The Sulfur Oxidation Operon Repressor Function is Influenced by the Product of its Adjacent Upstream ORF in <Emphasis Type="Italic">Pseudaminobacter salicylatoxidans</Emphasis> KCT001

The repressor of sulfur-oxidizing (sox) operon regulates expression of genes encoding a multienzyme complex that governs the chemolithotrophic sulfur oxidation in Pseudaminobacter salycylatoxidans KCT001. The inducer of sox operon viz., thiosulfate and other sulfur anions had no impact on in vitro repressor–operator interaction which indicates an atypical derepression mechanism. The reduced repressor has higher affinity for its operator DNA. The sulfur oxidation repressor binds with operator regions and led to efficient repression in trans, however, increased repressor concentration resulted in higher gene expression. Using a reporter system in E. coli, the present study established that the thioredoxin-like protein, encoded in immediate upstream ORF, could nullify the observed reversal of the repression at higher repressor concentration. In this context, the involvement of the upstream gene product in the regulation of the sulfur oxidation gene expression has been reported.     

Organic Carbon Utilization by Resting Cells of Thiosulfate-Oxidizing Marine Heterotrophs

Two thiosulfate-oxidizing marine heterotrophs, strains 12W and 16B, were tested for utilization of [¹⁴C]glucose and [¹⁴C]acetate, respectively, in the presence or absence of thiosulfate. Thiosulfate oxidation caused an increase in organic carbon incorporation and a corresponding decrease in respiration at pH 6.5, near the optimum pH for thiosulfate oxidation and thiosulfate-stimulated growth in these bacteria. The amount of glucose or acetate metabolized remained virtually unaffected by thiosulfate oxidation. The metabolic shift in carbon utilization was diminished by increasing the initial pH to 8.0. The results indicate that marine heterotrophs 12W and 16B exhibit a type of mixotrophic metabolism which differs from that observed in the thiobacilli.     

Some Properties of Thiosulfate-Oxidizing Enzyme from Marine Heterotroph 16B

Thiosulfate-oxidizing enzyme has been demonstrated in cell-free extracts of the marine, thiosulfate-oxidizing pseudomonad strain 16B. The enzyme, partially purified by ion-exchange chromatography and calcium phosphate gel treatment, catalyzed the oxidation of thiosulfate to tetrathionate with the concomitant reduction of ferricyanide. Native but not mammalian cytochrome c was also reduced by the enzyme in the presence of thiosulfate. The enzyme was located exclusively in the supernatant of ultracentrifuged cell extracts. The most purified enzyme preparation, like intact cells, exhibited a temperature optimum of 30 to 31°C. However, it exhibited no definite pH optimum. At pH 6.1 to 6.3 and 30°C, the K_m for thiosulfate was 1.57 mM. At lower temperatures, the apparent K_m for thiosulfate increased, but the apparent maximum velocity remained virtually unchanged. Thiosulfate oxidation in intact cells exhibited an increase in the pH optimum at lower temperatures. The thiosulfate-oxidizing enzyme of marine heterotroph 16B is compared with thiosulfate-oxidizing enzymes from other bacteria, and the effect of temperature on the relationship between pH and thiosulfate oxidation is discussed with reference to the natural habitat of the bacterium.     

Enzymic comparisons of the inorganic sulfur metabolism in autotrophic and heterotrophic Thiobacillus ferrooxidans.

Activities of enzymes which mediate the oxidation of thiosulfate to sulfate and the assimilation of sulfate to sulfide were assayed in various cell-free fractions of Thiobacillus ferrooxidans grown autotrophically on either ferrous iron or thiosulfate or heterotrophically on glucose. There was no activity of the thiosulfate-oxidizing enzyme in extracts of bacteria grown with ferrous iron. Comparable activities for ATP-sulfurylase (EC 2.7.7.4), ADP-sulfurylase (EC 2.7.7.5), and adenylate kinase (EC 2.7.4.3) were found in the bacteria grown autotrophically with either Fe2+ or S2O32- or heterotrophically with glucose.     

