SCREENING OF HYDROGENASE ACCESSORY GENES hypA AND hypB IN FEW ACID TOLERANT BACILLUS SP

Loading...
Thumbnail Image
Date
2017-07
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
AAU, Jorhat
Abstract
The present investigation reports on the screening of hydrogenase accessory genes viz., hypA and hypB in previously isolated acid tolerant Bacillus sp. followed by differential expression study (pH 7.0 and pH 4.5) of the both genes in three selected strains and subsequent cloning of hypA and hypB gene of Bacillus megaterium into pET28b+ vector. A total of 50 previously isolated acid tolerant Bacillus strains were considered for screening of hypA and hypB genes. Among the 50 isolates, 10 isolates tested positive for hypA & hypB genes. Three isolates viz., B. megaterium (GG1), B. cereus (GG2) and Lysinibacillus fusiformis (GG11) were considered for further study due to their ubiquitousness and higher acid tolerance. Bacterial RNA was isolated followed by synthesis of cDNA. A q-RT-PCR was performed in the above mentioned isolates to analyse the expression level of the said genes in acidic condition. The expression of hypA and hypB genes were significantly (P ≤ 0.05) up-regulated at pH 4.5 than in pH 7.0 in all the 3 isolates viz., GG1, GG2, GG11. An 18 fold increase in the expression of hypA and a 6.1 fold increase in expression of hypB were observed in the isolate GG1 at pH 4.5 compared to pH 7.0. The expression of hypA and hypB genes in GG2 was found to be 4.6 and 3.8 fold higher respectively at pH 4.5 than pH 7.0. In GG11, hypA showed 5.3 fold and hypB showed 2.8 fold increased expression at pH 4.5 compared to pH 7.0. Maximum expression of both the genes was observed in Bacillus megaterium. These genes were further cloned into pET28b+ vector. The transformation frequency obtained for the hypA and hypB was 1.5 x 106 and 3.2 x 104 CFU/μg respectively. Successfully transformed colonies were further validated through colony PCR as well as PCR on isolated plasmid DNA. The cloned hypA gene was sequenced and BLAST analysis was performed. BLAST analysis revealed that the genes belonged to the respective genus. The results of the present study revealed that hypA and hypB genes may have role in conferring acid tolerance to soil bacteria.
Description
Keywords
Citation
Collections