Sole Sulfur Source Research Articles

Mycobacterium tuberculosis Rv3406 Is a Type II Alkyl Sulfatase Capable of Sulfate Scavenging

The genome of Mycobacterium tuberculosis (Mtb) encodes nine putative sulfatases, none of which have a known function or substrate. Here, we characterize Mtb’s single putative type II sulfatase, Rv3406, as a non-heme iron (II) and α-ketoglutarate-dependent dioxygenase that catalyzes the oxidation and subsequent cleavage of alkyl sulfate esters. Rv3406 was identified based on its homology to the alkyl sulfatase AtsK from Pseudomonas putida. Using an in vitro biochemical assay, we confirmed that Rv3406 is a sulfatase with a preference for alkyl sulfate substrates similar to those processed by AtsK. We determined the crystal structure of the apo Rv3406 sulfatase at 2.5 Å. The active site residues of Rv3406 and AtsK are essentially superimposable, suggesting that the two sulfatases share the same catalytic mechanism. Finally, we generated an Rv3406 mutant (Δrv3406) in Mtb to study the sulfatase’s role in sulfate scavenging. The Δrv3406 strain did not replicate in minimal media with 2-ethyl hexyl sulfate as the sole sulfur source, in contrast to wild type Mtb or the complemented strain. We conclude that Rv3406 is an iron and α-ketoglutarate-dependent sulfate ester dioxygenase that has unique substrate specificity that is likely distinct from other Mtb sulfatases.

Enhancement of desulfurization activity by enzymes of the Rhodococcus dsz operon through coexpression of a high sulfur peptide and directed evolution

Removal of sulfur from petroleum improves the performance of refineries and reduces air pollution from fuel combustion. Biodesulfurization can selectively remove sulfur from petroleum, but improved biocatalysts are needed. This study illustrates a new approach to obtain improved desulfurization biocatalysts. A synthetic gene (Sulpeptide, S1) encoding high proportions of the sulfur containing amino acids methionine and cysteine was designed, constructed and cloned, as part of the Rhodococcus dszABC (desulfurization) operon, in vector pRESX, under control of the Rhodococcus kstD promoter. pRESXdszABC and pRESXdszAS1BC were electroporated into desulfurization-negative Rhodococcus opacus and the strains were transferred through 40 passages in medium with dibenzothiophene (DBT) as the sole sulfur source. Modest increases in desulfurization activity were achieved after 10 passages selecting for rapid growth with DBT. After selection for 40 passages, both the dszABC and dszAS1BC expressing R. opacus strains showed a greater than 20-fold increase in specific desulfurization ability, and exceeded the specific activity of the control, desulfurization positive strain Rhodococcus erythropolis IGTS8. This study serves as a proof of concept that engineering with Sulpeptide genes, combined with selective pressure for rapid growth with DBT, can drive evolution of improved desulfurization biocatalysts.

Cystathionine gamma-synthase is essential for methionine biosynthesis in Fusarium graminearum

Methionine (Met) plays an important role in various cellular processes in both eukaryotes and prokaryotes. Cystathionine gamma-synthase encoded by STR2 gene is a key enzyme in Met biosynthesis in Saccharomyces cerevisiae. In this study, we identified FgMETB, a homologue of S. cerevisiae STR2, from Fusarium graminearum using the Protein Basic Local Alignment Search Tool (BLASTP) program. The FgMETB deletion mutants were unable to grow on fructose gelatin agar (FGA) medium containing SO42− as sole sulphur source. In addition, more than 90 % conidia of the mutants were not able to germinate in 2 % sucrose solution within 6 or 12 h of incubation. Supplementation of 1 mM Met or 0.5 mg ml−1 homocysteine, but not 1 mM cysteine or 0.5 mg ml−1 glutathione, rescued the defect of mycelial growth and spore germination of FgMETB deletion mutants. These results indicated that the enzyme encoded by FgMETB is involved in conversion of cysteine into homocysteine. Inoculation tests showed that the FgMETB deletion mutant exhibited decreased virulence significantly on wheat heads, which is consistent with a low level of deoxynivalenol (DON) production of the mutant in wheat kernels. Fungicide sensitivity assays revealed FgMETB deletion mutants showed increased sensitivity to the sterol demethylation inhibitor tebuconazole, but did not change their sensitivities to other fungicides. Taken together, results of this study indicated that FgMETB plays a critical role in the regulation of various cellular processes in F. graminearum.

