Genome-resolved metagenomics revealed metal-resistance, geochemical cycles in a Himalayan hot spring.

The hot spring microbiome is a complex assemblage of micro- and macro-organisms; however, the understanding and projection of enzymatic repertoire that access earth's integral ecosystem processes remains ambivalent. Here, the Khirganga hot spring characterized with white microbial mat and ions rich in sulfate, chlorine, sodium, and magnesium ions is investigated and displayed the examination of 41 high and medium qualified metagenome-assembled genomes (MAGs) belonged to at least 12 bacterial and 2 archaeal phyla which aids to drive sulfur, oxygen, iron, and nitrogen cycles with metabolic mechanisms involved in heavy metal tolerance. These MAGs possess over 1749 genes putatively involved in crucial metabolism of elements viz. nitrogen, phosphorus, and sulfur and 598 genes encoding enzymes for czc efflux system, chromium, arsenic, and copper heavy metals resistance. The MAGs also constitute 229 biosynthetic gene clusters classified abundantly as bacteriocins and terpenes. The metabolic roles possibly involved in altering linkages in nitrogen biogeochemical cycles and explored a discerned rate of carbon fixation exclusively in archaeal member Methanospirillum hungatei inhabited in microbial mat. Higher Pfam entropy scores of biogeochemical cycling in Proteobacteria members assuring their major contribution in assimilation of ammonia and sequestration of nitrate and sulfate components as electron acceptors. This study will readily improve the understanding of the composite relationship between bacterial species owning metal resistance genes (MRGs) and underline the exploration of adaptive mechanism of these MAGs in multi-metal contaminated environment. KEY POINTS: • Identification of 41 novel bacterial and archaeal species in habitats of hot spring • Genome-resolved metagenomics revealed MRGs (n = 598) against Cr, Co, Zn, Cd, As, and Cu • Highest entropies of N (0.48) and Fe (0.44) cycles were detected within the MAGs.

Taxonomic and genomic characterization of Sporosarcina cyprini sp. nov., moderately tolerant of Cr+6 and Cd+2 isolated from the gut of invasive fish Cyprinus carpio var. communis (Linn., 1758).

A Gram-stain-positive, motile, and rod-shaped bacterium, designated as strain MB25T, was isolated from the gut of Cyprinus carpio from the highly polluted river Yamuna, India. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain MB25T belonged to the genus Sporosarcina, sharing the highest sequence similarity with S. luteola Y1T (98.98%) and S. koreensis S-K12T (98.91%). Digital DNA-DNA hybridization and average nucleotide identity values of strain MB25T with strain Y1T and S-K12T were 18.9, 77.69, and 18.2, 76.80 respectively. Genome analysis of strain MB25T revealed its biotechnological properties such as tolerance to potent heavy metals, genes for the production of carbohydrate-active enzymes, antimicrobial compounds, and also degradation of aromatic compounds. The G + C content of strain MB25T genome was 45%. Growth observed at 10-40 °C (optimum, 28-30 °C), pH 6.0-8.5 (optimum pH 7.5-8.0); NaCl concentrations up to 6.0% (w/v). The dominant respiratory quinone was MK-7, cell wall peptidoglycan is of the A-4 type containing amino acids Lys-Glu and the major fatty acids are anteiso-C11:0 and iso-C15: 0. The major polar lipids of strain MB25T are diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. On the basis of phenotypic, chemotaxonomic, phylogenetic, and phylogenomic data, strain MB25T represents a novel species of the genus Sporosarcina, for which the name Sporosarcina cyprini sp. nov. is proposed. The type strain is MB25T (= MCC 4366 T = JCM 34521 T = CCM 9113 T).

Metagenomic insights to understand the role of polluted river Yamuna in shaping the gut microbial communities of two invasive fish species.

