Molecular Cloning and Genetic Analysis of Functional merB Gene from Indian Isolates of Escherichia coli

Abstract

Studies were carried out to characterize organomercurial lyase genes from wild type mercury-resistant Escherichia coli isolates, previously collected from five geographically distinct regions of the Indian subcontinent. PCR amplification followed by DNA sequencing of amplified fragments showed three merB identical to the previously characterized mer B from E. coli pR831b that were thus considered as the same gene. The remaining two genes derived from E. coli isolates of an almost mercury-free site (Dal lake, Kashmir) and designated as pIAAD3 merB and pIAAD14 merB showed slight variation (2%) at base. However, this variation in pIAAD3 due to the absence of base "T" at 479 position results in complete frame shift and the predicted MerB-like polypeptide derived from it showed 21.53% divergent at its C terminal end from the previously characterized pR831b MerB. The expression profile of pIAAD3 merB in pQE30 and pUC18 vectors each demonstrated 22.2 kDa proteins. The induced DH5alpha E. coli cells possessing pIAAD3 merB cloned in pUC18 vector split phenyl mercuric acetate (PMA) into benzene and inorganic mercury efficiently, thus giving a clue that the expressed gene product is biologically active. The current study suggests that such genetic changes may take place in the continued absence of mercury pressure, and with such modifications, they finally break down to act as vestigial remnants. Further work is going on in our lab to exploit pIAAD3 merB for the bioremediation of mercury-polluted sites.