枯草芽胞杆菌NCD-2中调控因子PhoP对fengycin合成的调控作用
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摘要
PhoR/PhoP双因子调控系统是枯草芽胞杆菌中一个重要的全局性调控系统,在低磷环境下,感应激酶PhoR自磷酸化,并且将获得的磷酸基团转移到调控因子PhoP上,从而激活PhoP的调控活性。为了明确调控因子PhoP对枯草芽胞杆菌NCD-2菌株中主要抑菌活性物质-fengycin合成能力的调控作用,本研究通过同源重组技术缺失突变NCD-2菌株中的phoP基因,拮抗活性测定结果表明,phoP基因缺失菌株显著降低了对立枯丝核菌的抑菌活性。通过快速蛋白质液相色谱(FPLC)技术比较了NCD-2菌株野生型及其phoP基因突变子fengycin的合成能力,结果证明,phoP基因突变菌株降低了fengycin的合成能力。对突变子进行phoP基因互补发现,互补phoP基因可使突变子的相关性状恢复到野生型菌株水平。以上结果证明,phoP基因对枯草芽胞杆菌NCD-2菌株中fengycin的合成具有正调控功能。
PhoR /PhoP two-component is a global regulatory system in Bacillus subtilis. Under the phosphate starvation condition,the sensor protein kinase PhoR self-phosphorylates and transfers its phosphate group to the regulator PhoP. The phosphorylated PhoP impresses or activates the expression of phosphate-regulated genes. With the purpose of clarifying the function of regulator PhoP on the fengycin production in B. subtilis NCD-2, the phoP was in-frameless deleted by homologous recombination. The testing showed that the phoP mutant decreased the antifungal ability against the growth of Rhizoctonia solani in vitro. The lipopeptides were extracted from strains NCD-2 wild type and phoP mutant,then analyzed by Fast Protein Liquid Chromatography(FPLC). The results indicated that the production of fengycin,the major antifungal activity compound in strain NCD-2, was significantly decreased in the phoP mutant compared to that of strain NCD-2 wild type. All of these characteristics were restored by complementation of phoP gene in the phoP deletion mutant. It was confirmed that the phoP was a positively regulation factor for fengycin production in strain NCD-2.
PhoR /PhoP two-component is a global regulatory system in Bacillus subtilis. Under the phosphate starvation condition,the sensor protein kinase PhoR self-phosphorylates and transfers its phosphate group to the regulator PhoP. The phosphorylated PhoP impresses or activates the expression of phosphate-regulated genes. With the purpose of clarifying the function of regulator PhoP on the fengycin production in B. subtilis NCD-2, the phoP was in-frameless deleted by homologous recombination. The testing showed that the phoP mutant decreased the antifungal ability against the growth of Rhizoctonia solani in vitro. The lipopeptides were extracted from strains NCD-2 wild type and phoP mutant,then analyzed by Fast Protein Liquid Chromatography(FPLC). The results indicated that the production of fengycin,the major antifungal activity compound in strain NCD-2, was significantly decreased in the phoP mutant compared to that of strain NCD-2 wild type. All of these characteristics were restored by complementation of phoP gene in the phoP deletion mutant. It was confirmed that the phoP was a positively regulation factor for fengycin production in strain NCD-2.
引文
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[20]Wang Y,Deng C,Peng Q,et al.Effect of quorum sensing response regulator nprR deletion on expression of cry protein in Bacillus thuringiensis(in Chinese)[J].Acta Microbiologica Sinica(微生物学报),2010,50(11):1550-1555.
[21]Martín J F,Santos-Beneit F,Rodríguez-García A,et al.Transcriptomic studies of phosphate control of primary and secondary metabolism in Streptomyces coelicolor[J].Applied Microbiology and Biotechnology,2012,95(1):61-75.
[22]Eder S,Liu W,Hulett F M.Mutational analysis of the phoD promoter in Bacillus subtilis:implications for PhoP binding and promoter activation of Pho regulon promoters[J].Journal of Bacteriology,1999,181(7):2017-2025.
[23]Ke W J,Chang B Y,Lin T P,et al.Activation of the promoter of fengycin synthetase operon by the UP element[J].Journal of Bacteriology,2009,191(14):4615-4623.
[24]Tsuge K,Ano T,Hirai M,et al.The Genes degQ,pps,and lpa-8(sfp)are responsible for conversion of Bacillus subtilis 168 to plipastatin production[J].Antimicrobial Agents and Chemotherapy,1999,43(9):2183-2192.
[2]Ongena M,Jacques P.Bacillus lipopeptides:versatile w eapons for plant disease biocontrol[J].Trends in Microbiology,2007,16:115-125.
[3]Stein T.Bacillus subtilis antibiotics:structures,syntheses and specific functions[J].Molecular Microbiology,2005,56(4):845-857.
[4]Velho R,Medina L F,Segalin J,et al.Production of lipopeptides among Bacillus strains show ing grow th inhibition of phytopathogenic fungi[J].Folia Micro biologica,2011,56(4):297-303.
