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Untargeted metabolomics analysis revealed changes in the composition of glycerolipids and phospholipids in Bacillus subtilis under 1-butanol stress

  • Genomics, transcriptomics, proteomics
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Abstract

1-Butanol has been utilized widely in industry and can be produced or transformed by microbes. However, current knowledge about the mechanisms of 1-butanol tolerance in bacteria remains quite limited. Here, we applied untargeted metabolomics to study Bacillus subtilis cells under 1-butanol stress and identified 55 and 37 ions with significantly increased and decreased levels, respectively. Using accurate mass determination, tandem mass spectra, and synthetic standards, 86 % of these ions were characterized. The levels of phosphatidylethanolamine, diglucosyldiacylglycerol, and phosphatidylserine were found to be upregulated upon 1-butanol treatment, whereas those of diacylglycerol and lysyl phosphatidylglycerol were downregulated. Most lipids contained 15:0/15:0, 16:0/15:0, and 17:0/15:0 acyl chains, and all were mapped to membrane lipid biosynthetic pathways. Subsequent two-stage quantitative real-time reverse transcriptase PCR analyses of genes in the two principal membrane lipid biosynthesis pathways revealed elevated levels of ywiE transcripts in the presence of 1-butanol and reduced expression levels of cdsA, pgsA, mprF, clsA, and yfnI transcripts. Thus, the gene transcript levels showed agreement with the metabolomics data. Lastly, the cell morphology was investigated by scanning electron microscopy, which indicated that cells became almost twofold longer after 1.4 % (v/v) 1-butanol stress for 12 h. Overall, the studies uncovered changes in the composition of glycerolipids and phospholipids in B. subtilis under 1-butanol stress, emphasizing the power of untargeted metabolomics in the discovery of new biological insights.

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References

  • Aono R, Kobayashi H (1997) Cell surface properties of organic solvent-tolerant mutants of Escherichia coli K-12. Appl Environ Microbiol 63(9):3637–3642

    CAS  PubMed Central  PubMed  Google Scholar 

  • Beaven MJ, Charpentier C, Rose AH (1982) Production and tolerance of ethanol in relation to phospholipid fatty-acyl composition in Saccharomyces cerevisiae NCYC 431. J Gen Microbiol 128(7):1447–1455

    CAS  Google Scholar 

  • Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G, Azevedo V, Bertero MG, Bessieres P, Bolotin A, Borchert S, Borriss R, Boursier L, Brans A, Braun M, Brignell SC, Bron S, Brouillet S, Bruschi CV, Caldwell B, Capuano V, Carter NM, Choi SK, Codani JJ, Connerton IF, Cummings NJ, Daniel RA, Denizot F, Devine KM, Dusterhoft A, Ehrlich SD, Emmerson PT, Entian KD, Errington J, Fabret C, Ferrari E, Foulger D, Fritz C, Fujita M, Fujita Y, Fuma S, Galizzi A, Galleron N, Ghim SY, Glaser P, Goffeau A, Golightly EJ, Grandi G, Guiseppi G, Guy BJ, Haga K, Haiech J, Harwood CR, Henaut A, Hilbert H, Holsappel S, Hosono S, Hullo MF, Itaya M, Jones L, Joris B, Karamata D, Kasahara Y, Klaerr-Blanchard M, Klein C, Kobayashi Y, Koetter P, Koningstein G, Krogh S, Kumano M, Kurita K, Lapidus A, Lardinois S, Lauber J, Lazarevic V, Lee SM, Levine A, Liu H, Masuda S, Mauel C, Medigue C, Medina N, Mellado RP, Mizuno M, Moestl D, Nakai S, Noback M, Noone D, O’Reilly M, Ogawa K, Ogiwara A, Oudega B, Park SH, Parro V, Pohl TM, Portetelle D, Porwollik S, Prescott AM, Presecan E, Pujic P, Purnelle B, Rapoport G, Rey M, Reynolds S, Rieger M, Rivolta C, Rocha E, Roche B, Rose M, Sadaie Y, Sato T, Scanlan E, Schleich S, Schroeter R, Scoffone F, Sekiguchi J, Sekowska A, Seror SJ, Serror P, Shin BS, Soldo B, Sorokin A, Tacconi E, Takagi T, Takahashi H, Takemaru K, Takeuchi M, Tamakoshi A, Tanaka T, Terpstra P, Tognoni A, Tosato V, Uchiyama S, Vandenbol M, Vannier F, Vassarotti A, Viari A, Wambutt R, Wedler E, Wedler H, Weitzenegger T, Winters P, Wipat A, Yamamoto H, Yamane K, Yasumoto K, Yata K, Yoshida K, Yoshikawa HF, Zumstein E, Yoshikawa H, Danchin A (1997) The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390(6657):249–256

