acetyl-CoA acetyltransferase | 2.77 | 9.07E-04 | ||
SAR0514 | putative O-acetylserine (thiol)-lyase | 2.41 | 4.43E-03 | ||
SAR1142 | ornithine carbamoyltransferase | 2.03 | 5.43E-03 | ||
SAR2352 | putative molybdenum cofactor biosynthesis protein A | 3.73 | 6.34E-03 | ||
SAR2352 | putative molybdenum cofactor biosynthesis protein A | 2.25 | 9.64E-03 | ||
SAR2460 | putative acetyltransferase (GNAT) family protein | 5.37 | 6.22E-05 | ||
SAR2460 | putative acetyltransferase (GNAT) family protein | 5.20 | 2.03E-02 | ||
SAR2641 | putative aminotransferase | 2.12 | 8.47E-03 | ||
SAR2694 | putative anaerobic ribonucleotide reductase activating protein | 3.38 | 4.66E-04 | ||
SAR2694 | putative anaerobic ribonucleotide reductase activating protein | 2.48 | 3.43E-02 | ||
Hypothetical Proteins | SAR0246 | 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase | 3.37 | 2.20E-04 | |
SAR0246 | 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase | 2.90 | 1.69E-03 | ||
SAR0985 | putative RNA ligase protein | 2.11 | 1.68E-02 | ||
SAR1105 | hypothetical protein | 2.25 | 3.60E-03 | ||
SAR2063 | hypothetical phage protein | 2.06 | 1.41E-03 | ||
SAR2369 | putative acyl-CoA dehydrogenase | 2.61 | 2.43E-04 | ||
SAR2545 | M42 glutamyl aminopeptidase | 2.08 | 1.72E-02 | ||
SAR2674 | hypothetical protein | 2.45 | 5.46E-04 |
Group Functions | MRSA252 ORF | MRSA252 Gene | MRSA252 Gene Product | Fold Change Down Regulated | P-value |
Protein Synthesis | SAR0927 | signal peptidase Ib | 4.55 | 8.44E-03 | |
Peptidoglycan Synthesis | SAR0920 | NAD-specific glutamate dehydrogenase | 2.17 | 8.21E-03 | |
Miscellaneous | SAR2622 | family regulatory protein | 2.13 | 1.21E-02 | |
Metabolism | SAR0483 | putative thymidylate kinase | 2.27 | 1.04E-03 | |
SAR1399 | ABC transporter ATP-binding protein | 3.23 | 1.80E-03 |
Allelic replacement mutants were constructed in the genes
A Graph showing the percentage survival of WT and mutant variants of SH1000 when serial dilutions of the strains were plated on BHI agar containing 1 mM linoleic acid. Survival is expressed as a percentage of viable cell counts obtained for control plates lacking linoleic acid. Values are the mean of multiple independent experiments. Error bars indicate standard errors of the mean. p<0.005 for each mutant by Student's t-test. B Plates showing the relative survival of SH1000 and the
Cells grown in the presence of linoleic acid under constant growth conditions displayed reduced expression of the CtsR regulon, which is known to impact on cell autolysis
The result of growth of MRSA252 and SH1000 in the absence (closed triangle) or presence (open box) of 0.01mM linoleic acid on autolysis is shown in A and B, respectively. Survival is expressed as a percentage of OD600 at T = 0. Values are from three independent experiments. Error bars indicate standard errors of the mean. **p<0.01, *p<0.05 by Student's t test. C Relative hydrophobicity of the MRSA252 and SH1000 strains following overnight growth in BHI +/− 0.1 mM linoleic acid. Values are from three independent experiments. Error bars indicate standard errors of the mean. **p<0.01, *p<0.05 by Student's t test.
