Abstract
The ability of bacteria to tolerate acid stress plays an important role in their growth and survival. In particular, aciduric bacteria have several survival systems that prevent cell damage from acid stress. In this study, the effect of the bacterial stress induced by pre-adaptation at different pH values on the cellular macromolecules of Lactobacillus plantarum was investigated using Raman spectroscopy and Fourier transform infrared spectroscopy. The expression of key genes was also quantified to provide understanding of the transcriptional response of the cells to lethal acid stress conditions. Principal component analysis of the spectra exhibited marked differences in the spectral regions associated with carbohydrates, lipids, proteins, and nucleic acids for all acid-stressed cells compared to those of untreated control cells. The changes in spectroscopic and transcriptomic profiles that were observed revealed alterations in bacterial cell wall composition after acid treatment. The results suggest the existence of a complex bacterial stress response in which modifications of cellular compounds from pre-adaption at low pH are involved. This study demonstrates the potential application of vibrational spectroscopy techniques to discriminate between intact and injured bacterial cells as well as to study their stress responses after exposure to acid environments during food processing.
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References
Alvarez-Ordonez A, Mouwen DJM, Lopez M, Prieto M (2011) Fourier transform infrared spectroscopy as a tool to characterize molecular composition and stress response in foodborne pathogenic bacteria. J Microbiol Methods 84(3):369–378. https://doi.org/10.1016/j.mimet.2011.01.009
Ashton L, Lau K, Winder CL, Goodacre R (2011) Raman spectroscopy: lighting up the future of microbial identification. Future Microbiol 6(9):991–997. https://doi.org/10.2217/fmb.11.89
Bocklitz T, Walter A, Hartmann K, Roesch P, Popp J (2011) How to pre-process Raman spectra for reliable and stable models? Anal Chim Acta 704(1–2):47–56. https://doi.org/10.1016/j.aca.2011.06.043
Bore E, Langsrud S, Langsrud O, Rode TM, Holck A (2007) Acid-shock responses in Staphylococcus aureus investigated by global gene expression analysis. Microbiology-Sgm 153:2289–2303. https://doi.org/10.1099/mic.0.2007/005942-0
Cadusch PJ, Hlaing MM, Wade SA, McArthur SL, Stoddart PR (2013) Improved methods for fluorescence background subtraction from Raman spectra. J Raman Spectrosc 44(11):1587–1595
Cagno RD, Coda R, Angelis M, Gobbetti M (2013) Exploitation of vegetables and fruits through lactic acid fermentation. Food Microbiol 33(1):1–10
Cappa F, Cattivelli D, Cocconcelli PS (2005) The uvrA gene is involved in oxidative and acid stress responses in Lactobacillus helveticus CNBL1156. Res Microbiol 156(10):1039–1047. https://doi.org/10.1016/j.resmic.2005.06.003
Carvalho AL, Cardoso FS, Bohn A, Neves AR, Santos H (2011) Engineering trehalose synthesis in Lactococcus lactis for improved stress tolerance. Appl Environ Microbiol 77(12):4189–4199. https://doi.org/10.1128/aem.02922-10
Chung HJ, Bang W, Drake MA (2006) Stress response of Escherichia coli. Compr Rev Food Sci Food Saf 5(3):52–64. https://doi.org/10.1111/j.1541-4337.2006.00002.x
Cotter PD, Hill C (2003) Surviving the acid test: responses of gram-positive bacteria to low pH. Microbiol Mol Biol Rev 67(3):429–453. https://doi.org/10.1128/mmbr.37.3.429-453.2003
Foster JW, Hall HK (1991) Inducible pH homeostasis and the acid tolerance response of Salmonella typhimurium. J Bacteriol 173(16):5129–5135
Fozo EA, Quivey RG (2004) Shifts in the membrane fatty acid profile of Streptococcus mutans enhance survival in acidic environments. Appl Environ Microbiol 70(2):929–936. https://doi.org/10.1128/aem.70.2.929-936.2004
G-Alegria E, Lopez I, Ruiz JI, Saenz J, Fernandez E, Zarazaga M, Dizy M, Torres C, Ruiz-Larrea F (2004) High tolerance of wild Lactobacillus plantarum and Oenococcus oeni strains to lyophilisation and stress environmental conditions of acid pH and ethanol. FEMS Microbiol Lett 230(1):53–61. https://doi.org/10.1016/s0378-1097(03)00854-1
Harz A, Roesch P, Popp J (2009) Vibrational spectroscopy—a powerful tool for the rapid identification of microbial cells at the single-cell level. Cytometry Part A 75A(2):104–113. https://doi.org/10.1002/cyto.a.20682
