Bacterial community distribution and functional potentials provide key insights into their role in the ecosystem functioning of a retreating Eastern Himalayan glacier.

Himalayan glaciers are receding at an exceptional rate, perturbing the local biome and ecosystem processes. Understanding the microbial ecology of an exclusively microbe-driven biome provides insights into their contributions to the ecosystem functioning through biogeochemical fluxes. Here, we investigated the bacterial communities and their functional potential in the retreating East Rathong Glacier (ERG) of Sikkim Himalaya. Amplicon-based taxonomic classification revealed the dominance of the phyla Proteobacteria, Bacteroidota, and candidate Patescibacteria in the glacial sites. Further, eight good-quality metagenome-assembled genomes (MAGs) of Proteobacteria, Patescibacteria, Acidobacteriota, and Choloflexota retrieved from the metagenomes elucidated the microbial contributions to nutrient cycling. The ERG MAGs showed aerobic respiration as a primary metabolic feature, accompanied by carbon fixation and complex carbon degradation potentials. Pathways for nitrogen metabolism, chiefly dissimilatory nitrate reduction and denitrification, and a complete sulphur oxidation enzyme complex for sulphur metabolism were identified in the MAGs. We observed that DNA repair and oxidative stress response genes complemented with osmotic and periplasmic stress and protein chaperones were vital for adaptation against the intense radiation and stress conditions of the extreme Himalayan niche. Current findings elucidate the microbiome and associated functional potentials of a vulnerable glacier, emphasizing their significant ecological roles in a changing glacial ecosystem.

Red bioactive pigment from Himalayan Janthinobacterium sp. ERMR3:09: optimization, characterization, and potential applications.

Prodigiosin is a red pigment commonly produced as a secondary metabolite by Serratia marcescens. It exhibits inherent bioactivities, including antimicrobial and anticancer, with low to no toxic effects on normal cells. The present study investigates a bioactive prodigiosin production from an atypical, red-pigmented, potentially novel Janthinobacterium sp. ERMR3:09 isolated from a glacial moraine. Statistically optimized culture parameters, i.e., w/v 1.0% glucose and 0.08% peptone as carbon and nitrogen sources, temperature 20 °C, and media pH 7, resulted in a four-fold increase in the pigment yield. The upscaled production in an 8 L volume resulted in higher pigment production within a shorter period of 48 h. The ultra-performance liquid chromatography (UPLC) analysis validated the identity of the purified pigment as prodigiosin that showed thermostability at 75 °C for 3 h. Evaluation of antimicrobial activity showed potent inhibitory effects (> 50%) against the opportunistic pathogenic fungal and Gram-positive bacterial strains. The pigment showed significant cytotoxicity (p < 0.05) towards A549 and HeLa cell lines with IC50 values of 42.2 µM and 36.11 µM, respectively. The study demonstrated that microbial communities from extreme niches can be ideal sources of bioactive pigments with immense pharmaceutical potential vital for the development of non-synthetic therapeutic agents.

Essential oil composition and antimicrobial potential of aromatic plants grown in the mid-hill conditions of the Western Himalayas.

