Discrimination of Bacillus subtilis from Other Bacillus Species Using Specific Oligonucleotide Primers for the Pyruvate Carboxylase and Shikimate Dehydrogenase Genes
0
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
Bacillus subtilis is a useful bacterium in the food industry with applications as a starter strain for fermented food and as a probiotic. However, it is difficult to discriminate B. subtilis from other Bacillus species because of high phenotypic and genetic similarity. In this study, we employed five previously constructed multilocus sequence typing (MLST) methods for the discrimination of B. subtilis from other Bacillus species and all five MLST assays clearly distinguished B. subtilis. Additionally, the 17 housekeeping genes used in the five MLST assays also clearly distinguished B. subtilis. The pyruvate carboxylase (pyrA) and shikimate dehydrogenase (aroE) genes were selected for the discrimination of B. subtilis because of their high number of polymorphic sites and the fact that they displayed the lowest homology among the 17 housekeeping genes. Specific primer sets for the pyrA and aroE genes were designed and PCR products were specifically amplified from B. subtilis, demonstrating the high specificity of the two housekeeping genes for B. subtilis. This species-specific PCR method provides a quick, simple, powerful, and reliable alternative to conventional methods in the detection and identification of B. subtilis.
Abstract
Bacillus subtilis is a useful bacterium in the food industry with applications as a starter strain for fermented food and as a probiotic. However, it is difficult to discriminate B. subtilis from other Bacillus species because of high phenotypic and genetic similarity. In this study, we employed five previously constructed multilocus sequence typing (MLST) methods for the discrimination of B. subtilis from other Bacillus species and all five MLST assays clearly distinguished B. subtilis. Additionally, the 17 housekeeping genes used in the five MLST assays also clearly distinguished B. subtilis. The pyruvate carboxylase (pyrA) and shikimate dehydrogenase (aroE) genes were selected for the discrimination of B. subtilis because of their high number of polymorphic sites and the fact that they displayed the lowest homology among the 17 housekeeping genes. Specific primer sets for the pyrA and aroE genes were designed and PCR products were specifically amplified from B. subtilis, demonstrating the high specificity of the two housekeeping genes for B. subtilis. This species-specific PCR method provides a quick, simple, powerful, and reliable alternative to conventional methods in the detection and identification of B. subtilis.
참고문헌 (34)
- Earl AM, "Ecology and genomics of Bacillus subtilis", Trends Microbiol, 16, pp.269~275 (2008)
- Batista MT, "Gut adhesive Bacillus subtilis spores as a platform for mucosal delivery of antigens", Infect. Immun, 82, pp.1414~1423 (2014)
- Leser TD, "Germination and outgrowth of Bacillus subtilis and Bacillus licheniformis spores in the gastrointestinal tract of pigs", J. Appl. Microbiol, 104, pp.1025~1033 (2008)
- Hong HA, "The use of bacterial spore formers as probiotics", FEMS Microbiol. Rev, 29, pp.813~835 (2005)
- Chen H, "Progress in Bacillus subtilis spore surface display technology towards environment, vaccine development, and biocatalysis", J. Mol. Microbiol. Biotechnol, 27, pp.159~167 (2017)
- 이나경, "Bacillus strains as human probiotics: characterization, safety, microbiome, and probiotic carrier", Food Science and Biotechnology, 28(5), pp.1297~1305 (2019), 한국식품과학회[DOI]
- Lee JH, "Determination of microbial diversity in meju, fermented cooked soya beans, using nested PCR-denaturing gradient gel electrophoresis", Lett. Appl. Microbiol, 51, pp.388~394 (2010)
- Kim TW, "Analysis of microbial communities in doenjang, a Korean fermented soybean paste, using nested PCR-denaturing gradient gel electrophoresis", Int. J. Food Microbiol, 131, pp.265~271 (2009)
- Chen T, "Molecular identification of microbial community in surface and undersurface douchi during postfermentation", J. Food Sci, 79, pp.653~658 (2014)[DOI]
- Homma K, "Treatment of natto, a fermented soybean preparation, to prevent excessive plasma vitamin K concentrations in patients taking warfarin", J. Nutr. Sci. Vitaminol, 52, pp.297~301 (2006)
