Abstract
Problem statement: This study was an attempt to isolate anaerobic microbes with potential for DNA double strand break repair using methanogen specific medium (DSMZ 120) from East Calcutta Wetland in India. It also intended to verify the specificity of the medium for isolation of the desired family of microbe. Approach: Culture based technique was used to obtain the pure isolate that was further characterized in details. For double strand break repair studies, isolate was irradiated with different doses of 60Co gamma rays and its subsequent repair was observed using pulse field gel electrophoresis and asymmetric field inversion gel electrophoresis. Inhibitor was used to predict the mechanism of repair. Results: In this study we isolated and characterized a metal sensitive anaerobic microbial strain obtained using methanogen specific medium (DSMZ 120) from East Calcutta Wetland in India. The strain was one of the members of the group of uncultivated bacterium as evident from phylogenetic analysis, thus indicating the successful cultivation of an as yet uncultivable novel microbe (GenBank Acc. No. FJ 930097) and also the non-specific growth of microbes in prescribed medium. It was a Gram positive Bacilli, member of Fermicutes with optimum growth at 25°C and pH-7. The growth curve analysis showed a lag phase up to 24 h, log phase from 24-48 h, an early stationary phase from 96 h onwards. The strain could repair the DNA double strand break caused by irradiation with 60Co γ rays. The dose profile study revealed maximum repair at 60 Grays and thereafter a drop in repair ability with increase in irradiation dose. The time required for repair showed an essential incubation period of 4 h. The DNA polymerase inhibitor, Arabinose CTP inhibited the repair indicating the involvement of polymerase in the repair process and thus pointing towards homologous recombination as the underlying mechanism. Conclusion: In this study we were able to cultivate an as yet uncultivable anaerobic bacterial isolate and predict the growth conditions for the isolate. On irradiation with 60Co γ rays the isolate showed maximum repair following 60 Gray damage. DNA polymerase inhibitor arabinose CTP inhibited the repair mechanism completely. This indicated that DNA polymerase took active part in repair process and thus the mechanism was that of homologous recombination repair.
Highlights
DNA molecules can be damaged in many ways
Isolation: Microbial growth was obtained in methanogen specific media from water of Captain bheri of East Calcutta Wetland
Arabinose CTP inhibited the repair of the isolate (Fig. 11). This finding proves that DNA polymerase was involved in the repair process and the repair takes place through homologous recombination
Summary
DNA molecules can be damaged in many ways. Spontaneous damage may occur due to replication errors, deamination, depurination and oxidation. Methanogen is a single celled archaea that produce methane as a metabolic end product in an anaerobic environment. They are commonly founds in wetland, paddy fields and marshlands. Methanogen are used worldwide to reduce and detoxify agricultural, industrial and urban waste to generate methane from waste biomass to be used as fuel. They are very important from the point of view of biotechnology and bioremediation. In this study we have characterized the isolate studying the DNA-DSB repair mechanism in it
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