Essential oils are highly concentrated natural extracts obtained from plants, rich in bioactive constituents with antimicrobial properties, but the distinctive climate of the Western Himalayan region influences the same. Aromatic and medicinal plants, viz., Origanum majorana, Origanum vulgare, Cymbopogon winterianus, Pelargonium graveolens, and Nepeta cataria were grown in the foothills of the Western Himalayan condition and evaluated for essential oil content, composition, and their effect on some of the most common pathogenic microorganisms. The essential oil content (%) was 0.77, 0.45, 1.37, 0.15 and 0.17% in O. majorana, O. vulgare, C. winterianus, P. graveolens, and N. cataria, respectively. The major essential oil constituents of the isolated oils were terpinen-4-ol, thymol, citronellal, citronellol, and nepetalactone, contributing 41.24%, 31.81%, 43.13%, 43.35% and 91.43% in O. majorana, O. vulgare, C. winterianus, P. graveolens, and N. cataria, respectively. Well-diffusion assay revealed that the essential oil of O. majorana and O. vulgare was active against both the tested Gram-positive, viz., Bacillus subtilis MTCC 121, Micrococcus luteus MTCC 2470, and Staphylococcus aureus MTCC 96; and Gram-negative, viz., Escherichia coli MTCC 43, Klebsiella pneumoniae MTCC 109, and Pseudomonas aeruginosa MTCC 2453 bacteria, while the essential oil of C. winterianus, P. graveolens, and N. cataria showed activity against only some Gram-positive bacteria. Minimum inhibitory concentration (v/v) values indicated the highest efficacy of O. majorana essential oil against B. subtilis (0.5%), M. luteus (1%), and S. aureus (1%), while O. vulgare was most efficient to E. coli (2%) and K. pneumoniae (2%). C. winterianus essential oil did not inhibit any bacterial strains. M. luteus was susceptible to the essential oil of P. graveolens (1%) and N. cataria (0.5%) at low concentrations. Present findings showed the association between the chemical constituents' profile of isolated essential oils from the Himalayan region and their antimicrobial activity, indicating their perspective to be utilized as antibacterial means.

Antioxidant prodigiosin-producing cold-adapted Janthinobacterium sp. ERMR3:09 from a glacier moraine: Genomic elucidation of cold adaptation and pigment biosynthesis.

Janthinobacterium from cold niches has been studied broadly for bioactive violacein production. However, reports on the atypical red-pigmented Janthinobacterium strains are shallow. The bioactive red prodigiosin pigment has immense pharmacological significance, including antioxidant, antimicrobial and anticancer potential. Here, we report the first complete genome of a prodigiosin-producing Janthinobacterium sp. ERMR3:09 from Sikkim Himalaya in an attempt to elucidate its cold adaptation and prodigiosin biosynthesis. Nanopore sequencing and Flye assembly of the ERMR3:09 genome resulted in a single contig of 6,262,330 bp size and 62.26% GC content. Phylogenomic analysis and genome indices indicate that ERMR3:09 is a potentially novel species of the genus Janthinobacterium. The multicopy cold-responsive genes and gene upregulation under cold stress denoted its cold adaptation mechanisms. Genome analysis identified the unique genes, gene cluster and pathway for prodigiosin biosynthesis in ERMR3:09. Considering the notable antioxidant activity, it can be the next powerhouse of bioactive prodigiosin production.

Indirect organogenesis-mediated high frequency conversion of non-embryonic synthetic seeds, essential oil profiling and antibacterial activity in genetically stable plants of Patchouli.

Patchouli is a prized tropical medicinal herb with broad-spectrum therapeutic importance. The present research work describes development of an efficient callus-mediated plant regeneration protocol along with associated germplasm portability system (via alginate-encapsulation). Using 1.5 mg/l α-naphthalene acetic acid (NAA) and 1.0 mg/l 2, 4-dichlorophenoxy acetic acid (2, 4-D), highly proliferative friable calli were produced that subsequently underwent organogenesis in combinatorial cytokinin treatment to yield multiple shoot clusters. The highest frequency of shoot formation was achieved using 1.5 mg/l NAA with 1.5 mg/l 6-benzylaminopurine (BAP) in Murashige and Skoog (MS) medium. In vitro-derived shoot tips were encapsulated with 3% sodium alginate and 100 mM CaCl2 solution. The encapsulated beads were germinated in MS media with various concentrations of polyamines, where the highest regeneration frequency was observed with 1.5 mg/l spermidine. The regenerated shoots were rooted in basal MS medium and were successfully acclimatized with 96% survival rate. Genetic homogeneity amongst the regenerated plantlets was validated using Start Codon Targeted polymorphism (SCoT) and CAAT box-derived polymorphism (CBDP) ascertaining a high degree of clonal fidelity. The essential oil (EO) profiling of the donor plant and the in vitro-derived plantlets revealed identical composition. Furthermore, the antibacterial activities of various tissue extracts and extracted EOs were evaluated against the opportunistic pathogens viz. Klebsiella pneumoniae (MTCC 109), Salmonella typhii (MTCC 733), Micrococcus luteus (MTCC 2470) and Staphylococcus aureus (MTCC 96). The minimum inhibitory concentration (MIC) ranged from 0.31 to 5.0 mg/ml and 2.5 to 5.0 mg/ml against Gram-positive and Gram-negative bacteria, respectively. Eventually, the present research provides a holistic insight into the rapid regeneration of quality planting material as well as pharmacological bioprospection of patchouli along with the scope of further qualitative improvement via genetic transformation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03302-3.