  • Boch J, Kempf B, Bremer E (1994) Osmoregulation in Bacillus subtilis: synthesis of the osmoprotectant glycine betaine from exogenously provided choline. J Bacteriol 176(17):5364–5371

    CAS  PubMed Central  PubMed  Google Scholar 

  • Bohin JP, Rigomier D, Schaeffer P (1976) Ethanol sensitivity of sporulation in Bacillus subtilis: a new tool for the analysis of the sporulation process. J Bacteriol 127(2):934–940

    CAS  PubMed Central  PubMed  Google Scholar 

  • Borden JR, Papoutsakis ET (2007) Dynamics of genomic-library enrichment and identification of solvent tolerance genes for Clostridium acetobutylicum. Appl Environ Microbiol 73(9):3061–3068. doi:10.1128/AEM.02296-06

  • Bustard MT, Whiting S, Cowan DA, Wright PC (2002) Biodegradation of high-concentration isopropanol by a solvent-tolerant thermophile, Bacillus pallidus. Extremophiles 6(4):319–323. doi:10.1007/s00792-001-0260-5

  • Clejan S, Krulwich TA, Mondrus KR, Seto-Young D (1986) Membrane lipid composition of obligately and facultatively alkalophilic strains of Bacillus spp. J Bacteriol 168(1):334–340

    CAS  PubMed Central  PubMed  Google Scholar 

  • Connor MR, Liao JC (2009) Microbial production of advanced transportation fuels in non-natural hosts. Curr Opin Biotechnol 20(3):307–315. doi:10.1016/j.copbio.2009.04.002

    Article  CAS  PubMed  Google Scholar 

  • Durre P (2011) Fermentative production of butanol—the academic perspective. Curr Opin Biotechnol 22(3):331–336. doi:10.1016/j.copbio.2011.04.010

    Article  PubMed  Google Scholar 

  • Fischer CR, Klein-Marcuschamer D, Stephanopoulos G (2008) Selection and optimization of microbial hosts for biofuels production. Metab Eng 10(6):295–304. doi:10.1016/j.ymben.2008.06.009

    Article  CAS  PubMed  Google Scholar 

  • Fortman JL, Chhabra S, Mukhopadhyay A, Chou H, Lee TS, Steen E, Keasling JD (2008) Biofuel alternatives to ethanol: pumping the microbial well. Trends in Biotechnology 26(7):375–381. doi:http://dx.doi.org/10.1016/j.tibtech.2008.03.008

  • Gidden J, Denson J, Liyanage R, Ivey DM, Lay JO (2009) Lipid compositions in Escherichia coli and Bacillus subtilis during growth as determined by MALDI-TOF and TOF/TOF mass spectrometry. Int J Mass Spectrom 283(1–3):178–184. doi:10.1016/j.ijms.2009.03.005

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Green EM (2011) Fermentative production of butanol—the industrial perspective. Curr Opin Biotechnol 22(3):337–343. doi:10.1016/j.copbio.2011.02.004

    Article  CAS  PubMed  Google Scholar 

  • Grundling A, Schneewind O (2007) Synthesis of glycerol phosphate lipoteichoic acid in Staphylococcus aureus. Proc Natl Acad Sci U S A 104(20):8478–8483. doi:10.1073/pnas.0701821104

    Article  PubMed Central  PubMed  Google Scholar 

  • Ingram LO (1976) Adaptation of membrane lipids to alcohols. J Bacteriol 125(2):670–678

    CAS  PubMed Central  PubMed  Google Scholar 

  • Jervis AJ, Thackray PD, Houston CW, Horsburgh MJ, Moir A (2007) SigM-responsive genes of Bacillus subtilis and their promoters. J Bacteriol 189(12):4534–4538. doi:10.1128/Jb.00130-07

  • Kabelitz N, Santos PM, Heipieper HJ (2003) Effect of aliphatic alcohols on growth and degree of saturation of membrane lipids in Acinetobacter calcoaceticus. FEMS Microbiol Lett 220(2):223–227

    Article  CAS  PubMed  Google Scholar 

  • Kataoka N, Tajima T, Kato J, Rachadech W, Vangnai AS (2011) Development of butanol-tolerant Bacillus subtilis strain GRSW2-B1 as a potential bioproduction host. AMB Express 1(1):10. doi:10.1186/2191-0855-1-10

  • Kilelee E, Pokorny A, Yeaman MR, Bayer AS (2010) Lysyl-phosphatidylglycerol attenuates membrane perturbation rather than surface association of the cationic antimicrobial peptide 6W-RP-1 in a model membrane system: implications for daptomycin resistance. Antimicrob Agents Chemother 54(10):4476–4479. doi:10.1128/AAC.00191-10

  • Kongpol A, Kato J, Vangnai AS (2008) Isolation and characterization of Deinococcus geothermalis T27, a slightly thermophilic and organic solvent-tolerant bacterium able to survive in the presence of high concentrations of ethyl acetate. FEMS Microbiol Lett 286(2):227–235. doi:10.1111/j.1574-6968.2008.01273.x

