苏云金芽胞杆菌XL6 BTXL6＿11095基因对生物被膜相关表型的调控
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摘要
在细菌生物被膜(bacterial biofilm, BBF)状态下,细菌往往具有更强的抗紫外线能力、环境适应性和耐药性。本课题组在前期工作中通过转座子插入突变强生物被膜产生菌苏云金芽胞杆菌(Bacillus thuringiensis,Bt)XL6,筛选出了一个可能调控生物被膜的基因BTXL6_11095。本文以该基因为研究对象,通过生物信息学手段分析该基因功能,构建了BTXL6_11095基因敲除菌株,并对其生物被膜相关表型进行了分析。生物信息学分析结果表明BTXL6_11095基因的结构域为PAS-GGDEF-GGDEF-EAL。生物被膜相关表型分析表明,与野生菌株相比,基因敲除菌株的生长曲线基本不变,群游能力上升,生物被膜形成能力减弱、UV-B抗性降低。本研究通过对BTXL6_11095基因表型变化综合评估,从功能角度揭示此基因对Bt XL6生物被膜相关表型的影响,为进一步解析Bt XL6生物被膜调控网络和构建高抗UV的工程生物被膜奠定了基础。
Under the condition of bacterial biofilm(BBF), bacteria tend to have higher anti-UV-B ability, better environmental adaptability and stronger pesticide resistance. In our previous studies, the BTXL6_11095 gene putatively with the function regulating biofilm formation was screened by transposon insertion into the genome of Bacillus thuringiensis(Bt) XL6 with strong biofilm formation ability. In this study, the potential functions of this gene were analyzed using bioinformatics using BTXL6_11095 knockout mutant and through analysis of the biofilm related phenotypes of the mutant. Bioinformatics analysis showed that BTXL6_11095 gene had the PASGGDEF-GGDEF-EAL motif. BTXL6_11095 knockout mutant showed no different growth curve relative to the wild type, but exhibited higher swimming ability, lower biofilm formation ability, and lower resistance to UV-B. This study, through comprehensive evaluation of phenotypic changes of BTXL6_11095 knockout mutant, has revealed the effect of this gene on the biofilm formation ability of Bt XL6, which serves to elucidate biofilm regulation network of Bt XL6 and construct its engineered biofilm with high biofilm formation ability.
Under the condition of bacterial biofilm(BBF), bacteria tend to have higher anti-UV-B ability, better environmental adaptability and stronger pesticide resistance. In our previous studies, the BTXL6_11095 gene putatively with the function regulating biofilm formation was screened by transposon insertion into the genome of Bacillus thuringiensis(Bt) XL6 with strong biofilm formation ability. In this study, the potential functions of this gene were analyzed using bioinformatics using BTXL6_11095 knockout mutant and through analysis of the biofilm related phenotypes of the mutant. Bioinformatics analysis showed that BTXL6_11095 gene had the PASGGDEF-GGDEF-EAL motif. BTXL6_11095 knockout mutant showed no different growth curve relative to the wild type, but exhibited higher swimming ability, lower biofilm formation ability, and lower resistance to UV-B. This study, through comprehensive evaluation of phenotypic changes of BTXL6_11095 knockout mutant, has revealed the effect of this gene on the biofilm formation ability of Bt XL6, which serves to elucidate biofilm regulation network of Bt XL6 and construct its engineered biofilm with high biofilm formation ability.
引文
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[22]李澜,陈锦灿,杨兆元,等.纳米Mg(OH)2对苏云金芽胞杆菌蛋白杀虫活性及抗紫外能力影响的研究[J].农业生物技术学报,2015,23(11):1452-1457.
[23]Kobayashi K.Gradual activation of the response regulator DegU controls serial expression of genes for flagellum formation and biofilm formation in Bacillus subtilis[J].Molecular Microbiology,2007,66(2):395-409.
[24]Panzer S,Losick R,Sun D,et al.Evidence for an additional temporal class of gene expression in the forespore compartment of sporulating Bacillus subtilis[J].Journal of Bacteriology,1989,171(1):561-564.