Psychrotrophic plant beneficial bacteria from the glacial ecosystem of Sikkim Himalaya: Genomic evidence for the cold adaptation and plant growth promotion.

Commercial biofertilizers tend to be ineffective in cold mountainous regions due to reduced metabolic activity of the microbial inoculants under low temperatures. Cold-adapted glacier bacteria with plant growth-promoting (PGP) properties may prove significant in developing cold-active biofertilizers for improving mountain agriculture. With this perspective, the cultivable bacterial diversity was documented from the East Rathong glacier ecosystem lying above 3900 masl of Sikkim Himalaya. A total of 120 bacterial isolates affiliated to Gammaproteobacteria (53.33%), Bacteroidetes (16.66%), Actinobacteria (15.83%), Betaproteobacteria (6.66%), Alphaproteobacteria (4.16%), and Firmicutes (3.33%) were recovered. Fifty-two isolates showed many in vitro PGP activities of phosphate solubilization (9-100 µg/mL), siderophore production (0.3-100 psu) and phytohormone indole acetic acid production (0.3-139 µg/mL) at 10 °C. Plant-based bioassays revealed an enhancement of shoot length by 21%, 22%, and 13% in ERGS5:01, ERMR1:04, and ERMR1:05, and root length by 14%, 17%, 11%, and 22% in ERGS4:06, ERGS5:01, ERMR1:04, and ERMR1:05 treated seeds respectively. An increased shoot dry weight of 4-29% in ERMR1:05 and ERMR1:04, and root dry weight of 42-98% was found in all the treatments. Genome analysis of four bacteria from diverse genera predicted many genes involved in the bacterial PGP activity. Comparative genome study highlighted the presence of PGP-associated unique genes for glucose dehydrogenase, siderophore receptor, tryptophan synthase, phosphate metabolism (phoH, P, Q, R, U), nitrate and nitrite reductase, TonB-dependent receptor, spermidine/putrescine ABC transporter etc. in the representative bacteria. The expression levels of seven cold stress-responsive genes in the cold-adapted bacterium ERGS4:06 using real-time quantitative PCR (RT-qPCR) showed an upregulation of all these genes by 6-17% at 10 °C, and by 3-33% during cold-shock, which indicates the cold adaptation strategy of the bacterium. Overall, this study signifies the psychrotrophic bacterial diversity from an extreme glacier environment as a potential tool for improving plant growth under cold environmental stress.

Seasonal variability in essential oil composition and biological activity of Rosmarinus officinalis L. accessions in the western Himalaya.

Rosmarinus officinalis L. is an imperative herb used in pharmaceutical yet knowledge on chemical and activity profile of essential oil (EO) to harvest seasons and accessions from the Himalayan region is limited. Thus, accessions were evaluated to determine the EO content, compositional, antimicrobial, and cytotoxic potential of rosemary in different harvest seasons during 2018‒2019. EO content was 30.5% higher in IHBT/RMAc-1 compared with IHBT/RMAc-2 accession while 27.9% and 41.6% higher in the autumn as compared with summer and rainy season, respectively. Major EO compound was 1,8-cineole; ranged from 32.50‒51.79% during harvest seasons and 38.70‒42.20% in accessions. EO was active against both the tested Gram-positive bacteria (Micrococcus luteus MTCC 2470 and Staphylococcus aureus MTCC 96). EOs showed inhibition of Gram-negative bacteria (Salmonella typhi MTCC 733), while Klebsiella pneumoniae MTCC 109 was found to be resistant. The rosemary EO of T1 (Rainy season IHBT/RMAc-1) was most effective against S. aureus MTCC 96 with the minimum inhibitory concentration (MIC) of 4% (v/v). In vitro cytotoxicity evaluation showed no potential anti-proliferative activity of EO. The rosemary EO profile in the western Himalayan region was influenced by harvesting seasons and genetic variability within the accessions; furthermore, a promising antibacterial agent in pharmaceutical and flavour industries.