IsdA reduces cell surface hydrophobicity and acts to increase staphylococcal resistance to LC-uFFAs
A murine arthritis model of infection was used to determine a role for the LC-uFFA survival genes
A Effect of WT SH1000 (open box) and mutations of
Analysing the response of MRSA252, an EMRSA-16 clone, to the LC-uFFAs linoleic [C18:2 (n-6,9)] and oleic [C18:1 (n-6)] acid revealed modulated expression of many genes, including those encoding virulence determinants. After exposure of exponentially growing cells to linoleic acid there was a very large increase in RNAIII compared to control cells, and this was also observed at all stages of growth when either linoleic or oleic acid were present from the time of inoculation. This observed up-regulation of RNAIII synthesis was unexpected given previous reports on the effects of GML, a lauric acid monoester, and the LC-uFFA hexadecenoic acid [C16:1 (n-6)] on
Microarray analysis revealed further virulence factors exhibiting increased transcription, including the
The
A screen for additional mutants of SH1000 that were defective for survival in the presence of linoleic acid identified several Tn
The identification of cell envelope mutants correlated with the gene expression and proteomic data, in which altered levels of cell wall synthesis and regulation components was observed (
Sections A, B, C and D highlight the various genes involved in peptidoglycan, carotenoid, menaquinone and energy metabolism respectively. Genes in red and blue boxes are up- and down-regulated, respectively. See text for details.
Previous studies have used gene expression profiling to determine the cellular pathway targeted by antimicrobial agents
A consequence of LC-uFFAs inserting in the cell membrane could be the disruption of the electron transport chain. This would explain the numerous changes in expression of genes associated with energy creation within the cell (
Strains and plasmids used in this study are listed in
Strain or Plasmid | Comment | Reference or Source |
Strains: | ||
DH5α | ||
SH1000 | Functional |
|
MRSA252 | Wild-type clinical isolate | |
RN4220 | Restriction-deficient transformation recipient mutant of 8325-4 | |
N315 | Wild-type pharyngeal smear clinical isolate | |
MSSA476 | Wild-type clinical isolate | |
Liv033 ( |
8325-4 |
|
Liv038 ( |
SH1000 |
|
Liv039 ( |
SH1000 |
|
Liv101 ( |
SH1000 |
|
Liv130 ( |
SH1000 |
|
Liv142 ( |
SH1000 |
|
Liv405 ( |
8325-4 |
T. Foster |
Liv442 ( |
SH1000 |
This Study |
Liv671 ( |
SH1000 |
|
Liv673 ( |
Newman |
|
Liv681 |
SH1000 |
This study |
Liv684 ( |
RN4220 |
This Study |
Liv686 ( |
RN4220 |
This Study |
Liv692 ( |
SH1000 |
This Study |
Liv694 ( |
SH1000 |
This Study |
Liv718 ( |
SH1000 |
This Study |
Liv723 ( |
RN4220 |
This Study |
Liv750 ( |
SH1000 |
This study |
Liv753 ( |
SH1000 |
This Study |
Liv766 ( |
SH1000 |
This Study |
Liv994 | RN4220 pSK5630+ |
This Study |
Liv995 | RN4220 pSK5630+ |
This Study |
Liv996 | RN4220 pSK5630+ |
This Study |
Liv997 | RN4220 pSK5630+ |
This Study |
Liv1000 ( |
Liv694 complemented with pSK5630+ |
This Study |
Liv1001 ( |
Liv692 complemented with pSK5630+ |
This Study |
Liv1002 ( |
Liv753 complemented with pSK5630+ |
This Study |
Liv1003 ( |
Liv766 complemented with pSK5630+ |
This Study |
Plasmids: | ||
pLTV1 | Temperature-sensitive plasmid harbouring Tn |
|
pAZ106 | Promoterless |
|
pDG1513 | pMTL22 derivative [tetr] | |
pSK5630 | Low copy number |
Transposon mutagenesis was performed on the SH1000 strain of
Construction of
Oligonucleotides | Sequence (5′ to 3′) |
