Microbial machinery dealing diverse aromatic compounds: Decoded from pelagic sediment ecogenomics in the gulfs of Kathiawar Peninsula and Arabian Sea.

Aromatic hydrocarbons are persistent pollutants in aquatic systems as endocrine disruptors, significantly impacting natural ecosystems and human health. Microbes perform as natural bioremediators to remove and regulate aromatic hydrocarbons in the marine ecosystem. The present study focuses upon the comparative diversity and abundance of various hydrocarbon-degrading enzymes and their pathways from deep sediments along the Gulf of Kathiawar Peninsula and Arabian Sea, India. The elucidation of large number of degradation pathways in the study area under the presence of a wide range of pollutants whose fate needs to be addressed. Sediment core samples were collected, and the whole microbiome was sequenced. Analysis of the predicted ORFs (open reading frames) against the AromaDeg database revealed 2946 aromatic hydrocarbon-degrading enzyme sequences. Statistical analysis portrayed that the Gulfs were more diverse in degradation pathways compared to the open sea, with the Gulf of Kutch being more prosperous and more diverse than the Gulf of Cambay. The vast majority of the annotated ORFs belonged to groups of dioxygenases that included catechol, gentisate, and benzene dioxygenases, along with Rieske (2Fe-2S) and vicinal oxygen chelate (VOC) family proteins. From the sampling sites, only 960 of the total predicted genes were given taxonomic annotations, which mention the presence of many under-explored marine microorganism-derived hydrocarbon degrading genes and pathways. Through the present study, we tried to unveil the array of catabolic pathways of aromatic hydrocarbon degradation and genes from a marine ecosystem that upholds economic and ecological significance in India. Thus, this study provides vast opportunities and strategies for microbial resource recovery in marine ecosystems, which can be investigated to explore aromatic hydrocarbon degradation and their potential mechanisms under various oxic or anoxic environments. Future studies should focus on aromatic hydrocarbon degradation by considering degradation pathways, biochemical analysis, enzymatic, metabolic, and genetic systems, and regulations.

Evolutionary and Antigenic Profiling of the Tendentious D614G Mutation of SARS-CoV-2 in Gujarat, India.

Humankind has suffered many pandemics in history including measles, SARS, MERS, Ebola, and recently the novel Coronavirus disease caused by SARS-CoV-2. As of September 2021, it has affected over 200 million people and caused over 4 million deaths. India is the second most affected country in the world. Up to this date, more than 38 Lakh viral genomes have been submitted to public repositories like GISAID and NCBI to analyze the virus phylogeny and mutations. Here, we analyzed 2349 genome sequences of SARS-CoV-2 submitted in GISAID by a single institute pertaining to infections from the Gujarat state to know their variants and phylogenetic distributions with a major focus on the spike protein. More than 93% of the genomes had one or more mutations in the spike glycoprotein. The D614G variant in spike protein is reported to have a very high frequency of >95% globally followed by the L452R and P681R, thus getting significant attention. The antigenic propensity of a small peptide of 29 residues from 597 to 625 of the spike protein variants having D614 and G614 showed that G614 has a little higher antigenic propensity. Thus, the D614G is the cause for higher viral antigenicity, however, it has not been reported to be effective to be causing more deaths.

In-Silico analysis reveals lower transcription efficiency of C241T variant of SARS-CoV-2 with host replication factors MADP1 and hnRNP-1.

Novel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has claimed more than 3.3 million lives worldwide and still counting. As per the GISAID database, the genomics of SARS-CoV-2 has been extensively studied, with more than 500 genome submissions per day. Out of several hotspot mutations within the SARS-CoV-2 genome, recent research has focused mainly on the missense variants. Moreover, significantly less attention has been accorded to delineate the role of the untranslated regions (UTRs) of the SARS-CoV-2 genome in the disease progression and etiology. One of the most frequent 5' UTR variants in the SARS-CoV-2 genome is the C241T, with a global frequency of more than 95 %. In the present study, the effect of the C241T mutation has been studied with respect to the changes in RNA structure and its interaction with the host replication factors MADP1 Zinc finger CCHC-type and RNA-binding motif 1 (hnRNP1). The results obtained from molecular docking and molecular dynamics simulation indicated weaker interaction of C241T mutant stem-loops with the host transcription factor MADP1, indicating a reduced replication efficiency. The results are also correlated with increased recovery rates and decreased death rates of global SARS-CoV-2 cases.

