Isolation of local bacterial capable of degrading halogenated compounds and analysis of putative haloacid permease gene

Abstract

3-chloropropionic acid and 2,2-dichloropropionic acid are synthetic halogenated compounds used in herbicide. A bacterium isolated from a soil sample and characterised as Rhodococcus sp. by 16S rRNA analysis, was able to degrade and utilised 3-chloropropionate as the sole source of carbon and energy. This was supported by the ability of the bacterium to grow on 20 mM 3-chloropropionate with a doubling time of 11.72 hours. The utilisation of 3-chloropropionate was also confirmed by detection of 3-chloropropionate depletion in the medium using HPLC. Cell free extract of Rhodococcus sp. had an enzyme specific activity of 0.013 µmol Cl-/min/mg protein towards 3-chloropropionate. Another bacterium isolated from the same soil sample and identified as Methylobacterium sp. by 16S rRNA analysis was found to be able to degrade 2,2-dichloropropionate. The bacterium grew in 20mM 2,2-dichloropropionate minimal medium with a doubling time of 20.32 hours. Degradation of 2,2-dichloropropionate was further confirmed by detection of 2,2- dichloropropionate depletion in growth medium by HPLC. Cell free extract prepared from the cell showed 0.039 µmol Cl-/min/mg protein specific activity towards 2,2-dichloropropionate. A putative haloacid permease gene (dehrP) from Rhizobium sp. was subcloned into Novagen pET 43.1a plasmid. The newly constructed plasmid was designated as pHJ. The cloned gene was sequenced and analysed using various online analysis tools. DehrP has a calculated molecular weight of 45 kDa and an isoelectric point of 9.78. The nucleotide sequence of dehrP showed significant homology (86%) with the putative mono-chloropropionic acid permease from Agrobacterium sp. NHG3 and 62 % homology with the haloacid specific transferase from Burkholderia sp.