Bacillus subtilis strain NCD-2 is an excellent biocontrol agent for plant soil-borne diseases, and the lipopeptide fengycin is one of the active antifungal compounds in strain NCD-2. The regulator PhoP and its sensor ...Bacillus subtilis strain NCD-2 is an excellent biocontrol agent for plant soil-borne diseases, and the lipopeptide fengycin is one of the active antifungal compounds in strain NCD-2. The regulator PhoP and its sensor kinase PhoR compose a two-component system in B. subtilis. In this study, the phoR- and phoP-knockout mutants were constructed by in-frame deletion and the role of PhoR/PhoP on the production of fengycin was determined. Inactivation of phoR or phoP in B. subtilis decreased its inhibition ability against Botrytis cinerea growth in vitro compared to the strain NCD-2 wild type. The lipopeptides were extracted from strain NCD-2 wild type and its mutant strains by hydrochloric acid precipitate, and the lipopeptides from phoR-null mutant orphoP-null mutant almost lost the inhibition ability against B. cinerea growth compared to the lipopeptides from strain NCD-2 wild type. Fast protein liquid chromatography （FPLC） analysis of the lipopeptides showed that inactivation of phoR or phoP genes reduced the production of fengycin by strain NCD-2. The fengycin production abilities were compared for bacteria under low-phosphate medium （LPM） and high-phosphate medium （HPM）, respectively. Results indicated that the regulation of fengycin production by the PhoR/PhoP two-component system occurred in LPM but not in HPM. Reverse transcriptionaI-PCR confirmed that the fengycin synthetase gene fenC was positively regulated by phoP when cultured in LPM. All of these characteristics could be partially restored by complementation of intact phoR or phoP gene in the mutant. These data indicated that the PhoR/PhoP two-component system greatly regulated fengycin production and antifungal ability in B. subtilis NCD-2 mainly under low-phosphate conditions.展开更多
[Objectives]To amplify the DNA-binding response regulator PhoP in Vibrio alginolyticus and analyze its sequence characteristics and subunit structure.[Methods]According to the sequence of the DNA-binding response regu...[Objectives]To amplify the DNA-binding response regulator PhoP in Vibrio alginolyticus and analyze its sequence characteristics and subunit structure.[Methods]According to the sequence of the DNA-binding response regulator PhoP in V.alginolyticus,a pair of specific primers was designed for PCR amplification,and the bioinformatics of the sequence amplified was analyzed.Using MEGA 5.0 software,the phoP phylogenetic tree was constructed by the neighbor-joining method.Using SWISS-MODEL software,the three-dimensional structural model of the PhoP subunit was simulated.[Results]The full-length phoP gene was 732 bp,encoding a total of 243 amino acids.The predicted theoretical molecular weight of the protein is about 27.67 kD,and the isoelectric point is 5.09.The prediction results of protein subcellular localization,SignalP 4.0,TMHMM Server 2.0 and SoftBerry-Psite show that PhoP is located in the cytoplasm,and is stable and hydrophobic;there is a signal peptide cleavage site between amino acids 29 and 30,and there is no transmembrane region.The amino acid sequence contains one Asn-glycosylation site,one protein kinase C phosphorylation site,seven casein kinase II phosphorylation sites,one tyrosine kinase phosphorylation site,three myristoylation sites,and seven C-terminal microbody targeting signal sites.The PhoP of V.alginolyticus has high homology with that of Vibrio campbellii.The PhoP subunit of V.alginolyticus has similar configuration to the single-subunit RegX3 protein of Mycobacterium tuberculosis.[Conclusions]This study has a positive effect on the prevention and control of vibriosis and the improvement of the current aquatic economic animal breeding environment.展开更多
基金funded by the earmarked fund for the China Agriculture Research System (CARS-18-15)the National Natural Science Foundation of China (31272085,31572051)the Special Fund for Agro-scientific Research in the Public Interest,China (201503109)
文摘Bacillus subtilis strain NCD-2 is an excellent biocontrol agent for plant soil-borne diseases, and the lipopeptide fengycin is one of the active antifungal compounds in strain NCD-2. The regulator PhoP and its sensor kinase PhoR compose a two-component system in B. subtilis. In this study, the phoR- and phoP-knockout mutants were constructed by in-frame deletion and the role of PhoR/PhoP on the production of fengycin was determined. Inactivation of phoR or phoP in B. subtilis decreased its inhibition ability against Botrytis cinerea growth in vitro compared to the strain NCD-2 wild type. The lipopeptides were extracted from strain NCD-2 wild type and its mutant strains by hydrochloric acid precipitate, and the lipopeptides from phoR-null mutant orphoP-null mutant almost lost the inhibition ability against B. cinerea growth compared to the lipopeptides from strain NCD-2 wild type. Fast protein liquid chromatography （FPLC） analysis of the lipopeptides showed that inactivation of phoR or phoP genes reduced the production of fengycin by strain NCD-2. The fengycin production abilities were compared for bacteria under low-phosphate medium （LPM） and high-phosphate medium （HPM）, respectively. Results indicated that the regulation of fengycin production by the PhoR/PhoP two-component system occurred in LPM but not in HPM. Reverse transcriptionaI-PCR confirmed that the fengycin synthetase gene fenC was positively regulated by phoP when cultured in LPM. All of these characteristics could be partially restored by complementation of intact phoR or phoP gene in the mutant. These data indicated that the PhoR/PhoP two-component system greatly regulated fengycin production and antifungal ability in B. subtilis NCD-2 mainly under low-phosphate conditions.
基金Outstanding Graduate Entering Laboratory Project of College of Fisheries,Guangdong Ocean UniversityNational Natural Science Foundation of China(32073015)+3 种基金Natural Science Foundation of Guangdong Province(2021A1515011078)Special Fund for Science and Technology Innovation Strategy of Guangdong Province(Undergraduate Science and Technology Innovation Cultivation)(pdjh2021b0239)Undergraduate Innovation and Entrepreneurship Training Program of Guangdong Ocean University(CXXL2021122)Undergraduate Innovation Team of Guangdong Ocean University(CCTD-201802).
文摘[Objectives]To amplify the DNA-binding response regulator PhoP in Vibrio alginolyticus and analyze its sequence characteristics and subunit structure.[Methods]According to the sequence of the DNA-binding response regulator PhoP in V.alginolyticus,a pair of specific primers was designed for PCR amplification,and the bioinformatics of the sequence amplified was analyzed.Using MEGA 5.0 software,the phoP phylogenetic tree was constructed by the neighbor-joining method.Using SWISS-MODEL software,the three-dimensional structural model of the PhoP subunit was simulated.[Results]The full-length phoP gene was 732 bp,encoding a total of 243 amino acids.The predicted theoretical molecular weight of the protein is about 27.67 kD,and the isoelectric point is 5.09.The prediction results of protein subcellular localization,SignalP 4.0,TMHMM Server 2.0 and SoftBerry-Psite show that PhoP is located in the cytoplasm,and is stable and hydrophobic;there is a signal peptide cleavage site between amino acids 29 and 30,and there is no transmembrane region.The amino acid sequence contains one Asn-glycosylation site,one protein kinase C phosphorylation site,seven casein kinase II phosphorylation sites,one tyrosine kinase phosphorylation site,three myristoylation sites,and seven C-terminal microbody targeting signal sites.The PhoP of V.alginolyticus has high homology with that of Vibrio campbellii.The PhoP subunit of V.alginolyticus has similar configuration to the single-subunit RegX3 protein of Mycobacterium tuberculosis.[Conclusions]This study has a positive effect on the prevention and control of vibriosis and the improvement of the current aquatic economic animal breeding environment.
