RT-qPCR analysis of putative beer-spoilage gene expression during growth of Lactobacillus brevis BSO 464 and Pediococcus claussenii ATCC BAA-344T in beer

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• 作者：Jordyn Bergsveinson (1)
  Vanessa Pittet (1)
  Barry Ziola (1) b.ziola@usask.ca
• 关键词：Beer spoilage – ; Gene expression – ; Hop resistance – ; Lactic acid bacteria – ; Reference gene normalization – ; RT ; qPCR
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2012
• 出版时间：October 2012
• 年：2012
• 卷：96
• 期：2
• 页码：461-470
• 全文大小：374.1 KB
• 参考文献：1. Bamforth CW (2006) Scientific principles of malting and brewing. American Society of Brewing Chemists, St. Paul
  2. Behr J, G盲nzle MG, Vogel RF (2006) Characterization of a highly hop-resistant Lactobacillus brevis strain lacking hop transport. Appl Environ Microbiol 72:6483–6492
  3. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622
  4. De Man JC, Rogosa M, Sharpe ME (1960) A medium for the cultivation of Lactobacilli. J Appl Bacteriol 23:130–135
  5. Derveaux S, Vandesompele J, Hellemans J (2010) How to do successful gene expression analysis using real-time PCR. Methods 50:227–230
  6. Desroche N, Beltramo C, Guzzo J (2005) Determination of an internal control to apply reverse transcription quantitative PCR to study stress response in the lactic acid bacterium Oenococcus oeni. J Microbiol Methods 60:325–333
  7. Dheda K, Huggett JF, Bustin SA, Johnson MA, Rook G, Zumla A (2004) Validation of housekeeping genes for normalizing RNA expression in real-time PCR. BioTechniques 37:112–119
  8. Dobson CM, Deneer H, Lee S, Hemmingsen S, Glaze S, Ziola B (2002) Phylogenetic analysis of the genus Pediococcus, including Pediococcus claussenii sp. nov., a novel lactic acid bacterium isolated from beer. Int J Syst Evol Microbiol 52:2003–2010
  9. Fiocco D, Crisetti E, Capozzi V, Spano G (2008) Validation of an internal control gene to apply reverse transcription quantitative PCR to study heat, cold, and ethanol stresses in Lactobacillus plantarum. World J Microbiol Biotechnol 24:899–902
  10. Fujii T, Nakashima K, Hayashi N (2005) Random amplified polymorphic DNA-PCR based cloning of markers to identify the beer-spoilage strains of Lactobacillus brevis, Pediococcus damnosus, Lactobacillus collinoides and Lactobacillus coryniformis. J Appl Microbiol 98:1209–1220
  11. Haakensen MC, Butt L, Chaban B, Deneer H, Ziola B, Dowgiert T (2007) horA-specific real-time PCR for detection of beer-spoilage lactic acid bacteria. J Am Soc Brew Chem 65:157–165
  12. Haakensen M, Schubert A, Ziola B (2008) Multiplex PCR for putative Lactobacillus and Pediococcus beer-spoilage genes and ability of gene presence to predict growth in beer. J Am Soc Brew Chem 66:63–70
  13. Haakensen M, Pittet V, Morrow K, Schubert A, Ferguson J, Ziola B (2009) Ability of novel ATP-binding cassette multidrug resistance genes to predict growth of Pediococcus isolates in beer. J Am Soc Brew Chem 67:170–176
  14. Hayashi N, Ito M, Horiike S, Taguchi H (2001) Molecular cloning of a putative divalent-cation transporter gene as a new genetic marker for the identification of Lactobacillus brevis strains capable of growing in beer. Appl Microbiol Biotechnol 55:596–603
  15. Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8:R19
  16. Huggett J, Dheda K, Bustin S, Zumla A (2005) Real-time RT-PCR normalisation; strategies and considerations. Genes Immun 6:279–284
  17. Iijima K, Suzuki K, Ozaki K, Yamashita H (2006) horC confers beer-spoilage ability on hop-sensitive Lactobacillus brevis ABBC45cc. J Appl Microbiol 100:1282–1288
  18. Kandler O (1983) Carbohydrate metabolism in lactic acid bacteria. Anton Leeuw 49:209–224
  19. Marco ML, Kleerebezem M (2008) Assessment of real-time RT-PCR for quantification of Lactobacillus plantarum gene expression during stationary phase and nutrient starvation. J Appl Microbiol 104:587–594
  20. Pittet V, Abegunde T, Marfleet T, Haakensen M, Morrow K, Jayaprakash T, Schroeder K, Trost B, Byrns S, Bergsveinson J, Kusalik A, Ziola B (2012) Complete genome sequence of the beer spoilage organism Pediococcus claussenii ATCC BAA-344T. J Bacteriol 194:1271–1272
  21. Preissler P, Behr J, Vogel RF (2010) Detection of beer-spoilage Lactobacillus brevis strains by reduction of resazurin. J Inst Brew 116:399–404
  22. Priest FG, Campbell I (2003) Brewing microbiology. Kluwer Academic/Plenum Publishers, New York
  23. Sakamoto K, Konings WN (2003) Beer spoilage bacteria and hop resistance. Int J Food Microbiol 89:105–124
  24. Sakamoto K, van Veen HW, Saito H, Kobayashi H, Konings WN (2002) Membrane-bound ATPase contributes to hop resistance of Lactobacillus brevis. Appl Environ Microbiol 68:5374–5378
  25. Sami M, Yamashita H, Hirono T, Kadokura H, Kitamoto K, Yoda K, Yamasaki M (1997) Hop-resistant Lactobacillus brevis contains a novel plasmid harboring a multidrug resistance-like gene. J Ferment Bioeng 84:1–6
  26. Simpson WJ (1993a) Studies on the sensitivity of lactic acid bacteria to hop bitter acids. J Inst Brew 99:405–411
  27. Simpson WJ (1993b) Ionophoric action of trans-isohumulone on Lactobacillus brevis. J Gen Microbiol 139:1041–1045
  28. Simpson WJ, Fernandez JL (1992) Selection of beer-spoilage lactic acid bacteria and induction of their ability to grow in beer. Lett Appl Microbiol 14:13–16
  29. Simpson WJ, Fernandez JL (1994) Mechanism of resistance of lactic acid bacteria to trans-isohumulone. J Am Soc Brew Chem 52:9–11
  30. Suzuki K, Sami M, Kadokura H, Nakajima H, Kitamoto K (2002) Biochemical characterization of horA-independent hop resistance mechanism in Lactobacillus brevis. Int J Food Microbiol 76:223–230
  31. Suzuki K, Koyanagi M, Yamashita H (2004) Genetic characterization of non-spoilage variant isolated from beer-spoilage Lactobacillus brevis ABBC45. J Appl Microbiol 96:946–953
  32. Suzuki K, Iijima K, Ozaki K, Yamashita H (2005) Isolation of a hop-sensitive variant of Lactobacillus lindneri and identification of genetic markers for beer spoilage ability of lactic acid bacteria. Appl Environ Microbiol 71:5089–5097
  33. Suzuki K, Iijima K, Sakamoto K, Sami M, Yamashita H (2006) A review of hop resistance in beer spoilage lactic acid bacteria. J Inst Brew 112:173–191
  34. Theis T, Skurray RA, Brown MH (2007) Identification of suitable internal controls to study expression of a Staphylococcus aureus multidrug resistance system by quantitative real-time PCR. J Microbiol Methods 70:355–362
  35. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:0034.1–0034.11
  36. Willems E, Leyns L, Vandesompele J (2008) Standardization of real-time PCR gene expression data from independent biological replicates. Anal Biochem 379:127–129
• 作者单位：1. Department of Pathology and Laboratory Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
• ISSN：1432-0614