The gastrointestinal microbial community plays a crucial role in host health, immunity, protection, development and provides nutrients to the host. The rising human-induced pollution and heavy metal contamination in all aquatic systems globally has led us to explore the gut microbial diversity of two exotic invasive fish Cyprinus carpio (Linnaeus, 1858) and Oreochromis niloticus (Linnaeus,1857) from river Yamuna, India. These fishes are aquatic bioindicators with high demographic resilience. Exploring these associations would pave the way for addressing problems that inhabitant fishes are facing due to the increasing pollution load in the River Yamuna. Based on 16S rRNA gene amplicon sequencing, our results deliver comparative information on the gut microbiome of these fishes and highlight connotations between the microbiome of gut and water samples. The gut of C. carpio and O. niloticus was dominated by phyla Proteobacteria whereas Bacteroidetes dominated the water sample. Microbial communities showed predicted roles such as pathogenicity (Escherichia-Shigella, Aeromonas veronii, Vibrio cholerae, Streptococcus iniae, Flavobacterium columnare, Klebsiella pneumoniae, Mycobacterium sp.), probiotic applications (Bacillus velezensis, Lactobacillus plantarum, Enterococcus faecalis, Bifidobacterium longum, Lactococcus lactis, Leuconostoc falkenbergense) and involvement in sewage and organic matter decomposition (Nitrosomonas sp., Methanosaeta harundinacea, Dechloromonas agitata, Thauera humireducens, Zoogloea ramigera). Heavy metal degrading members (Leucobacter chromiireducens, Pseudomonas fluorescens, P. aeruginosa, Klebsiella pneumoniae, and Micrococcus luteus) were detected in gut microbiome samples thus supporting the notion that fish shapes its gut microbiota with changing ecology. Functional profiling showed that microbial communities are specialized in metabolic functions thus reflecting the dietary profile of these invasive fishes.

Fish Gut Microbiome: Current Approaches and Future Perspectives.

In recent years, investigations of microbial flora associated with fish gut have deepened our knowledge of the complex interactions occurring between microbes and host fish. The gut microbiome not only reinforces the digestive and immune systems in fish but is itself shaped by several host-associated factors. Unfortunately, in the past, majority of studies have focused upon the structure of fish gut microbiome providing little knowledge of effects of these factors distinctively and the immense functional potential of the gut microbiome. In this review, we have highlighted the recently gained insights into the diversity and functions of the fish gut microbiome. We have also delved on the current approaches that are being employed to study the fish gut microbiome with an aim to collate all the knowledge gained and make accurate conclusions for their application based perspectives. The literature reviewed indicated that the future research should shift towards functional microbiomics to improve the maximum sustainable yield in aquaculture.

Microbial taxonomy in the era of OMICS: application of DNA sequences, computational tools and techniques.

The current prokaryotic taxonomy classifies phenotypically and genotypically diverse microorganisms using a polyphasic approach. With advances in the next-generation sequencing technologies and computational tools for analysis of genomes, the traditional polyphasic method is complemented with genomic data to delineate and classify bacterial genera and species as an alternative to cumbersome and error-prone laboratory tests. This review discusses the applications of sequence-based tools and techniques for bacterial classification and provides a scheme for more robust and reproducible bacterial classification based on genomic data. The present review highlights promising tools and techniques such as ortho-Average Nucleotide Identity, Genome to Genome Distance Calculator and Multi Locus Sequence Analysis, which can be validly employed for characterizing novel microorganisms and assessing phylogenetic relationships. In addition, the review discusses the possibility of employing metagenomic data to assess the phylogenetic associations of uncultured microorganisms. Through this article, we present a review of genomic approaches that can be included in the scheme of taxonomy of bacteria and archaea based on computational and in silico advances to boost the credibility of taxonomic classification in this genomic era.

Comparative analyses of gut microbiota reveal ammonia detoxification and nitrogen assimilation in Cyprinus carpio var. specularis.

The complex niche of fish gut is often characterized by the associated microorganisms that have implications in fish gut-health nexus. Although efforts to distinguish the microbial communities have highlighted their disparate structure along the gut length, remarkably little information is available about their distinct structural and functional profiles in different gut compartments in different fish species. Here, we performed comparative taxonomic and predictive functional analyses of the foregut and hindgut microbiota in an omnivorous freshwater fish species, Cyprinus carpio var. specularis, commonly known as mirror carp. Our analyses showed that the hindgut microbiota could be distinguished from foregut based on the abundance of ammonia-oxidizing, denitrifying, and nitrogen-fixing commensals of families such as Rhodospirillaceae, Oxalobacteraceae, Nitrosomonadaceae, and Nitrospiraceae. Functionally, unique metabolic pathways such as degradation of lignin, 2-nitrobenzoate, vanillin, vanillate, and toluene predicted within hindgut also hinted at the ability of hindgut microbiota for assimilation of nitrogen and detoxification of ammonia. The study highlights a major role of hindgut microbiota in assimilating nitrogen, which remains to be one of the limiting nutrients within the gut of mirror carp.