- Onda T, "Time series analysis of aerobic bacterial flora during miso fermentation", Lett. Appl. Microbiol, 37, pp.162~168 (2003)
- Dash BK, "Molecular identification of a newly isolated Bacillus subtilis BI19 and optimization of production conditions for enhanced production of extracellular amylase", Biomed Res. Int, 2015, pp.859805~ (2015)
- Barbieri G, "Interplay of CodY and ScoC in the regulation of major extracellular protease genes of Bacillus subtilis", J. Bacteriol, 198, pp.907~920 (2016)
- Yi SH, "Characteristics of bacterial strains with desirable flavor compounds from Korean traditional fermented soybean paste(Doenjang)", Molecules, 26, pp.5067~ (2021)
- Nguyen T, "Determination of factors affecting the protease content generated in fermented soybean by Bacillus subtilis 1423", Energy Rep, 6, pp.831~836 (2020)
- Shukla S, "Effect of novel starter culture on reduction of biogenic amines, quality improvement, and sensory properties of doenjang, a traditional Korean soybean fermented sauce variety", J. Food Sci, 80, pp.1794~1803 (2015)
- Dajanta K, "Improvement of thua nao production using protein-rich soybean and Bacillus subtilis TN51 starter culture", Ann. Microbiol, 62, pp.785~795 (2012)
- Caulier S, "Overview of the antimicrobial compounds produced by members of the Bacillus subtilis group", Front. Microbiol, 10, pp.302~ (2019)
- nKubo Y, "Phylogenetic analysis of Bacillus subtilis strains applicable to natto(fermented soybean)production", Appl. Environ. Microbiol, 77, pp.6463~6469 (2011)
- Su Y, "Bacillus subtilis : a universal cell factory for industry, agriculture, biomaterials and medicine", Microb. Cell Fact, 19, pp.173~ (2020)
- Cihan AC, "The genetic diversity of genus Bacillus and the related genera revealed by 16S rRNA gene sequences and ardra analyses isolated from geothermal regions of turkey", Braz. J. Microbiol, 43, pp.309~324 (2012)
- Di J, "Evaluation of the potential probiotic Bacillus subtilis isolated from two ancient sturgeons on growth performance, serum immunity and disease resistance of Acipenser dabryanus", Fish Shellfish Immunol, 93, pp.711~719 (2019)
- Gonzalez MJ, "Polyphasic identification of closely related Bacillus subtilis and Bacillus amyloliquefaciens isolated from dairy farms and milk powder", J. Microbiol. Biotechnol. Food Sci, 2, pp.2326~2331 (2013)
- Xie S, "Two rapid and sensitive methods based on TaqMan qPCR and droplet digital PCR assay for quantitative detection of Bacillus subtilis in rhizosphere", J. Appl. Microbiol, 128, pp.518~527 (2020)
- Roman F, "Multilocus sequence typing : genetic diversity in Trypanosoma cruzi I(TcI)isolates from Brazilian didelphids", Parasit. Vectors, 11, pp.107~ (2018)
- Agarwal A, "Molecular and phylogenetic analysis of Chikungunya virus in central India during 2016 and 2017 outbreaks reveal high similarity with recent New Delhi and Bangladesh strains", Infect. Genet. Evol, 75, pp.103940~ (2019)
- Ashe S, "Specific oligonucleotide primers for detection of endoglucanase positive Bacillus subtilis by PCR", 3 Biotech, 4, pp.461~465 (2014)
- Huang CH, "Development of a novel PCR assay based on the gyrase B gene for species identification of Bacillus licheniformis", Mol. Cell. Probes, 26, pp.215~217 (2012)
- Boers SA, "High-throughput multilocus sequence typing : bringing molecular typing to the next level", PLoS One, 7, pp.e39630~ (2012)
- Jolley KA, "Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications", Wellcome Open Res, 3, pp.124~ (2018)
- Xuan The Le, "Exploration of genetic diversity of Bacillus spp. from industrial shrimp ponds in Vietnam by multi-locus sequence typing", Fisheries and Aquatic Sciences, 22(3), pp.1~9 (2019), 한국수산과학회[DOI]
- Boka B, "Genome analysis of a Bacillus subtilis strain reveals genetic mutations determining biocontrol properties", World J. Microbiol. Biotechnol, 35, pp.52~ (2019)
- Kabore D, "Rapid screening of starter cultures for maari based on antifungal properties", Microbiol. Res, 207, pp.66~74 (2018)
- 정도원, "Urease Characteristics and Phylogenetic Status of Bacillus paralicheniformis", Journal of Microbiology and Biotechnology, 28(12), pp.1992~1998 (2018), 한국미생물·생명공학회