The Reg1-interacting proteins, Bmh1, Bmh2, Ssb1, and Ssb2, have roles in maintaining glucose repression in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, a type 1 protein phosphatase complex composed of the Glc7 catalytic subunit and the Reg1 regulatory subunit represses expression of many glucose-regulated genes. Here we show that the Reg1-interacting proteins Bmh1, Bmh2, Ssb1, and Ssb2 have roles in glucose repression. Deleting both BMH genes causes partially constitutive ADH2 expression without significantly increasing the level of Adr1 protein, the major activator of ADH2 expression. Adr1 and Bcy1, the regulatory subunit of cAMP-dependent protein kinase, are both required for this effect indicating that constitutive expression in Deltabmh1Deltabmh2 cells uses the same activation pathway that operates in Deltareg1 cells. Deletion of both BMH genes and REG1 causes a synergistic relief from repression, suggesting that Bmh proteins also act independently of Reg1 during glucose repression. A two-hybrid interaction with the Bmh proteins was mapped to amino acids 187-232, a region of Reg1 that is conserved in different classes of fungi. Deleting this region partially releases SUC2 from glucose repression. This indicates a role for the Reg1-Bmh interaction in glucose repression and also suggests a broad role for Bmh proteins in this process. An in vivo Reg1-Bmh interaction was confirmed by copurification of Bmh proteins with HA(3)-TAP-tagged Reg1. The nonconventional heat shock proteins Ssb1 and Ssb2 are also copurified with HA(3)-TAP-tagged Reg1. Deletion of both SSB genes modestly decreases repression of ADH2 expression in the presence of glucose, suggesting that Ssb proteins, perhaps through their interaction with Reg1, play a minor role in glucose repression.     

Glucose represses the lactose-galactose regulon in Kluyveromyces lactis through a SNF1 and MIG1- dependent pathway that modulates galactokinase (GAL1) gene expression.

Expression of the lactose-galactose regulon in Kluyveromyces lactis is induced by lactose or galactose and repressed by glucose. Some components of the induction and glucose repression pathways have been identified but many remain unknown. We examined the role of the SNF1 (KlSNF1) and MIG1 (KlMIG1) genes in the induction and repression pathways. Our data show that full induction of the regulon requires SNF1; partial induction occurs in a Klsnf1 -deleted strain, indicating that a KlSNF1 -independent pathway(s) also regulates induction. MIG1 is required for full glucose repression of the regulon, but there must be a KlMIG1 -independent repression pathway also. The KlMig1 protein appears to act downstream of the KlSnf1 protein in the glucose repression pathway. Most importantly, the KlSnf1-KIMig repression pathway operates by modulating KlGAL1 expression. Regulating KlGAL1 expression in this manner enables the cell to switch the regulon off in the presence of glucose. Overall, our data show that, while the Snf1 and Mig1 proteins play similar roles in regulating the galactose regulon in Saccharomyces cerevisiae and K.lactis , the way in which these proteins are integrated into the regulatory circuits are unique to each regulon, as is the degree to which each regulon is controlled by the two proteins.     

Molecular analysis of the distribution and phylogeny of the soxB gene among sulfur-oxidizing bacteria - evolution of the Sox sulfur oxidation enzyme system

The soxB gene encodes the SoxB component of the periplasmic thiosulfate-oxidizing Sox enzyme complex, which has been proposed to be widespread among the various phylogenetic groups of sulfur-oxidizing bacteria (SOB) that convert thiosulfate to sulfate with and without the formation of sulfur globules as intermediate. Indeed, the comprehensive genetic and genomic analyses presented in the present study identified the soxB gene in 121 phylogenetically and physiologically divergent SOB, including several species for which thiosulfate utilization has not been reported yet. In first support of the previously postulated general involvement of components of the Sox enzyme complex in the thiosulfate oxidation process of sulfur-storing SOB, the soxB gene was detected in all investigated photo- and chemotrophic species that form sulfur globules during thiosulfate oxidation (Chromatiaceae, Chlorobiaceae, Ectothiorhodospiraceae, Thiothrix, Beggiatoa, Thiobacillus, invertebrate symbionts and free-living relatives). The SoxB phylogeny reflected the major 16S rRNA gene-based phylogenetic lineages of the investigated SOB, although topological discrepancies indicated several events of lateral soxB gene transfer among the SOB, e.g. its independent acquisition by the anaerobic anoxygenic phototrophic lineages from different chemotrophic donor lineages. A putative scenario for the proteobacterial origin and evolution of the Sox enzyme system in SOB is presented considering the phylogenetic, genomic (sox gene cluster composition) and geochemical data.     