309 metagenome assembled microbial genomes from deep sediment samples in the Gulfs of Kathiawar Peninsula.

Prokaryoplankton genomes from the deep marine sediments are less explored compared to shallow shore sediments. The Gulfs of Kathiawar peninsula experience varied currents and inputs from different on-shore activities. Any perturbations would directly influence the microbiome and their normal homeostasis. Advancements in reconstructing genomes from metagenomes allows us to understand the role of individual unculturable microbes in ecological niches like the Gulf sediments. Here, we report 309 bacterial and archaeal genomes assembled from metagenomics data of deep sediments from sites in the Gulf of Khambhat and Gulf of Kutch as well as a sample from the Arabian Sea. Phylogenomics classified them into 5 archaeal and 18 bacterial phyla. The genomes will facilitate understanding of the physiology, adaptation and impact of on-shore anthropogenic activities on the deep sediment microbes.

Viral footprints across Gulfs of Kathiawar Peninsula and Arabian Sea: Unraveled from pelagic sediment metagenomic data.

Marine biosphere is one of the largest, diverse and dynamic system hosting numerous of microorganisms. Viruses being the most abundant under explored lifeforms in ocean, represent a reservoir of great genetic diversity. We report the metagenomic insights on the viral communities in the deep sediments of the two Gulfs of Gujarat i.e. Gulf of Khambhat and Gulf of Kutch, with one sample from Arabian Sea, treated as open sea control. The viral reads were filtered from the whole dataset, assembled and studied for viral diversity, which was visualized by Pavian. The sequences were checked for the viral abundance, diversity and functionality. The resulting viral taxonomic classification contained 6 orders, 8 families and 47 genera. The results revealed that the phages infecting Cyanobacterium, Bacillus and Vibrio dominated the sediments. Further, it was observed that majority of viral sequences belonged to double-stranded DNA phages. The present study attempts to provide a primary insight of the viral signals and potential genetic content in the Gulfs of Kathiawar.

Fungi with high ability to crunch multiple Polycyclic Aromatic Hydrocarbons (PAHs) from the pelagic sediments of Gulfs of Gujarat.

Marine ecosystem harbors diverse microbial diversity adapted to varied environmental conditions and stress. Gujarat possesses a wide coastline with unique and diverse niche in its two Gulfs. PAHs enter marine environments through various anthropogenic discharges and act as a threat to environment due to their xenobiotic nature. In the present study, sediment cores were collected across 4 coordinates, each from Gulf of Kutch and Khambhat; while one from Arabian sea. These samples were enriched for fungal growth in basal medium supplemented with naphthalene, pyrene, phenanthrene, anthracene and fluoranthene. Eight isolates were obtained from 3 samples and checked for tolerance against 5 PAHs followed by assessment of their biodegradation ability. Penicillium ilerdanum NPDF1239-K3-F21 and Aspergillus versicolor NPDF190-C1-26 showed >75% ability to degrade multiple PAHs. The results reveal the potential of fungal isolates from pelagic sediment for further in situ optimization and application in PAH removal from contaminated soil and sediment.

Sediment Plasmidome of the Gulfs of Kathiawar Peninsula and Arabian Sea: Insights Gained from Metagenomics Data.

Plasmidomes have become the research area of interest for ecologists exploring bacteria rich ecosystems. Marine environments are among such niche that host a huge number of microbes and have a complex environment which pose the need to study these bacterial indicators of horizontal gene transfer events for survival and stability. The plasmid content of the metagenomics data from 8 sediment samples of the Gulfs of Kathiawar and an open Arabian Sea sample was screened. The reads corresponding to hits against the plasmid database were assembled and studied for diversity using Kraken and functional content using MG-RAST. The sequences were also checked for resistome and virulence factors. The replicon hosts were overall dominated by Proteobacteria, Firmicutes, and Actinobacteria while red algae specific to the Kutch samples. The genes encoded were dominant in the flagella motility and type VI secretion systems. Overall, results from the study confirmed that the plasmids encoded traits for metal, antibiotic, and phage resistance along with virulence systems, and these would be conferring benefit to the hosts. The study throws insights into the environmental role of the plasmidome in adaptation of the microbes in the studied sites to the environmental stresses.

