Screen a dehalogenase gene using isolated bacterium from sea-shore soil of caspian sea

Abstract

Halogenated organic compound are widely used in agriculture and industry over the past 100 years. The use of these compounds in the environment led to human health problems and environmental pollution because of their persistence and toxicity. Dalapon or 2,2-dichloropropionic acid is widely used as herbicides and plant growth regulator which cause environmental pollution. In this research, bacterium NR1 was isolated from soil sample taken from Bandar-e Anzali seashore in north of Iran. The result has shown that this bacterium grow in minimal media containing 20mM 2, 2-DCP with doubling time of 0.89 hours, which indicated its ability to degrade 2, 2-DCP. Based on microscopic observation and Gram staining, strain NR1 was identified as rod Gram positive bacterium. Biochemical tests for the bacterium NR1 were positive for oxidase, catalase, gelatin liquefaction, nitrate reduction, TSI, oxidation- fermentation glucose, starch and casein test, while the tests result were negative for lactose utilization, citrate, indole, and urease test. Genomic DNA from bacterium NR1 was extracted and 16S rRNA PCR amplification was carried out using universal primers, Fd1 (5‟ - AGA GTT TGA TCC TGGCTC AG - 3‟) and rP1 (5‟- ACG GTC ATA CCT TGT TAC GAC TT - 3‟) before sending for sequencing. NR1 strain 16S rRNA sequences were applied for Basic Local Alignment Search Tool nucleotide (BLASTn) and further analyzed using phylogenetic tree of Neighbour-Joining method (MEGA 5). Phylogenetic analysis indicated that NR1 strain clearly shared 97% homology to the genus of Bacillus cereus and therefore designated as Bacillus cereus sp. NR1. The PCR analysis of dehalogenase using dhlb_F_314/dhlB_R_637 primers showed a band with approximate size of >200 bp, suggesting this bacterium carries dehalogenase from class I.