Identification Of Novel Small Regulatory RNAs In Deep-Sea Microbes Through Genomic,Metagenomic And Metatranscriptomic Analysis

Understanding the adaptation mechanisms of deep-sea microorganisms have always been an elusive area of system biology.These microorganisms survive and thrive themselves in harsh conditions where they are constantly exposed to wide range of environmental changes by regulating their gene expression.Small regulatory RNAs(sRNAs)are short untranslated transcripts(50-500 nucleotide [nt]),known for modulating the gene expression at transcriptional and posttranscriptional levels in prokaryotes.They have been shown to involve in almost every global response in prokaryotes including environmentally significant processes.This thesis is aimed to explore the occurrence,role towards environmental adaptation and ecological relevance of sRNAs in the deep-sea environment.Therefore,this thesis revolves around the identification,characterization and function deciphering of sRNAs in deep-sea cultured and uncultured microbes using their genomic,metagenomic and metatranscriptomic data.Shewanella are one of the most abundant Proteobacteria in the deep-sea and are renowned for their versatile electron accepting capacities.To understand the potential roles of sRNAs in the adaptation of Shewanella towards deep-sea environments,Shewanella piezotolerans WP3,a piezotolerant and psychrotolerant deep-sea iron reducing bacterium is proposed here as a good candidate because of its ability to adapt in wide range of physical environmental conditions.As it can grow in the pressure range of 0.1-50 MPa with optimal growth at 20 MPa,and in the temperature range of 0-28°C with an optimum growth temperature of 20°C.A comparative genomics based in-silico approach was utilized to detect the sRNAs in the genome of Shewanella piezotolerans WP3.In total,209 sRNA candidates were identified with high confidence in S.piezotolerans WP3.About 92%(193 out of 209)of these putative sRNAs belong to the class trans-encoded RNAs,suggesting that trans-regulatory RNAs are the dominant class of sRNAs in S.piezotolerans WP3.The remaining 16 were belonging to class cis-encoded sRNAs and were validated experimentally using qPCR.The expression analysis of these cis-sRNAs under different environmental conditions proved that five of them act as coldregulated sRNAs.Being the less-understood type of sRNAs,cis-sRNAs are usually considered to entail in fundamental biological processes,however,our study demonstrates that cis-sRNAs could also play their roles in adaption to extreme conditions.sRNAs have also appeared to regulate environmentally significant processes including quorum sensing,iron homeostasis,amino acid and vitamin biosynthesis,stress responses and photosynthesis.Previous sRNA identification studies have mainly been carried out on and remain limited to a few model and cultured microorganisms,so little is known about their diversity and ecological relevance in deep-sea microbial communities.To study the environmental sRNAs and improve our understanding of their diversity and ecological relevance in deep-sea environment,this thesis demonstrates the sRNAs derived from metagenome and metatranscriptome samples recovered from Guaymas Basin sediments.Guaymas Basin represents unique features as compared to other hydrothermal vent sites as these accumulating sediments release huge amounts of petroleum-like products,the vent fluid also exhibits unique chemistry,i.e,nearly neutral pH and release a vast quantity of methane.Anaerobic methane-oxidizing(ANME)archaea which are the known activate group therein,undergo sulfate-dependent,anaerobic oxidation of methane(AOM)to maintain the global flux of methane.In this thesis,a metagenome and metatranscriptome based integrated approach was utilized to identifying the active sRNAs in Guaymas Basin sediments.The same approach was specifically used to investigate the sRNAs in ANME.To broaden the scope of sRNAs in ANME,the nearly complete genomes of the deep-sea dwelling anaerobic methane oxidizing archaea(ANME-2a and ANME-2d)were scanned using two independent computational approaches.This study reported the presence of hundreds of environmental sRNAs comprising of several groups,of which about 18% were more likely to be novel sRNA motifs.Interestingly,a putative cis-acting sRNA of methyl coenzyme M reductase alpha subunit(mcrA),a key enzyme in methanogenesis,and AOM,was also discovered in ANME-1 and discussed in this thesis.Furthermore,six and one sRNAs were identified in ANME-2a and ANME-2d respectively,using their nearly complete genome.Although,bacterial and archaeal sRNAs are somewhat structurally and functionally similar,however,prediction of fewer sRNAs in archaea using comparative genomics-based approaches suggests that sRNAs in archaea don't tend to be evolutionary as conserve as bacterial sRNAs.We believe that our findings will contribute to an improved understanding of environmental sRNAs,thereby,in microbial adaptation strategies in deep-sea environments.