Cloning and analysis of the antagonistic related genes of Enterobacter cloacae B8

Abstract

To understand the antagonistic mechanism of the broad spectrum antagonistic Enterobacter cloacae B8, Tn5 transposon-mediated mutagenesis is performed using suicide plasmid pZJ25. Two mutant strains that lost antagonistic character are isolated. Tagging with kan r gene on Tn5, an antagonistic related DNA fragment, the F fragment, right of the Tn5 insertion site is cloned in a plasmid named pTLF, from one of the mutant strains B8F. The 735 bp F fragment is then sequenced after subcloning. Genomic DNA of the original B8 strain is isolated, digested with Pst I and ligated to Pst I cassette. DNA fragments left and right of the F fragment are amplified from the Pst I cassette library using cassette primer and specific primers designed according to known sequence. 1106 bp sequence left of the F fragment and 664bp sequence right of the F fragment are finally obtained. Bioinformatics analysis shows that the contig assembled from the sequences of the cloned antagonistic related DNA fragments of B8 encodes three ORFs and is homogeneous to admM, admN and admO genes of Pantoea agglomerans andrimid biosynthetic gene cluster (AY192157). The ORF, named anrF gene which encodes a polyketide synthase, knocked out by Tn5 insertion, is a homology of admM and the insertion site of Tn5 is at 214 bp upstream of the stop codon. It is concluded that the anrF gene is a gene related to the antagonistic activity of E. cloacae B8, and speculated that the antagonistic substance produced by B8 is an andrimid.