To explore the natural microbial community of any ecosystems by high-resolution molecular approaches including next generation sequencing, it is extremely important to develop a sensitive and reproducible DNA extraction method that facilitate isolation of microbial DNA of sufficient purity and quantity from culturable and uncultured microbial species living in that environment. Proper lysis of heterogeneous community microbial cells without damaging their genomes is a major challenge. In this study, we have developed an improved method for extraction of community DNA from different environmental and human origin samples. We introduced a combination of physical, chemical and mechanical lysis methods for proper lysis of microbial inhabitants. The community microbial DNA was precipitated by using salt and organic solvent. Both the quality and quantity of isolated DNA was compared with the existing methodologies and the supremacy of our method was confirmed. Maximum recovery of genomic DNA in the absence of substantial amount of impurities made the method convenient for nucleic acid extraction. The nucleic acids obtained using this method are suitable for different downstream applications. This improved method has been named as the THSTI method to depict the Institute where the method was developed.

The gastric microbiome is suspected to have a role in the causation of diseases by Helicobacter pylori. Reports on their relative abundance vis-à-vis H. pylori are available from various ethnic and geographic groups, but little is known about their interaction patterns. Endoscopic mucosal biopsy samples from the gastric antrum and corpus of 39 patients with suspected H. pylori infection were collected and microbiomes were analyzed by 16S rDNA profiling. Four groups of samples were identified, which harbored Helicobacter as well as a diverse group of bacteria including Lactobacillus, Halomonas and Prevotella. There was a negative association between the microbiome diversity and Helicobacter abundance. Network analyses showed that Helicobacter had negative interactions with members of the gastric microbiome, while other microbes interacted positively with each other, showing a higher tendency towards intra-cluster co-occurrence/co-operation. Cross-geographic comparisons suggested the presence of region-specific microbial abundance profiles. We report the microbial diversity, abundance variation and interaction patterns of the gastric microbiota of Indian patients with H. pylori infection and present a comparison of the same with the gastric microbial ecology in samples from different geographic regions. Such microbial abundance profiles and microbial interactions can help in understanding the pathophysiology of gastric ailments and can thus help in development of new strategies to curb it.The acidic pH in the stomach lumen impedes bacterial growth 1 . However, it is now known that the human stomach is not sterile but is rather colonized by diverse microbiota 2 . High-throughput sequencing of gastric biopsy samples suggests that the human stomach may harbor 128 phyla, although mainly dominated by five phyla, namely, Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Fusobacteria 2-5 . Among these, several species of Helicobacter are known to be natural inhabitants of the human stomach. Helicobacter pylori (H. pylori), a Gram-negative bacterium and frequent isolate of stomach specimens, with reported links to the causation of chronic/atrophic gastritis, duodenal ulcers, gastric mucosa-associated lymphoid tissue (MALT) lymphoma and adenocarcinoma 2,6,7 , has coevolved with its human host.While H. pylori has been incriminated in disease, an aspect that has only been recently investigated is the role of the stomach microbiome in the causation of these diseases, especially adenocarcinoma. Studies focusing on the microbial composition of the stomach in healthy individuals 4,[8][9][10] have indicated the presence of genera like Streptococcus, Prevotella, Veillonella, Fusobacterium, Haemophilus and Clostridium. H. pylori is known to utilize specific molecular mechanisms to modulate host immune response and create a local micro-environment that aids its colonization 5,11 . The influence of Helicobacter abundance on the populations of other genera and vice versa, and any interactions between them in the causation ...

The genesis of toxigenic Vibrio cholerae involves acquisition of CTX, a single-stranded DNA (ssDNA) filamentous phage that encodes cholera toxin (CT). The phage exploits host-encoded tyrosine recombinases (XerC and XerD) for chromosomal integration and lysogenic conversion. The replicative genome of CTX produces ssDNA by rolling-circle replication, which may be used either for virion production or for integration into host chromosome. Fine-tuning of different ssDNA binding protein (Ssb) levels in the host cell is crucial for cellular functioning and important for CTX integration. In this study, we mutated the master regulator gene of SOS induction, lexA, of V. cholerae because of its known role in controlling levels of Ssb proteins in other bacteria. CTX integration decreased in cells with a ⌬lexA mutation and increased in cells with an SOS-noninducing mutation, lexA (Ind ؊ ). We also observed that overexpression of host-encoded Ssb (VC0397) decreased integration of CTX. We propose that LexA helps CTX integration, possibly by fine-tuning levels of host-and phage-encoded Ssbs. IMPORTANCECholera toxin is the principal virulence factor responsible for the acute diarrheal disease cholera. CT is encoded in the genome of a lysogenic filamentous phage, CTX. Vibrio cholerae has a bipartite genome and harbors single or multiple copies of CTX prophage in one or both chromosomes. Two host-encoded tyrosine recombinases (XerC and XerD) recognize the folded ssDNA genome of CTX and catalyze its integration at the dimer resolution site of either one or both chromosomes. Fine-tuning of ss-DNA binding proteins in host cells is crucial for CTX integration. We engineered the V. cholerae genome and created several reporter strains carrying ⌬lexA or lexA (Ind ؊ ) alleles. Using the reporter strains, the importance of LexA control of Ssb expression in the integration efficiency of CTX was demonstrated.

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