Research Aoubt Genes Related To Biocontrol Activity In Bacillus Amyloliquefaciens B3 And A Cyclodipeptide Synthetase

Bacillus spp.is Gram-positive,spore-forming,and rod-shaped organisms with an aerobic or facultatively anaerobic metabolism and distribute ubiquitously in many environments.And it has a powerful production and secretion system of big amounts of secondary metabolites such as phytase,extracellular proteases and antibiotics.Because of this system,Bacillus has been widely used as bioconctol agent.Our lab developed a biocontro agent called "MaiFengning" using B.amyloliquifaciens B3 as active principle.And we also sequenced the genome DNA of B3 strain.Cyclicodipeptide?CDPs?is a newly found bio-active compound and has antibacterial,antifungal and plant growth promotion activity.Also,CDPs could be developed to be a good biocontrol agent.Heterogeneous expression,radio-active labeling and HPLC-MS were used to study a plasmid-borne Rap-Phr system and a new NRPSs gene cluster found in B3 and a CDPSs gene cluster found in Parachlamydia acanthamoebae UV-7.The main contents include:?1?Regulation mechanism of the plasmid-borne Rap-Phr found in B3;?2?Substrate specificity of A domains in the new NRPSs and the construction of the heterogeneous expression system for NRPSs gene cluster;?3?Product,activity sites identity and biological function analysis of the new CDPSs.?1?Phenomenon experiment?sporulation efficiency,surfactin yield and transformation efficiency?results showed that expression of rapQ in B.subtilis OKB105strongly suppressed its sporulation efficiency,transformation efficiency and surfactin production.Co-expression of phrO or addition of synthesized PhrQ pentapeptide in vitro could compensate for the supressive effects caused by rapQ.We also found that expression of rapQ decreased the transcriptional level of the sporulation-related gene spoIIE and surfactin synthesis-related gene srfA;meanwhile,the transcriptional levels of these genes could be rescued by co-expression of phrQ and in vitro addition of PhrQ pentapeptide.Then we constructed the recombinant protein expression strain of ComA and RapQ and purified the target protein.EMSA result also showed that RapQ could bind to ComA without interacting with ComA binding to DNA,and PhrQ pentapeptide antagonized RapQ activity in vitro.These results indicate that this new plasmid-born RapQ-PhrQ system controls sporulation,competent cell formation and surfactin production in Bacillus amyloliquifaciens B3.?2?Bcillus produces many kinds of antibiotics,such as lipopeptides,polyketide,peptides,phospholipids and polyene.And products synthesized by nonribosomal peptide synthetase?NRPSs?take a big part of it,such as lipopeptides and polyketide.We found a new NRPSs gene cluster inside Bacillus amyloliquifaciens B3 strain by genome DNA sequencing.Sequence analysis result shows that there are two NRPSs,a TE domain,two putative modification enzymes coding genes and other genes related with products transformation and expression regulation locating inside this gene cluster.Radio activate label,enzyme in vitro activity assay and mass spectrometer assay were used to find the product of this new gene cluster.We constructed the recombinant protein expression strains for six A domains found in the two NRPSs and got the purified recombinant protein.Then,we got the substrate amino acid of these six A domains via ATP-PPi exchange experiment.Based on the sequence of the amino acid residue sequence of the product,we purified the putative modification enzyme but we did not get any result.Because B3 strain can hardly be transformed,we constructed a TAR cloning system and tried to capture the whole gene cluster and express it heterogeneously in yeast strain.?3?Cyclodipeptide synthetase uses aminoacyl-tRNAs as substrates and catalyze cyclic dipeptide?CDPs?formation.Owing to the diverse and interesting bioactivities observed for many naturally occurring cyclic dipeptide,such as antibacterial,antifungal and plant growth promotion,CDPs attract more and more attention.Via PSI-BLAST,we found a CDPSs coding gene pah?puv₁7450?in the genome DNA of Parachlamydia acanthamoebae UV-7.And we also found a luxR gene at the downstream of pah.We found the product of Pah was cPP?cyclic di-proline?by heterogeneous expression in E.coli BL21.And our results suggested that positive charged amino acid residues at both ends of a-4helix play an important role in the interaction between Pah and tRNA and S46,D188 and Q192 are part of catalyze center of Pah.The LC-MS result showed that mutant of negative charged residues on the loop between a-6 and a-7 helix could both increase the yield of"side products"?cPF and cPV?and cPP.We also expressed LuxR heterogeneously in E.coli BL21.Gel filtration result showed that LuxR was dimer in the solution and very unstable at high concentration.LuxR dimer could bind with cPP and cPP existence could make LuxR dimer bind more tighter and delay the its retention volume on gel filtration column.In vivo results also showed that LuxR could sense cPP and use it as a signal molecular.