Biology subjects

Narayanan, A. K.

Publications and source records attributed to Narayanan, A. K..

6 recordsLinked to original sources

bioRxiv2025

Illuminating the active virosphere with BONCAT and single virus genomic sequencing technologies

Marine viruses impact biogeochemical cycles through cell lysis, releasing organic matter and nutrients that fuel ocean productivity. Identifying and quantifying the specific viruses active in these processes remains a priority in the field. Here, we introduce a click-chemistry method to fluorescently label, sort, and sequence the genomes of newly produced viral particles released from transcriptionally active host microbial cells, alongside the analysis of co-occurring inactive cells and viruses in environmental samples. This approach, called viral BONCAT-FACS, combines biorthogonal non-canonical amino acid tagging (BONCAT) with environmental sample incubation, followed by single-virus and single-cell sorting by flow cytometry (FACS). Genomic analysis of translationally-active cells and new viral progeny in coastal seawater incubations confirmed BONCAT labeling and successful sorting of diverse marine bacteria, microeukaryotic cells, and virioplankton, with stark differences in the predicted turnover of specific groups of infecting viruses, including Pelagiphages, Methylophages, a Flavobacteriales-associated novel "Far-T4" clade, algae-infecting giant NCLDV viruses, and parasitic virophages. Sequenced BONCAT-active cells showed a strong enrichment in viral contigs relative to the inactive cell fraction, suggestive of a large proportion of translationally-active virocells. This study illustrates the effectiveness of viral BONCAT-FACS for uncovering genome-resolved viral-host dynamics. By providing a direct approach for tracking active viral infections in natural environments, this method enhances our ability to investigate behavior and interactions of these nanoscale predators, expanding our understanding of their role in ecosystem dynamics.

microbiology↗

bioRxiv2025

Viral communities from long-term anaerobic alkane-oxidizing enrichments may promote cell surface adhesion

The anaerobic oxidation of methane and higher C2+ alkanes is a dominant metabolism within hydrocarbon-rich deep-sea sediments and is largely mediated by alkane-oxidizing archaea in metabolic partnership with syntrophic sulfate-reducing bacteria. While these processes feed a diverse ecosystem, the viral component of alkane-rich sediments has historically been overlooked. We analyzed the viral community in long-term sediment-free enrichments of alkane-degrading organisms and found that abiotic factors such as incubation temperature had a greater correlation with community composition than with the phylogenetic patterns among individual viral species. No auxiliary metabolic genes directly involved in hydrocarbon oxidation or sulfate reduction were found, but the presence of AMGs involved in heme synthesis pathways common in methane oxidizers hints at a possible viral impact on alkane degradation. We also found evidence supporting the presence of a viral genus infecting several phyla across both the bacterial and archaeal domains, including one of the sulfate-reducing bacterial partners in the alkane-oxidizing syntrophy. Lastly, we report the presence of nosD-like proteins in viruses from sediment-derived systems that are not present in water column datasets; their distribution, genomic context, and lack of canonical nosD characteristics suggest an alternate adhesion-related role in sediment communities. The number of novel viruses obtained from these enrichment cultures and their potential roles in mediating host physiology illustrate the importance of studying the viral component in laboratory and environmental systems.

ecology↗

bioRxiv2025

Distinct Microbial Communities Within and On Seep Carbonates Support Long-term Anaerobic Oxidation of Methane and Novel pMMO Diversity

At methane seeps worldwide, syntrophic anaerobic methane-oxidizing archaea and sulfate-reducing bacteria (ANME-SRB) promote carbonate precipitation and rock formation, acting as methane and carbon sink. While maintenance of active anaerobic oxidation of methane (AOM) within seep carbonates has been documented, the ANME-SRB reactivity to methane exposure remains uncertain. Surface-associated microbes may metabolize AOM-derived sulfide, maintain carbonate anoxia, and contribute to carbonate dissolution and higher trophic levels; however, these microbial communities are poorly described thus far. Here we provide new insights into microbial diversity, metabolic potential, activity, and resiliency within and on Southern Californian methane seep carbonates, by combining 16S rRNA and metagenomic sequencing, laboratory incubations, and BONCAT-FISH. Ca. Methanophaga (ANME-1) dominated the carbonate interiors across different seepage activities, based on sequencing, while the dominant SRB was Ca. Desulfaltia, potentially a new ANME partner. BONCAT-FISH revealed differences in ANME-1 cell activity, suggesting cell dormancy or DNA preservation at less active seep sites. Carbonate incubations from low activity seeps ([≥]24 months) showed an exponential AOM reactivation (44-day doubling time), suggesting seep carbonates remain potential methane sinks over dynamic seepage conditions. The surface-associated communities were distinct from the carbonate interior and other seep habitats, and highly heterogeneous. Surface ANME-SRB biofilms and sulfide-oxidizing bacterial mats were associated with high and intermediate AOM carbonates, potentially influencing carbonate precipitation/dissolution. Carbonate surfaces shared diverse aerobic methanotrophs with invertebrates, potentially serving as pool for animal epibionts. Besides particulate methane monooxygenases from aerobic methanotrophs, we found divergent forms including within a Methylophagaceae (GCA-002733105) MAG suggesting a new function within Methylophagaceae.

