枯草芽孢杆菌SX3411产羊毛硫细菌素subtilomycin的初步鉴定与理化特性分析
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摘要
为了确定分离得到的枯草芽孢杆菌SX3411(Bacillus subtilis SX3411)具有产羊毛硫细菌素subtilomycin的能力和subtilomycin的理化特性。利用滤纸片扩散法检测到菌株SX3411发酵液对猪链球菌(Streptococcus suis)、枯草芽孢杆菌(非本筛选菌株)、金黄色葡萄球菌(Staphylococcus aureus)和嗜水性单胞杆菌(Aeromonas hydrophila)具有抑菌作用。通过基因簇克隆和测序,表明其中抑菌物质主要为羊毛硫细菌素subtilomycin。理化特性分析表明菌株SX3411发酵液中抑菌物质具耐热和耐酸碱特性。SDS-PAGE检测表明subtilomycin分子质量在4 k Da左右。模拟胃液对subtilomycin抑菌活性影响不大,但不耐蛋白酶K和模拟肠液的处理。该研究为进一步开发应用羊毛硫细菌素subtilomycin奠定了基础。
This study was conducted to identify and characterize the lantibiotic subtilomycin produced by Bacillus subtilis SX3411. The fermentation broth was found to have anti-bacterial activities against Streptococcus suis,B. subtilis(not B. subtilis SX3411),Staphylococcus aureus,and Aeromonas hydrophila. By cloning and sequencing the gene cluster,it was preliminarily determined that the main antimicrobial substance was subtilomycin,which had a molecular weight of 4 kDa. Moreover,the subtilomycin was resistant to heat,acid and alkali,while simulated gastric juice had less effect on its anti-bacterial activity. In comparison,the subtilomycin was more sensitive to protease K and simulated intestinal juice treatment. In conclusion,this study lays a foundation for further applications of lantibiotic subtilomycin.
This study was conducted to identify and characterize the lantibiotic subtilomycin produced by Bacillus subtilis SX3411. The fermentation broth was found to have anti-bacterial activities against Streptococcus suis,B. subtilis(not B. subtilis SX3411),Staphylococcus aureus,and Aeromonas hydrophila. By cloning and sequencing the gene cluster,it was preliminarily determined that the main antimicrobial substance was subtilomycin,which had a molecular weight of 4 kDa. Moreover,the subtilomycin was resistant to heat,acid and alkali,while simulated gastric juice had less effect on its anti-bacterial activity. In comparison,the subtilomycin was more sensitive to protease K and simulated intestinal juice treatment. In conclusion,this study lays a foundation for further applications of lantibiotic subtilomycin.
引文
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[27]DENG Y,LI C Z,ZHU Y G,et al.Apn I,a transmembrane protein responsible for subtilomycin immunity,unveils a novel model for lantibiotic immunity[J].Applied and Environmental Microbiology,2014,80(20):6 303-6 315.
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[2]VLAMAKIS H,CHAI Y,BEAUREGARD P,et al.Sticking together:Building a biofilm the Bacillus subtilis way[J].Nat Rev Microbiol,2013,11(3):157-168.
[3]RAO C V,GLEKAS G D,ORDAL G W.The three adaptation systems of Bacillus subtilis chemotaxis[J].Trends Microbiol,2008,16(10):480-487.
[4]CAIRNS L S,HOBLEY L,STANLEY-WALL N R.Biofilm formation by Bacillus subtilis:New insights into regulatory strategies and assembly mechanisms[J].Molecular Microbiology,2014,93(4):587-598.
[5]HONG H A,TO E,FAKHRY S,et al.Defining the natural habitat of Bacillus spore-formers[J].Res Microbiol,2009,160(6):375-379.
[6]EARL A M,LOSICK R,KOLTER R.Ecology and genomics of Bacillus subtilis[J].Trends Microbiol,2008,16(6):269-275.
[7]CHEN Y,CAO S,CHAI Y,et al.A Bacillus subtilis sensor kinase involved in triggering biofilm formation onthe roots of tomato plants[J].Mol Microbiol,2012,85(3):418-430.
[8]CHEN Y,YAN F,CHAI Y,et al.Biocontrol of tomato wilt disease by Bacillus subtilis isolates from naturalenvironments depends on conserved genes mediating biofilm formation[J].Environ Microbiol,2013,15(3):848-864.
