Aquificae overcomes competition by archaeal thermophiles, and crowding by bacterial mesophiles, to dominate the boiling vent-water of a Trans-Himalayan sulfur-borax spring.

Trans-Himalayan hot spring waters rich in boron, chlorine, sodium and sulfur (but poor in calcium and silicon) are known based on PCR-amplified 16S rRNA gene sequence data to harbor high diversities of infiltrating bacterial mesophiles. Yet, little is known about the community structure and functions, primary productivity, mutual interactions, and thermal adaptations of the microorganisms present in the steaming waters discharged by these geochemically peculiar spring systems. We revealed these aspects of a bacteria-dominated microbiome (microbial cell density ~8.5 × 104 mL-1; live:dead cell ratio 1.7) thriving in the boiling (85°C) fluid vented by a sulfur-borax spring called Lotus Pond, situated at 4436 m above the mean sea-level, in the Puga valley of eastern Ladakh, on the Changthang plateau. Assembly, annotation, and population-binning of >15-GB metagenomic sequence illuminated the numeral predominance of Aquificae. While members of this phylum accounted for 80% of all 16S rRNA-encoding reads within the metagenomic dataset, 14% of such reads were attributed to Proteobacteria. Post assembly, only 25% of all protein-coding genes identified were attributable to Aquificae, whereas 41% was ascribed to Proteobacteria. Annotation of metagenomic reads encoding 16S rRNAs, and/or PCR-amplified 16S rRNA genes, identified 163 bacterial genera, out of which 66 had been detected in past investigations of Lotus Pond's vent-water via 16S amplicon sequencing. Among these 66, Fervidobacterium, Halomonas, Hydrogenobacter, Paracoccus, Sulfurihydrogenibium, Tepidimonas, Thermus and Thiofaba (or their close phylogenomic relatives) were presently detected as metagenome-assembled genomes (MAGs). Remarkably, the Hydrogenobacter related MAG alone accounted for ~56% of the entire metagenome, even though only 15 out of the 66 genera consistently present in Lotus Pond's vent-water have strains growing in the laboratory at >45°C, reflecting the continued existence of the mesophiles in the ecosystem. Furthermore, the metagenome was replete with genes crucial for thermal adaptation in the context of Lotus Pond's geochemistry and topography. In terms of sequence similarity, a majority of those genes were attributable to phylogenetic relatives of mesophilic bacteria, while functionally they rendered functions such as encoding heat shock proteins, molecular chaperones, and chaperonin complexes; proteins controlling/modulating/inhibiting DNA gyrase; universal stress proteins; methionine sulfoxide reductases; fatty acid desaturases; different toxin-antitoxin systems; enzymes protecting against oxidative damage; proteins conferring flagellar structure/function, chemotaxis, cell adhesion/aggregation, biofilm formation, and quorum sensing. The Lotus Pond Aquificae not only dominated the microbiome numerically but also acted potentially as the main primary producers of the ecosystem, with chemolithotrophic sulfur oxidation (Sox) being the fundamental bioenergetic mechanism, and reductive tricarboxylic acid (rTCA) cycle the predominant carbon fixation pathway. The Lotus Pond metagenome contained several genes directly or indirectly related to virulence functions, biosynthesis of secondary metabolites including antibiotics, antibiotic resistance, and multi-drug efflux pumping. A large proportion of these genes being attributable to Aquificae, and Proteobacteria (very few were ascribed to Archaea), it could be worth exploring in the future whether antibiosis helped the Aquificae overcome niche overlap with other thermophiles (especially those belonging to Archaea), besides exacerbating the bioenergetic costs of thermal endurance for the mesophilic intruders of the ecosystem.

Extremely oligotrophic and complex-carbon-degrading microaerobic bacteria from Arabian Sea oxygen minimum zone sediments.

