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 .

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.

Kinetic enrichment of 34S during proteobacterial thiosulfate oxidation and the conserved role of SoxB in S-S bond breaking.

During 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.

Whole-genome shotgun sequence of the sulfur-oxidizing chemoautotroph Pseudaminobacter salicylatoxidans KCT001.

The facultatively sulfur-oxidizing chemolithoautotrophic alphaproteobacterium Pseudaminobacter salicylatoxidans KCT001 (MTCC 7265) belongs to the family Phyllobacteriaceae of the order Rhizobiales. Analysis of its genome offers valuable insight into the adaptive specializations and evolution of free-living soil bacteria that are phylogenetically closely related to symbiotic and invasive rhizobacteria.

Whole-genome shotgun sequencing of the sulfur-oxidizing chemoautotroph Tetrathiobacter kashmirensis.

The chemolithoautotrophic betaproteobacterium Tetrathiobacter kashmirensis belongs to the family Alcaligenaceae and is phylogenetically closely related to pathogens such as Taylorella and Bordetella species. While a complete inorganic sulfur oxidation gene cluster, soxCDYZAXWB, is present in its genome, pathogenicity islands or genes associated with virulence, disease, cellular invasion, and/or intracellular resistance are completely absent.

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.

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.

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.

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.

Resilience and receptivity worked in tandem to sustain a geothermal mat community amidst erratic environmental conditions.

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.

Genome implosion elicits host-confinement in Alcaligenaceae: evidence from the comparative genomics of Tetrathiobacter kashmirensis, a pathogen in the making.

This study elucidates the genomic basis of the evolution of pathogens alongside free-living organisms within the family Alcaligenaceae of Betaproteobacteria. Towards that end, the complete genome sequence of the sulfur-chemolithoautotroph Tetrathiobacter kashmirensis WT001(T) was determined and compared with the soil isolate Achromobacter xylosoxidans A8 and the two pathogens Bordetella bronchiseptica RB50 and Taylorella equigenitalis MCE9. All analyses comprehensively indicated that the RB50 and MCE9 genomes were almost the subsets of A8 and WT001(T), respectively. In the immediate evolutionary past Achromobacter and Bordetella shared a common ancestor, which was distinct from the other contemporary stock that gave rise to Tetrathiobacter and Taylorella. The Achromobacter-Bordetella precursor, after diverging from the family ancestor, evolved through extensive genome inflation, subsequent to which the two genera separated via differential gene losses and acquisitions. Tetrathiobacter, meanwhile, retained the core characteristics of the family ancestor, and Taylorella underwent massive genome degeneration to reach an evolutionary dead-end. Interestingly, the WT001(T) genome, despite its conserved architecture, had only 85% coding density, besides which 578 out of its 4452 protein-coding sequences were found to be pseudogenized. Translational impairment of several DNA repair-recombination genes in the first place seemed to have ushered the rampant and indiscriminate frame-shift mutations across the WT001(T) genome. Presumably, this strain has just come out of a recent evolutionary bottleneck, representing a unique transition state where genome self-degeneration has started comprehensively but selective host-confinement has not yet set in. In the light of this evolutionary link, host-adaptation of Taylorella clearly appears to be the aftereffect of genome implosion in another member of the same bottleneck. Remarkably again, potent virulence factors were found widespread in Alcaligenaceae, corroborating which hemolytic and mammalian cell-adhering abilities were discovered in WT001(T). So, while WT001(T) relatives/derivatives in nature could be going the Taylorella way, the lineage as such was well-prepared for imminent host-confinement.