[9]BARBOSA T M,SERRA C R,LA-RAGIONE R M,et al.Screening for Bacillus isolates in the broiler gastrointestinal tract[J].Appl Environ Microbiol,2005,71(2):968-978.
[10]HUYNH H,KHANEJA R,BARNES I,et al.Bacillus subtilis isolated from the human gastrointestinal tract[J].Res Microbiol,2009,160(2):134-143.
[11]FACUNDO A,CARLOS B,SEBASTIAN C,et al.Microbial flora,probiotics,Bacillus subtilis and the search for a long and healthy human longevity[J].Microbial Cell,2017,4(4):133-136.
[12]赵朋超,王建华,权春善,等.枯草芽孢杆菌抗菌肽生物合成的研究进展[J].中国生物工程杂志,2010,30(10):108-113.
[13]SUMI C D,YANG B W,YEO I C,et al.Antimicrobial peptides of the genus Bacillus:A new era for antibiotics[J].Can J Microbiol,2015,61(2):93-103.
[14]白杰,贠建民,祝发明,等.枯草芽孢杆菌菌株B-3抗菌肽的分离纯化与鉴定[J].食品与发酵工业,2018,44(8):82-89.
[15]TAREQ F S,LEE M A,LEE H S,et al.Gageotetrins A-C,noncytotoxic antimicrobial linear lipopeptides from a marine bacterium Bacillus subtilis[J].Org Lett,2014,16(3):928-931.
[16]GONZALO C V G D,ZHU L,OMAN T J,et al.NMRstructure of the S-linked glycopeptide sublancin 168[J].ACS Chemical Biology,2014,9(3):796-801.
[17]ZHAO X,KUIPERS O P.Identification and classification of known and putative antimicrobial compounds produced by a wide variety of Bacillales species[J].BMC Genomics,2016,17(1):882.
[18]SZEKAT C,JACK R W,SKUTLAREK D,et al.Construction of an expression system for site-directed mutagenesis of the lantibiotic mersacidin[J].Applied and Environmental Microbiology,2003,69(7):3 777-3 783.
[19]SCHNELL N,ENTIAN K D,SCHNEIDER U,et al.Prepeptide sequence of epidermin,a ribosomally synthesized antibiotic with four sulphide-rings[J].Nature,1988,333(6 170):276-278.
[20]AMISON P G,BIBB M J,BIERBAUM G,et al.Ribosomally synthesized and post-translationally modified peptide natural products:Overview and recommendations for a universal nomenclature[J].Natural Product Reports,2013,30:108-160.
[21]MATHUR H,FIELD D,REA M C,et al.Fighting biofilms with lantibiotics and other groups of bacteriocins[J].NPJ Biofilms and Microbiomes,2018,4(1):9.
[22]LIU W,HANSEN J N.The antimicrobial effect of a structural variant of subtilin against outgrowing Bacillus cereus T spores and vegetative cells occurs by different mechanisms[J].Appl Environ Microbiol,1993,59(2):648-651.
[23]STEIN T,BORCHERT S,CONRAD B,et al.Two different lantibiotic-like peptides originate from the ericin gene cluster of Bacillus subtilis A1/3[J].J Bacteriol,2002,184(6):1 703-1 711.
[24]FUCHS S W,JASKOLLA T W,BOCHMANN S,et al.Entianin,a novel subtilin-like lantibiotic from Bacillus subtilis subsp.spizizenii DSM 15029T with high antimicrobial activity[J].Applied and Environmental Microbiology,2011,77(5):1 698-1 707.
[25]SANDIFORD S K.Genome database mining for the discovery of novel lantibiotics[J].Expert Opinion on Drug Discovery,2017,12(5):489-495.
[26]PHELAN R W,BARRET M,COTTER P D,et al.Subtilomycin:A new lantibiotic from Bacillus subtilis strain MMA7 isolated from the marine sponge Haliclona simulans[J].Mar Drugs,2013,11(6):1 878-1 898.
[27]DENG Y,LI C Z,ZHU Y G,et al.Apn I,a transmembrane protein responsible for subtilomycin immunity,unveils a novel model for lantibiotic immunity[J].Applied and Environmental Microbiology,2014,80(20):6 303-6 315.
[28]国家药典委员会.中华人民共和国药典:二部[M].北京:北京工业出版社,2005:771-792.
[29]R.E.布坎南,N.E.吉本斯,著.中国科学院微生物所《伯杰细菌鉴定手册》翻译组,译.(第八版)[M].北京:科学出版社,1984:362-366.