Desulfurization of mucin by Pseudomonas aeruginosa: influence of sulfate in the lungs of cystic fibrosis patients

Pseudomonas aeruginosa is a common cause of chronic respiratory infection in cystic fibrosis (CF) patients. Infection is established within the lung epithelial mucus layer through adhesion to mucins. Terminal residues on mucin oligosaccharide chains are highly sulfated and sialylated, which increases their resistance to degradation by bacterial enzymes. However, a number of microbes, including P. aeruginosa, display mucin sulfatase activity. Using ion chromatography, the levels of sulfation on different respiratory mucins and the availability of inorganic sulfate to pathogens in sputum from CF patients were quantified. The ability of clinical isolates of P. aeruginosa to desulfate mucin was tested by providing mucin as a sole sulfur source for growth. All tested P. aeruginosa strains isolated from the lungs of CF patients were able to use human respiratory mucin as a source of sulfur for growth, whereas other non-clinical species of the genus Pseudomonas were not. However, measured levels of inorganic sulfate in sputum from CF patients suggested that bacteria resident in the lung have sufficient inorganic sulfate for growth and are unlikely to require access to mucin sulfur as a sulfur source during chronic infection. This was confirmed when expression of sulfate-repressed P. aeruginosa genes atsK and msuE was found to be repressed in the sputum of CF patients, which was detected by using quantitative RT-PCR. These results indicate that sulfate starvation is unlikely to occur in pathogens residing in the sputum of CF patients and, therefore, mucin desulfation may have an alternative purpose in the association between P. aeruginosa and the airways of CF patients.

Nutrient requirements for growth of the extreme oligotroph ‘Candidatus Pelagibacter ubique’ HTCC1062 on a defined medium

Chemoheterotrophic marine bacteria of the SAR11 clade are Earth's most abundant organisms. Following the first cultivation of a SAR11 bacterium, 'Candidatus Pelagibacter ubique' strain HTCC1062 (Ca. P. ubique) in 2002, unusual nutritional requirements were identified for reduced sulfur compounds and glycine or serine. These requirements were linked to genome streamlining resulting from selection for efficient resource utilization in nutrient-limited ocean habitats. Here we report the first successful cultivation of Ca. P. ubique on a defined artificial seawater medium (AMS1), and an additional requirement for pyruvate or pyruvate precursors. Optimal growth was observed with the collective addition of inorganic macro- and micronutrients, vitamins, methionine, glycine and pyruvate. Methionine served as the sole sulfur source but methionine and glycine were not sufficient to support growth. Optimal cell yields were obtained when the stoichiometry between glycine and pyruvate was 1:4, and incomplete cell division was observed in cultures starved for pyruvate. Glucose and oxaloacetate could fully replace pyruvate, but not acetate, taurine or a variety of tricarboxylic acid cycle intermediates. Moreover, both glycine betaine and serine could substitute for glycine. Interestingly, glycolate partially restored growth in the absence of glycine. We propose that this is the result of the use of glycolate, a product of phytoplankton metabolism, as both a carbon source for respiration and as a precursor to glycine. These findings are important because they provide support for the hypothesis that some micro-organisms are challenging to cultivate because of unusual nutrient requirements caused by streamlining selection and gene loss. Our findings also illustrate unusual metabolic rearrangements that adapt these cells to extreme oligotrophy, and underscore the challenge of reconstructing metabolism from genome sequences in organisms that have non-canonical metabolic pathways.