Hemert, Sv, Meijerink M, Molenaar D, Bron PA, Vos Pd, Kleerebezem M, Wells JM, Marco ML (2010) Identification of Lactobacillus plantarum genes modulating the cytokine response of human peripheral blood mononuclear cells. BMC Microbiol 10(Nov.):13pp.-13pp
Hlaing MM, Dunn M, McArthur SL, Stoddart PR (2014) Raman spectroscopy for bacterial identification: effects of sample preparation and storage. Int J Integr Biol 15(1):11–17
Hlaing MM, Dunn M, Stoddart PR, McArthur SL (2016) Raman spectroscopic identification of single bacterial cells at different stages of their lifecycle. Vib Spectrosc 86:81–89. https://doi.org/10.1016/j.vibspec.2016.06.008
Hlaing MM, Wood BR, McNaughton D, Ying D, Dumsday G, Augustin MA (2017) Effect of drying methods on protein and DNA conformation changes in Lactobacillus rhamnosus GG cells by Fourier transform infrared spectroscopy. J Agric Food Chem 65(8):1724–1731. https://doi.org/10.1021/acs.jafc.6b05508
Huang WE, Griffiths RI, Thompson IP, Bailey MJ, Whiteley AS (2004) Raman microscopic analysis of single microbial cells. Anal Chem 76(15):4452–4458. https://doi.org/10.1021/ac049753k
Huang WE, Li M, Jarvis RM, Goodacre R, Banwart SA (2010) Shining light on the microbial world: the application of Raman microspectroscopy. In: Laskin AI, Sariaslani S, Gadd GM (eds) Advances in applied microbiology, Vol 70. Advances in Applied Microbiology, vol 70, pp 153–186
Kaushik JK, Kumar A, Duary RK, Mohanty AK, Grover S, Batish VK (2009) Functional and probiotic attributes of an indigenous isolate of Lactobacillus plantarum. PLoS One 4(12). https://doi.org/10.1371/journal.pone.0008099
Kawahata M, Masaki K, Fujii T, Iefuji H (2006) Yeast genes involved in response to lactic acid and acetic acid: acidic conditions caused by the organic acids in Saccharomyces cerevisiae cultures induce expression of intracellular metal metabolism genes regulated by Aft1p. FEMS Yeast Res 6(6):924–936. https://doi.org/10.1111/j.1567-1364.2006.00089.x
Kleerebezem M, Boekhorst J, van Kranenburg R, Molenaar D, Kuipers OP, Leer R, Tarchini R, Peters SA, Sandbrink HM, Fiers M, Stiekema W, Lankhorst RMK, Bron PA, Hoffer SM, Groot MNN, Kerkhoven R, de Vries M, Ursing B, de Vos WM, Siezen RJ (2003) Complete genome sequence of Lactobacillus plantarum WCFS1. Proc Natl Acad Sci U S A 100(4):1990–1995. https://doi.org/10.1073/pnas.0337704100
Kong J, Yu S (2007) Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochim Biophys Sin 39(8):549–559. https://doi.org/10.1111/j.1745-7270.2007.00320.x
Koponen J, Laakso K, Koskenniemi K, Kankainen M, Savijoki K, Nyman TA, de Vos WM, Tynkkynen S, Kalkkinen N, Varmanen P (2012) Effect of acid stress on protein expression and phosphorylation in Lactobacillus rhamnosus GG. J Proteome 75(4):1357–1374. https://doi.org/10.1016/j.jprot.2011.11.009
Kumar AM, Murugalatha N (2012) Isolation of Lactobacillus plantarum from cow milk and screening for the presence of sugar alcohol producing gene. J Microbiol Antimicrob 4(1):16–22
Leja K, Dembczynski R, Bialas W, Jankowski T (2009) Production of dry Lactobacillus rhamnosus GG preparations by spray drying and lyophilization in aqueous two-phase systems. Acta Sci Pol Technol Aliment 8(4):39–49
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method. Methods 25(4):402–408. https://doi.org/10.1006/meth.2001.1262
Maurer LM, Yohannes E, Bondurant SS, Radmacher M, Slonczewski JL (2005) pH regulates genes for flagellar motility, catabolism, and oxidative stress in Escherichia coli K-12. J Bacteriol 187(1):304–319. https://doi.org/10.1128/jb.187.1.304-319.2005
Moat AG, Foster JW, Spector MP (2003) Microbial stress responses microbial physiology, 1st edn. John Wiley & Sons, Inc
Moritz TJ, Taylor DS, Polage CR, Krol DM, Lane SM, Chan JW (2010) Effect of cefazolin treatment on the nonresonant Raman signatures of the metabolic state of individual Escherichia coli cells. Anal Chem 82(7):2703–2710. https://doi.org/10.1021/ac902351a
Naumann D (2001) FT-infrared and FT-Raman spectroscopy in biomedical research. Appl Spectrosc Rev 36(2–3):239–298. https://doi.org/10.1081/asr-100106157