Riverine pollution influences the intraspecific variation in the gut microbiome of an invasive fish, Cyprinus carpio (Linn., 1758).

Humans are significantly impacting riverine systems worldwide, prompting us to investigate the effects of water pollution on the gut microbiome of Cyprinus carpio (common carp). Using 16S rRNA gene sequencing, we compared the gut microbiomes of common carp from two sites along river Yamuna with different pollution levels. Water pollution significantly altered the fish gut microbiome structure and microbial composition. Proteobacteria dominated in both sampling sites, while Bacteroidota prevailed in polluted water samples, indicating sewage and fecal contamination. Less polluted samples exhibited Verrucomicrobiae and Planctomycetes, negatively correlated with pollution levels. The polluted site had higher prevalence of potentially pathogenic and heavy metal-resistant bacteria, as well as microbial communities associated with wastewater treatment systems. Functional prediction highlighted the significant role of the gut microbiome in digestion and metabolism, with active enzymes for breaking down various organic substances. Biosynthetic pathways for leucine, valine, and isoleucine were present in both sites, known to be involved fish immunity. The host maintained a stable and diverse bacterial consortium, while microbial diversity became more specialized due to human activities, adapting to anthropogenic stress and selection pressures. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03747-0.

Monkeypox virus: phylogenomics, host-pathogen interactome and mutational cascade.

While the world is still recovering from the Covid-19 pandemic, monkeypox virus (MPXV) awaits to cause another global outbreak as a challenge to all of mankind. However, the Covid-19 pandemic has taught us a lesson to speed up the pace of viral genomic research for the implementation of preventive and treatment strategies. One of the important aspects of MPXV that needs immediate insight is its evolutionary lineage based on genomic studies. Utilizing high-quality isolates from the GISAID (Global Initiative on Sharing All Influenza Data) database, primarily sourced from Europe and North America, we employed a SNP-based whole-genome phylogeny method and identified four major clusters among 628 MPXV isolates. Our findings indicate a distinct evolutionary lineage for the first MPXV isolate, and a complex epidemiology and evolution of MPXV strains across various countries. Further analysis of the host-pathogen interaction network revealed key viral proteins, such as E3, SPI-2, K7 and CrmB, that play a significant role in regulating the network and inhibiting the host's cellular innate immune system. Our structural analysis of proteins E3 and CrmB revealed potential disruption of stability due to certain mutations. While this study identified a large number of mutations within the new outbreak clade, it also reflected that we need to move fast with the genomic analysis of newly detected strains from around the world to develop better prevention and treatment methods.

Microbial Ecology of Sulfur Biogeochemical Cycling at a Mesothermal Hot Spring Atop Northern Himalayas, India.

Sulfur related prokaryotes residing in hot spring present good opportunity for exploring the limitless possibilities of integral ecosystem processes. Metagenomic analysis further expands the phylogenetic breadth of these extraordinary sulfur (S) metabolizing microorganisms as well as their complex metabolic networks and syntrophic interactions in environmental biosystems. Through this study, we explored and expanded the microbial genetic repertoire with focus on S cycling genes through metagenomic analysis of S contaminated hot spring, located at the Northern Himalayas. The analysis revealed rich diversity of microbial consortia with established roles in S cycling such as Pseudomonas, Thioalkalivibrio, Desulfovibrio, and Desulfobulbaceae (Proteobacteria). The major gene families inferred to be abundant across microbial mat, sediment, and water were assigned to Proteobacteria as reflected from the reads per kilobase (RPKs) categorized into translation and ribosomal structure and biogenesis. An analysis of sequence similarity showed conserved pattern of both dsrAB genes (n = 178) retrieved from all metagenomes while other S disproportionation proteins were diverged due to different structural and chemical substrates. The diversity of S oxidizing bacteria (SOB) and sulfate reducing bacteria (SRB) with conserved (r)dsrAB suggests for it to be an important adaptation for microbial fitness at this site. Here, (i) the oxidative and reductive dsr evolutionary time-scale phylogeny proved that the earliest (but not the first) dsrAB proteins belong to anaerobic Thiobacillus with other (rdsr) oxidizers, also we confirm that (ii) SRBs belongs to δ-Proteobacteria occurring independent lateral gene transfer (LGT) of dsr genes to different and few novel lineages. Further, the structural prediction of unassigned DsrAB proteins confirmed their relatedness with species of Desulfovibrio (TM score = 0.86, 0.98, 0.96) and Archaeoglobus fulgidus (TM score = 0.97, 0.98). We proposed that the genetic repertoire might provide the basis of studying time-scale evolution and horizontal gene transfer of these genes in biogeochemical S cycling.

