Characterization of Hexachlorocyclohexane (Hch) Degradation Pathway Genes in Two Strains of Sphingobium Bacteria Previosuly Isolated From Hch Contaminated Soil in Kitengela

Abstract

Hexachlorocyclohexane (HCH) continues to pose threat to the environment despite the restricted use or complete ban in most parts of the world due to its toxicity effects, environmental persistence, and bioaccumulation within the food chain. The extensive use of lindane (99% pure γ-HCH isomer) in agriculture has resulted in contamination of soil and water environments on a global scale. Microbes, particularly sphingomonads, can degrade HCH residues into non-toxic and environmentally safe metabolites. A variety of enzymes participate in the lindane degradation pathway, including dehydrochlorinase (LinA), dehalogenase (LinB), dehydrogenase (LinC & LinX), dechlorinase (LinD), dioxygenase (LinE), and transcriptional regulator (LinR). To develop efficient technologies for sustainable bioremediation of lindane, information on the organization and diversity of Lin genes among sphingomonads with the potential to degrade lindane is a prerequisite. Therefore, this study aimed to characterize Lin genes involved in the degradation of Hexachlorocyclohexane (HCH) in two strains of Sphingobium bacteria (Sphingobium sp. S6 and Sphingobium sp. S8). DNA was extracted from broth cultures of Sphingobium strains S6 and S8, and DNA hybridization was carried out to detect the Lin genes in the two strains using Digoxigenin (DIG)-labeled DNA probes synthesized by PCR. The Lin genes detected were amplified by PCR using their respective primers and purified PCR products sequenced by the Sanger sequencing method. The resulting DNA sequences were analysed by homology and phylogenetic analysis. Proteins of the respective Lin genes were modeled via homology modeling to predict their 3D structure and active sites. Both Sphingobium strains S6 and S8 were found to contain LinA, LinB, LinC, LinD, LinE, LinR, and LinX gene and IS6100, which were conserved. Single copies of LinA, LinB, and LinD gene, two copies of LinC, LinE, and LinX gene, and multiple copies of LinR gene and IS6100 occurred within the genome of Sphingobium strain S6. Moreover, the DNA sequences of LinA to LinX from Sphingobium sp. S6 produced full-length polypeptides (LinA, LinB, LinC, LinD, LinE, LinR, and LinX) containing 156, 296, 250, 346, 321, 301, and 250 amino acid residues, respectively, whereas those of Sphingobium sp. S8 contained 150, 291, 250, 344, 317, 293, and 250 amino acids, respectively. The predicted protein models of LinA, LinB, LinC, LinD, and LinE comprised of one to four chains. Furthermore, the active binding site of LinA contained three conserved catalytic residues (Lys20, Asp25, and His73), that of LinB possessed a quintet consisting of the nucleophile−Asp108, catalytic acid−Glu132, and catalytic base−His272 and the halide stabilizing residues Asn38 and Trp109. Putative residues (Trp109, Val134, Phe143, Pro144, Gln146, Asp147, Phe151, Phe169, Val173, Leu177, Trp207, Pro208, Ile211, Ala247, Leu248, and Phe273) surrounding the active site of LinB were also conserved. The protein models of LinC, LinD, and LinE contained between two to four active binding sites whose catalytic residues have not been elucidated yet. This study demonstrated that Sphingobium sp. S6 and Sphingobium sp. S8 possess Lin genes with their copy numbers ranging from one to two to multiple copies and are highly conserved. Based on the presence of catalytic sites, information on the substrate-binding properties of lindane by the various Lin proteins need to be elucidated, which could form the basis for designing enzyme mutants with improved lindane degradation capabilities. These can then be applied in the enzymatic bioremediation of HCH stockpiles and liquid contamination.