Identification of anrF gene, a homology of admM of andrimid biosynthetic gene cluster related to the antagonistic activity of Enterobacter cloacae B8

AIM： To identify the gene （s） related to the antagonistic activity of Enterobacter cloacae B8 and to elucidate its antagonistic mechanism. METHODS： Transposon-mediated mutagenesis and tagging method and cassette PCR-based chromosomal walking method were adopted to isolate the mutant strain （s） of B8 that lost the antagonistic activity and to clone DNA fragments around Tn5 insertion site. Sequence compiling and open reading frame （ORF） finding were done with DNAStar program and homologous sequence and conserved domain searches were performed with BlastN or BlastP programs at www.ncbi.nlm.nih.gov. To verify the gene involved in the antagonistic activity, complementation of a full-length clone of the anrFgene to the mutant B8F strain was used. RESULTS： A 3 321 bp contig around the Tn5 insertion site was obtained and an ORF of 2 634 bp in length designated as anrFgene encoding for a 877 aa polyketide synthase-like protein was identified. It had a homology of 83% at the nucleotide level and 79% ID/87% SIM at the protein level, to the admM gene of Pantoea agglomerans andrimid biosynthetic gene cluster （AY192157）. The Tn5 was inserted at 2 420 bp of the gene corresponding to the COG3319 （the thioesterase domain of type I polyketide synthase） coding region on BSF. The antagonistic activity against Xanthomonas oryzae pv. oryzae was resumed with complementation of the full-length anrFgene to the mutant B8F. CONCLUSION： The anrFgene obtained is related to the antagonistic activity of BS, and the antagonistic substances produced by B8 are andrimid and/or its analogs.

Preliminary study on the role of novel Lys R family gene kp05372 in Klebsiella pneumoniae of forest musk deer

Lys R-type transcriptional regulators are involved in the regulation of numerous cellular metabolic processes in Klebsiella pneumoniae,leading to severe infection.Earlier,we found a novel Lys R family gene,named kp05372,in a strain of K.pneumoniae(designated GPKP)isolated from forest musk deer.To study the function of this gene in relation to the biological characteristics of GPKP,we used the suicide plasmid and conjugative transfer methods to construct deletion mutant strain GPKP-Δkp05372;moreover,we also constructed the GPKP-Δkp05372+complemented strain.The role of this gene was determined by comparing the following characteristics of three strains:growth curves,biofilm formation,drug resistance,stress resistance,median lethal dose(LD50),organ colonization ability,and the histopathology of GPKP.Real-time polymerase chain reaction(RT-PCR)was used to test the expression level of seven genes upstream of kp05372.There was no significant difference in the growth rates when comparing the three bacterial strains,and no significant difference was recorded at different osmotic pressures,temperatures,salt contents,or hydrogen peroxide concentrations.The GPKP-Δkp05372 mutant formed a weak biofilm,and the other two strains formed medium biofilm.The drug resistance of the GPKP-Δkp05372 mutant toward cephalothin,cotrimoxazole,and polymyxin B was changed.The acid tolerance of the deletion strain was stronger than that of the other two strains.The LD50 values of the wild-type and complemented strains were 174-fold and 77-fold higher than that of the GPKP-Δkp05372 mutant,respectively.The colonization ability of the GPKP-Δkp05372 mutant in the heart,liver,spleen,kidney,and intestine was the weakest.The three strains caused different histopathological changes in the liver and lungs.In the GPKP-Δkp05372 mutant,the relative expression levels of kp05374 and kp05379 were increased to 1.32-fold and 1.42-fold,respectively,while the level of kp05378 was decreased by 42%.Overall,the deletion of kp05372 gene leads to changes in the following:drug resistance and acid tolerance;decreases in virulence,biofilm formation,and colonization ability of GPKP;and regulation of the upstream region of adjacent genes.