Regulation Mechanisms Of Exoprotease Synthesis And Antimicrobial Function In B. Subtilis

Bacillus, as one of the best understood genus of prokaryotes, has received much attention for its widely applications in fermentation industry and agriculture. The research on mechanism of metabolite gene regulation in Bacillus has long been a focus of applied microbiology. In this research, insertional mutagenesis of two wild type strains with transposon Mini-Tn10 were carried out to discover genes involeved in metabolite production. Some mutant strains were isolated from their abnormal phenotypes. The insertional sites were identified and the affect mechanisms were analyzed. The work can be summarized as followed.1. Plasmid pIC333 with Mini-Tn10 was used as a tool to carry out insertional mutagenesis of B.amyloliquefaciens strain NK10.BAhjaWT and B. subtilis strain NK1010, respectively. The transformation efficiency of the plasmid was about 8.7×102 CFU/μg DNA. The results showed that the mutation rate can be up to 0.49% and 97% cells lost the plasmids while growth at 37℃for 4 hours.2. With the phenotype of protein hydrolysis ability, one mutant strain, GWH1023 (citB-), was isolated from the transposon libraries of NK1010 with increased exoprotease productivity. Complemented with the citB gene coloned from the genome DNA of the wild type strain, the exoprotease productivity could partially be restored. In the mutant strain, the swarming motility and biofilm formation ability were also tested and the results indicated that the abnormal phenotypes were caused by the increased DegU level in cells. In addition, a site-directed mutagenesis of the citB was constructed, in which the Arg741 and Gln745 were both changed into Glu. The resulting strain GWH1026 (citBm) exhibited increased enzymatic activity of aconitase comparing to that of the wild type strain. Western blot showed that the aconitase protein expression level was significantly increased in strain GWH1026. Byβ-Galactosidase activity assay, the transcription level of citB was also increased.3. Compared with the antibacterial and antifungal phenotypes of the strain NK10.BAhjaWT, some mutant strains were identified. In these mutant strains, L1 (secE), L3 (yukB) and L4 (purL) exhibited increased antibacterial ability, while the antibacterial ability of strain L7 (pksN), L8 (wapA) and L10 (ywqC) decreased. Another two mutants, G3(citB) and G7 (mycB) were deprived the antifungal ability.4. Further detection of the mutant strain L4 (purL) showed that only the antagonist ability against G+ bacteria increased, and the antagonist ability against G-bacteria (E.coli) and fungi (Aspergillus niger) did not changed. Further analysis indicated that the increased antibacterial ability may be caused by the blockage of biosynthesis pathway of thiamine pyrophosphate (TPP). By adding TPP to the medium, the antibacterial ability of L4 (purL) could be restored to normal level.5. With acid precipitation and RP-HPLC purification, the results showed that L4 (purL) and the wild type strains contained same components of the lipopeptide products. The isolated components from RP-HPLC were analyzed by antibacterial ability assay and MALDI-TOF-MS. The results indicated that the high antibacterial activity may come from the synergy effect of several kinds of lipopeptides.