Wriddhiman Ghosh

Biochemistry and molecular biology of lithotrophic sulfur oxidation by taxonomically and ecologically diverse bacteria and archaea.

Lithotrophic sulfur oxidation is an ancient metabolic process. Ecologically and taxonomically diverged prokaryotes have differential abilities to utilize different reduced sulfur compounds as lithotrophic substrates. Different phototrophic or chemotrophic species use different enzymes, pathways and mechanisms of electron transport and energy conservation for the oxidation of any given substrate. While the mechanisms of sulfur oxidation in obligately chemolithotrophic bacteria, predominantly belonging to Beta- (e.g. Thiobacillus) and Gammaproteobacteria (e.g. Thiomicrospira), are not well established, the Sox system is the central pathway in the facultative bacteria from Alphaproteobacteria (e.g. Paracoccus). Interestingly, photolithotrophs such as Rhodovulum belonging to Alphaproteobacteria also use the Sox system, whereas those from Chromatiaceae and Chlorobi use a truncated Sox complex alongside reverse-acting sulfate-reducing systems. Certain chemotrophic magnetotactic Alphaproteobacteria allegedly utilize such a combined mechanism. Sulfur-chemolithotrophic metabolism in Archaea, largely restricted to Sulfolobales, is distinct from those in Bacteria. Phylogenetic and biomolecular fossil data suggest that the ubiquity of sox genes could be due to horizontal transfer, and coupled sulfate reduction/sulfide oxidation pathways, originating in planktonic ancestors of Chromatiaceae or Chlorobi, could be ancestral to all sulfur-lithotrophic processes. However, the possibility that chemolithotrophy, originating in deep sea, is the actual ancestral form of sulfur oxidation cannot be ruled out.

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.

A Novel Gene Cluster soxSRT Is Essential for the Chemolithotrophic Oxidation of Thiosulfate and Tetrathionate by Pseudaminobacter salicylatoxidans KCT001

Chemolithotrophic sulfur oxidation (Sox) in the α-proteobacterium Pseudaminobacter salicylatoxidans KCT001 was found to be governed by the gene cluster soxSRT–soxVWXYZABCD. Independent transposon-insertion mutations in the genes soxB, soxC, soxD, and also in a novel open reading frame (ORF), designated as soxT, afforded revelation of the entire sox locus of this bacterium. The deduced amino acid sequence of the novel ORF soxT comprised 362 residues and exhibited significant homology with hypothetical proteins of diverse origin, including a permease-like transport protein of Escherichia coli. Two contiguous ORFs, soxR and soxS, immediately preceded the soxT gene. The gene cluster soxSRT was located upstream of soxVWXYZABCD and was transcribed divergently with respect to the latter. Chemolithotrophic utilization of both thiosulfate and tetrathionate was observed to have been impaired in all of these Sox− mutants, implicating the involvement of the gene cluster soxSRT–soxVWXYZABCD in the oxidation of both thiosulfate and tetrathionate.

Origin of the Sox multienzyme complex system in ancient thermophilic bacteria and coevolution of its constituent proteins.

The multienzyme complex SoxXABYZ(CD)(2), characteristic of facultatively chemolithotrophic Alphaproteobacteria, oxidizes both sulfone and sulfane sulfur species directly to sulfate, while a truncated SoxXABYZ oxidizes only sulfone sulfur in species of Chromatiaceae and Chlorobi. Here we phylogenetically analyzed SoxXA, SoxYZ and SoxCD sequences, correlated the results with earlier SoxB-based data, and postulated that the system originated in putatively common ancestors of Aquificae and Epsilonproteobacteria, and evolved through extensive horizontal gene transfer, accompanied by gain and/or loss of constituents by different lineages. However, in several Sox systems, particularly those from Alphaproteobacteria (and also Chromatiaceae and Chlorobi), there has been no extra gain or loss of constituents and all their proteins have similar evolutionary paths. This implies that the components of these systems have coevolved parallel to each other without any shuffling with other divergent systems. This, however, holds good only for those Sox systems, which render sulfur oxidation functions equivalent to the typical alphaproteobacterial process. We postulate that coevolution of all the proteins is essential for the typical modular function of Sox. Conversely, mosaic Sox systems (where constituents have disparate phylogenetic paths) are either nonfunctional or with activities deviated from typical systems. Monomeric Sox subunits of the mosaic systems, however, possess almost all the motifs and conserved domains critical for their designated activity and heterodimer formation. So what could be the basis of the functional discrepancies of the mosaic Sox systems? It appears that their discretely evolved heterodimers cannot interact among themselves in the same way as ideally envisaged in the modular Sox system, which in turn, may in some cases lead to novel adventitious reactions.

