Isolation & Characterization Of HCH-Degrading Bacterial Strains And Cloning Of Their Degradation Related Genes

In the present study, three bacterial strains (designated as BHC-B, BHC-C and BHC-D) capable of degrading and utilizing y-HCH as a sole carbon source for growth were isolated from the soil of the wasteland around a warehouse which was used to store HCHs long time ago. Their 16S rRNA gene sequences were sequenced and deposited in the GenBank under accession numbers of KC 191573, KC 191574 and KC 191575, respectively. They have high similarity in 16S rRNA gene sequences and form a distinct clade in the phylogenetic tree. Their common nearest neighbors are Sphingobium japonicum UT26T, Sphingobium francense Sp+T and Sphingobium indicum B90AT, with sequence similarities ranging from 99.10% to 99.72%, which are all HCH-degrading strains and have been investigated intensively.As the16S rRNA gene sequence similarities among theses strains exceed 99%, it is difficult to distinguish them. ERIC-PCR is a method used to analyze the intestinal microbial flora communities, which was first used in this study to distinguish different strains from the genus of Sphingobium, which have high similarity in 16S rRNA gene sequences. Their ERIC-PCR products showed different band patterns on the DNA agarose electrophoresis gel and the phylogenetic tree based on band patterns analysis is very similar, indicating that ERIC-PCR analysis has some potential in distinguishing Sphingobium strains with high similarity.To distinguish these strains further, we also investigated their morphological physiological and biochemical characteristics. All of the three strains can grow on LB agar and form smooth and wet colonies within 2-3 days at 30℃. The colony color of BHC-B, BHC-C and BHC-D are yellow, pale yellow and bright yellow, respectively. They all grow optimally at pH 7.0 and 30℃. However, they differ from each other in two or more physiologic and biochemical features. Based on the above results, strains BHC-B, BHC-C and BHC-D were all identified as Sphingobium sp.As for the degradation of γ-HCH, the increasing inocula of BHC-B.BHC-C and BHC-D could accelerate the degradation, and the aeration had little influence on the degrading rate. All of them showed highest degrading efficiency at pH 7.0 and 30℃. BHC-B, BHC-C and BHC-D could also degrade other three isomers(α-, β-and δ-isomer) of HCH, but strain BHC-C showed the highest average removal rates against the same concentration (5 mg/L) of four isomers with 12 h, being 60.1%,30.2%,100% and 38.6% for α-,β-, γ- and δ-isomer, resprectively. Strain BHC-D showed the lowest average removal rates, being 34.3%,6.6%,98% and 20.3% for α-,β-,γ- and δ-isomer, resprectively. For the different isomers, the removal rates of the three strains were in the following order: γ-HCH>α-HCH>δ-HCH>β-HCH. For the same isomer, the removal rates were in the following order:BHC-C> BHC-B> BHC-D.We designed the primers according to the reported lin genes sequences,and PCR amplification results revealed that strains BHC-B, BHC and BHC-D all had six key lin genes (linABCDER). LinA, LinC, LinD, LinE and LinR genes had 99%-100% similarities with the corresponding reported enzymes in the HCH-degrading strains. By contrast, LinB showed 2%-3% divergence. The detailed analysis on the amino acids of LinB of three strains revealed that D108, E132 and H272 was highly conserved. However, several unique amino acid residues appeared differences. In strain BHC-B, they were N88, V123, H190and T274. In strain BHC-D, they were S137, F173, A177and V248. The differences in LinB may be responsible for the differences in the degradation rate of the different strains against the same HCH isomer and further studies are needed to verify this hypothesis.Strain BHC-A is isolated and stored by our lab, it could degrade four isomers of HCH. Its degradation pathway of δ-HCH has been partially investigated. The initial degrading steps were catalyzed by LinA and LinB2 and formed a conversion network. In oder to further study the complete pathway, the linA knockout strain BHC-A-ΔlinA was constructed and used to transform δ-HCH. The analysis of the metabolites showed that a new compound with the peak at RT=8.19min appeared at the GC chromatogram, but it is difficult to identify the structure of the compound through the mass spectrogram, which needs further study.