Cloning and expression of dnaK gene from Bacillus pumilus of hot water spring origin

Abstract

A set of thermotolerant strains isolated from hot springs of Manikaran and Bakreshwar (India) were selected with an aim to isolate dnak gene which encodes DnaK protein. The gene dnaK along with its flanking region was successfully amplified from 5 different strains (4 from Bakreshwar and one from Manikaran). Restriction fragment length polymorphism (RFLP) revealed that amplicons were almost identical in sequence. The dnak gene from one representative, Bacillus pumilus strain B3 isolated from Bakreshwar hot springs was successfully cloned and sequenced. The dnaK gene was flanked by gene grpE on one side. The dnaK gene was 1842bp in length encoding a polypeptide of 613 amino acid residues. Calculated molecular weight and pI of the protein were 66,128.36Da and 4.72 respectively. The deduced amino acid sequence of this gene shared high sequence homology with other DnaK proteins and its homologue Hsp 70 from other microorganisms, but possessed 36 substitutions and two insertions, as compared to DnaK protein of Bacillus subtilis. The dnaK gene of B. pumilus was successfully expressed in Escherichia coli BL 21 (DE3) using pET expression systems. Heterologous expression of dnaK of B. pumilus in E. coli BL 21 (DE3) allowed for the growth of E. coli up to 50°C and survival up to 60°C for 16h, suggesting that dnak from B. pumilus imparts tolerance to host cells under high temperature. This novel gene can be an important component for possible utilization in abiotic stress management of plants.