Sediments underlying marine hypoxic zones are huge sinks of unreacted complex organic matter, where despite acute O2 limitation, obligately aerobic bacteria thrive, and steady depletion of organic carbon takes place within a few meters below the seafloor. However, little knowledge exists about the sustenance and complex carbon degradation potentials of aerobic chemoorganotrophs in these sulfidic ecosystems. We isolated and characterized a number of aerobic bacterial chemoorganoheterotrophs from across a ~ 3 m sediment horizon underlying the perennial hypoxic zone of the eastern Arabian Sea. High levels of sequence correspondence between the isolates' genomes and the habitat's metagenomes and metatranscriptomes illustrated that the strains were widespread and active across the sediment cores explored. The isolates catabolized several complex organic compounds of marine and terrestrial origins in the presence of high or low, but not zero, O2. Some of them could also grow anaerobically on yeast extract or acetate by reducing nitrate and/or nitrite. Fermentation did not support growth, but enabled all the strains to maintain a fraction of their cell populations over prolonged anoxia. Under extreme oligotrophy, limited growth followed by protracted stationary phase was observed for all the isolates at low cell density, amid high or low, but not zero, O2 concentration. While population control and maintenance could be particularly useful for the strains' survival in the critically carbon-depleted layers below the explored sediment depths (core-bottom organic carbon: 0.5-1.0% w/w), metagenomic data suggested that in situ anoxia could be surmounted via potential supplies of cryptic O2 from previously reported sources such as Nitrosopumilus species.

Thermal Endurance by a Hot-Spring-Dwelling Phylogenetic Relative of the Mesophilic Paracoccus.

High temperature growth/survival was revealed in a phylogenetic relative (SMMA_5) of the mesophilic Paracoccus isolated from the 78 to 85°C water of a Trans-Himalayan sulfur-borax spring. After 12 h at 50°C, or 45 min at 70°C, in mineral salts thiosulfate (MST) medium, SMMA_5 retained ~2% colony forming units (CFUs), whereas comparator Paracoccus had 1.5% and 0% CFU left at 50°C and 70°C, respectively. After 12 h at 50°C, the thermally conditioned sibling SMMA_5_TC exhibited an ~1.5 time increase in CFU count; after 45 min at 70°C, SMMA_5_TC had 7% of the initial CFU count. 1,000-times diluted Reasoner's 2A medium, and MST supplemented with lithium, boron, or glycine-betaine, supported higher CFU-retention/CFU-growth than MST. Furthermore, with or without lithium/boron/glycine-betaine, a higher percentage of cells always remained metabolically active, compared with what percentage formed single colonies. SMMA_5, compared with other Paracoccus, contained 335 unique genes: of these, 186 encoded hypothetical proteins, and 83 belonged to orthology groups, which again corresponded mostly to DNA replication/recombination/repair, transcription, secondary metabolism, and inorganic ion transport/metabolism. The SMMA_5 genome was relatively enriched in cell wall/membrane/envelope biogenesis, and amino acid metabolism. SMMA_5 and SMMA_5_TC mutually possessed 43 nucleotide polymorphisms, of which 18 were in protein-coding genes with 13 nonsynonymous and seven radical amino acid replacements. Such biochemical and biophysical mechanisms could be involved in thermal stress mitigation which streamline the cells' energy and resources toward system-maintenance and macromolecule-stabilization, thereby relinquishing cell-division for cell-viability. Thermal conditioning apparently helped inherit those potential metabolic states which are crucial for cell-system maintenance, while environmental solutes augmented the indigenous stability-conferring mechanisms. IMPORTANCE For a holistic understanding of microbial life's high-temperature adaptation, it is imperative to explore the biology of the phylogenetic relatives of mesophilic bacteria which get stochastically introduced to geographically and geologically diverse hot spring systems by local geodynamic forces. Here, in vitro endurance of high heat up to the extent of growth under special (habitat-inspired) conditions was discovered in a hot-spring-dwelling phylogenetic relative of the mesophilic Paracoccus species. Thermal conditioning, extreme oligotrophy, metabolic deceleration, presence of certain habitat-specific inorganic/organic solutes, and potential genomic specializations were found to be the major enablers of this conditional (acquired) thermophilicity. Feasibility of such phenomena across the taxonomic spectrum can well be paradigm changing for the established scopes of microbial adaptation to the physicochemical extremes. Applications of conditional thermophilicity in microbial process biotechnology may be far reaching and multifaceted.

Draft Genome Sequences from Two Gram-Negative Bacteria, Serratia sp. Strain EWG9 and Leclercia sp. Strain EMC7, Isolated from the Earthworm Eisenia fetida.