Bacterial line of defense in Dirinaria lichen from two different ecosystems: First genomic insights of its mycobiont Dirinaria sp. GBRC AP01.

Lichens have been widely studied for their symbiotic properties and for the secondary metabolites production by its fungal symbiont. Recent molecular studies have confirmed coexistence of bacteria along with the fungal and algal symbionts. Direct nucleic acid study by -omics approaches is providing better insights into their structural and functional dynamics. However, genomic analysis of individual members of lichen is difficult by the conventional approach. Hence, genome assembly from metagenome data needs standardization in the eukaryotic system like lichens. The present study aimed at metagenomic characterization of rock associated lichen Dirinaria collected from Kutch and Dang regions of Gujarat, followed by genome reconstruction and annotation of the mycobiont Dirinaria. The regions considered in the study are eco-geographically highly variant. The results revealed higher alpha diversity in the dry region Kutch as compared to the tropical forest associated lichen from Dang. Ascomycota was the most abundant eukaryote while Proteobacteria dominated the bacterial population. There were 23 genera observed only in the Kutch lichen (KL) and one genus viz., Candidatus Vecturithrix unique to the Dang lichen (DL). The exclusive bacterial genera in the Kutch mostly belonged to groups reported for stress tolerance and earlier isolated from lithobionts of extreme niches. The assembled data of KL & DL were further used for genome reconstruction of Dirinaria sp. using GC and tetra-pentamer parameters and reassembly that resulted into a final draft genome of 31.7 Mb and 9556 predicted genes. Twenty-eight biosynthesis gene clusters were predicted that included genes for polyketide, indole and terpene synthesis. Association analysis of bacteria and mycobiont revealed 8 pathways specific to bacteria with implications in lichen symbiosis and environment interaction. The study provides the first draft genome of the entire fungal Dirinaria genus and provides insights into the Dirinaria lichen metagenome from Gujarat region.

Antibiotic Resistome Biomarkers associated to the Pelagic Sediments of the Gulfs of Kathiawar Peninsula and Arabian Sea.

Antibiotic resistance has been one of the most persistent global issue. Specifically, marine microbiomes have served as complex reservoirs of antibiotic resistant genes. Molecular advancements have enabled exploration of the uncultured microbial portion from hitherto difficult to sample niches such as deeper oceans. The Gulfs of Kathiawar Peninsula have been known for their unique properties like extreme tidal variations, different sediment textures and physicochemical variations. Pelagic sediment cores across four coordinates each of the Gulf of Kutch, Gulf of Khambhat and an open Arabian Sea were collected, processed for metagenomic sequencing and assessed for antibiotic and metal resistome. The dominant genes were mostly resistant to macrolides, glycopeptides and tetracycline drugs. Studied samples divided into three clusters based on their resistome with carA, macB, bcrA, taeA, srmB, tetA, oleC and sav1866 among the abundant genes. Samples from creek of Gulf of Kutch and mouth of Gulf of Khambhat were most diverse in resistance gene profile. Biomarkers observed include gyrA mutation conferring resistance gene in the Arabian Sea; Proteobacteria species in Gulf of Kutch and Arabian sea; while Aquificae, Acidobacteria and Firmicutes species in the Gulf of Khambhat. Region-wise differentially abundant 23 genes and 3 taxonomic biomarkers were proposed for antibiotic resistance monitoring.

Antibiotic resistance genes allied to the pelagic sediment microbiome in the Gulf of Khambhat and Arabian Sea.

Antibiotics have been widely spread in the environments, imposing profound stress on the resistome of the residing microbes. Marine microbiomes are well established large reservoirs of novel antibiotics and corresponding resistance genes. The Gulf of Khambhat is known for its extreme tides and complex sedimentation process. We performed high throughput sequencing and applied bioinformatics techniques on pelagic sediment microbiome across four coordinates of the Gulf of Khambhat to assess the marine resistome, its corresponding bacterial community and compared with the open Arabian Sea sample. We identified a total of 2354 unique types of resistance genes, with most abundant and diverse gene profile in the area that had anthropogenic activities being carried out on-shore. The genes with >1% abundance in all samples included carA, macB, sav1866, tlrC, srmB, taeA, tetA, oleC and bcrA which belonged to the macrolides, glycopeptides and peptide drug classes. ARG enriched phyla distribution was quite varying between all the sites, with Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes among the dominant phyla. Based on the outcomes, we also propose potential biomarker candidates Desulfovibrio, Thermotaga and Pelobacter for antibiotic monitoring in the two of the Gulf samples probable contamination prone environments, and genera Nitrosocccus, Marinobacter and Streptomyces in the rest of the three studied samples. Outcomes support the concept that ARGs naturally originate in environments and human activities contribute to the dissemination of antibiotic resistance.