[25]凡肖,束长龙,关雄,等.细菌生物被膜在生物防治中的作用[J].中国生物防治学报,2018,34(5):791-801.
[26]Mata A R,Pacheco C M,Pérez J F C,et al.In silico comparative analysis of GGDEF and EAL domain signaling proteins from the Azospirillum genomes[J].BMC Microbiology,2018,18(1):20.
[27]Skariyachan S,Sridhar V S,Packirisamy S,et al.Recent perspectives on the molecular basis of biofilm formation by Pseudomonas aeruginosa and approaches for treatment and biofilm dispersal[J].Folia Microbiologica,2018,63(4):413-432.
[28]屈铁军.变形链球菌GGDEF结构域基因的克隆表达及功能初步研究[D].第四军医大学,2008:63-94.
[29]Fu Y,Yu Z,Liu S,et al.c-di-GMP regulates various phenotypes and insecticidal activity of gram-positive Bacillus thuringiensis[J].Frontiers in Microbiology,2018,18(1):20.
[30]Carlson H K,Vance R E,Marletta M A.H-NOX regulation of c-di-GMP metabolism and biofilm formation in Legionella pneumophila[J].Molecular Microbiology,2010,77(4):930-942.
[31]刘舒.苏云金芽胞杆菌BMB171中第二信使c-di-GMP合成与降解相关基因的研究[D].武汉:华中农业大学,2013,29-30.
[32]Strempel N,Nusser M,Neidig A,et al.The oxidative stress agent hypochlorite stimulates c-di-GMP synthesis and biofilm formation in Pseudomonas aeruginosa[J].Frontiers in Microbiology,2017,8(1):2311.
[2]彭琦,周子珊,张杰.苏云金芽胞杆菌杀虫晶体蛋白研究进展[J].中国生物防治学报,2015,31(5):712-722.
[3]刘梅,孙明,喻子牛.苏云金芽孢杆菌防治鞘翅目害虫的研究进展[J].中国生物防治,1998,14(1):39-43.
[4]邱丽娜,弓爱君,贾月.苏云金杆菌生物农药的优缺点及其改进[J].生物技术,2003,13(1):47-48.
[5]姚祖杰,吴松青,彭艳,等.一株高产杀虫蛋白苏云金杆菌的生物学特性研究以及其aiiA基因的克隆和生物信息学分析[J].生物技术进展,2013,3(2):124-131,157.
[6]陈维贤,张莉萍.细菌生物被膜(bacterial biofilm)的研究进展[J].微生物学杂志,2004,24(1):46-48.
[7]Vincent Z,Thomas A,Thomas T,et al.Subgingival biofilm structure[J].Frontiers of oral biology,2011,15(1):1.
[8]Furukawa S.Studies on formation,control and application of biofilm formed by food related microorganisms[J].Journal of the Agricultural Chemical Society of Japan,2015,79(7):1050-1056.
[9]Kviatkovski I,Mamane H,Lakretz A,et al.Resistance of a multiple-isolate marine culture to UV-C irradiation:inactivation vs.biofilm formation[J].Letters in Applied Microbiology,2018,67(3):78-84.
[10]Finkel J S,Mitchell A P.Genetic control of Candida albicans biofilm development[J].Nature Reviews Microbiology,2011,9(2):109-118.
[11]Bellows L E,Koestler B J,Karaba S M,et al.Hfq-dependent,co-ordinate control of cyclic diguanylate synthesis and catabolism in the plague pathogen Yersinia pestis[J].Molecular Microbiology,2012,86(3):661-674.
[12]Raposo M P,Inácio J M,Mota L J,et al.Transcriptional regulation of genes encoding arabinan-degrading enzymes in Bacillus subtilis[J].Journal of Bacteriology,2004,186(5):1287-1296.
[13]Zhu H,Mao X J,Guo X P,et al.The hmsT 3'untranslated region mediates c-di-GMP metabolism and biofilm formation in Yersinia pestis[J].Molecular Microbiology,2016,99(6):1167-1178.