Metabolism of thiosulfate and tetrathionate by heterotrophic bacteria from soil

Two heterotrophic bacteria that oxidized thiosulfate to tetrathionate were isolated from soil. The enzyme system in one of the isolates (C-3) was constitutive, but in the other isolate (A-50) it was induced by thiosulfate or tetrathionate. The apparent K(m) for oxygen for thiosulfate oxidation by A-50 was about 223 mum, but, for lactate oxidation by A-50 or thiosulfate oxidation by C-3, the apparent K(m) for oxygen was below 2 mm. The oxidation of thiosulfate by A-50 was first order with respect to oxygen from 230 mum. The rate of oxidation was greatest at pH 6.3 to 6.8 and at about 10 mm thiosulfate, and it was strongly inhibited by several metal-binding reagents. Extracts of induced A-50 reduced ferricyanide, endogenous cytochrome c, and mammalian cytochrome c in the presence of thiosulfate. A-50, once induced to oxidize thiosulfate, also reduced tetrathionate to thiosulfate in the presence of an electron donor such as lactate. The optimal pH for this reaction was at 8.5 to 9.5, and the reaction was first order with respect to tetrathionate. There was no correlation between the formation of the thiosulfate-oxidizing enzyme of A-50 and the incorporation of thiosulfate-sulfur into cell sulfur. Thiosulfate did not affect the growth rate or yield of A-50.     

Capacity of Azospirillum thiophilum for lithotrophic growth coupled to oxidation of reduced sulfur compounds

Capacity for lithotrophic growth coupled to oxidation of reduced sulfur compounds was revealed in an Azospirillum strain, A. thiophilum BV-S ^T . Oxygen concentration in the medium was the major factor determining the type of energy metabolism (organotrophic or lithotrophic) in the presence of thiosulfate. Under aerobic conditions, metabolism of A. thiophilum BV-S^T was organoheterotrophic, with thiosulfate oxidation to tetrathionate resulting from the interaction with reactive oxygen species, mostly H₂O₂, which was formed in the electron transport chain in the course of oxidation of organic electron donors. Under microaerobic conditions (2 mg/L O₂ in liquid medium), A. thiophilum BV-S^T carried out lithoheterotrophic (mixotrophic) metabolism; enzymes of the dissimilatory type of sulfur metabolism were responsible for thiosulfate oxidation to tetrathionate and sulfate. Two enzyme systems were found in the cells: thiosulfate dehydrogenase, which catalyzes incomplete oxidation of thiosulfate to tetrathionate and the thiosulfate-oxidizing Sox enzyme complex, which is involved in complete oxidation of thiosulfate to sulfate. The genetic determinant of a Sox complex component in A. thiophilum BV-S^T was revealed. The soxB gene was found, and its expression under microaerobic conditions was observed to increase 32-fold compared to aerobic cultivation.     

Cytochrome c551 from Starkeya novella: characterization, spectroscopic properties, and phylogeny of a diheme protein of the SoxAX family

Cytochromes from the SoxAX family have a major role in thiosulfate oxidation via the thiosulfate-oxidizing multi-enzyme system (TOMES). Previously characterized SoxAX proteins from Rhodovulum sulfidophilum and Paracoccus pantotrophus contain three heme c groups, two of which are located on the SoxA subunit. In contrast, the SoxAX protein purified from Starkeya novella was found to contain only two heme groups. Mass spectrometry showed that a disulfide bond replaced the second heme group found in the diheme SoxA subunits. Apparent molecular masses of 27,229 +/- 10.3 Da and 20,258.6 +/- 1 Da were determined for SoxA and SoxX with an overall mass of 49.7 kDa, indicating a heterodimeric structure. Optical redox potentiometry found that the two heme cofactors are reduced at similar potentials (versus NHE) that are as follows: +133 mV (pH 6.0); +104 mV (pH 7.0); +49 (pH 7.9) and +10 mV (pH 8.7). EPR spectroscopy revealed that both ferric heme groups are in the low spin state, and the spectra were consistent with one heme having a His/Cys axial ligation and the other having a His/Met axial ligation. The His/Cys ligated heme is present in different conformational states and gives rise to three distinct signals. Amino acid sequencing was used to unambiguously assign the protein to the encoding genes, soxAX, which are part of a complete sox gene cluster found in S. novella. Phylogenetic analysis of soxA- and soxX-related gene sequences indicates a parallel development of SoxA and SoxX, with the diheme and monoheme SoxA sequences located on clearly separated branches of a phylogenetic tree.     