Metagenome-assembled genomes recovered from the datasets of a high-altitude Himalayan hot spring Khirganga, Himachal Pradesh, India.

Khirganga, a pristine hot spring that lies in the Parvati Valley within the Northern Himalayas characterised with unique white colour microbial mat and divine water with healing abilities. Here, we report 41 metagenome-assembled genomes (MAGs) reconstructed from the microbial mat, sediment and water samples of hot spring passed through Genome Standards Consortium (GSC) and Minimum Information of Metagenome-assembled Genome (MIMAG).

Genome analyses of 174 strains of Mycobacterium tuberculosis provide insight into the evolution of drug resistance and reveal potential drug targets.

Mycobacterium tuberculosis is a known human pathogen that causes the airborne infectious disease tuberculosis (TB). Every year TB infects millions of people worldwide. The emergence of multi-drug resistant (MDR), extensively drug resistant (XDR) and totally drug resistant (TDR) M. tuberculosis strains against the first- and second-line anti-TB drugs has created an urgent need for the development and implementation of new drug strategies. In this study, the complete genomes of 174 strains of M. tuberculosis are analysed to understand the evolution of molecular drug target (MDT) genes. Phylogenomic placements of M. tuberculosis strains depicted close association and temporal clustering. Selection pressure analysis by deducing the ratio of non-synonymous to synonymous substitution rates (dN/dS) in 51 MDT genes of the 174 M. tuberculosis strains led to categorizing these genes into diversifying (D, dN/dS>0.70), moderately diversifying (MD, dN/dS=0.35-0.70) and stabilized (S, dN/dS<0.35) genes. The genes rpsL, gidB, pncA and ahpC were identified as diversifying, and Rv0488, kasA, ndh, ethR, ethA, embR and ddn were identified as stabilized genes. Furthermore, sequence similarity networks were drawn that supported these divisions. In the multiple sequence alignments of diversifying and stabilized proteins, previously reported resistance mutations were checked to predict sensitive and resistant strains of M. tuberculosis. Finally, to delineate the potential of stabilized or least diversified genes/proteins as anti-TB drug targets, protein-protein interactions of MDT proteins with human proteins were analysed. We predict that kasA (dN/dS=0.29), a stabilized gene that encodes the most host-interacting protein, KasA, should serve as a potential drug target for the treatment of TB.

Defining the Environmental Adaptations of Genus Devosia: Insights into its Expansive Short Peptide Transport System and Positively Selected Genes.

Devosia are well known for their dominance in soil habitats contaminated with various toxins and are best characterized for their bioremediation potential. In this study, we compared the genomes of 27 strains of Devosia with aim to understand their metabolic abilities. The analysis revealed their adaptive gene repertoire which was bared from 52% unique pan-gene content. A striking feature of all genomes was the abundance of oligo- and di-peptide permeases (oppABCDF and dppABCDF) with each genome harboring an average of 60.7 ± 19.1 and 36.5 ± 10.6 operon associated genes respectively. Apart from their primary role in nutrition, these permeases may help Devosia to sense environmental signals and in chemotaxis at stressed habitats. Through sequence similarity network analyses, we identified 29 Opp and 19 Dpp sequences that shared very little homology with any other sequence suggesting an expansive short peptidic transport system within Devosia. The substrate determining components of these permeases viz. OppA and DppA further displayed a large diversity that separated into 12 and 9 homologous clusters respectively in addition to large number of isolated nodes. We also dissected the genome scale positive evolution and found genes associated with growth (exopolyphosphatase, HesB_IscA_SufA family protein), detoxification (moeB, nifU-like domain protein, alpha/beta hydrolase), chemotaxis (cheB, luxR) and stress response (phoQ, uspA, luxR, sufE) were positively selected. The study highlights the genomic plasticity of the Devosia spp. for conferring adaptation, bioremediation and the potential to utilize a wide range of substrates. The widespread toxin-antitoxin loci and 'open' state of the pangenome provided evidence of plastic genomes and a much larger genetic repertoire of the genus which is yet uncovered.

