Molecular Characterization of camphor utilizing bacterial isolates from refinery sludge and detection of target loci-Cytochrome P-450 cam mono oxygenase (cam C gene) by PCR and gene probe

Abstract

This study presents the isolation and molecular characterization of bacterial strains utilizing 1, 7, 7-Trimethylbicyclo (2.2.1) heptane-2-one (camphor) as a sole source of carbon, isolated from the biomass sludge sample collected from an effluent treatment plant of Mathura Refinery Limited (MRL), India. Initial screening was carried out where the 16S rDNA PCR was performed using reported eubacterial primer set followed by Amplified Ribosomal DNA Restriction Analysis (ARDRA). About 47% of the isolates have shown unique ARDRA pattern based on which, 15 distinct isolates were selected and tested for the presence of cam C gene that was successfully demonstrated by PCR using gene specific primers. A Dot-Blot experiment was designed to detect the cam C loci in the plasmid DNA of all camphor isolates based on non-radioactive "Biotin-Streptavidin" detection system. The bacterial identity with respect to partial 16S rDNA gene sequences of all camphor isolates placed them in 9 major genera viz., Pseudomonas sp., Staphylococcus sp., Alcaligenes sp., Agromyces sp., Stenotrophomonas sp., Reichenowia sp., Achromobacter sp., Brevibacterium sp. and Pseudaminobacter sp. A detailed phylogentic tree was also constructed to establish their evolutionary status from the gene sequence data. © 2013 Kumar et al.