We present the draft genome sequences of two bacterial strains that are putatively unique species and belong to two different Gram-negative genera: Serratia sp. EWG9 and Leclercia sp. EMC7, recovered from the gut and cast, respectively, of the compost worm Eisenia fetida.

Inducible boron resistance via active efflux in Lysinibacillus and Enterococcus isolates from boron-contaminated agricultural soil.

Phylogenetically diverse bacteria tolerate high boron concentrations while others require it for metabolic purposes despite the metalloid being toxic beyond a threshold. Boron resistance and plant growth promoting attributes of two bacterial strains, Lysinibacillus sp. OL1 and a novel Enterococcus sp. OL5, isolated from boron-fertilizer-amended cauliflower fields were investigated in this study. OL1 and OL5 grew efficiently in the presence of 210-230 mM boron, and resistance was found to be inducible by small amounts of the element: 5 to 50 mM boron pre-exposure progressively shortened the lag phase of growth in the presence of 200 mM boron. Intracellular boron accumulation was also found to be regulated by the level of pre-exposure: no induction or induction by small amounts led to higher levels of intracellular accumulation, whereas induction by high concentrations led to lower accumulation. These data, in the context of the strains' overall resistance towards 200 mM boron, indicated that induction by higher boron concentrations turned potential efflux mechanisms on, while resistance was eventually achieved by continuous cellular entry and exit of the ions. Involvement of solute efflux in boron resistance was corroborated by the genome content of the isolates (genes encoding proteins of the ATP-binding cassette, major facilitator, small multidrug resistance, multi antimicrobial extrusion, and resistance-nodulation-cell division, family/superfamily). Bacteria such as OL1 and OL5, which resist boron via influx-efflux, potentially lower boron bioavailability, and therefore toxicity, for the soil microbiota at large. These bacteria, by virtue of their plant-growth-promoting attributes, can also be used as biofertilizers.

Draft Genome Sequences of Four Novel Strains of Microbes Isolated from Lepidocephalichthys guntea.

We report the draft genome sequences of four bacterial strains (all of which are putatively novel species) belonging to four different genera. The Gram-positive Bacillus sp. strain GG161 and Rhodococcus sp. strain GG48 and the Gram-negative Achromobacter sp. strain GG226 and Shigella sp. strain GCP5 were all isolated from the gut of the optionally intestine-breathing freshwater fish Lepidocephalichthys guntea.

34S enrichment as a signature of thiosulfate oxidation in the "Proteobacteria".

Kinetics of thiosulfate oxidation, product and intermediate formation, and 34S fractionation, were studied for the members of Alphaproteobacteria Paracoccus sp. SMMA5 and Mesorhizobium thiogangeticum SJTT, the Betaproteobacteria member Pusillimonas ginsengisoli SBO3, and the Acidithiobacillia member Thermithiobacillus sp. SMMA2, during chemolithoautotrophic growth in minimal salts media supplemented with 20 mM thiosulfate. The two Alphaproteobacteria oxidized thiosulfate directly to sulfate, progressively enriching the end-product with 34S; Δ34Sthiosulfate-sulfate values recorded at the end of the two processes (when no thiosulfate was oxidized any further) were -2.9‰ and -3.5‰, respectively. Pusillimonas ginsengisoli SBO3 and Thermithiobacillus sp. SMMA2, on the other hand, oxidized thiosulfate to sulfate via tetrathionate intermediate formation, with progressive 34S enrichment in the end-product sulfate throughout the incubation period; Δ34Sthiosulfate-sulfate, at the end of the two processes (when no further oxidation took place), reached -3.5‰ and -3.8‰, respectively. Based on similar 34S fractionation patterns recorded previously during thiosulfate oxidation by strains of Paracoccus pantotrophus, Advenella kashmirensis and Hydrogenovibrio crunogenus, it was concluded that progressive reverse fractionation, enriching the end-product sulfate with 34S, could be a characteristic signature of bacterial thiosulfate oxidation.

Trends of mutation accumulation across global SARS-CoV-2 genomes: Implications for the evolution of the novel coronavirus.