ISOLATION AND SCREENING OF ENDOSULFANDEGRADING FUNGI FROM SOIL

Fungal colonies capable of tolerating and degrading endosulfan were enriched and isolated from soil with history of endosulfan application. The microorganisms were enriched using technical-grade endosulfan with gradual increase in concentration from 10 to 100 mgl-1, each enrichment run lasting for 210 h. Four (4) fungal isolates, labeled as P701, P702, P601 and P801, were obtained by successive plating of enriched colonies. The isolates were screened based on α- and β-endosulfan degradation in nutrient media with endosulfan as sole-carbon source (NM-SCS) and as sole-sulfur source (NM-SSS). Results indicated that P601 grown in the NM-SSS exhibited the highest endosulfan degradation after 14 d (93.85% for α-endosulfan and 90.29% for β-endosulfan). Isolate P601 was identified as Candida tropicalis using the API C AUX yeast identification system.

Enhancement of bunker oil biodesulfurization by adding surfactant

Biodesulfurization (BDS) is a promising method to remove sulfur compounds from diesel and gasoline. However, the information on BDS of heavy oil is scanty, which might be due to their "undesirable" physical properties and more complicated sulfur diversities. In this study, the BDS of one kind of heavy oil, bunker oil MFO380 was investigated. The biocatalyst was obtained by the enrichment with oil sludge as the seed and using dibenzothiophene (DBT) as the sole sulfur source. The enriched biocatalyst (microbial mixed culture) could selectively remove sulfur from DBT and DBT was transformed into 2-hydroxybiphenyl, which indicates that the BDS process is beneficial to non-destructive carbon bonds and thus can maintain the calorific value. The bunker oil BDS results showed that after 7 days of incubation, the removal efficiency of sulfur in MFO380 was only 2.88 %, but this could be significantly improved by adding surfactants Triton X-100 or Tween 20. This effect could be attributed to greatly reduced viscosity of heavy oil and increased mass transfer of sulfur compounds in heavy oil into water. Adding Triton X-100 achieved the highest removal efficiency of sulfur, up to 51.7 % after 7 days of incubation. The optimal amount of Triton X-100 was 0.5 g/50 ml medium. When toluene was added as an organic solvent for MFO380, the BDS activity was improved, while lower than the effect of adding surfactants.

Agrobacterium tumefaciens can obtain sulphur from an opine that is synthesized by octopine synthase using S‐methylmethionine as a substrate

Agrobacterium tumefaciens incites plant tumours that produce nutrients called opines, which are utilized by the bacteria during host colonization. Various opines provide sources of carbon, nitrogen and phosphorous, but virtually nothing was previously known about how A. tumefaciens acquires sulphur during colonization. Some strains encode an operon required for the catabolism of the opine octopine. This operon contains a gene, msh, that is predicted to direct the conversion of S-methylmethionine (SMM) and homocysteine (HCys) to two equivalents of methionine. Purified Msh carried out this reaction, suggesting that SMM could be an intermediate in opine catabolism. Purified octopine synthase (Ocs, normally expressed in plant tumours) utilized SMM and pyruvate to produce a novel opine, designated sulfonopine, whose catabolism by the bacteria would regenerate SMM. Sulfonopine was produced by tobacco and Arabidopsis when colonized by A. tumefaciens and was utilized as sole source of sulphur by A. tumefaciens. Purified Ocs also used 13 other proteogenic and non-proteogenic amino acids as substrates, including three that contain sulphur. Sulfonopine and 11 other opines were tested for induction of octopine catabolic operon and all were able to do so. This is the first study of the acquisition of sulphur, an essential element, by this pathogen.