Functional gene profiling through metaRNAseq approach reveals diet-dependent variation in rumen microbiota of buffalo (Bubalus bubalis).

Recent advances in next generation sequencing technology have enabled analysis of complex microbial community from genome to transcriptome level. In the present study, metatranscriptomic approach was applied to elucidate functionally active bacteria and their biological processes in rumen of buffalo (Bubalus bubalis) adapted to different dietary treatments. Buffaloes were adapted to a diet containing 50:50, 75:25 and 100:0 forage to concentrate ratio, each for 6 weeks, before ruminal content sample collection. Metatranscriptomes from rumen fiber adherent and fiber-free active bacteria were sequenced using Ion Torrent PGM platform followed by annotation using MG-RAST server and CAZYmes (Carbohydrate active enzymes) analysis toolkit. In all the samples Bacteroidetes was the most abundant phylum followed by Firmicutes. Functional analysis using KEGG Orthology database revealed Metabolism as the most abundant category at level 1 within which Carbohydrate metabolism was dominating. Diet treatments also exerted significant differences in proportion of enzymes involved in metabolic pathways for VFA production. Carbohydrate Active Enzyme(CAZy) analysis revealed the abundance of genes encoding glycoside hydrolases with the highest representation of GH13 CAZy family in all the samples. The findings provide an overview of the activities occurring in the rumen as well as active bacterial population and the changes occurring through different dietary treatments.

Mining of Ruminant Microbial Phytase (RPHY1) from Metagenomic Data of Mehsani Buffalo Breed: Identification, Gene Cloning, and Characterization.

Phytases have been widely used as animal feed supplements to increase the availability of digestible phosphorus, especially in monogastric animals fed cereal grains. The present study describes the identification of a full-length phytase gene of Prevotella species present in Mehsani buffalo rumen. The gene, designated as RPHY1, consists of 1,251 bp and is expressed into protein with 417 amino acids. A homology search of the deduced amino acid sequence of the RPHY1 phytase gene in a nonredundant protein database showed that it shares 92% similarity with the histidine acid phosphatase domain. Subsequently, the RPHY1 gene was expressed using a pET32a expression vector in Escherichia coli BL21 and purified using a His60 Ni-NTA gravity column. The mass of the purified RPHY1 was estimated to be approximately 63 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal RPHY1 enzyme activity was observed at 55°C (pH 5) and exhibited good stability at 5°C and within the acidic pH range. Significant inhibition of RPHY1 activity was observed for Mg2+ and K+ metal ions, while Ca2+, Mn2+, and Na+ slightly inhibited enzyme activity. The RPHY1 phytase was susceptible to SDS, and it was highly stimulated in the presence of EDTA. Overall, the observed comparatively high enzyme activity levels and characteristics of the RPHY1 gene mined from rumen prove its promising candidature as a feed supplement enzyme in animal farming.

Draft Genome Sequence of Commercial Textile Dye-Decolorizing and -Degrading Bacillus subtilis Strain C3 Isolated in India.

Bacillus subtilis C3, a commercial textile dye-decolorizing and -degrading bacterium, was isolated from the common effluent treatment plant (CEPT) of the Jetpur textile dyeing and printing industrial sector situated in the district of Rajkot, Gujarat, India. Here, we present the annotated 4.18-Mb draft genome sequence of B. subtilis C3, providing information about the metabolic pathways involved in decolorization and degradation of several commercial textile azo dyes. Thus, we confirm B. subtilis C3 as a potential candidate for bioremediation of textile effluents.

Identification and quantification of novel RNA isoforms in horn cancer of Bos indicus by comprehensive RNA-Seq.

