Genomic Sequence Analysis Of Bacillus Amyloliquefaciens PEBA20 And Characteristics Of The Sfp Gene

Bacillus amyloliquefaciens PEBA20 was an endophytic bacterium isolated from branches of poplar,which has strong antimicrobial activity against many plant pathogenic fungi and bacteria.In this study,PEBA20 genome was sequenced and analyzed,and we found nine gene clusters related with synthesizing antibiotics.We also got the mutant strain?sfp using the method of homologous recombination.The?sfp mutant cannot form the robust and complicated biofilm,and also lost the swarming motility and antimicrobial activity.Finally,we constructed green fluorescent expressing vector in order to get the fluorescein-labeled recombinant strain.The results were as follows:The genome of PEBA20 is 4,249,176-bp,containing an estimated 3,728,166-bp protein-coding sequences.It was predicted that PEBA20 had 4,487 genes,including4,304 protein-coding genes and 183 RNA genes.We also built phylogenetic trees using gyrA and cheA protein sequences.We defined that it belongs to Bacillus amyloliquefaciens subsp.plantarum.Different gene functions with function was predicted in PEBA20 genomic annotation,which containing 3,425 genes with pfam domains,185 genes with signal peptides,1084 genes with transmembrane helices,282 genes with Genome islands,1358genes with virulence,255 genes with antibiotic resistance,833 genes with secondary metabolism.Nine gene clusters related with synthesizing antibiotics were found in PEBA20genome.These gene clusters were srf A,bmy,fen,dhb,bac,mln,bae,dfn,mrs,with production Surfactin,BacillomycinD,Fengycin,Bacillibactin,Bacilysin/anticapsin,Macrolactin,Bacillaene,Difficidin,Amylocyclicin,Mersacidin.We made the genomic re-sequencing of variant SCEV.Compared with PEBA20,SCEV was detected 36 SNPs,including 1 SNP with non synonymous coding.That mutagenic deoxynucleotide belongs to gene Yux H with T>A,leading to I¹⁴¹>N¹⁴¹.We also got the mutant strain?sfp using the method of homologous recombination.Two homologous recombinant fragments,sfpF and sfpB,were amplified by PCR from PEBA20,and connected using EcoRI.The sfpFB fragment was inserted between the HindIII and Bam HI sites of pMUTIN4 plasmid.The resulting plasmid was used to transform the PEBA20 wild type strain by electroporation,and the sfp locus was deleted and replaced with the sfp FB via al elic exchange by homologous recombination.The effect of sfp on biofilm formation and on swarming motility was similar to Bacillus subtilis as previously reported.The?sfp mutant was unable to form robust and complicated biofilm,and the colony became flat and slick on solid media.In air-liquid interface,the pellicle was fragile,and dispersion delayed.The deletion sfp also affect the antimicrobial activity against p lant pathogenic fungi compared with the wild type PEBA20.And because the?sfp mutant cannot synthesize surfactin,it was non-motile with swarming motility.Finally,we amplified the fragments of p43 promotor and gfpmut3a gene from Bacillus subtilis B3 and pS4GFP plasmid.And both the fragments were cloned to the amyE locus in pMUTIN4 plasmid.The resulting plasmid was expressed in E.coli,and the labelled cells were detected green fluorescence on Fluorescence Microscopy of N ikon90I.The construction wild type PEBA20 labelled with GFP are underway.