Characterizing the roles of Met31 and Met32 in coordinating Met4-activated transcription in the absence of Met30

Yeast sulfur metabolism is transcriptionally regulated by the activator Met4. Met4 lacks DNA-binding ability and relies on interactions with Met31 and Met32, paralogous proteins that bind the same cis-regulatory element, to activate its targets. Although Met31 and Met32 are redundant for growth in the absence of methionine, studies indicate that Met32 has a prominent role over Met31 when Met30, a negative regulator of Met4 and Met32, is inactive. To characterize different roles of Met31 and Met32 in coordinating Met4-activated transcription, we examined transcription in strains lacking either Met31 or Met32 upon Met4 induction in the absence of Met30. Microarray analysis revealed that transcripts involved in sulfate assimilation and sulfonate metabolism were dramatically decreased in met32Δ cells compared to its wild-type and met31Δ counterparts. Despite this difference, both met31Δ and met32Δ cells used inorganic sulfur compounds and sulfonates as sole sulfur sources in minimal media when Met30 was present. This discrepancy may be explained by differential binding of Met31 to Cbf1-dependent promoters between these two conditions. In the absence of Met30, genome-wide chromatin immunoprecipitation analyses found that Met32 bound all Met4-bound targets, supporting Met32 as the main platform for Met4 recruitment. Finally, Met31 and Met32 levels were differentially regulated, with Met32 levels mimicking the profile for active Met4. These different properties of Met32 likely contribute to its prominent role in Met4-activated transcription when Met30 is absent.

Glutathione Degradation Is a Key Determinant of Glutathione Homeostasis

Glutathione (GSH) has several important functions in eukaryotic cells, and its intracellular concentration is tightly controlled. Combining mathematical models and (35)S labeling, we analyzed Saccharomyces cerevisiae sulfur metabolism. This led us to the observation that GSH recycling is markedly faster than previously estimated. We set up additional in vivo assays and concluded that under standard conditions, GSH half-life is around 90 min. Sulfur starvation and growth with GSH as the sole sulfur source strongly increase GSH degradation, whereas cadmium (Cd(2+)) treatment inhibits GSH degradation. Whatever the condition tested, GSH is degraded by the cytosolic Dug complex (composed of the three subunits Dug1, Dug2, and Dug3) but not by the γ-glutamyl-transpeptidase, raising the question of the role of this enzyme. In vivo, both DUG2/3 mRNA levels and Dug activity are quickly induced by sulfur deprivation in a Met4-dependent manner. This suggests that Dug activity is mainly regulated at the transcriptional level. Finally, analysis of dug2Δ and dug3Δ mutant cells shows that GSH degradation activity strongly impacts on GSH intracellular concentration and that GSH intracellular concentration does not affect GSH synthesis rate. Altogether, our data led us to reconsider important aspects of GSH metabolism, challenging notions on GSH synthesis and GSH degradation that were considered as established.

Isolation and Characterization of An Endosulfan-Degrading Strain, Stenotrophomonas sp. LD-6, and its Potential in Soil Bioremediation

Aerobic bacteria degrading endosulfan were isolated from contaminated sludge. One of the isolates, LD-6, was identified as Stenotrophomonas sp. The bacterium could utilize endosulfan as the sole source of carbon and sulfur. 100 mg/l endosulfan was completely degraded within 10 days, and endosulfan diol and endosulfan ether were detected as major metabolites with a slight decrease in culture pH. The results indicated that Stenotrophomonas. sp. LD-6 might degrade endosulfan by a non-oxidative pathway. Biodegradation of both isomers was relatively better at a temperature range of 25-35 degrees C, with a maximum at 30 degrees C. In addition, cell crude extract of strain LD-6 could metabolize endosulfan rapidly, and degradative enzymes were intracellular distributed and constitutively expressed. Besides, application of the strain was found to promote the removal of endosulfan in soil. This study might help with the future research in better understanding of the biodegradation.