Horn cancer (HC) is a squamous cell carcinoma of horn, commonly observed in Bos indicus of the Asian countries. To elucidate the complexity of alternative splicing present in the HC, high-throughput sequencing and analysis of HC and matching horn normal (HN) tissue were carried out. A total of 535,067 and 849,077 reads were analysed after stringent quality filtering for HN and HC, respectively. Cufflinks pipeline for transcriptome analysis revealed 4786 novel splice isoforms comprising 2432 exclusively in HC, 2055 exclusively in HN and 298 in both the conditions. Based on pathway clustering and in silico verification, 102 novel splice isoforms were selected and further analysed with respect to change in protein sequence using Blastp. Finally, fourteen novel splicing events supported both by Cufflinks and UCSC genome browser were selected and confirmed expression by RT-qPCR. Future studies targeted at in-depth characterization of these potential candidate splice isoforms might be helpful in the development of relevant biomarkers for early diagnosis of HC. The results reported in this study refine the available information on transcriptome repertoire of bovine species and boost the research in the line of development of relevant biomarkers for early diagnosis of HC.

Effect of roughage on rumen microbiota composition in the efficient feed converter and sturdy Indian Jaffrabadi buffalo (Bubalus bubalis).

BACKGROUND: The rumen microbiota functions as an effective system for conversion of dietary feed to microbial proteins and volatile fatty acids. In the present study, metagenomic approach was applied to elucidate the buffalo rumen microbiome of Jaffrabadi buffalo adapted to varied dietary treatments with the hypothesis that the microbial diversity and subsequent in the functional capacity will alter with diet change and enhance our knowledge of effect of microbe on host physiology. Eight adult animals were gradually adapted to an increasing roughage diet (4 animals each with green and dry roughage) containing 50:50 (J1), 75:25 (J2) and 100:0 (J3) roughage to concentrate proportion for 6 weeks. Metagenomic sequences of solid (fiber adherent microbiota) and liquid (fiber free microbiota) fractions obtained using Ion Torrent PGM platform were analyzed using MG-RAST server and CAZymes approach. RESULTS: Taxonomic analysis revealed that Bacteroidetes was the most abundant phylum followed by Firmicutes, Fibrobacter and Proteobacteria. Functional analysis revealed protein (25-30 %) and carbohydrate (15-20 %) metabolism as the dominant categories. Principal component analysis demonstrated that roughage proportion, fraction of rumen and type of forage affected rumen microbiome at taxonomic as well as functional level. Rumen metabolite study revealed that rumen fluid nitrogen content reduced in high roughage diet fed animals and pathway analysis showed reduction in the genes coding enzymes involved in methanogenesis pathway. CAZyme annotation revealed the abundance of genes encoding glycoside hydrolases (GH), with the GH3 family most abundant followed by GH2 and GH13 in all samples. CONCLUSIONS: Results reveals that high roughage diet feed improved microbial protein synthesis and reduces methane emission. CAZyme analysis indicated the importance of microbiome in feed component digestion for fulfilling energy requirements of the host. The findings help determine the role of rumen microbes in plant polysaccharide breakdown and in developing strategies to maximize productivity in ruminants.

In silico analysis of consequences of non-synonymous SNPs of Slc11a2 gene in Indian bovines.

The aim of our study was to analyze the consequences of non-synonymous SNPs in Slc11a2 gene using bioinformatic tools. There is a current need of efficient bioinformatic tools for in-depth analysis of data generated by the next generation sequencing technologies. SNPs are known to play an imperative role in understanding the genetic basis of many genetic diseases. Slc11a2 is one of the major metal transporter families in mammals and plays a critical role in host defenses. In this study, we performed a comprehensive analysis of the impact of all non-synonymous SNPs in this gene using multiple tools like SIFT, PROVEAN, I-Mutant and PANTHER. Among the total 124 SNPs obtained from amplicon sequencing of Slc11a2 gene by Ion Torrent PGM involving 10 individuals of Gir cattle and Murrah buffalo each, we found 22 non-synonymous. Comparing the prediction of these 4 methods, 5 nsSNPs (G369R, Y374C, A377V, Q385H and N492S) were identified as deleterious. In addition, while tested out for polar interactions with other amino acids in the protein, from above 5, Y374C, Q385H and N492S showed a change in interaction pattern and further confirmed by an increase in total energy after energy minimizations in case of mutant protein compared to the native.