Phylogenetic Relationships and Potential Functional Attributes of the Genus Parapedobacter: A Member of Family Sphingobacteriaceae.

The genus Parapedobacter was established to describe a novel genus within the family Sphingobacteriaceae and derives its name from Pedobacter, with which it is shown to be evolutionarily related. Despite this, Parapedobacter and Pedobacter do not share very high 16S rRNA gene sequence similarities. Therefore, we hypothesized whether these substantial differences at the 16S rRNA gene level depict the true phylogeny or that these genomes have actually diverged. Thus, we performed genomic analysis of the four available genomes of Parapedobacter to better understand their phylogenomic position within family Sphingobacteriaceae. Our results demonstrated that Parapedobacter is more closely related to species of Olivibacter, as opposed to the genus Pedobacter. Further, we identified a significant class of enzymes called pectinases with potential industrial applications within the genomes of Parapedobacter luteus DSM 22899T and Parapedobacter composti DSM 22900T. These enzymes, specifically pectinesterases and pectate lyases, are presumed to have largely different catalytic activities based on very low sequence similarities to already known enzymes and thus may be exploited for industrial applications. We also determined the complete Bacteroides aerotolerance (Bat) operon (batA, batB, batC, batD, batE, hypothetical protein, moxR, and pa3071) within the genome of Parapedobacter indicus RK1T. This expands the definition of genus Parapedobacter to containing members that are able to tolerate oxygen stress using encoded oxidative stress responsive systems. By conducting a signal propagation network analysis, we determined that BatD, BatE, and hypothetical proteins are the major controlling hubs that drive the expression of Bat operon. As a key metabolic difference, we also annotated the complete iol operon within the P. indicus RK1T genome for utilization of all three stereoisomers of inositol, namely myo-inositol, scyllo-inositol, and 1D-chiro-inositol, which are abundant sources of organic phosphate found in soils. The results suggest that the genus Parapedobacter holds promising applications owing to its environmentally relevant genomic adaptations, which may be exploited in the future.

Comparative Genomic Analysis of Rapidly Evolving SARS-CoV-2 Reveals Mosaic Pattern of Phylogeographical Distribution.

The outbreak of coronavirus disease 2019 (COVID-19) that started in Wuhan, China, in December 2019 has spread worldwide, emerging as a global pandemic. The severe respiratory pneumonia caused by novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has so far claimed more than 0.38 million lives and has impacted human lives worldwide. However, as the novel SARS-CoV-2 virus displays high transmission rates, the underlying genomic severity is required to be fully understood. We studied the complete genomes of 95 SARS-CoV-2 strains from different geographical regions worldwide to uncover the pattern of the spread of the virus. We show that there is no direct transmission pattern of the virus among neighboring countries, suggesting that its spread is a result of travel of infected humans to different countries. We revealed unique single nucleotide polymorphisms (SNPs) in nonstructural protein 13 (nsp13), nsp14, nsp15, and nsp16 (ORF1b polyproteins) and in the S-protein within 10 viral isolates from the United States. These viral proteins are involved in RNA replication and binding with the human receptors, indicating that the viral variants that are circulating in the population of the United States are different from those circulating in the populations of other countries. In addition, we found an amino acid addition in nsp16 (mRNA cap-1 methyltransferase) of a U.S. isolate (GenBank accession no. MT188341.1) leading to a shift in the amino acid frame from position 2540 onward. Through comparative structural analysis of the wild-type and mutant proteins, we showed that this addition of a phenylalanine residue renders the protein in the mutant less stable, which might affect mRNA cap-1 methyltransferase function. We further analyzed the SARS-CoV-2-human interactome, which revealed that the interferon signaling pathway is targeted by orf1ab during infection and that it also interacts with NF-κB-repressing factor (NKRF), which is a potential regulator of interleukin-8 (IL-8). We propose that targeting this interaction may subsequently improve the health condition of COVID-19 patients. Our analysis also emphasized that SARS-CoV-2 manipulates spliceosome machinery during infection; hence, targeting splicing might affect viral replication. In conclusion, the replicative machinery of SARS-CoV-2 is targeting interferon and the notch signaling pathway along with spliceosome machinery to evade host challenges.IMPORTANCE The COVID-19 pandemic continues to storm the world, with over 6.5 million cases worldwide. The severity of the disease varies with the territories and is mainly influenced by population density and age factor. In this study, we analyzed the transmission pattern of 95 SARS-CoV-2 genomes isolated from 11 different countries. Our study also revealed several nonsynonymous mutations in ORF1b and S-proteins and the impact on their structural stability. Our analysis showed the manipulation of host system by viral proteins through SARS-CoV-2-human protein interactome, which can be useful to understand the impact of virus on human health.