To understand SARS-CoV-2 microevolution, this study explored the genome-wide frequency, gene-wise distribution, and molecular nature of all point-mutations detected across its 71,703 RNA-genomes deposited in GISAID till 21 August 2020. Globally, nsp1/nsp2 and orf7a/orf3a were the most mutation-ridden non-structural and structural genes respectively. Phylogeny of 4618 spatiotemporally-representative genomes revealed that entities belonging to the early lineages are mostly spread over Asian countries, including India, whereas the recently-derived lineages are more globally distributed. Of the total 20,163 instances of polymorphism detected across global genomes, 12,594 and 7569 involved transitions and transversions, predominated by cytidine-to-uridine and guanosine-to-uridine conversions, respectively. Positive selection of nonsynonymous mutations (dN/dS >1) in most of the structural, but not the non-structural, genes indicated that SARS-CoV-2 has already harmonized its replication/transcription machineries with the host metabolism, while it is still redefining virulence/transmissibility strategies at the molecular level. Mechanistic bases and evolutionary/pathogenicity-related implications are discussed for the predominant mutation-types.

Aerobic microbial communities in the sediments of a marine oxygen minimum zone.

The ecology of aerobic microorganisms is never explored in marine oxygen minimum zone (OMZ) sediments. Here we reveal aerobic bacterial communities along ∼3 m sediment-horizons of the eastern Arabian Sea OMZ. Sulfide-containing sediment-cores retrieved from 530 mbsl (meters beneath the sea-level) and 580 mbsl were explored at 15-30 cm intervals, using metagenomics, pure-culture-isolation, genomics and metatranscriptomics. Genes for aerobic respiration, and oxidation of methane/ammonia/alcohols/thiosulfate/sulfite/organosulfur-compounds, were detected in the metagenomes from all 25 sediment-samples explored. Most probable numbers for aerobic chemolithoautotrophs and chemoorganoheterotrophs at individual sample-sites were up to 1.1 × 107 (g sediment)-1. The sediment-sample collected from 275 cmbsf (centimeters beneath the seafloor) of the 530-mbsl-core yielded many such obligately aerobic isolates belonging to Cereibacter, Guyparkeria, Halomonas, Methylophaga, Pseudomonas and Sulfitobacter which died upon anaerobic incubation, despite being provided with all possible electron acceptors and fermentative substrates. High percentages of metatranscriptomic reads from the 275 cmbsf sediment-sample, and metagenomic reads from all 25 sediment-samples, matched the isolates' genomic sequences including those for aerobic metabolisms, genetic/environmental information processing and cell division, thereby illustrating the bacteria's in-situ activity, and ubiquity across the sediment-horizons, respectively. The findings hold critical implications for organic carbon sequestration/remineralization, and inorganic compounds oxidation, within the sediment realm of global marine OMZs.

Microbiome and ecology of a hot spring-microbialite system on the Trans-Himalayan Plateau.

Little is known about life in the boron-rich hot springs of Trans-Himalayas. Here, we explore the geomicrobiology of a 4438-m-high spring which emanates ~70 °C-water from a boratic microbialite called Shivlinga. Due to low atmospheric pressure, the vent-water is close to boiling point so can entropically destabilize biomacromolecular systems. Starting from the vent, Shivlinga's geomicrobiology was revealed along the thermal gradients of an outflow-channel and a progressively-drying mineral matrix that has no running water; ecosystem constraints were then considered in relation to those of entropically comparable environments. The spring-water chemistry and sinter mineralogy were dominated by borates, sodium, thiosulfate, sulfate, sulfite, sulfide, bicarbonate, and other macromolecule-stabilizing (kosmotropic) substances. Microbial diversity was high along both of the hydrothermal gradients. Bacteria, Eukarya and Archaea constituted >98%, ~1% and <1% of Shivlinga's microbiome, respectively. Temperature constrained the biodiversity at ~50 °C and ~60 °C, but not below 46 °C. Along each thermal gradient, in the vent-to-apron trajectory, communities were dominated by Aquificae/Deinococcus-Thermus, then Chlorobi/Chloroflexi/Cyanobacteria, and finally Bacteroidetes/Proteobacteria/Firmicutes. Interestingly, sites of >45 °C were inhabited by phylogenetic relatives of taxa for which laboratory growth is not known at >45 °C. Shivlinga's geomicrobiology highlights the possibility that the system's kosmotrope-dominated chemistry mitigates against the biomacromolecule-disordering effects of its thermal water.

