The Chrarcteristics Of Fus Gene Cluster And Genome Analysis Of Paenibacilus Polymyxa SQR-21

Paenibacillus polymyxa is considered as an important group of plant growth-promoting rhizobacteria (PGPR), and has been widely used in agriculture. Paenibacillus polymyxa strain is also widely and commercially used as bio-fertilizaers for plant growth promotion and biological control of soil-borne plant diseases. P. polymyxa SQR-21, isolated by the Jiangsu Province Key Lab of Organic Solid Waste Utilization, China, was found to be an outstanding PGPR by its abilities to suppress plant pathogens and decrease the incidence of cucumber wilt.This paper determined the antibiotics produced by P. polymyxa SQR-21and study the colonizations behavior of the SQR-21strain on cucumber root. A fosmid genomic library of SQR-21was constructed, and fusaricidin gene cluster was screened by colony hybridization. Promoters of fusaricidin gene cluster were also sreened and different transcriptional activities of five promoters with different lengths in E. coli and SQR-21were studied. SQR-21genome was sequenced and analyzed. The main results obtained are lisetd as follows.1. The wide-type SQR-21strain was tested for antagonistic ability to Fusarium oxysporum on potato-dextrose-agar (PDA). The SQR-21showed the strong inhibiton activity against F. oxysporum. The extracted products of SQR-21in KL medium were analyzed by MALDI-TOF and four ion peaks with [M+H]+883,897,947and961were detected. Based on molecular weight, the four ion peaks could be fusaricidin A, B, C and D respectively. P. polymyxa SQR-21could also produce the phytase and indole-3-acetic acid, which may promote the plant growth.2. The SQR-21was marked with gfp by transformantion of gfp-carrying plasmid pHAPII. gfp marked strain SQR-21-gfp showed bright green fluorescence under fluorescence microscopy. In hydroponic system, SQR-21-gfp strain was inoculated to cucumber roots. After24h, the roots were analyzed through CLSM. The images revealed that colonization occurred preferentially in the elongation aerea and root tips as microcolonies. In the soil system, the CLSM images also showed that the colonization of GFP-tagged SQR-21mainly occurred on the elongation aerea of cucumber root and the junctions between roots.3. We constructed a fosmid genomic library of P. polymyxa SQR-21with the CopyControlTM Fosmid Library Production kits, which contained more than three thousand clones. Partial fragments of fusA, fusB, fusC and fusF ORFs were chosen as the probes for subsequent screening of the fus gene cluster. Twenty-five fosmids clones were obtained by probe hybridization. Seven fosmids clones containing the fusaricidin gene cluster were confirmed after PCR and sequence analysis, among which three fosmid clones,4G1,5D8and16A10contained the full length of fusaricidin gene cluster and were selected for further study. According to the analysis, seven ORFs were found in the fusaricidin synthesis.4. In order to monitor the transcriptional activity of fusaricidin gene cluster promoter, five different lengths of Pfus were fused to gfp gene with no promoter respectively, generating the reporter plasmids PfusGO to PfusG4. These reporter plasmids were introduced into E. coli Top10and SQR-21, and their expression abilities were detected by monitoring theis green fluorescence intensity. The results indicated that different lengths of Pfus showed different transcriptional activity in both of E. coli Top10and SQR-21and the same lengths of Pfus showed different transcriptional activity in E. coli Top10and SQR-21, which was possibly due to the different regulators and their binding regions in various hosts.5. The transcriptional factors of fusaricidin gene cluster in SQR-21were screened by pull-down assay. After a series of processes, we finally got a specific protein about18-20KDa. But the sequence analysis result showed that it was not involved in transcriptional regulation.6. The SQR-21genome was sequenced and analyzed. The SQR-21genome was circular with5,828,436bp, and contained the13rRNA operons and111tRNA. The functional genes related to utilization of various rhizosphere carbon sources, transport system, cell motility and chemotaxis, plant growth promotion and antibiotic synthesis were identified from the genome of SQR-21.25gene island were found in SQR-21genome, some of which were not found in other strains of P. polymyxa Especially,6.1%of sequences in P. polymyxa SQR-21genome were related to the synthesis of antibiotics. A new PKS-NRPS gene cluster was found in the SQR-21genome. Genome analysis provided useful information for deeply understanding SQR-21as a potential PGPR strain, and provided new insights into the functions and evolution of the Paenibacillus polymyxa.