ecology↗

bioRxiv2024

Functional characterization of two glycosyltransferases from Withania somnifera illuminates their role in withanosides biosynthesis and defence against bacteria

The medicinal properties of Ashwagandha (Withania somnifera L. Dunal) are attributed to the presence of unique class of natural products called as withanolides and their glycosylated forms, withanosides. Withanosides are proposed to be formed from withanolides by the action of glycosyltransferases (GTs). This study reports the functional characterization of two GTs (WsGT4 and WsGT6) from W. somnifera that exhibited induced expression in response to methyl jasmonate treatment and showed highest expression in leaves compared to other tissues. Biochemical assays with recombinant WsGT proteins showed that WsGT4 and WsGT6 formed glycosylated products with four and one of the seven tested withanolides substrates, respectively. WsGT4 catalyzed product formation using withanolide A, withanolide B, withanone, and 12-deoxywithastramonolide as substrates, with UDP-glucose serving as the glucose donor, while WsGT6 catalyzed the product formation only with withaferin A as substrate employing either UDP-glucose or UDP-galactose as sugar donors. Moreover, in planta studies through virus-induced gene silencing and transient overexpression of WsGT4 and WsGT6 in W. somnifera leaves modulated the levels of withanolides and withanosides, indicating their role in withanosides biosynthesis. Furthermore, while individual silencing of both WsGT4 and WsGT6 in W. somnifera reduced the tolerance to Pseudomonas syringae DC3000 growth, their overexpression enhanced the tolerance to the bacterium in W. somnifera. Taken together, these results shed light on the roles of WsGT4 and WsGT6 in withanoside biosynthesis and defence against model bacterial pathogen in W. somnifera.

plant biology↗

bioRxiv2023

Analysis of root volatiles and functional characterization of a root-specific germacrene A synthase in Artemisia pallens

Davana (Artemisia pallens) is a valuable aromatic herb within the Asteraceae family, highly prized for its essential oil (EO) produced in the aerial parts. However, the root volatiles and their specific composition, and genes responsible for root volatiles have remained unexplored until now. Here, we show that A. pallens roots possess distinct oil bodies and yields [~]0.05% of EO, which is primarily composed of sesquiterpenes {beta}-elemene, neryl isovalerate, {beta}-selinene, and -selinene, and trace amounts of monoterpenes {beta}-myrcene, D-limonene. This shows that, besides aerial parts, roots of davana can also be a source of unique EO. Moreover, we functionally characterized a terpene synthase (ApTPS1) that exhibited high in silico expression in the root transcriptome. The recombinant ApTPS1 showed the formation of {beta}-elemene and germacrene A with E,E-farnesyl diphosphate (FPP) as a substrate. Further, detailed analysis of assay products revealed that {beta}-elemene was the thermal rearrangement product of germacrene A. Furthermore, the functional expression of ApTPS1 in Saccharomyces cerevisiae confirmed the in vivo {beta}-elemene/germacrene A synthase activity of ApTPS1. At the transcript level, ApTPS1 displayed predominant expression in root, with significantly lower level of expression in other tissues. This expression pattern of ApTPS1 positively correlated with the tissue-specific accumulation level of {beta}-elemene. Overall, these findings provide fundamental insights into the EO profile of davana roots, and the contribution of ApTPS1 in the formation of a major root volatile. Main conclusionThe study demonstrated that Artemisia pallens roots can be a source of terpene-rich essential oil and root-specific ApTPS1 forms germacrene-A contributing to major root volatiles.

plant biology↗

bioRxiv2023

The cytochrome P450 enzyme WsCYP71B35 from Withania somnifera has a role in withanolides biosynthesis and defense against bacteria

The medicinal properties of Ashwagandha (Withania somnifera L. Dunal) are attributed to withanolides, which belong to the triterpenoid steroidal lactones class of compounds. Though it is proposed that intermediates of the universal phytosterol pathway are utilized by cytochrome P450 (CYP450) enzymes to form withanolides, studies on functional characterization of these enzymes has been sparse. This study reports the functional characterization of a CYP450 candidate from W. somnifera (WsCYP71B35) that exhibited induced expression in response to methyl jasmonate treatment and showed higher expression in tissues that accumulate withanolides. Biochemical assay with yeast microsomal fraction expressing recombinant WsCYP71B35 indicated no activity when phytosterols and their intermediate 24-methylene cholesterol were used as substrates. However, WsCYP71B35 catalyzed product formation with withaferin A, withanolide A, withanolide B, and withanoside IV among the tested substrates. Moreover, virus-induced gene silencing (VIGS) and transient overexpression of WsCYP71B35 in W. somnifera leaves modulated the levels of withaferin A, withanolide A, and withanolide B, indicating the role of WsCYP71B35 in withanolides pathway. Furthermore, VIGS of WsCYP71B35 in W. somnifera reduced its tolerance to Pseudomonas syringae (DC3000) infection, whereas overexpression enhanced the tolerance to the bacterium in W. somnifera and transgenic tobacco. Overall, these results provide insights into the role of W. somnifera WsCYP71B35 in withanolides biosynthesis and bacterial defense.

plant biology↗