Molecular mechanism of sulfur chemolithotrophy in the betaproteobacterium Pusillimonas ginsengisoli SBSA.

Chemolithotrophic sulfur oxidation represents a significant part of the biogeochemical cycling of this element. Due to its long evolutionary history, this ancient metabolism is well known for its extensive mechanistic and phylogenetic diversification across a diverse taxonomic spectrum. Here we carried out whole-genome sequencing and analysis of a new betaproteobacterial isolate, Pusillimonas ginsengisoli SBSA, which is found to oxidize thiosulfate via the formation of tetrathionate as an intermediate. The 4.7 Mb SBSA genome was found to encompass a soxCDYZAXOB operon, plus single thiosulfate dehydrogenase (tsdA) and sulfite : acceptor oxidoreductase (sorAB) genes. Recombination-based knockout of tsdA revealed that the entire thiosulfate is first converted to tetrathionate by the activity of thiosulfate dehydrogenase (TsdA) and the Sox pathway is not functional in this bacterium despite the presence of all necessary sox genes. The ∆soxYZ and ∆soxXA knockout mutants exhibited a wild-type-like phenotype for thiosulfate/tetrathionate oxidation, whereas ∆soxB, ∆soxCD and soxO::KanR mutants only oxidized thiosulfate up to tetrathionate intermediate and had complete impairment in tetrathionate oxidation. The substrate-dependent O2 consumption rate of whole cells and the sulfur-oxidizing enzyme activities of cell-free extracts, measured in the presence/absence of thiol inhibitors/glutathione, indicated that glutathione plays a key role in SBSA tetrathionate oxidation. The present findings collectively indicate that the potential glutathione : tetrathionate coupling in P. ginsengisoli involves a novel enzymatic component, which is different from the dual-functional thiol dehydrotransferase (ThdT), while subsequent oxidation of the sulfur intermediates produced (e.g. glutathione : sulfodisulfane molecules) may proceed via the iterative action of soxBCD .

Draft Genome Sequences of Two Boron-Tolerant, Arsenic-Resistant, Gram-Positive Bacterial Strains, Lysinibacillus sp. OL1 and Enterococcus sp. OL5, Isolated from Boron-Fortified Cauliflower-Growing Field Soils of Northern West Bengal, India.

Two novel boron-tolerant, arsenic-resistant, Gram-positive bacterial strains, Lysinibacillus sp. OL1 and Enterococcus sp. OL5, were isolated from boron fertilizer-amended cauliflower plantation field soils in India. Here, we report the draft genome sequences of OL1 (4.87 Mb) and OL5 (3.93 Mb) to explore the intricacies of boron tolerance in bacteria.

Two pathways for thiosulfate oxidation in the alphaproteobacterial chemolithotroph Paracoccus thiocyanatus SST.

Chemolithotrophic bacteria oxidize various sulfur species for energy and electrons, thereby operationalizing biogeochemical sulfur cycles in nature. The best-studied pathway of bacterial sulfur-chemolithotrophy involves direct oxidation of thiosulfate (S2O32-) to sulfate (SO42-) without any free intermediate. This pathway mediated by SoxXAYZBCD is apparently the exclusive mechanism of thiosulfate oxidation in facultatively chemolithotrophic alphaproteobacteria. Here we explore the molecular mechanisms of sulfur oxidation in the thiosulfate- and tetrathionate(S4O62-)-oxidizing alphaproteobacterium Paracoccus thiocyanatus SST, and compare them with the prototypical Sox process of Paracoccus pantotrophus. Our results reveal a unique case where an alphaproteobacterium has Sox as its secondary pathway of thiosulfate oxidation converting ∼10% of the thiosulfate supplied, whilst ∼90% of the substrate is oxidized via a pathway that produces tetrathionate as an intermediate. Sulfur oxidation kinetics of a deletion mutant showed that thiosulfate-to-tetrathionate conversion, in SST, is catalyzed by a thiosulfate dehydrogenase (TsdA) homolog that has far-higher substrate-affinity than the Sox system of this bacterium, which in turn is also less efficient than the P. pantotrophus Sox. Deletion of soxB abolished sulfate-formation from thiosulfate/tetrathionate, while thiosulfate-to-tetrathionate conversion remained unperturbed. Physiological studies revealed the involvement of glutathione in SST tetrathionate oxidation. However, zero impact of the insertional mutation of a thiol dehydrotransferase (thdT) homolog, together with the absence of sulfite as an intermediate, indicated that SST tetrathionate oxidation is mechanistically novel, and distinct from its betaproteobacterial counterpart mediated by glutathione, ThdT, SoxBCD and sulfite:acceptor oxidoreductase. The present findings highlight extensive functional diversification of sulfur-oxidizing enzymes across phylogenetically close, as well as distant, bacteria.