sasF_BamHI | |
sasF_NotI | |
sasF_KpnI | |
sasF_EcoRI | |
arcA_BamHI | |
arcA_NotI | |
arcA_KpnI | |
arcA_EcoRI | |
vraS_BamHI | |
vraS_NotI | |
vraS_KpnI | |
vraS_EcoRI | |
Tet_NotI | |
Tet_KpnI | |
Complementation | |
Sar2725_SasF_For | |
Sar2725_SasF_Rev | |
arcA_For | |
arcA_Rev | |
Sar2632_For | |
Sar2632_Rev | |
Sar2782_vraE_For | |
Sar2782_vraE_Rev |
Complementation of the
To ascertain the transcriptional responses of MRSA252 to fatty acids, overnight cultures (18 h) of MRSA252 were used to inoculate 100 ml of BHI (Merck) with or without 10 µM of oleic or linoleic acid in 250 ml conical flasks. These 100 ml cultures were placed in a shaking water bath at 37°C at 250 rpm and 10 ml samples were taken from the flask when the cultures reached late exponential phase (OD600 = 3). Identical inoculations were performed to 100 ml of BHI lacking additional fatty acids. 100 µM of linoleic acid in ethanol or an equal volume of the ethanol used to dilute the fatty acid was added to these cultures at an OD600 = 3.0 and the RNA extracted from treated and untreated cells 20 min post-treatment. Each treatment and control culture was performed in biological triplicate. The concentrations of fatty acids used in these experiments did not alter the pH of the media. RNA was extracted from 10 ml samples of culture taken at the indicated time intervals and stabilised by the addition of 20 ml of RNA Bacteria Protect (Qiagen). The cells were subsequently harvested by centrifugation at 5000 rpm for 10 min and cell pellets resuspended in lysis buffer (10 mM Tris, pH8.0) containing 200 U ml−1 of lysostaphin and 400 U ml−1 of mutanolysin, and incubated for 90 min at 37°C with gentle shaking every 10–15 min. The RNA was subsequently extracted using the RNeasy Midi kit (Qiagen) and DNase treated whilst on the purification column using the RNase-Free DNase Set according to manufacturers instructions (Qiagen). The quantity and quality of the RNA was assessed on an Agilent 2100 bioanalyzer by using the RNA 6000 Nano LabChip Kit. The RNA was converted to cDNA and labelled by incorporation of Cy5 dCTP during reverse transcription of RNA using the enzyme Superscript II (Amersham). DNA used in the microarray hybridisations was extracted from 5 ml of an overnight culture (18 h) of MRSA252 using the Edge Biosystems Bacterial Genomic DNA purification kit according to manufacturer's instructions. The DNA was labeled by the incorporation of Cy3 dCTP using Klenow (Invitrogen). cDNA derived from RNA and genomic DNA were pooled and hybridized on whole-genome microarrays supplied by the Bacterial Microarray Group at St. George's Hospital (BμG@S [
To confirm the validity of microarray data gene specific mRNAs were quantified from treated and untreated cultures by quantitative real-time PCR (qRT-PCR). Cells were grown in biological triplicate exactly as described above for the microarray experiments and bacterial RNA was isolated using the Pro-Blue Fast RNA kit (MP Biomedicals). DNA was removed from the samples by DNase I treatment (Ambion) according to manufacturer's instructions. The purified RNA was quantified using the nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific) and the integrity assessed by electrophoresis. 0.5 µg of RNA was reverse transcribed with 100 U of Bioscript Reverse Transcriptase (Bioline) using 0.2 µg of random hexamer primers (Promega) according to manufacturer's instructions. qRT-PCR was performed using the 7500 Fast System (Applied Biosystems) and the QuantiFast SYBR Green PCR kit (Qiagen) according to manufacturer's instructions. The relative levels of gene expression in fatty acid treated cells and the non-treated controls were calculated by relative quantification using