Sulfur‐36S stable isotope labeling of amino acids for quantification (SULAQ)

We introduce a universal metabolic labeling strategy using elemental heavy 36Sulfur (36S) called 36Sulfur stable isotope labeling of amino acids for quantification (SULAQ). In the proof of principle experiment, Pseudomonas putida KT2440 was grown in defined minimal medium with sodium benzoate or sodium succinate as the sole carbon and 32S- or 36S-sodium sulfate as the sole sulfur sources. Quantification using mass spectrometry resulted in 562 proteins with 1991 unique peptides. SULAQ technology can be a valuable alternative strategy for the quantitative comparisons in MS-based proteomics approaches characterizing bacterial and other biological samples in different growth conditions.

Metabolic engineering of hydrophobic Rhodococcus opacus for biodesulfurization in oil–water biphasic reaction mixtures

An organic solvent-tolerant bacterium, Rhodococcus opacus B-4, was metabolically engineered to remove sulfur from dibenzothiophene (DBT), a component of crude oil. The resulting recombinant strain ROD2-8 constitutively expressed the Rhodococcus erythropolis IGTS8 genes dszA, dszB, and dszC, encoding dibenzothiophene sulfone monooxygenase, 2-(2'-hydroxyphenyl) benzenesulfinate desulfinase, and dibenzothiophene monooxygenase, respectively, of the 4S pathway to avoid transcriptional inhibition by the sulfate end-product. Unlike the wild-type strain, ROD2-8 grew in mineral salts medium containing DBT as the sole sulfur source. Under aqueous conditions, ROD2-8 resting cells converted greater than 85% of DBT to 2-hydroxybiphenyl (2-HBP), although the consumption rate by ROD2-8 cells precultured on DBT as the sole sulfur source was 3.3-fold higher than that of cells cultured in complex medium. Notably, DBT consumption rates increased by 80% in oil-water biphasic reaction mixtures with n-hexadecane as the organic solvent, and resting cells were predominantly localized in the emulsion layer. Desulfurization activity in biphasic reaction mixtures increased with increasing concentrations of DBT and was not markedly inhibited by 2-HBP accumulation. Intracellular concentrations of DBT and 2-HBP were significantly lower under biphasic conditions than aqueous conditions. Our findings suggest that the enhanced desulfurization activity under biphasic conditions results from the combined effects of attenuated feedback inhibition and reduced mass transfer limitations due to 2-HBP diffusion from cells and accumulation of both substrate and biocatalyst in the emulsion layer, respectively. Therefore, the solvent-tolerant and hydrophobic bacterium R. opacus B-4 appears suitable for biodesulfurization reactions in solvents containing a minimum ratio of water.

Isolation of an endosulfan-degrading bacterium from a coffee farm soil: Persistence and inhibitory effect on its biological functions

Endosulfan is a lypophilic persistent organic pollutant (POP) that has caused widespread concern due to its persistence in the environment, toxicity and bioaccumulation in living organisms. The aim of this study is to isolate endosulfan-degrading bacteria taken from five coffee farms historically exposed to this insecticide which could be used in future remediation strategies. The biodegradation capability of the isolated strain as well as endosulfan's impact on some of the strain's biological functions was studied. Endosulfan and its metabolites were analyzed using TLC and GC-MS. The isolated strain, capable of growing in a liquid culture treated with this insecticide as the sole sulfur source rather than a carbon source, was selected for further study. The isolated bacterium is Gram-negative, having the morphological and biochemical characteristics of Azotobacter sp. The remaining concentrations after 6 days, using 2 and 10 mg l − 1 of endosulfan, were 57.6 and 72.3% respectively, and the degradation constants were 0.12 d − 1 and 0.26 d − 1 . Four metabolites were detected, one of which was identified as endosulfan ether. Endosulfan reduced nitrogenase activity but had no impact on indole 3-acetic acid production. Thus, these results suggest that this strain has the potential to act as a biocatalyst in endosulfan degradation.