Suppressors Reveal Two Classes of Glucose Repression Genes in the Yeast Saccharomyces Cerevisiae

We selected and analyzed extragenic suppressors of mutations in four genes-GRR1, REG1, GAL82 and GAL83-required for glucose repression of the GAL genes in the yeast Saccharomyces cerevisiae. The suppressors restore normal or nearly normal glucose repression of GAL1 expression in these glucose repression mutants. Tests of the ability of each suppressor to cross-suppress mutations in the other glucose repression genes revealed two groups of mutually cross-suppressed genes: (1) REG1, GAL82 and GAL83 and (2) GRR1. Mutations of a single gene, SRG1, were found as suppressors of reg1, GAL83-2000 and GAL82-1, suggesting that these three gene products act at a similar point in the glucose repression pathway. Mutations in SRG1 do not cross-suppress grr1 or hxk2 mutations. Conversely, suppressors of grr1 (rgt1) do not cross-suppress any other glucose repression mutation tested. These results, together with what was previously known about these genes, lead us to propose a model for glucose repression in which Grr1p acts early in the glucose repression pathway, perhaps affecting the generation of the signal for glucose repression. We suggest that Reg1p, Gal82p and Gal83p act after the step(s) executed by Grr1p, possibly transmitting the signal for repression to the Snf1p protein kinase.     

Effect of Thiosulfate on the Photosynthetic Growth of Rhodopseudomonas palustris

Cell yields of Rhodopseudomonas palustris grown photoheterotrophically in pyruvate-mineral salts medium were increased by the photooxidation of added thiosulfate. However, thiosulfate had no effect on cell yields of cultures grown aerobically in darkness, although thiosulfate was also oxidized. The presence of thiosulfate increased photosynthetic cell yields on a variety of other organic substrates. Growth of cells in thiosulfate-containing medium, or the addition of thiosulfate to cells grown in thiosulfate-free medium, induced the formation of a thiosulfate-oxidizing system which quantitatively photooxidized thiosulfate to sulfate. R. palustris grew photoautotrophically with thiosulfate as an oxidizable substrate. Large amounts of supplemental bicarbonate carbon were incorporated when cells were grown photosynthetically in pyruvate-thiosulfate medium. Cells harvested after photoautotrophic or photoheterotrophic growth in fumarate-thiosulfate medium fixed (14)CO(2) at an 8- to 10-fold greater rate when provided with thiosulfate. The evolution of (14)CO(2) from pyruvate-1-(14)C during photoassimilation by R. palustris was greatly suppressed by the presence of thiosulfate. The increase in photoheterotrophic cell yields of R. palustris caused by the oxidation of thiosulfate may result from assimilation of substrate carbon which is normally evolved as carbon dioxide.     

Diversity of thiosulfate-oxidizing bacteria from marine sediments and hydrothermal vents

Species diversity, phylogenetic affiliations, and environmental occurrence patterns of thiosulfate-oxidizing marine bacteria were investigated by using new isolates from serially diluted continental slope and deep-sea abyssal plain sediments collected off the coast of New England and strains cultured previously from Galapagos hydrothermal vent samples. The most frequently obtained new isolates, mostly from 10(3)- and 10(4)-fold dilutions of the continental slope sediment, oxidized thiosulfate to sulfate and fell into a distinct phylogenetic cluster of marine alpha-Proteobacteria. Phylogenetically and physiologically, these sediment strains resembled the sulfate-producing thiosulfate oxidizers from the Galapagos hydrothermal vents while showing habitat-related differences in growth temperature, rate and extent of thiosulfate utilization, and carbon substrate patterns. The abyssal deep-sea sediments yielded predominantly base-producing thiosulfate-oxidizing isolates related to Antarctic marine Psychroflexus species and other cold-water marine strains of the Cytophaga-Flavobacterium-Bacteroides phylum, in addition to gamma-proteobacterial isolates of the genera Pseudoalteromonas and Halomonas-Deleya. Bacterial thiosulfate oxidation is found in a wide phylogenetic spectrum of Flavobacteria and Proteobacteria.