Identification and characterization of genes conferring salt tolerance to Escherichia coli from pond water metagenome

Metagenomics provides culture-independent access to gene pool of the whole microbial communities. To identify genes responsible for salt tolerance in unculturable bacteria, Escherichia coli clones were enriched with an ability to grow at inhibitory NaCl concentrations (750 mM) from a pond water metagenomic library. From two unique clones, genes encoding for proteins with similarity to a putative general stress protein (GspM) harbouring GsiB domain and a putative enoyl-CoA hydratase (EchM) were identified to be responsible for salt tolerance. The gspM was expressed by its native promoter whereas the echM was expressed from the lacZ promoter of the plasmid. EchM was overexpressed with a hexahistidyl tag. Purified EchM showed crotonyl-CoA hydratase activity. These genes have potential application in generating salt tolerant recombinant bacteria or transgenic plants.<br /

Gut microbial diversity in health and disease: experience of healthy Indian subjects, and colon carcinoma and inflammatory bowel disease patients

Background: The intestinal microbiota, through complex interactions with the gut mucosa, play a key role in the pathogenesis of colon carcinoma and inflammatory bowel disease (IBD). The disease condition and dietary habits both influence gut microbial diversity. Objective: The aim of this study was to assess the gut microbial profile of healthy subjects and patients with colon carcinoma and IBD. Healthy subjects included ‘Indian vegetarians/lactovegetarians’, who eat plant produce, milk and milk products, and ‘Indian non-vegetarians’, who eat plant produce, milk and milk products, certain meats and fish, and the eggs of certain birds and fish. ‘Indian vegetarians’ are different from ‘vegans’, who do not eat any foods derived wholly or partly from animals, including milk products. Design: Stool samples were collected from healthy Indian vegetarians/lactovegetarians and non-vegetarians, and colon cancer and IBD patients. Clonal libraries of 16S ribosomal DNA (rDNA) of bacteria were created from each sample. Clones were sequenced from one representative sample of each group. Approximately 500 white colonies were picked at random from each sample and 100 colonies were sequenced after amplified rDNA restriction analysis. Results: The dominant phylum from the healthy vegetarian was Firmicutes (34%), followed by Bacteroidetes (15%). The balance was reversed in the healthy non-vegetarian (Bacteroidetes 84%, Firmicutes 4%; ratio 21:1). The colon cancer and IBD patients had higher percentages of Bacteroidetes (55% in both) than Firmicutes (26% and 12%, respectively) but lower Bacteroidetes:Firmicutes ratios (3.8:1 and 2.4:1, respectively) than the healthy non-vegetarian. Bacterial phyla of Verrucomicrobiota and Actinobacteria were detected in 23% and 5% of IBD and colon patients, respectively. Conclusions: Ribosomal Database Project profiling of gut flora in this study population showed remarkable differences, with unique diversity attributed to different diets and disease conditions

Sequence analysis of a salt tolerant metagenomic clone

Metagenome represent an unlimited resource for discovery of novel genes. Here we report, sequence analysis of a salt tolerant metagenomic clone (6B4) from a pond water metagenomic library. Clone 6B4 had an insert of 2254 bp with G+C composition of 64.06%. DNA sequence from 6B4 showed homology to DNA sequences from proteobacteria indicating origin of 6B4 metagenomic insert from a yet uncharacterized proteobacteria. Two encoded proteins from clone 6B4 showed match with ATP-dependent Clp protease adaptor protein (ClpS) and phasin, while two truncated encoded proteins showed match with poly-3-hydroxybutyrate synthase and permease. Clp complex is known to play a role in stress tolerance. Expression of ClpS from metagenomic clone is proposed to be responsible for salt tolerance of the metagenomic clone 6B4.</p