Genomic Analysis Of B9601-Y2Strain And Partial Functions Confirmation

Plant growth-promoting rhizobacteria （PGPR）, is one kind of bacteria living in the plant rhizospheric soil and could promote crop growth and control plant diseases. Bacillus amyloliquefaciens B9601-Y2（Y2）, belonging to plantarum group, was isolated from rhizospheric soil of wheat and stored in our laboratory. In vitro, Y2has strong antagonistic activity against many common phytopathogenic fungi and bacteria, and could promote a number of crops growth and increase yield in the field. To understand its mechanisms, Y2genome was sequenced and analyzed. Y2’s ability to produce antibiotic metabolites and colonize in maize and its rhizospheric soil was confirmed by MALDI-TOF-MS and through GFP-tagged technology, respectively. Y2natural competence was recovered with gene-engineering method. The results obtained in this study were as follows:1. The genome size of Y2is4238624bp（base pairs） with accession number NC17912.1in NCBI database. It was predicted that Y2had45.76%GC content and4159CDS with the overall length3704557bp, accounting for87.4%of the whole genome. The number of tRNA gene, rDNA operon, tandem repeated region, transposon and inserted sequence element are91,10,69,73and24, respectively.2. Many living strategies were found in Y2through genomic annotation, which indicate that Y2could tolerate harsh environments and survive. Y2has conserved core genes related to the DNA repair system in Bacillus, such as alkA （MUS₄255）, ung（MUS₄184）,mutM（MUS₃183） and other genes acquired in excision repair system; mutS（MUS₁860）, mutL（MUS₁861）, XseA（MUS₂727） and XseB （MUS₂726） in mismatch repair system and some essential genes in non-homologous end joining and homologous repair system. Members of gene utilizing polysaccharides such as cellulose, hemicellulose, pectin and starch were discovered in Y2genome, such as MUS₂161, ydhP（MUS₄265）, bglS （MUS₄304）, pelB （MUS₄319） and amyE （MUS₀286）, suggesting that Y2could make use of limit nutrient in the soil without appropriate plant hosts to increase its own population rapidly. Meanwhile, some genes encoding the chitin-degrading enzymes were also found, partially explaining why Y2has so strong antagonism against some pathogenic fungi. Y2owns the synthetic clusters and regulatory genes of biofilm, which shows Y2has a good mutual benefit relationship with plant host. 3. Many antibiotic biosynthetic clusters were found in Y2genome, whose products were detected by MALDT-TOF-MS. Besides the production of some common lipopeptides fengycin, iturin, bacillibactin, bacilysin and polyketides bacillaene, macrolactin and difficidin, Y2could secrete bacteriocins such as mersacidin to inhibit the growth of phytopathogenic fungi and reduce the occurrence of plant disease. The alkaline serine protease gene and metabolic pathways of some plant growth-promoting volatile compound2,3-butanediol and acetoin discovered in Y2genome provide molecular evidence for Y2to control diseases caused by fungi and root-knot nematode and promote plant growth.4. An amazing phenomenon we discovered is that Y2is a surfactin-deficient strain because of the deletion of srfAB, the second gene member in surfactin operon srfA. The indirect result of srfAB deletion leads to the loss of comS, an essential gene in natural competence, which makes Y2deficient in natural transformation. comX, an upstream regulatory gene of comS, was also absent. At the same time, Y2contains type I restriction-modification system hsdRMS （MUS₃105～MUS₃103）. All of that leads to difficulty in molecular manipulation of Y2.5. Several types of Escherichia coli-Bacillus shuttle vector in GFP expression had been constructed based on the backbone of Bacillus expression plasmid pHT01. These vectors were transformed into transient hosts B. subtilis168and B. amyloliquefaciens FZB42by natural transformation. Y2electro-competent cells were electroporated with the plasmid DNA extracted from B168and FZB42.3GFP-tagged Y2strains were obtained and named as Y2-pHAPII, Y2-pGFP4412and Y2-P43GFPmut3a, respectively.6. The colonization dynamics and endophytic characteristic of Y2in maize root and rhizo spheric soil were studied primarily, with the GFP-tagged strain Y2-P43GFPmut3a as inoculant. The results showed that Y2had a good colonization in maize roots, root surface and rhizospheric soil, whose population number has been10～104,106～107and105～106cfu/g （root or soil）, respectively, and hasn’t changed sharply during two months. Furthermore, Y2could colonize in maize stem and leaf although the number was only102cfu/g （fresh tissue）. The tissue slices were observed under the confocal laser scanning microscopy. And the results showed2days after inoculation （DAI）, Y2-P43GFPmut3a could colonize the root hair, elongation region and matured zone of maize root and in maize stem and leaf tissue at5DAI and9DAI, respectively. The phenomenon was proved by fluorescence observation. 7. Inducible expression vector pHT01-comK was constructed, based on pHT01skeleton. The natural competence of Y2was recovered through the inducible expression of ComK, a key factor in Bacillus natural competence. The condition of natural competence formation was optimized primarily, and resulted in the highest transformation efficiencies3.06±0.22×104cfu/μg DNA when using GCHE medium as growth medium, and inducibly expressing1h with a final concentration of lmM IPTG with0.5-0.6OD6oo, recovering3h after the addition of exogenous plasmid DNA. So far, it is the first report to recover the natural transformation of undomesticated B. amyloliquefaciens deficient in natural competence, which could provide a technological strategy for gene manipulation in these strains.