The SoxXAYZB(CD) -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.

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 progressivelydrying 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. The microbial ecologies of habitats that are hydrothermal, or hypersaline, have been well-characterized, and can give insights into the origins of early life on Earth 1-3. Both chaotrope-rich hypersaline brines and high-temperature freshwater systems can entropically disorder the macromolecules of cellular systems, and are in this way analogous as microbial habitats 4-7. Indeed, highly-chaotropic and hydrothermal habitats are comparable at various scales of biology: the biomacromolecule, cellular system, and functional ecosystem 8,9. Chaotropic, hypersaline habitats include the MgCl 2-constrained ecosystems located at the interfaces of some of the stratified deep-sea hypersaline brines and their overlying seawater. Biophysical, culture-based, and metagenomic studies of the steep haloclines found at these interfaces have revealed that macromolecule-disordering (chaotropic) activities of MgCl 2 not only determine microbial community composition, but also limit Earth's functional biosphere 5,7,10 in such locations, as in situ microbial communities stop functioning at 2.2-2.4 M MgCl 2

bDuring chemolithoautotrophic thiosulfate oxidation, the phylogenetically diverged proteobacteria Paracoccus pantotrophus, Tetrathiobacter kashmirensis, and Thiomicrospira crunogena rendered steady enrichment of 34 S in the end product sulfate, with overall fractionation ranging between ؊4.6‰ and ؉5.8‰. The fractionation kinetics of T. crunogena was essentially similar to that of P. pantotrophus, albeit the former had a slightly higher magnitude and rate of 34 S enrichment. In the case of T. kashmirensis, the only significant departure of its fractionation curve from that of P. pantotrophus was observed during the first 36 h of thiosulfate-dependent growth, in the course of which tetrathionate intermediate formation is completed and sulfate production starts. The almost-identical 34 S enrichment rates observed during the peak sulfate-producing stage of all three processes indicated the potential involvement of identical S-S bond-breaking enzymes. Concurrent proteomic analyses detected the hydrolase SoxB (which is known to cleave terminal sulfone groups from SoxYZ-bound cysteine S-thiosulfonates, as well as cysteine S-sulfonates, in P. pantotrophus) in the actively sulfate-producing cells of all three species. The inducible expression of soxB during tetrathionate oxidation, as well as the second leg of thiosulfate oxidation, by T. kashmirensis is significant because the current Sox pathway does not accommodate tetrathionate as one of its substrates. Notably, however, no other Sox protein except SoxB could be detected upon matrix-assisted laser desorption ionization mass spectrometry analysis of all such T. kashmirensis proteins as appeared to be thiosulfate inducible in 2-dimensional gel electrophoresis. Instead, several other redox proteins were found to be at least 2-fold overexpressed during thiosulfate-or tetrathionate-dependent growth, thereby indicating that there is more to tetrathionate oxidation than SoxB alone.

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 10 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.

To elucidate how geothermal irregularities affect the sustainability of high-temperature microbiomes we studied the synecological dynamics of a geothermal microbial mat community (GMMC) vis-à-vis fluctuations in its environment. Spatiotemporally-discrete editions of a photosynthetic GMMC colonizing the travertine mound of a circum-neutral hot spring cluster served as the model-system. In 2010 a strong geyser atop the mound discharged mineral-rich hot water, which nourished a GMMC continuum from the proximal channels (PC) upto the slope environment (SE) along the mound’s western face. In 2011 that geyser extinguished and consequently the erstwhile mats disappeared. Nevertheless, two relatively-weaker vents erupted in the southern slope and their mineral-poor outflow supported a small GMMC patch in the SE. Comparative metagenomics showed that this mat was a relic of the 2010 community, conserved via population dispersal from erstwhile PC as well as SE niches. Subsequently in 2012, as hydrothermal activity augmented in the southern slope, ecological niches widened and the physiologically-heterogeneous components of the 2011 “seed-community” split into PC and SE meta-communities, thereby reclaiming either end of the thermal gradient. Resilience of incumbent populations, and the community’s receptiveness towards immigrants, were the key qualities that ensured the GMMC’s sustenance amidst habitat degradation and dispersal to discrete environments.