Functional Identification Of Dif Sequence From Bacillus Licheniformis

Bacillus licheniformis is one of the most important strains for industrial biotechnological processes, and widely used in fields such as medicine, plant diseases, food, feed, and environment. A simple and effecient method for the gene deletion of Bacillus licheniformis is fundamental for its genetic improvement.The Xer/dif system is a site-specific recombination system which is ubiquitous in prokaryotes, and the dif site consists of a specific sequence that can be recognized by the Xer recombinases. However the successful application of Escherichia coli difEco sequence and the B. subtilis difBs sequence for multiple genes disruption in E. coli and B. subtilis, respectively, the characterization and functional idetification of Xer/dif system for genetic manipulation in B. licheniformis has not been reported.In this paper, B. licheniformis difBLi, a B. subtilis difBs-homolog sequence, has been cloned and identified from the genome of B. licheniformis ATCC14580. Two recombinant plasmids pMD19-difGm and pMD19-xi were constructed by inserting a gentamicin resistant gene flanking difBLi sequence and inserting a xylose isomerase gene (xi) obtained by PCR amplified from B. licheniformis ATCC14580 respectively. The fragment dif-Gm-dif digested by SmaI from pMD19-difGm was inserted into the sites of SmaI and StuI of pMD19-xi, constructed the recombinant plasmid pMD19-xi′::difGm. The fragment xi′::difGm digested by EcoRI from pMD19-xi′::difGm and then subcloned into the site of EcoRI of pHY300 PLK, finally constructed the gene deletion plasmid pHY-xi′::difGm. The plasmid pHY-xi′::difGm was transformed into B. licheniformis ATCC14580 by electroporation method. Following regeneration of the transformants, the gentamicin resistant cassette was efficiently removed by homologous recombination mediated with the native Xer recombinase, which acting to resolve the two directly repeated dif sites to a single site. The function of the dif sequence in B. licheniformis has been confirmed, which may supply a new method for the deletion of the multiple genes in B. licheniformis genome with limitied antibiotic resistance genes.To improve the electro-transformation efficiency, the effect of osmotic agents and voltage on electro-transformation efficiency of B. licheniformis ATCC14580 were investigated. With the growth medium LB complemented 0.5 mol/L sorbitol, the recovery medium LB containing 0.5 mol/L sorbitol and 0.38 mol/L mannitol, the voltage was 1800 V, the optimal transformation efficiency of 6.3 CFU/μg was obtained.