[5]Eldakak A,Hulett F M.Cys303 in the histidine kinase PhoR is crucial for the phosphotransfer reaction in the PhoPR tw o-component system in Bacillus subtilis[J].Journal of Bacteriology,2007,189(2):410-421.
[6]Martín J F.Phosphate control of the biosynthesis of antibiotics and other secondary metabolites is mediated by the PhoR-PhoP system:an unfinished story[J].Journal of Bacteriology,2004,186(16):5197-5201.
[7]Allenby N E E,O’Connor N,Prágai Z,et al.Genome-w ide transcriptional analysis of the phosphate starvation stimulon of Bacillus subtilis[J].Journal of Bacteriology,2005,187(23):8063-8080.
[8]Fernandez-Martinez L T,Santos-Beneit F,Martín J F.Is PhoR-PhoP partner fidelity strict?PhoR is required for the activation of the pho regulon in Streptomyces coelicolor[J].Molecular Genetics and Genomics,2012,287(7):565-573.
[9]Santos-Beneit F,Rodríguez-García A,Martín J F.Complex transcriptional control of the antibiotic regulator afsS in Streptomyces:PhoP and AfsR are overlapping,competitive activators[J].Journal of Bacteriology,2011,193(9):2242-2251.
[10]Santos-Beneit F,Rodríguez-García A,Martín J F.Identification of different promoters in the absA1-absA2tw o-component system,a negative regulator of antibiotic production in Streptomyces coelicolor[J].Molecular Genetics and Genomics,2013,288(1-2):39-48.
[11]Asensio J G,Maia C,Ferrer N L,et al.The virulence-associated tw o component PhoP-PhoR system controls the biosynthesis of polyketide-derived lipids in Mycobacterium tuberculosis[J].Journal of Biological Chemistry,2006,281(3):1313-1316.
[12]Liu W,Hulett F M.Comparison of PhoP binding to the tuaA promoter w ith PhoP binding to other Phoregulon promoters establishes a Bacillus subtilis Pho core binding site[J].Microbiology,1998,144:1443-1450.
[13]Howell A,Dubrac S,Noone D,et al.Interactions betw een the YycFG and PhoPR tw o-component systemsin Bacillus subtilis:The PhoR kinase phosphorylates the non-cognate YycF response regulator upon phosphate limitation[J].Molecular Microbiology,2005,59(4):1199-1215.
[14]Li S Z,Lu X Y,Ma P.Evaluation of biocontrol potential of a bacterial strain NCD-2 against Cotton Verticillium w ilt in field trials(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2005,35(5):451-455.
[15]Guo Q G,Li S Z,Lu X Y,et al.PhoR/PhoP two component regulatory system affects biocontrol capability of Bacillus subtilis NCD-2[J].Genetics and Molecular Biology,2010,33(2):333-340.
[16]Guo Q G,Li S Z,Lu X Y,et al.Mapping and cloning function genes from antigonistic bacterium NCD-2 against Verticillium dahliae(in Chinese)[J].Acta Agricultural Boreali Sinica(华北农学报),2007,22(6):190-194.
[17]Dong W X,Li B Q,Li S Z,et al.The fengycin lipopeptides are major components in the Bacillus subtilis strain NCD-2 against the grow th of Botrytis cinerea(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报)2013,43(4):401-410.
[18]Martin P,Lohr J,Dean D.Transformation of Bacillus thuringiensis protoplasts by plasmid deoxyribonucleic acid[J].Journal of Bacteriology,1981,145(2):980-983.
[19]Arnaud M,Chastanet A,DébarbouilléM.New vector for efficient allelic replacement in naturally nontransformable,low-GC-content,gram-positive bacteria[J].Applied and Environmental Microbiology,2004,70(11):6887-6891.
[20]Wang Y,Deng C,Peng Q,et al.Effect of quorum sensing response regulator nprR deletion on expression of cry protein in Bacillus thuringiensis(in Chinese)[J].Acta Microbiologica Sinica(微生物学报),2010,50(11):1550-1555.
[21]Martín J F,Santos-Beneit F,Rodríguez-García A,et al.Transcriptomic studies of phosphate control of primary and secondary metabolism in Streptomyces coelicolor[J].Applied Microbiology and Biotechnology,2012,95(1):61-75.
[22]Eder S,Liu W,Hulett F M.Mutational analysis of the phoD promoter in Bacillus subtilis:implications for PhoP binding and promoter activation of Pho regulon promoters[J].Journal of Bacteriology,1999,181(7):2017-2025.
[23]Ke W J,Chang B Y,Lin T P,et al.Activation of the promoter of fengycin synthetase operon by the UP element[J].Journal of Bacteriology,2009,191(14):4615-4623.
[24]Tsuge K,Ano T,Hirai M,et al.The Genes degQ,pps,and lpa-8(sfp)are responsible for conversion of Bacillus subtilis 168 to plipastatin production[J].Antimicrobial Agents and Chemotherapy,1999,43(9):2183-2192.