  • Lee AG (2004) How lipids affect the activities of integral membrane proteins. Biochim Biophys Acta 1666(1–2):62–87. doi:10.1016/j.bbamem.2004.05.012

    Article  CAS  PubMed  Google Scholar 

  • Lepage C, Fayolle F, Hermann M, Vandecasteele JP (1987) Changes in membrane lipid composition of Clostridium acetobutylicum during acetone-butanol fermentation: effects of solvents, growth temperature and pH. J Gen Microbiol 133(1):103–110

    CAS  Google Scholar 

  • Liu S, Qureshi N (2009) How microbes tolerate ethanol and butanol. New Biotechnology 26(3–4):117–121. doi:10.1016/j.nbt.2009.06.984

  • Lopez CS, Alice AF, Heras H, Rivas EA, Sanchez-Rivas C (2006) Role of anionic phospholipids in the adaptation of Bacillus subtilis to high salinity. Microbiology 152(Pt 3):605–616. doi:10.1099/mic.0.28345-0

    Article  CAS  PubMed  Google Scholar 

  • Matias VR, Beveridge TJ (2008) Lipoteichoic acid is a major component of the Bacillus subtilis periplasm. J Bacteriol 190(22):7414–7418. doi:10.1128/JB.00581-08

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Matsumoto M, de Bont JA, Isken S (2002) Isolation and characterization of the solvent-tolerant Bacillus cereus strain R1. J Biosci Bioeng 94(1):45–51

    Article  CAS  PubMed  Google Scholar 

  • Matsuoka S, Hashimoto M, Kamiya Y, Miyazawa T, Ishikawa K, Hara H, Matsumoto K (2011) The Bacillus subtilis essential gene dgkB is dispensable in mutants with defective lipoteichoic acid synthesis. Genes Genet Syst 86(6):365–376

    Article  CAS  PubMed  Google Scholar 

  • Nakano MM, Dailly YP, Zuber P, Clark DP (1997) Characterization of anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end products and genes required for growth. J Bacteriol 179(21):6749–6755

    CAS  PubMed Central  PubMed  Google Scholar 

  • Neumann G, Veeranagouda Y, Karegoudar TB, Sahin O, Mausezahl I, Kabelitz N, Kappelmeyer U, Heipieper HJ (2005) Cells of Pseudomonas putida and Enterobacter sp. adapt to toxic organic compounds by increasing their size. Extremophiles 9(2):163–168. doi:10.1007/s00792-005-0431-x

    Article  CAS  PubMed  Google Scholar 

  • Ni Y, Sun Z (2009) Recent progress on industrial fermentative production of acetone-butanol-ethanol by Clostridium acetobutylicum in China. Appl Microbiol Biotechnol 83(3):415–423. doi:10.1007/s00253-009-2003-y

    Article  CAS  PubMed  Google Scholar 

  • Nicolaou SA, Gaida SM, Papoutsakis ET (2010) A comparative view of metabolite and substrate stress and tolerance in microbial bioprocessing: from biofuels and chemicals, to biocatalysis and bioremediation. Metab Eng 12(4):307–331. doi:10.1016/j.ymben.2010.03.004

    Article  CAS  PubMed  Google Scholar 

  • Nielsen LE, Kadavy DR, Rajagopal S, Drijber R, Nickerson KW (2005) Survey of extreme solvent tolerance in Gram-positive cocci: membrane fatty acid changes in Staphylococcus haemolyticus grown in toluene. Appl Environ Microbiol 71(9):5171–5176. doi:10.1128/AEM.71.9.5171-5176.2005

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Petersohn A, Brigulla M, Haas S, Hoheisel JD, Volker U, Hecker M (2001) Global analysis of the general stress response of Bacillus subtilis. J Bacteriol 183(19):5617–5631. doi:10.1128/JB.183.19.5617-5631.2001

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Rigomier D, Bohin JP, Lubochinsky B (1980) Effects of ethanol and methanol on lipid metabolism in Bacillus subtilis. J Gen Microbiol 121(1):139–149

    CAS  PubMed  Google Scholar 

  • Saghatelian A, Trauger SA, Want EJ, Hawkins EG, Siuzdak G, Cravatt BF (2004) Assignment of endogenous substrates to enzymes by global metabolite profiling. Biochemistry 43(45):14332–14339

    Article  CAS  PubMed  Google Scholar 

  • Salzberg LI, Helmann JD (2008) Phenotypic and transcriptomic characterization of Bacillus subtilis mutants with grossly altered membrane composition. J Bacteriol 190(23):7797–7807. doi:10.1128/JB.00720-08