Conjugative Type 4 Secretion System of a Novel Large Plasmid from the Chemoautotroph Tetrathiobacter kashmirensis and Construction of Shuttle Vectors for Alcaligenaceae

ABSTRACT Tetrathiobacter spp. and other members of the Alcaligenaceae are metabolically versatile and environmentally significant. A novel, ∼60-kb conjugative plasmid, pBTK445, from the sulfur chemolithoautotroph Tetrathiobacter kashmirensis, was identified and characterized. This plasmid exists at a low copy number of 2 to 3 per host chromosome. The portion of pBTK445 sequenced so far (∼25 kb) harbors genes putatively involved in replication, transfer functions, partition, and UV damage repair. A 1,373-bp region was identified as the minimal replicon. This region contains a repA gene encoding a protein belonging to the RPA (replication protein A) superfamily and an upstream, iteron-based oriV. A contiguous 11-gene cluster homologous to various type 4 secretion systems (T4SSs) was identified. Insertional inactivation demonstrated that this cluster is involved in the conjugative transfer functions of pBTK445, and thus, it was named the tagB (transfer-associated gene homologous to virB) locus. The core and peripheral TagB components show different phylogenetic affinities, suggesting that this system has evolved by assembling components from evolutionarily divergent T4SSs. A virD4 homolog, putatively involved in nucleoprotein transfer, is also present downstream of the tagB locus. Although pBTK445 resembles IncP plasmids in terms of its genomic organization and the presence of an IncP-specific trbM homolog, it also shows several unique features. Unlike that of IncP, the oriT of pBTK445 is located in close proximity to the oriV, and a traL homolog, which is generally present in the TraI locus of IncP, is present in pBTK445 in isolation, upstream of the tagB locus. A significant outcome of this study is the construction of conjugative shuttle vectors for Tetrathiobacter and related members of the Alkaligenaceae.

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 34S 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 34S 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 34S 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.

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 WT001T 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 WT001T, 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 WT001T 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 WT001T 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 WT001T. So, while WT001T relatives/derivatives in nature could be going the Taylorella way, the lineage as such was well-prepared for imminent host-confinement.

Optimization of a phenol extraction‐based protein preparation method amenable to downstream 2DE and MALDI‐MS based analysis of bacterial proteomes

2DE is one of the most efficient and widely used methods for resolving complex protein mixtures. For efficient analysis of complex samples, high‐resolution separation of proteins on 2D gel is essential, and for that purpose good sample preparation is crucial. In this study, we have improvized a method for preparing bacterial total cellular proteome, from a strategy applied earlier to recalcitrant plant tissues, which gave high‐quality resolution on 2DE. The method involving phenol extraction followed by methanol/ammonium acetate precipitation was first optimized for the chemolithotrophic proteobacteria Tetrathiobacter kashmirensis WT001 and Pseudaminobacter salicylatoxidans KCT001 that did not yield quality protein preps in conventional trichloroacetic acid/acetone precipitation method. Subsequently, to validate its general applicability, the method was evaluated against the trichloroacetic acid/acetone precipitation method for two other model bacteria, i.e. Escherichia coli DH5α and Mycobacterium smegmatis mc26. Identification of at least four proteins each from the outer membrane, periplasm, and cytoplasm of T. kashmirensis by MALDI‐MS not only proved the efficiency of the method in extracting proteins from the different cellular compartments but also the amenability of the obtained protein spots toward MALDI‐MS based identification.

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.