Multilocus sequence based identification and adaptational strategies of Pseudomonas sp. from the supraglacial site of Sikkim Himalaya.

Microorganisms inhabiting the supraglacial ice are biotechnologically significant as they are equipped with unique adaptive features in response to extreme environmental conditions of high ultraviolet radiations and frequent freeze-thaw. In the current study, we obtained eleven strains of Pseudomonas from the East Rathong supraglacial site in Sikkim Himalaya that showed taxonomic ambiguity in terms of species affiliation. Being one of the most complex and diverse genera, deciphering the correct taxonomy of Pseudomonas species has always been challenging. So, we conducted multilocus sequence analysis (MLSA) using five housekeeping genes, which concluded the taxonomic assignment of these strains to Pseudomonas antarctica. This was further supported by the lesser mean genetic distances with P. antarctica (0.73%) compared to P. fluorescens (3.65%), and highest ANI value of ~99 and dDDH value of 91.2 of the representative strains with P. antarctica PAMC 27494. We examined the multi-tolerance abilities of these eleven Pseudomonas strains. Indeed the studied strains displayed significant tolerance to freezing for 96 hours compared to the mesophilic control strain, while except for four strains, seven strains exhibited noteworthy tolerance to UV-C radiations. The genome-based findings revealed many cold and radiation resistance-associated genes that supported the physiological findings. Further, the bacterial strains produced two or more cold-active enzymes in plate-based assays. Owing to the polyadaptational attributes, the strains ERGC3:01 and ERGC3:05 could be most promising for bioprospection.

Industrial applications of cold-adapted enzymes: challenges, innovations and future perspective.

Extreme cold environments are potential reservoirs of microorganisms producing unique and novel enzymes in response to environmental stress conditions. Such cold-adapted enzymes prove to be valuable tools in industrial biotechnology to meet the increasing demand for efficient biocatalysts. The inherent properties like high catalytic activity at low temperature, high specific activity and low activation energy make the cold-adapted enzymes well suited for application in various industries. The interest in this group of enzymes is expanding as they are the preferred alternatives to harsh chemical synthesis owing to their biodegradable and non-toxic nature. Irrespective of the multitude of applications, the use of cold-adapted enzymes at the industrial level is still limited. The current review presents the unique adaptive features and the role of cold-adapted enzymes in major industries like food, detergents, molecular biology and bioremediation. The review highlights the significance of omics technology i.e., metagenomics, metatranscriptomics and metaproteomics in enzyme bioprospection from extreme environments. It further points out the challenges in using cold-adapted enzymes at the industrial level and the innovations associated with novel enzyme prospection strategies. Documentations on cold-adapted enzymes and their applications are abundant; however, reports on the role of omics tools in exploring cold-adapted enzymes are still scarce. So, the review covers the aspect concerning the novel techniques for enzyme discovery from nature.

Physiological and genomic evidence supports the role of Serratia quinivorans PKL:12 as a biopriming agent for the biohardening of micropropagated Picrorhiza kurroa plantlets in cold regions.

The medicinal herb, Picrorhiza kurroa Royle ex Benth has become endangered because of indiscriminate over-harvesting. Although micropropagation has been attempted for mass propagation of the plant, survival of in vitro plantlets under green house/open field poses a major challenge. Biopriming of micropropagated plantlets with plant growth-promoting rhizobacteria (PGPR) are among the successful methods to combat this problem. Serratia quinivorans PKL:12 was the best-characterized PGPR from rhizospheric soil of P. kurroa as it increased the vegetative growth and survival of the micropropagated plantlets most effectively. Complete genome (5.29 Mb) predicted genes encoding proteins for cold adaptation and plant growth-promoting traits in PKL:12. Antibiotic and biosynthetic gene cluster prediction supported PKL:12 as a potential biocontrol agent. Comparative genomics revealed 226 unique genes with few genes associated with plant growth-promoting potential. Physiological and genomic evidence supports S. quinivorans PKL:12 as a potential agent for bio-hardening of micropropagated P. kurroa plantlets in cold regions.