Phylogenomics of an uncultivated, aerobic and thermophilic, photoheterotrophic member of Chlorobia sheds light into the evolution of the phylum Chlorobi.

All cultivated members of the phylum Chlorobi are classified under the two classes Chlorobia and Ignavibacteria. The recently-reported, uncultivated genome-species of Chlorobi have not suggested any alteration in the dichotomy of the two classes, but have hypothesized the existence of a distinct, aerobic and photoheterotrophic, order/family level lineage within Chlorobia, which otherwise was considered to be a monophyletic group of anaerobic sulfur-photolithoautotrophs. Here we report the discovery of a novel population genome bin (named Chlorobi-445) from the combined metagenomes of three spatially-contiguous but visually-distinct microbial mats growing along the 65-41 °C hydrothermal gradient of a boron-rich microbialite spring located in the Puga geothermal area of Eastern Ladakh, India. 1.3, 8.2 and 3.8% metagenomic reads from the mat communities located at 65 °C, 52 °C and 41 °C sample-sites respectively, were found to map-back to the 2,809,852 bp genome of Chlorobi-445. Phylogenomically, and therefore in terms of potential metabolic attributes, Chlorobi-445 showed close relationship with Ca. Thermochlorobacter aerophilum. Gene content suggested Chlorobi-445 to be an aerobic photoorganoheterotroph. Although this new lineage encodes all the proteins necessary for the biosynthesis of bacteriochlorophylls and the photosynthetic reaction centre, it is potentially devoid of genes concerned with lithotrophic sulfur oxidation and carbon-fixation. Individual Chlorobi phylogenies based on the sequence similarities of 16S rRNA genes, 22 ribosomal proteins, and 56 conserved marker-proteins that are encoded from single-copy genes, unanimously suggested that the class Chlorobia encompasses two major branches/clades. Whereas the Clade-I is a homogeneous cluster of culturable, anaerobic sulfur-/iron-oxidizing photolithoautotrophs, Clade-II harbors (i) Chloroherpeton species, and (ii) uncultivated aerobic photoheterotrophs such as Chlorobi-445, Chlorobium sp. GBChlB &Ca. T. aerophilum, in its two sub-clades. Distribution of bioenergetic attributes over the different branches of Chlorobi, together with the aerobic chemoorganoheterotrophic nature of the deepest-branching genome-species NICIL-2, indicated that the early Chlorobi were aerobic chemoorganoheterotrophs, while anaerobicity, phototrophy, lithotrophy, and autotrophy were all potentially added in the course of evolution.

Pradoshia eiseniae gen. nov., sp. nov., a spore-forming member of the family Bacillaceae capable of assimilating 3-nitropropionic acid, isolated from the anterior gut of the earthworm Eisenia fetida.