Interactions between bicarbonate, potassium, and magnesium, and sulfur‐dependent induction of luminescence in Vibrio fischeri

In spite of its central importance in research efforts, the relationship between seawater compounds and bacterial luminescence has not previously been investigated in detail. Thus, in this study, we investigated the effect of cations (Na(+) , K(+) , NH(4) (+) , Mg(2+) , and Ca(2+) ) and anions (Cl(-) , HCO(3) (-) , CO(3) (2-) , and NO(3) (-) ) on the induction of both inorganic (sulfate, sulfite, and thiosulfate) and organic (L-cysteine and L-cystine) sulfur-dependent luminescence in Vibrio fischeri. We found that HCO(3) (-) (bicarbonate) and CO(3) (2-) (carbonate), in the form of various compounds, had a stimulatory effect on sulfur-dependent luminescence. The luminescence induced by bicarbonate was further promoted by the addition of magnesium. Potassium also increased sulfur-dependent luminescence when sulfate or thiosulfate was supplied as the sole sulfur source, but not when sulfite, L-cysteine, or L-cystine was supplied. The positive effect of potassium was accelerated by the addition of magnesium and/or calcium. Furthermore, the additional supply of magnesium improved the induction of sulfite- or L-cysteine-dependent luminescence, but not the l-cystine-dependent type. These results suggest that sulfur-dependent luminescence of V. fischeri under nutrient-starved conditions is mainly controlled by bicarbonate, carbonate, and potassium. In addition, our results indicate that an additional supply of magnesium is effective for increasing V. fischeri luminescence.

The ChrA homologue from a sulfur-regulated gene cluster in cyanobacterial plasmid pANL confers chromate resistance

The cyanobacterium Synechococcus elongatus strain PCC 7942 possesses pANL, a plasmid rich in genes related to sulfur metabolism. One of these genes, srpC, encodes the SrpC protein, a homologue of the CHR chromate ion transporter superfamily. The srpC gene was cloned and expressed in Escherichia coli and its role in relation to sulfate and chromate was analyzed. srpC was unable to complement the growth of an E. coli cysA sulfate uptake mutant when sulfate was utilized as a sole sulfur source, suggesting that SrpC is not a sulfate transporter. Expression of srpC in E. coli conferred chromate resistance and caused diminished chromate uptake. These results suggest that the S. elongatus SrpC protein functions as a transporter that extrudes chromate ions from the cell's cytoplasm, and further demonstrate the close relationship between sulfate and chromate metabolism in this organism.

Identification and characterization of feather-degrading bacteria from keratin-rich wastes

In this study we aimed to isolate and identify bacteria showing high keratinolytic activity from different sources of decomposed poultry waste and soil in Shiraz, Iran. Initial screening identified 30 bacteria showing high proteolytic activity, which were selected for transfer to basal feather medium using feathers as the sole source of carbon, nitrogen and sulfur. The keratinolytic activity of the 13 isolates growing well on this medium was assayed using keratin azure as substrate. All isolates were identified based on 16S rDNA as a molecular marker. Of these, Bacillus sp. MKR5 exhibited the highest keratinolytic activity (225 U/ml), and was selected for further characterization. MKR5 demonstrated growth over a wide range of temperature (20–50°C). Complete feather degradation was achieved within 24 h at 40°C. This strain produced a thermostable keratinase with optimum activity at 70°C and pH 8.0. Enzyme activity was increased significantly by using 2-mercaptoethanol as a reducing agent. The keratinase was activated substantially in the presence of Co2+, Mg2+, TritonX-100, Tween-80 and EDTA, whereas SDS had a negative effect on enzyme activity. The thermostability of this enzyme makes it feasible to take advantage of this bacterium in biotechnological processes.

