Identification of anrF gene, a homology of admM of andrimid biosynthetic gene cluster related to the antagonistic activity of Enterobacter cloacae B8.
OBJECTIVE
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 anrF gene 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 anrF gene 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 B8F. The antagonistic activity against Xanthomonas oryzae pv. oryzae was resumed with complementation of the full-length anrF gene to the mutant B8F.


CONCLUSIONS
The anrF gene obtained is related to the antagonistic activity of B8, and the antagonistic substances produced by B8 are andrimid and/or its analogs.
Yu
Xu-Ping
XP
Veterinary Medicine Department, Animal Science College, Zhejiang University, Hangzhou 310029, Zhejiang Province, China. xpyu@zju.edu.cn
Zhu
Jun-Li
JL
Yao
Xue-Ping
XP
He
Shi-Cheng
SC
Huang
Hai-Ning
HN
Chen
Wei-Liang
WL
Hu
Yong-Hao
YH
Li
De-Bao
DB
D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics; D016972:Enterobacter cloacae / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family; D016254:Mutagenesis, Insertional; D011090:Polyenes; D011758:Pyrroles
D004251:DNA Transposable Elements; D011090:Polyenes; D011758:Pyrroles; C088069:andrimid