A Gram-stain-positive, spore-forming bacterium, EAG3T, capable of growing on 3-nitropropionic acid as the sole source of carbon, nitrogen and energy, was isolated from the anterior gut of an earthworm (Eisenia fetida) reared at the Centre of Floriculture and Agribusiness Management of the University of North Bengal at Siliguri (26.7072° N, 88.3558° E), West Bengal, India. The DNA G+C content of strain EAG3T was 42.5 mol%. Strain EAG3T contained MK-7 and MK-8 as predominant menaquinones. The predominant polar lipids were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The major cellular fatty acids were 13-methyltetradecanoic acid, (9Z)-9-hexadecen-1-ol, 12-methyltetradecanoic acid and 14-methylpentadecanoic acid. The draft genome of strain EAG3T, distributed in 57 contigs, was found to be 3.8 Mb. A total of 3811 potential coding sequences or genes were predicted, including 3672 protein-coding and 108 RNA-coding ones together with 31 pseudogenes. One hundred and thirty-five genes encoded hypothetical proteins with no meaningful homologies with known proteins. The EAG3T genome encompassed two nitronate monooxygenase and one methylmalonate-semialdehyde dehydrogenase (CoA acylating) homologues. 16S rRNA gene sequence-based phylogeny revealed that the closest relative of strain EAG3T was Bacillus methanolicus NCIMB 13113T (95.7 % similarity). Phylogenetic, physiological and biochemical characteristics differentiated strain EAG3T from B. methanolicus, as well as from the other close taxonomic relatives Planococcus rifietoensis M8T, Bhargavaea cecembensis DSE10T, Planomicrobium flavidum ISL-41Tand Fermentibacilluspolygoni IEB3T, with which strain EAG3T had 93.3-94.2 % 16S rRNA gene sequence similarities. The new isolate, therefore, was considered as representing a novel genus of family Bacillaceae, for which the name Pradoshia eiseniae gen. nov., sp. nov. is proposed, with EAG3T (=LMG 30312T=JCM 32460T) as the type strain.

Enhanced carbon-sulfur cycling in the sediments of Arabian Sea oxygen minimum zone center.

Biogeochemistry of oxygen minimum zone (OMZ) sediments, which are characterized by high input of labile organic matter, have crucial bearings on the benthic biota, gas and metal fluxes across the sediment-water interface, and carbon-sulfur cycling. Here we couple pore-fluid chemistry and comprehensive microbial diversity data to reveal the sedimentary carbon-sulfur cycle across a water-depth transect covering the entire thickness of eastern Arabian Sea OMZ, off the west coast of India. Geochemical data show remarkable increase in average total organic carbon content and aerial sulfate reduction rate (JSO42-) in the sediments of the OMZ center coupled with shallowing of sulfate methane transition zone and hydrogen sulfide and ammonium build-up. Total bacterial diversity, including those of complex organic matter degraders, fermentative and exoelectrogenic bacteria, and sulfate-reducers (that utilize only simple carbon compounds) were also found to be highest in the same region. The above findings indicate that higher organic carbon sequestration from the water-columns (apparently due to lower benthic consumption, biodegradation and biotransformation) and greater bioavailability of simple organic carbon compounds (apparently produced by fermetative microflora of the sediments) are instrumental in intensifying the carbon-sulfur cycle in the sediments of the OMZ center.

NaCl-saturated brines are thermodynamically moderate, rather than extreme, microbial habitats.

NaCl-saturated brines such as saltern crystalliser ponds, inland salt lakes, deep-sea brines and liquids-of-deliquescence on halite are commonly regarded as a paradigm for the limit of life on Earth. There are, however, other habitats that are thermodynamically more extreme. Typically, NaCl-saturated environments contain all domains of life and perform complete biogeochemical cycling. Despite their reduced water activity, ∼0.755 at 5 M NaCl, some halophiles belonging to the Archaea and Bacteria exhibit optimum growth/metabolism in these brines. Furthermore, the recognised water-activity limit for microbial function, ∼0.585 for some strains of fungi, lies far below 0.755. Other biophysical constraints on the microbial biosphere (temperatures of >121°C; pH > 12; and high chaotropicity; e.g. ethanol at >18.9% w/v (24% v/v) and MgCl2 at >3.03 M) can prevent any cellular metabolism or ecosystem function. By contrast, NaCl-saturated environments contain biomass-dense, metabolically diverse, highly active and complex microbial ecosystems; and this underscores their moderate character. Here, we survey the evidence that NaCl-saturated brines are biologically permissive, fertile habitats that are thermodynamically mid-range rather than extreme. Indeed, were NaCl sufficiently soluble, some halophiles might grow at concentrations of up to 8 M. It may be that the finite solubility of NaCl has stabilised the genetic composition of halophile populations and limited the action of natural selection in driving halophile evolution towards greater xerophilicity. Further implications are considered for the origin(s) of life and other aspects of astrobiology.

Homologs from sulfur oxidation (Sox) and methanol dehydrogenation (Xox) enzyme systems collaborate to give rise to a novel pathway of chemolithotrophic tetrathionate oxidation.

