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 1842 bp in length encoding a polypeptide of 613 amino acid residues. Calculated molecular weight and pI of the protein were 66,128.36 Da 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 16 h, 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.

Carbohydrate Hydrolytic Activity, Antibiotic Resistance and Stress Tolerance ofLacticaseibacillus paracaseiBCRC-16100 andLacticaseibacillus paracaseiZFM54 for Probiotics Using Genomic and Biochemical Approaches

“…The growth of Lacticaseibacillus paracasei ZFM54 is less influenced by temperature and higher frequency of TFs that may potentially bind to its promoter region to a thermoregulatory gene dnaK can play a significant role in this tolerance. A previous study has shown that in known thermotolerant Bacillus pumilus, dnaK is highly expressed (Kumar et al , 2013). Although it cannot be ruled out that there might be differences in dnaK of different species and strains and other genes present in the genome of the two isolates play synergistic or antagonistic roles in tolerance to certain temperatures.…”

“…There are several genes that confer thermotolerance in both isolates which allows growth in a wide range of temperature where dnaK and dnaJ are likely responsible for this trait these genes show the highest potential to be regulated due to the association of promoter regions to multiple transcription factors (TFs) ( Figure 4a ). Both dnaK and dnaJ belong to a chaperone system in microbes known to confer wider tolerance to temperatures (Kumar et al , 2013 and Kim et al , 2021). The two isolates grow optimally at pH 5-7, however, promoter analysis of genes responsible for this trait only suggested clpB as may be responsible in differential growth in pH.…”

“…Genetic map is generated using Proksee v1.0.0a6. expressed (Kumar et al, 2013). Although it cannot be ruled out that there might be differences in dnaK of different species and strains and other genes present in the genome of the two isolates play synergistic or antagonistic roles in tolerance to certain temperatures.…”

Carbohydrate Hydrolytic Activity, Antibiotic Resistance and Stress Tolerance of <em>Lacticaseibacillus paracasei </em>BCRC-16100 and <em>Lacticaseibacillus paracasei</em> ZFM54 for Probiotics Using Genomic and Biochemical Approaches

“…There are several genes that confer thermotolerance in both isolates which allows growth in a wide range of temperature where dnaK and dnaJ are likely responsible for this trait these genes show the highest potential to be regulated due to the association of promoter regions to multiple transcription factors (TFs) (Figure 4a). Both dnaK and dnaJ belong to a chaperone system in microbes known to confer wider tolerance to temperatures [44,57]. The two isolates grow optimally at pH 5-7, however, promoter analysis of genes responsible for this trait only suggested clpB as may be responsible in differential growth in pH.…”

“…The growth of L. paracasei ZFM54 is less influenced by temperature and higher frequency of TFs that may potentially bind to its promoter region to a thermoregulatory gene dnaK can play a significant role in this tolerance. A previous study has shown that in known thermotolerant Bacillus pumilus, dnaK is highly expressed [44]. Although it cannot be ruled out that there might be differences in dnaK of different species and strains and other genes present in the genome of the two isolates play synergistic or antagonistic roles in tolerance to certain temperatures.…”