Expression and Functional Roles ofBradyrhizobium japonicumGenes Involved in the Utilization of Inorganic and Organic Sulfur Compounds in Free-Living and Symbiotic Conditions

Strains of Bradyrhizobium spp. form nitrogen-fixing symbioses with many legumes, including soybean. Although inorganic sulfur is preferred by bacteria in laboratory conditions, sulfur in agricultural soil is mainly present as sulfonates and sulfur esters. Here, we show that Bradyrhizobium japonicum and B. elkanii strains were able to utilize sulfate, cysteine, sulfonates, and sulfur-ester compounds as sole sulfur sources for growth. Expression and functional analysis revealed that two sets of gene clusters (bll6449 to bll6455 or bll7007 to bll7011) are important for utilization of sulfonates sulfur source. The bll6451 or bll7010 genes are also expressed in the symbiotic nodules. However, B. japonicum mutants defective in either of the sulfonate utilization operons were not affected for symbiosis with soybean, indicating the functional redundancy or availability of other sulfur sources in planta. In accordance, B. japonicum bacteroids possessed significant sulfatase activity. These results indicate that strains of Bradyrhizobium spp. likely use organosulfur compounds for growth and survival in soils, as well as for legume nodulation and nitrogen fixation.

Effect of glutathione on growth of the probiotic bacterium Lactobacillus reuteri

Glutathione (GSH) is an abundant nonprotein thiol that plays numerous roles within the cell. Previously, we showed that Lactobacillus salivarius has the capacity to mount a glutathione-mediated acid-tolerance response. In the present work we provide evidence of a requirement for GSH by Lactobacillus reuteri and have studied the role of GSH during cell growth. Medium supplementation with 0.5 mM GSH as the sole sulfur source enhanced cell growth, resulting in an increase in glucose consumption, and increased cell GSH and protein contents compared with levels seen in the absence of supplementation. Moreover, L. reuteri showed enhanced amino acid consumption when grown with 0.5 mM GSH. These findings indicate that glutathione is a nutrient for bacterial growth.

An Extracellular Tetrathionate Hydrolase from the Thermoacidophilic Archaeon Acidianus Ambivalens with an Activity Optimum at pH 1.

Background: The thermoacidophilic and chemolithotrophic archaeon Acidianus ambivalens is routinely grown with sulfur and CO2-enriched air. We had described a membrane-bound, tetrathionate (TT) forming thiosulfate:quinone oxidoreductase. Here we describe the first TT hydrolase (TTH) from Archaea. Results: A. ambivalens cells grown aerobically with TT as sole sulfur source showed doubling times of 9 h and final cell densities of up to 8 × 108/ml. TTH activity (≈0.28 U/mg protein) was found in cell-free extracts of TT-grown but not of sulfur-grown cells. Differential fractionation of freshly harvested cells involving a pH shock showed that about 92% of the TTH activity was located in the pseudo-periplasmic fraction associated with the surface layer, while 7.3% and 0.3% were present in the soluble and membrane fractions, respectively. The enzyme was enriched 54-fold from the cytoplasmic fraction and 2.1-fold from the pseudo-periplasmic fraction. The molecular mass of the single subunit was 54 kDa. The optimal activity was at or above 95°C at pH 1. Neither PQQ nor divalent cations had a significant effect on activity. The gene (tth1) was identified following N-terminal sequencing of the protein. Northern hybridization showed that tth1 was transcribed in TT-grown cells in contrast to a second paralogous tth2 gene. The deduced amino acid sequences showed similarity to the TTH from Acidithiobacillus and other proteins from the PQQ dehydrogenase superfamily. It displayed a β-propeller structure when being modeled, however, important residues from the PQQ-binding site were absent. Conclusion: The soluble, extracellular, and acidophilic TTH identified in TT-grown A. ambivalens cells is essential for TT metabolism during growth but not for the downstream processing of the TQO reaction products in S°-grown cells. The liberation of TTH by pH shock from otherwise intact cells strongly supports the pseudo-periplasm hypothesis of the S-layer of Archaea.