The SoxXAYZB(CD)2 -mediated pathway of bacterial sulfur-chemolithotrophy explains the oxidation of thiosulfate, sulfide, sulfur and sulfite but not tetrathionate. Advenella kashmirensis, which oxidizes tetrathionate to sulfate, besides forming it as an intermediate during thiosulfate oxidation, possesses a soxCDYZAXOB operon. Knock-out mutations proved that only SoxBCD is involved in A. kashmirensis tetrathionate oxidation, whereas thiosulfate-to-tetrathionate conversion is Sox independent. Expression of two glutathione metabolism-related proteins increased under chemolithotrophic conditions, as compared to the chemoorganotrophic one. Substrate-dependent oxygen consumption pattern of whole cells, and sulfur-oxidizing enzyme activities of cell-free extracts, measured in the presence/absence of thiol inhibitors/glutathione, corroborated glutathione involvement in tetrathionate oxidation. Furthermore, proteome analyses detected a sulfite:acceptor oxidoreductase (SorAB) exclusively under chemolithotrophic conditions, while expression of a methanol dehydrogenase (XoxF) homolog, subsequently named thiol dehydrotransferase (ThdT), was found to increase 3- and 10-fold during thiosulfate-to-tetrathionate conversion and tetrathionate oxidation respectively. A thdT knock-out mutant did not oxidize tetrathionate but converted half of the supplied 40 mM S-thiosulfate to tetrathionate. Knock-out of another thiosulfate dehydrogenase (tsdA) gene proved that both ThdT and TsdA individually converted ∼ 20 mM S-thiosulfate to tetrathionate. The overexpressed and isolated ThdT protein exhibited PQQ-dependent thiosulfate dehydrogenation, whereas its PQQ-independent thiol transfer activity involving tetrathionate and glutathione potentially produced a glutathione:sulfodisulfane adduct and sulfite. SoxBCD and SorAB were hypothesized to oxidize the aforesaid adduct and sulfite respectively.

A novel soxO gene, encoding a glutathione disulfide reductase, is essential for tetrathionate oxidation in Advenella kashmirensis.

Molecular mechanisms of chemolithotrophic tetrathionate oxidation are not clearly understood. Here we used transposon(Tn5-mob)-insertion mutagenesis to search for novel tetrathionate oxidation genes in the facultatively chemolithoautotrophic betaproteobacterium Advenella kashmirensis that not only oxidizes tetrathionate, but also produces the same as an intermediate during thiosulfate oxidation. Genome-wide random insertion of Tn5-mob occurred at a frequency of one per 104 donor E. coli cells. A library of 8000 transconjugants yielded five tetrathionate-oxidation-impaired mutants, of which, the one named Ak_Tn_16 was studied here in detail. When grown chemolithoautotrophically on thiosulfate, Ak_Tn_16 converted the total thiosulfate supplied to equivalent amount of tetrathionate, exactly in the same way as the wild type. It could not, however, oxidize the intermediary tetrathionate to sulfate; Ak_Tn_16 could not also oxidize tetrathionate when it was supplied as the starting chemolithotrophic substrate. In the Ak_Tn_16 genome, Tn5-mob was found to have transposed in a novel soxO gene, located just-upstream of soxB, within the sox gene cluster. SoxO was predicted, via iterative threading assembly simulation, to be a glutathione-disulfide (GSSG) reductase. When Ak_Tn_16 was grown in tetrathionate-based chemolithoautotrophic medium supplemented with reduced glutathione (GSH) its tetrathionate-oxidation deficiency, remarkably, was ameliorated. Implications for a key role of GSH in tetrathionate oxidation are discussed in the light of other molecular evidences available for A. kashmirensis.

Genome Sequence of the Multiple-Protease-Producing Strain Geobacillus thermoleovorans N7, a Thermophilic Bacterium Isolated from Paniphala Hot Spring, West Bengal, India.

Here, we present the draft genome sequence of Geobacillus thermoleovorans strain N7 (MCC 3175), isolated from Paniphala Hot Spring, West Bengal, India, which contains genes that encode several industrially and medically important thermostable enzymes like neutral protease, xylose isomerase, rhamnogalacturonan acetylesterase, nitrate and nitrite reductase, l-asparaginase, glutaminase, and RNase P.