Oligonucleotide | SAR Number | Sequence (5′ to 3′) |
gyrB_For | ||
gyrB_Rev | ||
spa_For | ||
spa_Rev | ||
fadA_For | ||
fadA_Rev | ||
fadD_For | ||
fadD_Rev | ||
lytR_For | ||
lytR_Rev | ||
sarA_For | ||
sarA_Rev | ||
clpB_For | ||
clpB_Rev | ||
fabI_For | ||
fabI_Rev | ||
hla_For | ||
hla_Rev | ||
katA_For | ||
katA_Rev | ||
murG_For | ||
murG_Rev | ||
fabZ_For | ||
fabZ_Rev | ||
cidA_For | ||
cidA_Rev | ||
crtM_For | ||
crtM_Rev | ||
arcA_For | ||
arcA_Rev | ||
sasF-For | ||
sasF_Rev | ||
RNAIII_For | RNAIII | |
RNAIII_Rev | RNAIII |
Cultures of MRSA252 (100 ml) were grown to late exponential phase (OD600 = 3.0) and exposed to 0.1 mM linoleic acid or 0.1 mM hexadecenoic acid as described above. Cells were harvested by centrifugation at 5000 g for 10 min at 4°C. After two washes in PBS the cells were resuspended in 2 ml of lysis buffer (PBS, 1 mg/ml DNase I, 100 µM benzamidine, 100 µM PMSF, 1 mg/ml RNase, 2 mg/ml lysostaphin) and incubated at 37°C for 20 min before chilling on ice. Cell debris and insoluble material was pelleted by centrifugation at 4°C for 20,000 g for 20 min. The supernatant was stored at −20°C. Protein samples were quantified using the BioRad Protein assay. The protein samples were desalted using Slide-A-Lyzer Mini Dialysis Units with a 3.5 kDa MWCO (Thermo Scientific).
Soluble protein (120 µg ) was brought up to 320 µl with rehydration buffer (8 M urea, 2M thiourea, 4% (w/v) CHAPS, 20 mM DTT, 1% (v/v) ASB 14 detergent and 0.5% (v/v) carrier ampholytes (Bio-lyte 3/10, Bio-Rad)). Samples were incubated for an hour at room temperature with gentle shaking, before centrifugation at 8,000 g for 5 min. Samples were in-gel rehydrated and focused on 17 cm, pH 4–7 IPG strips (Bio-Rad) for a total of 40000 V h (150V for 1h, 300V for 1h, 600V for 1h, 1200V for 1h, 1200–8000V over 1h (linear gradient), 8000 V to 40000 v (steady state)), using a Protean IEF Cell (Bio-Rad). After focusing, strips were equilibrated in 50 mM Tris (pH 6.8), 6 M urea, 2% (w/v) SDS, 30% (w/v) glycerol, and bromophenol blue, containing 20 mM DTT in the reduction step (15 min) and 25 mM iodoacetamide in the alkylation step (15 min). IPG strips were run in the second dimension on 20×20cm 12.5% SDS-PAGE gels using a Protean II xi 2D Cell (Bio-Rad). Gels were run in triplicate, silver-stained
Spots for identification were excised and digested in-gel with trypsin. Gel Plugs were destained in 50% (v/v) acetonitrile:50% (v/v) 50 mM ammonium bicarbonate (37°C), dehydrated in 100% acetonitrile (37°C), and rehydrated overnight (37°C) in 10 µl of 50mM ammonium bicarbonate containing trypsin (1 µl of 100 ng trypsin stock reconstituted in 50 mM acetic acid (Promega)). Supernatants containing the extracted peptides were removed and analyzed by MALDI-TOF.
Peptide Mass Fingerprinting (PMF) was conducted on a reflectron MALDI-TOF instrument (
Cell surface hydrophobicity of
Cell autolysis rates were determined on cells exposed to linoleic acid using an assay modified from that described by Blackman
A well described mouse model of septic arthritis was used to test the
We would like to thank Alan McCarthy and Heather Allison for critical reading of the manuscript and Timothy Foster, Dorte Frees, Friedrich Götz and Simon Foster for kindly supplying mutant strains.