Sabyasachi Bhattacharya

Sediments underlying marine hypoxic zones are huge sinks of unreacted complex organic matter, where despite acute O ₂ -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, O ₂ . 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, O ₂ 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 O ₂ from previously-reported sources such as Nitrosopumilus species.

Kinetics of thiosulfate oxidation, product and intermediate formation, and ³⁴S fractionation, were studied for the members of Alphaproteobacteria Paracoccus sp. SMMA5 and Mesorhizobium thiogangeticum SJT^T, 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; Δ³⁴S_{thiosulfate-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; Δ³⁴S_{thiosulfate-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.

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.

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 calledShivlinga. 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 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.

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::Kan^R 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 .

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.

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 (J_SO4²⁻) 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.

Exploration of the aquatic microbiota of several circum-neutral (6.0-8.5 pH) mid-temperature (55-85 OC) springs revealed rich diversities of phylogenetic relatives of mesophilic bacteria, which surpassed the diversity of the truly-thermophilic taxa. To gain insight into the potentially-thermophilic adaptations of the phylogenetic relatives of Gram-negative mesophilic bacteria detected in culture-independent investigations we attempted pure-culture isolation by supplementing the enrichment media with 50 µg ml-1 vancomycin. Surprisingly, this Gram-positive-specific antibiotic eliminated the entire culturable-diversity of chemoorganotrophic and sulfur-chemolithotrophic bacteria present in the tested hot water inocula. Moreover, it also killed all the Gram-negative hot-spring isolates that were obtained in vancomycin-free media. Concurrent literature search for the description of Gram-negative thermophilic bacteria revealed that at least 16 of them were reportedly vancomycin-susceptible. While these data suggested that vancomycin-susceptibility could be a global trait of thermophilic bacteria (irrespective of their taxonomy, biogeography and Gram-character), MALDI Mass Spectroscopy of the peptidoglycans of a few Gram-negative thermophilic bacteria revealed that tandem alanines were present in the fourth and fifth positions of their muropeptide precursors (MPPs). Subsequent phylogenetic analyses revealed a close affinity between the D-alanine-D-alanine ligases (Ddl) of taxonomically-diverse Gram-negative thermophiles and the thermostable Ddl protein of Thermotoga maritima, which is well-known for its high specificity for alanine over other amino acids. The Ddl tree further illustrated a divergence between the homologs of Gram-negative thermophiles and mesophiles, which broadly coincided with vancomycin-susceptibility and vancomycin-resistance respectively. It was thus hypothesized that thermophilic Ddls have been evolutionarily selected to favor a D-ala-D-ala bonding. However, preference for D-ala-D-ala-terminated MPPs does not guarantee vancomycin susceptibility of thermophilic bacteria as the large and relatively-hydrophilic vancomycin molecule has to cross the outer membrane before it can inhibit peptidoglycan biosynthesis. Literature shows that many mesophilic Gram-negative bacteria also have D-ala-D-ala-terminated MPPs, but they still remain resistant to vancomycin due to the relative impermeability of their membranes. But the global vancomycin-susceptibility phenotype of thermophilic bacteria itself testifies that the drug crosses the membrane in all these cases. As a corollary, it seems quite likely that the outer membranes of thermophilic bacteria have some yet-unknown characteristic feature(s) that invariably ensures the entry of vancomycin.

The explosion of research in the cancer immunotherapy field has led to a quest for better animal models to validate and develop various immunotherapies. Syngeneic mouse tumor models are one of the best options currently available to select cancers where clinical efficacy may be expected, to test mechanism of action hypotheses, in providing clues for possible biomarkers of immune pharmacodynamic (PD) activity and most successfully for predicting best partners for synergistic combinations. In order to select an appropriate model to evaluate a specific immunotherapy, it is essential to ensure sufficient understanding of the tumor model especially the immunogenicity and the characteristics of the immune infiltrates in the tumor microenvironment. It is also important to understand the dynamics of tumor growth and its effect on the immune landscape in the various peripheral tissues compartments during cancer progression.