Comparative genomics and molecular adaptational analysis of Arthrobacter from Sikkim Himalaya provided insights into its survivability under multiple high-altitude stress.

Arthrobacter is a dominant aerobic bacterium under the class Actinobacteria, known for its nutritionally versatile nature and wide prevalence in stressful environments. In the current study representative two strains of Arthrobacter, ERGS1:01 and ERGS4:06, with efficient survivability under high altitude stress conditions were selected for comparative genomic studies with their mesophilic counterparts. Physiological analysis and genome insights supported the survival of these strains under multiple high-altitude stress conditions. Molecular cold-adaptation and substitution analysis of the studied strains supported the incidence of more cold-adapted proteins for functionality at low temperatures. Studied strains preferred amino acids like serine, asparagine, lysine, tryptophan for favoring increased flexibility supporting their broad temperature survivability. To the best of our knowledge, this is the first molecular cold adaptation analysis performed for the genus Arthrobacter and has revealed that 'aromaticity', one of the cold-adaptor indicators, should be carefully considered while evaluating cold adaptation strategies in psychrotrophic/psychrophilic bacteria.

A Broad Temperature Active Lipase Purified From a Psychrotrophic Bacterium of Sikkim Himalaya With Potential Application in Detergent Formulation.

Bacterial lipases with activity spanning over a broad temperature and substrate range have several industrial applications. An efficient enzyme-producing bacterium Chryseobacterium polytrichastri ERMR1:04, previously reported from Sikkim Himalaya, was explored for purification and characterization of cold-adapted lipase. Optimum lipase production was observed in 1% (v/v) rice bran oil, pH 7 at 20°C. Size exclusion and hydrophobic interaction chromatography purified the enzyme up to 21.3-fold predicting it to be a hexameric protein of 250 kDa, with 39.8 kDa monomeric unit. MALDI-TOF-MS analysis of the purified lipase showed maximum similarity with alpha/beta hydrolase (lipase superfamily). Biochemical characterization of the purified enzyme revealed optimum pH (8.0), temperature (37°C) and activity over a temperature range of 5-65°C. The tested metals (except Cu2+ and Fe2+) enhanced the enzyme activity and it was tolerant to 5% (v/v) methanol and isopropanol. The Km and Vmax values were determined as 0.104 mM and 3.58 U/mg, respectively for p-nitrophenyl palmitate. Bioinformatics analysis also supported in vitro findings by predicting enzyme's broad temperature and substrate specificity. The compatibility of the purified lipase with regular commercial detergents, coupled with its versatile temperature and substrate range, renders the given enzyme a promising biocatalyst for potential detergent formulations.

Complete genome sequence of Pseudomonas frederiksbergensis ERDD5:01 revealed genetic bases for survivability at high altitude ecosystem and bioprospection potential.

Pseudomonas frederiksbergensis ERDD5:01 is a psychrotrophic bacteria isolated from the glacial stream flowing from East Rathong glacier in Sikkim Himalaya. The strain showed survivability at high altitude stress conditions like freezing, frequent freeze-thaw cycles, and UV-C radiations. The complete genome of 5,746,824 bp circular chromosome and a plasmid of 371,027 bp was sequenced to understand the genetic basis of its survival strategy. Multiple copies of cold-associated genes encoding cold active chaperons, general stress response, osmotic stress, oxidative stress, membrane/cell wall alteration, carbon storage/starvation and, DNA repair mechanisms supported its survivability at extreme cold and radiations corroborating with the bacterial physiological findings. The molecular cold adaptation analysis in comparison with the genome of 15 mesophilic Pseudomonas species revealed functional insight into the strategies of cold adaptation. The genomic data also revealed the presence of industrially important enzymes.