Isolation of chitinolytic Clostridium sp. NCR from Mehsani buffalo rumen, its genomic analysis and potential role in rumen.

Genomic analysis of Clostridium sp. NCR, an anaerobic Gram positive bacterium which was isolated from rumen fluid of Mehsani breed of buffalo revealed presence of various environmental gene tags (EGTs) involved in pathways for utilizing a wide range of substrates. Here we report the sequence of this rumen isolate, its whole genome sequence has been deposited in DDBJ/EMBL/GenBank under the accession number JQHY00000000. The genome comprises of a 3.62-Mb draft genome with a G + C content of 28.10%, which encodes a total of 3126 proteins. Functional analysis provides information about the microbe's role in maintaining host homeostasis and its fiber degradation potential.

Functional Characterization Reveals Novel Putative Coding Sequences in Prevotella ruminicola Genome Extracted from Rumen Metagenomic Studies.

AIM: To reassemble Prevotella ruminicola genome from rumen metagenomic data of cattle and buffalo and compare with the published reference genome. METHOD: Rumen microbial communities from Mehsani buffaloes (n = 8) and Kankrej cattle (n = 8), each adapted to different proportions of a dry or green roughage diet, were subjected to metagenomic sequencing by Ion Torrent PGM, and subsequent reads were analyzed by MG-RAST. Using reference-guided assembly of the sequences against the published P. ruminicola strain 23, draft genomes of 2.56 and 2.46 Mb were reconstructed from Mehsani buffalo and Kankrej cows, respectively. The genomes were annotated using the RAST Server and carbohydrate active enzyme (CAZyme) analysis. RESULTS: Taxonomic analysis by MG-RAST revealed P. ruminicola to be the most abundant species present among the rumen microflora. Functional annotation of reconstructed genomes using the RAST Server depicted the maximum assignment of coding sequences involved in the subsystems amino acid and derivatives and carbohydrate metabolism. CAZyme profiling revealed the glycoside hydrolases (GH) family to be the most abundant. GH family subclassification revealed that the extracted genomes had more sequence hits for GH2, GH3, GH92 and GH97 as compared to the reference. CONCLUSION: The results reflect the metabolic significance of rumen-adapted P. ruminicola in utilizing a coarse diet for animals based on acquisition of novel genetic elements.

Exploring genetic polymorphism in innate immune genes in Indian cattle (Bos indicus) and buffalo (Bubalus bubalis) using next generation sequencing technology.

Activation of innate immunity initiates various cascades of reactions that largely contribute to defense against physical, microbial or chemical damage, prompt for damage repair and removal of causative organisms as well as restoration of tissue homeostasis. Genetic polymorphism in innate immune genes plays prominent role in disease resistance capabilities in various breeds of cattle and buffalo. Here we studied single nucleotide variations (SNP/SNV) and haplotype structure in innate immune genes viz CHGA, CHGB, CHGC, NRAMP1, NRAMP2, DEFB1, BNBD4, BNBD5, TAP and LAP in Gir cattle and Murrah buffalo. Targeted sequencing of exonic regions of these genes was performed by Ion Torrent PGM sequencing platform. The sequence reads obtained corresponding to coding regions of these genes were mapped to reference genome of cattle BosTau7 by BWA program using genome analysis tool kit (GATK). Further variant analysis by Unified Genotyper revealed 54 and 224 SNPs in Gir and Murrah respectively and also 32 SNVs was identified. Among these SNPs 43, 36, 11,32,81,21 and 22 variations were in CHGA, CHGB, CHGC, NRAMP1, NRAMP2, DEFB1 and TAP genes respectively. Among these identified 278 SNPs, 24 were found to be reported in the dbSNP database. Variant analysis was followed by structure formation of haplotypes based on multiple SNPs using SAS software revealed a large number of haplotypes. The SNP discovery in innate immune genes in cattle and buffalo breeds of India would advance our understanding of role of these genes in determining the disease resistance/susceptibility in Indian breeds. The identified SNPs and haplotype data would also provide a wealth of sequence information for conservation studies, selective breeding and designing future strategies for identifying disease associations involving samples from distinct populations.