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Schirner K, Marles-Wright J, Lewis RJ, Errington J (2009) Distinct and essential morphogenic functions for wall- and lipo-teichoic acids in Bacillus subtilis. EMBO J 28(7):830–842. doi:10.1038/emboj.2009.25

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Smith CA, O’Maille G, Want EJ, Qin C, Trauger SA, Brandon TR, Custodio DE, Abagyan R, Siuzdak G (2005) METLIN: a metabolite mass spectral database. Ther Drug Monit 27(6):747–751

    Article  CAS  PubMed  Google Scholar 

  • Smith CA, Want EJ, O’Maille G, Abagyan R, Siuzdak G (2006) XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal Chem 78(3):779–787. doi:10.1021/ac051437y

    Article  CAS  PubMed  Google Scholar 

  • Spizizen J (1958) Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc Natl Acad Sci U S A 44(10):1072–1078

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Thackray PD, Moir A (2003) SigM, an extracytoplasmic function sigma factor of Bacillus subtilis, is activated in response to cell wall antibiotics, ethanol, heat, acid, and superoxide stress. J Bacteriol 185(12):3491–3498. doi:10.1128/Jb.185.12.3491-3498.2003

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Torres S, Pandey A, Castro GR (2011) Organic solvent adaptation of Gram positive bacteria: applications and biotechnological potentials. Biotechnol Adv 29(4):442–452. doi:10.1016/j.biotechadv.2011.04.002

    Article  CAS  PubMed  Google Scholar 

  • Vinayavekhin N, Saghatelian A (2009) Regulation of alkyl-dihydrothiazole-carboxylates (ATCs) by iron and the pyochelin gene cluster in Pseudomonas aeruginosa. ACS Chem Biol 4(8):617–623. doi:10.1021/cb900075n

    Article  CAS  PubMed  Google Scholar 

  • Vinayavekhin N, Saghatelian A (2010) Untargeted metabolomics. Current protocols in molecular biology/edited by Frederick M Ausubel [et al] Chapter 30:Unit 30 1 1–24. doi:10.1002/0471142727.mb3001s90

  • Vinayavekhin N, Saghatelian A (2011) Discovery of a protein-metabolite interaction between unsaturated fatty acids and the nuclear receptor Nur77 using a metabolomics approach. J Am Chem Soc 133(43):17168–17171. doi:10.1021/ja208199h

    Article  CAS  PubMed  Google Scholar 

  • Weber FJ, de Bont JA (1996) Adaptation mechanisms of microorganisms to the toxic effects of organic solvents on membranes. Biochim Biophys Acta 1286(3):225–245

    Article  CAS  PubMed  Google Scholar 

  • Wormann ME, Corrigan RM, Simpson PJ, Matthews SJ, Grundling A (2011) Enzymatic activities and functional interdependencies of Bacillus subtilis lipoteichoic acid synthesis enzymes. Mol Microbiol 79(3):566–583. doi:10.1111/j.1365-2958.2010.07472.x

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Zingaro KA, Papoutsakis ET (2012) Toward a semisynthetic stress response system to engineer microbial solvent tolerance. MBio 3(5). doi:10.1128/mBio.00308-12

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Acknowledgments

We thank Sitthiporn Associates Co., Ltd. for providing us with the Waters Alliance e2695 high performance LC instrument; the Food Research and Testing Laboratory, Faculty of Science, Chulalongkorn University for access to the nitrogen concentrator; the Thai Government Stimulus Package 2 (TKK2555) for the financial support to acquire the Bruker MicrOTOF Q-II mass spectrometer; Poompat Aroonsri for his initial help with the project; and Dr. Robert Douglas John Butcher (Publication Counseling Unit, Chulalongkorn University) for his thorough checking of English usage and constructive comments in preparing this manuscript. This work was supported by the Thailand Research Fund (TRG5780194) and the Ratchadapisek Somphot Fund of Chulalongkorn University. The opinions expressed in this paper are the sole responsibility of the authors and do not necessarily reflect those of the Thailand Research Fund or Chulalongkorn University.

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand

    Nawaporn Vinayavekhin & Polkit Sangvanich

  2. Biological Sciences Program, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand

    Gumpanat Mahipant

  3. Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand

    Gumpanat Mahipant & Alisa S. Vangnai

  4. Center of Excellence on Hazardous Substance Management (HSM), Chulalongkorn University, Bangkok, 10330, Thailand

    Alisa S. Vangnai

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  1. Nawaporn Vinayavekhin
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Correspondence to Nawaporn Vinayavekhin.

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Vinayavekhin, N., Mahipant, G., Vangnai, A.S. et al. Untargeted metabolomics analysis revealed changes in the composition of glycerolipids and phospholipids in Bacillus subtilis under 1-butanol stress. Appl Microbiol Biotechnol 99, 5971–5983 (2015). https://doi.org/10.1007/s00253-015-6692-0

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