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.     

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

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.The genus Tetrathiobacter includes environmentally important betaproteobacteria belonging to the family Alcaligenaceae. Members of this family inhabit diverse habitats, ranging from animals and humans to soil, sewage, and sludge. They are also metabolically diverse and include facultative chemolithotrophs, versatile heterotrophs, xenobiotic degraders, fastidious parasites, and pathogens (15). While the type species, Tetrathiobacter kashmirensis, isolated from a temperate orchard soil, has been recognized as a thiosulfate- and tetrathionate-oxidizing facultative chemolithoautotroph (11, 15), Tetrathiobacter mimigardefordensis, isolated from compost, can utilize the organic disulfide 3,3′-dithiodipropionic acid for growth (42). More recently isolated soil-dwelling strains of T. kashmirensis can detoxify selenite by reducing it to insoluble elemental red selenium (18). Strains identified as T. kashmirensis on the basis of 16S rRNA gene sequence similarity (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"EU523111","term_id":"170674436","term_text":"EU523111"}}EU523111) are allegedly involved in the biodegradation of thiodiglycol, the hydrolysis product of yperite, a highly hazardous derivative of mustard gas used in chemical weapons. In addition, bacteria isolated from a deep-sea environment and phylogenetically identified as T. kashmirensis (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"EF619402","term_id":"148751435","term_text":"EF619402"}}EF619402) have been observed to degrade alkanes.Species of Alcaligenaceae possess a wide repertoire of plasmids (21, 32), a feature pertinent to their biodegradative and biogeochemical roles in the environment. Many of these plasmids are well known for harboring genes involved in biodegradation (14, 39, 44). However, not many of them have been studied at the molecular level. In the present study, we have identified, partially sequenced, and characterized a large (∼60-kb), low-copy-number, self-transmissible, novel plasmid, designated pBTK445, from T. kashmirensis strain WGT. We have characterized the minimal replication region of this plasmid and have subsequently constructed shuttle vectors that could be used for diverse members of the Alcaligenaceae, including Tetrathiobacter. A major part of the sequenced region was found to be occupied by genes homologous to constituents of various type 4 secretion systems (T4SSs) (5-7, 9). This locus was found to be involved in the conjugal transfer function of the new plasmid. Many features of pBTK445 resemble those of IncP plasmids, but the new plasmid also possesses several characteristics distinct from those of IncP plasmids. We discuss in detail those characteristics of pBTK445 that make it an interesting model for the study of the diversity and evolution of large plasmids.     

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.     

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.     

Chemolithoautotrophic oxidation of thiosulfate, tetrathionate and thiocyanate by a novel rhizobacterium belonging to the genus Paracoccus

Two tropical leguminous-rhizospheric strains, SST and JT 001, phylogenetically closest to Paracoccus thiocyanatus and Paracoccus pantotrophus, respectively, were isolated on reduced sulfur compounds as sole energy and electron sources. While SST had versatile chemolithotrophic abilities to oxidize thiosulfate, tetrathionate, thiocyanate, sulfide and elemental sulfur, JT 001 could oxidize thiosulfate, soluble sulfide, elemental sulfur and a relatively lesser amount of tetrathionate. Positive hybridization signals were detected for JT 001 but not SST, when their genomic DNAs were probed with DIG-labeled sulfur oxidation genes amplified from the chemolithotrophic alphaproteobacterium Pseudaminobacter salicylatoxidans KCT001. Though the new isolate SST exhibited high 16S rRNA gene sequence similarity with the monotypic species P. thiocyanatus, it was found to be considerably distinct from the latter in terms of phenotypic and chemotaxonomic characteristics. Polyphasic systematic analysis, however, confirmed that JT 001 was a strain of P. pantotrophus.     

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.     

Molecular and Cellular Fossils of a Mat-Like Microbial Community in Geothermal Boratic Sinters

Silica and travertine deposits, the two most common surface manifestations of terrestrial hot springs, have so far been the only markers universally helpful in locating and interpreting geothermal systems and past lifeforms potentially associated with them. In the current study, we for the first time report microbial fossils from a third type of geothermal sinter, viz. the boron mineral deposits, which are characteristic of the relatively uncommon silicate-poor hot springs. Organic biomarker analyses of the boratic sinters framing the hot springs of Puga valley, Ladakh, identified molecular fossils (viz. respiratory and photosynthetic isoprenoid quinones and photosynthetic pigments) of a mat-like microbial community putatively comprised of algae, fungi, cyanobacteria and other photosynthetic bacteria, some of which may be proteobacteria. So far as microfossil preservation is concerned, mineralized mat-like biofabrics with diverse cellular morphotypes could be identified in scanning electron microscopy of sub-recent sediments as well as hard and consolidated old sinters. Though this putative past community is quite unusual in volcanic niches, its ubiquitous and enduring presence in the Puga geothermal area is highlighted by the discovery of its biosignatures along all of the three dimensions of the explored boratic deposits. These findings usher a new paradigm of looking at past or present geothermal life in as well as outside the Earth.     

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.     

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. Pradosh Roy Deceased     

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.