Draft Genome Sequence of Deinococcus sp. Strain S9, Isolated from Microbial Mat Deposits of Hot Springs Located atop the Himalayan Ranges at Manikaran, India.

Here, we present the draft genome sequence of Deinococcus sp. strain S9, a red-pigmented and moderately thermophilic bacterium isolated from microbial mat deposits around the hot springs at Manikaran, Himachal Pradesh, India. The draft genome (3.34 Mb) contains 101 contigs with an average GC content of 66.4%.

Comparative Genomic Analysis Reveals Habitat-Specific Genes and Regulatory Hubs within the Genus Novosphingobium.

Species belonging to the genus Novosphingobium are found in many different habitats and have been identified as metabolically versatile. Through comparative genomic analysis, we identified habitat-specific genes and regulatory hubs that could determine habitat selection for Novosphingobium spp. Genomes from 27 Novosphingobium strains isolated from diverse habitats such as rhizosphere soil, plant surfaces, heavily contaminated soils, and marine and freshwater environments were analyzed. Genome size and coding potential were widely variable, differing significantly between habitats. Phylogenetic relationships between strains were less likely to describe functional genotype similarity than the habitat from which they were isolated. In this study, strains (19 out of 27) with a recorded habitat of isolation, and at least 3 representative strains per habitat, comprised four ecological groups-rhizosphere, contaminated soil, marine, and freshwater. Sulfur acquisition and metabolism were the only core genomic traits to differ significantly in proportion between these ecological groups; for example, alkane sulfonate (ssuABCD) assimilation was found exclusively in all of the rhizospheric isolates. When we examined osmolytic regulation in Novosphingobium spp. through ectoine biosynthesis, which was assumed to be marine habitat specific, we found that it was also present in isolates from contaminated soil, suggesting its relevance beyond the marine system. Novosphingobium strains were also found to harbor a wide variety of mono- and dioxygenases, responsible for the metabolism of several aromatic compounds, suggesting their potential to act as degraders of a variety of xenobiotic compounds. Protein-protein interaction analysis revealed ß-barrel outer membrane proteins as habitat-specific hubs in each of the four habitats-freshwater (Saro_1868), marine water (PP1Y_AT17644), rhizosphere (PMI02_00367), and soil (V474_17210). These outer membrane proteins could play a key role in habitat demarcation and extend our understanding of the metabolic versatility of the Novosphingobium species. IMPORTANCE This study highlights the significant role of a microorganism's genetic repertoire in structuring the similarity between Novosphingobium strains. The results suggest that the phylogenetic relationships were mostly influenced by metabolic trait enrichment, which is possibly governed by the microenvironment of each microbe's respective niche. Using core genome analysis, the enrichment of a certain set of genes specific to a particular habitat was determined, which provided insights on the influence of habitat on the distribution of metabolic traits in Novosphingobium strains. We also identified habitat-specific protein hubs, which suggested delineation of Novosphingobium strains based on their habitat. Examining the available genomes of ecologically diverse bacterial species and analyzing the habitat-specific genes are useful for understanding the distribution and evolution of functional and phylogenetic diversity in the genus Novosphingobium.