[14]Davies B W,Bogard R W,Young T S,et al.Coordinated regulation of accessory genetic elements produces cyclic di-nucleotides for Vibrio cholerae virulence[J].Cell,2012,149(2):358-370.
[15]Krasteva P V,Fong J C,Shikuma N J,et al.Vibrio cholerae vpsT regulates matrix production and motility by directly sensing cyclic di-GMP[J].Science,2010,327(5967):866-868.
[16]Tischler A D,Camilli A.Cyclic diguanylate(c-di-GMP)regulates Vibrio cholerae biofilm formation[J].Molecular Microbiology,2010,53(3):857-869.
[17]孙长坡,宋福平,张杰,等.苏云金芽胞杆菌G03 spoIVF操纵子敲除对芽胞和晶体形成的影响[J].微生物学报,2007,47(4):583-587.
[18]姚俊敏,张韶芮,金鑫,等.苏云金芽胞杆菌XL6-候选生物被膜调控基因00940序列分析及基因敲除突变体构建[J].农业生物技术学报,2018,26(8):1401-1409.
[19]Cuevas D A,Edwards R A.PMAnalyzer:a new web interface for bacterial growth curve analysis[J].Bioinformatics,2017,33(12):1905.
[20]Gnasekaran P,Subramaniam S.Mapping of the interaction between Agrobacterium tumefaciens and vanda kasem's delight orchid protocorm-like bodies[J].Indian Journal of Microbiology,2015,55(3):285-291.
[21]Bridier A,Duboisbrissonnet F,Boubetra A,et al.The biofilm architecture of sixty opportunistic pathogens deciphered using a high throughput CLSMmethod[J].Journal of Microbiological Methods,2010,82(1):64-70.
[22]李澜,陈锦灿,杨兆元,等.纳米Mg(OH)2对苏云金芽胞杆菌蛋白杀虫活性及抗紫外能力影响的研究[J].农业生物技术学报,2015,23(11):1452-1457.
[23]Kobayashi K.Gradual activation of the response regulator DegU controls serial expression of genes for flagellum formation and biofilm formation in Bacillus subtilis[J].Molecular Microbiology,2007,66(2):395-409.
[24]Panzer S,Losick R,Sun D,et al.Evidence for an additional temporal class of gene expression in the forespore compartment of sporulating Bacillus subtilis[J].Journal of Bacteriology,1989,171(1):561-564.
[25]凡肖,束长龙,关雄,等.细菌生物被膜在生物防治中的作用[J].中国生物防治学报,2018,34(5):791-801.
[26]Mata A R,Pacheco C M,Pérez J F C,et al.In silico comparative analysis of GGDEF and EAL domain signaling proteins from the Azospirillum genomes[J].BMC Microbiology,2018,18(1):20.
[27]Skariyachan S,Sridhar V S,Packirisamy S,et al.Recent perspectives on the molecular basis of biofilm formation by Pseudomonas aeruginosa and approaches for treatment and biofilm dispersal[J].Folia Microbiologica,2018,63(4):413-432.
[28]屈铁军.变形链球菌GGDEF结构域基因的克隆表达及功能初步研究[D].第四军医大学,2008:63-94.
[29]Fu Y,Yu Z,Liu S,et al.c-di-GMP regulates various phenotypes and insecticidal activity of gram-positive Bacillus thuringiensis[J].Frontiers in Microbiology,2018,18(1):20.
[30]Carlson H K,Vance R E,Marletta M A.H-NOX regulation of c-di-GMP metabolism and biofilm formation in Legionella pneumophila[J].Molecular Microbiology,2010,77(4):930-942.
[31]刘舒.苏云金芽胞杆菌BMB171中第二信使c-di-GMP合成与降解相关基因的研究[D].武汉:华中农业大学,2013,29-30.
[32]Strempel N,Nusser M,Neidig A,et al.The oxidative stress agent hypochlorite stimulates c-di-GMP synthesis and biofilm formation in Pseudomonas aeruginosa[J].Frontiers in Microbiology,2017,8(1):2311.