Nawaz H, Bonnier F, Knief P, Howe O, Lyng FM, Meade AD, Byrne HJ (2010) Evaluation of the potential of Raman microspectroscopy for prediction of chemotherapeutic response to cisplatin in lung adenocarcinoma. Analyst 135(12):3070–3076. https://doi.org/10.1039/c0an00541j
Nguyen TDT, Kang JH, Lee MS (2007) Characterization of Lactobacillus plantarum PH04, a potential probiotic bacterium with cholesterol-lowering effects. Int J Food Microbiol 113(3):358–361. https://doi.org/10.1016/j.ijfoodmicro.2006.08.015
Niedzielin K, Kordecki H, Birkenfeld B (2001) A controlled, double-blind, randomized study on the efficacy of Lactobacillus plantarum 299V in patients with irritable bowel syndrome. Eur J Gastroenterol Hepatol 13(10):1143–1147. https://doi.org/10.1097/00042737-200110000-00004
Prueckler M, Lorenz C, Endo A, Kraler M, Duerrschmid K, Hendriks K, FSd S, Auterith E, Kneifel W, Michlmayr H (2015) Comparison of homo- and heterofermentative lactic acid bacteria for implementation of fermented wheat bran in bread. Food Microbiol 49:211–219
Quivey RG, Faustoferri R, Monahan K, Marquis R (2000) Shifts in membrane fatty acid profiles associated with acid adaptation of Streptococcus mutans. FEMS Microbiol Lett 189(1):89–92. https://doi.org/10.1016/s0378-1097(00)00258-5
Rina W, Wenyi Z, Tiansong S, Junrui W, Xiqing Y, He M, Heping Z (2011) Proteomic analysis of responses of a new probiotic bacterium Lactobacillus casei Zhang to low acid stress. Int J Food Microbiol 147(3):181–187
Savitzky A, Golay MJE (1964) Smoothing and differentiation of data by simplified least squares procedures. Anal Chem 36(8):1627. https://doi.org/10.1021/ac60214a047
Sawitzki MC, Fiorentini AM, Bertol TM, Sant'anna ES (2009) Lactobacillus plantarum strains isolated from naturally fermented sausages and their technological properties for application as starter cultures. Ciencia E Tecnologia De Alimentos 29(2):340–345
Siezen RJ, Hylckama Vlieg, JETv (2011) Genomic diversity and versatility of Lactobacillus plantarum, a natural metabolic engineer. Microbial Cell Factories 10(Suppl. 1):13pp.-13pp
Small P, Blankenhorn D, Welty D, Zinser E, Slonczewski JL (1994) Acid and base resistance in Escherichia coli and Shigella flexneri: role of rpoS and growth pH. J Bacteriol 176(6):1729–1737
Thomas GJ (1999) Raman spectroscopy of protein and nucleic acid assemblies. Annu Rev Biophys Biomol Struct 28:1–27. https://doi.org/10.1146/annurev.biophys.28.1.1
van de Guchte M, Serror P, Chervaux C, Smokvina T, Ehrlich SD, Maguin E (2002) Stress responses in lactic acid bacteria. Antonie Van Leeuwenhoek 82(1–4):187–216
Van Montfort R, Slingsby C, Vierling E (2001) Structure and function of the small heat shock protein/alpha-crystallin family of molecular chaperones. Adv Protein Chem 59:105–156
Vankeirsbilck T, Vercauteren A, Baeyens W, Van der Weken G, Verpoort F, Vergote G, Remon JP (2002) Applications of Raman spectroscopy in pharmaceutical analysis. Trac-Trends in Analytical Chemistry 21(12):869–877. https://doi.org/10.1016/s0165-9936(02)01208-6
Williams AC, Edwards HGM (1994) Fourier-transform Raman-spectroscopy of bacterial cell walls. J Raman Spectrosc 25(7–8):673–677. https://doi.org/10.1002/jrs.1250250730
Xiaonan L, Al-Qadiri HM, Mengshi L, Rasco BA (2011) Application of mid-infrared and Raman spectroscopy to the study of bacteria. Food Bioprocess Technol 4(6):919–935
Yokoigawa K, Takikawa A, Okubo Y, Umesako S (2003) Acid tolerance and gad mRNA levels of Escherichia coli O157:H7 grown in foods. Int J Food Microbiol 82(3):203–211. https://doi.org/10.1016/S0168-1605(02)00305-7
Zhu SC, Ying DY, Sanguansri L, Tang JW, Augustin MA (2013) Both stereo-isomers of glucose enhance the survival rate of microencapsulated Lactobacillus rhamnosus GG during storage in the dry state. J Food Eng 116(4):809–813
Acknowledgements
We thank Thomas Stent (Department of Microbiology, Monash University) for assisting with the ddPCR experiments.
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Hlaing, M.M., Wood, B.R., McNaughton, D. et al. Vibrational spectroscopy combined with transcriptomic analysis for investigation of bacterial responses towards acid stress. Appl Microbiol Biotechnol 102, 333–343 (2018). https://doi.org/10.1007/s00253-017-8561-5
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DOI: https://doi.org/10.1007/s00253-017-8561-5