文摘

Lactic acid bacteria (LAB) contamination of beer presents a continual economic threat to brewers. Interestingly, only certain isolates of LAB can grow in the hostile beer environment (e.g., as studied here, Lactobacillus brevis BSO 464 (Lb464) and a non-ropy isolate of Pediococcus claussenii ATCC BAA-344T (Pc344NR)), indicating that significant genetic specialization is required. The genes hitA, horA, horB, horC, and bsrA, which have been proposed to confer beer-spoiling ability to an organism, are suspected of counteracting the antimicrobial effects of hops. However, these genes are not present in the same combination (if at all) across beer-spoiling organisms. As such, we sought to investigate the extent to which these genes participate during Lb464 and Pc344NR mid-logarithmic growth in beer through reverse transcription quantitative PCR analysis. We first determined the optimal reference gene set needed for data normalization and, for each bacterium, established that two genes were needed for accurate assessment of gene expression. Following this, we found that horA expression was induced for Pc344NR, but not for Lb464, during growth in beer. Instead, horC expression was dramatically increased in Lb464 when growing in beer, whereas no change was detected for the other putative beer-spoilage-related genes. This indicates that HorC may be one of the principle mediators enabling growth of Lb464 in beer, whereas in Pc344NR, this may be attributable to HorA. These findings not only reveal that Lb464 and Pc344NR are unique in their beer-specific genetic expression profile but also indicate that a range of genetic specialization exists among beer-spoilage bacteria.