Isolation And Characterization Of Biphenyl-Degrading Bacterium Achromobacter Sp. BP3 And Cloning Of Catabolic Genes

A bacterial strain capable of utilizing biphenyl as the sole carbon source for its growth was isolated from petroleum-contaminated soil.This bacterium was identified preliminarily as Achromobacter sp.based on its physiological & biochemical characters and 16S rDNA sequence analysis,and designated as BP3(GenBank Accession No.EF057390).Strain BP3 could transform 50 mg·L^-1 of biphenyl within 12h,and yellow meta-cleavage product(2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate,HOPDA) during biphenyl degradation was detected.The optimal pH for biphenyl degradation ranged from pH7.0 to pH9.0,and the optimal temperature was 35℃.The degradation rate correlated with the initial inoculum size positively.On the concentration of 3-50 mg·L^-1,biphenyl was degraded rapidly.No significant effect of Zn²⁺,Al³⁺,and Fe²⁺ was observed for biphenyl degradation by strain BP3,whereas Cu²⁺,Ni²⁺,and Cd²⁺ showed obvious inhibitory effect.Six positive clones with 2,3-dihydroxybiphenyl 1,2-dioxygenase(2,3-DHBPDO) activity were obtained by the construction of expression library.And three structural genes designated as bphC,bphC2 and xylE for meta-cleavage of 2,3-DHBP were predicted based on sequencing and ORF analysis online.The amino acid sequence of BphC2 and XylE showed 100%similarity with 2,3-DHBPDO and catechol 2,3-dioxygenase,respectively, while BphC exhibited the highest similarity of 49%with 2,3-DHBPDO.These three genes were ligated into expression vector pET29a,respectively,and functional expressed in E. coli BL21(DE3).Further analysis revealed that bphC2 and xylE located on the bph operon for biphenyl degradation and xyl operon for xylene degradation,respectively.A 16.7-kb DNA fragment consisted with entire bph cluster(bphRA1A2XA3A4BC2HJID) was obtained using normal PCR amplification and chromosome walking.Genes encoding integrase and transposon related sequences were detected upstream and downstream of bph cluster,respectively, indicating the bph cluster may locate on a big mobile genetic element(MGE) based on the literature available,xylE lied on a xyl operon compring xylZLTEG,the organization of which was in good agreement with xyl operons reported before(xylXYZLTEGFJQKIH).It was postulated that bph operon was responsible for the upper pathway and partial lower pathway in biphenyl degradation,while xyl cluster participated the degradation of benzoate formed during the upper pathway.Whether the bphC gene was involved in biphenyl degradation by strain BP3 needed to be further studied.