Metagenomic approach to assess diversity of bacterial community in saline pokkali habitats of kerala
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Date
2012
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College of Horticulture, Vellanikkara
Abstract
The use of traditional microbiological culturing methods for the study of microbes has its limitations. It has been estimated that 99 per cent of microbes cannot be cultured easily. Over the past decade, “Metagenomics,” which is culture-independent genomic analysis of microbes, has been developed to overcome the drawbacks in culture-based analysis of microbial communities. Soil is considered to be a complex environment and a major reservoir of microbial genetic diversity. Metagenomics is a powerful tool in the analysis of diversity of microbial communities in specific environments. An attempt was made to analyse the diversity of bacterial community in the acid saline Pokkali soil from Vyttila, through Metagenomic approach. Environmental DNA was isolated from the soil by direct method. 16S rDNA which encodes 16S rRNA found in the 30S subunit of prokaryotic ribosome is used as the barcode for bacterial identification. 1500bp fragment of 16S rDNA was amplified using specific primers through polymerase chain reaction (PCR). The amplicons were ligated in plasmid vector pGEMT and cloned in E. coli, to construct a Metagenomic library. Sequence analysis of 30 randomly picked colonies revealed that 23 clones (76.7%) were of unculturable bacteria and 7 clones (23.3%) were culturable. The most predominant phylum was Proteobacteria, which included Ectothiorhodospiraceae, Azospira sp., Stenotrophomonas maltophilia, Thiobacillus prosperus, Levilinea saccharolytica, Desulfobacteraceae, Thioalkalivibrio sp. and Rhodocylales. Other phyla included Chloroflexi, Acidobacteria and Cynobacteria. A range of bacteria including acidophiles, extreme halophiles, denitrifier, S oxidizers, sulphate reducers, aerobes, strict anaerobes, thermophiles, mesophiles, photosynthetic bacteria and even human pathogens were obtained, of the 23 unculturable bacteria detected, six did not show homology with any bacterial sequence available in NCBI database, indicating the possibility that these could be novel. The phylogenetic tree based on partial 16S rRNA gene placed 30 clones from Pokkali soil in three major cluster- Proteobacteria, Chloroflexi and Acidobacteria. Three predominant bacteria isolated by the traditional method belonged to the genus Bacillus. Metagenomic approach successfully revealed the composition and diversity of bacterial community in Pokkali soil. A function-derived strategy could be used for bioprospecting of gene related to acid and salt tolerance genes.
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