Sulfoquinovose is a select nutrient of prominent bacteria and a source of hydrogen sulfide in the human gut.

Responses of the microbiota to diet are highly personalized but mechanistically not well understood because many metabolic capabilities and interactions of human gut microorganisms are unknown. Here we show that sulfoquinovose (SQ), a sulfonated monosaccharide omnipresent in green vegetables, is a selective yet relevant substrate for few but ubiquitous bacteria in the human gut. In human feces and in defined co-culture, Eubacterium rectale and Bilophila wadsworthia used recently identified pathways to cooperatively catabolize SQ with 2,3-dihydroxypropane-1-sulfonate as a transient intermediate to hydrogen sulfide (H2S), a key intestinal metabolite with disparate effects on host health. SQ-degradation capability is encoded in almost half of E. rectale genomes but otherwise sparsely distributed among microbial species in the human intestine. However, re-analysis of fecal metatranscriptome datasets of four human cohorts showed that SQ degradation (mostly from E. rectale and Faecalibacterium prausnitzii) and H2S production (mostly from B. wadsworthia) pathways were expressed abundantly across various health states, demonstrating that these microbial functions are core attributes of the human gut. The discovery of green-diet-derived SQ as an exclusive microbial nutrient and an additional source of H2S in the human gut highlights the role of individual dietary compounds and organosulfur metabolism on microbial activity and has implications for precision editing of the gut microbiota by dietary and prebiotic interventions.

A glycyl radical enzyme enables hydrogen sulfide production by the human intestinal bacterium Bilophila wadsworthia.

Hydrogen sulfide (H2S) production in the intestinal microbiota has many contributions to human health and disease. An important source of H2S in the human gut is anaerobic respiration of sulfite released from the abundant dietary and host-derived organic sulfonate substrate in the gut, taurine (2-aminoethanesulfonate). However, the enzymes that allow intestinal bacteria to access sulfite from taurine have not yet been identified. Here we decipher the complete taurine desulfonation pathway in Bilophila wadsworthia 3.1.6 using differential proteomics, in vitro reconstruction with heterologously produced enzymes, and identification of critical intermediates. An initial deamination of taurine to sulfoacetaldehyde by a known taurine:pyruvate aminotransferase is followed, unexpectedly, by reduction of sulfoacetaldehyde to isethionate (2-hydroxyethanesulfonate) by an NADH-dependent reductase. Isethionate is then cleaved to sulfite and acetaldehyde by a previously uncharacterized glycyl radical enzyme (GRE), isethionate sulfite-lyase (IslA). The acetaldehyde produced is oxidized to acetyl-CoA by a dehydrogenase, and the sulfite is reduced to H2S by dissimilatory sulfite reductase. This unique GRE is also found in Desulfovibrio desulfuricans DSM642 and Desulfovibrio alaskensis G20, which use isethionate but not taurine; corresponding knockout mutants of D. alaskensis G20 did not grow with isethionate as the terminal electron acceptor. In conclusion, the novel radical-based C-S bond-cleavage reaction catalyzed by IslA diversifies the known repertoire of GRE superfamily enzymes and enables the energy metabolism of B. wadsworthia This GRE is widely distributed in gut bacterial genomes and may represent a novel target for control of intestinal H2S production.

Anaerobic Degradation of the Plant Sugar Sulfoquinovose Concomitant With H2S Production: Escherichia coli K-12 and Desulfovibrio sp. Strain DF1 as Co-culture Model.

Sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) is produced by plants and other phototrophs and its biodegradation is a relevant component of the biogeochemical carbon and sulfur cycles. SQ is known to be degraded by aerobic bacterial consortia in two tiers via C3-organosulfonates as transient intermediates to CO2, water and sulfate. In this study, we present a first laboratory model for anaerobic degradation of SQ by bacterial consortia in two tiers to acetate and hydrogen sulfide (H2S). For the first tier, SQ-degrading Escherichia coli K-12 was used. It catalyzes the fermentation of SQ to 2,3-dihydroxypropane-1-sulfonate (DHPS), succinate, acetate and formate, thus, a novel type of mixed-acid fermentation. It employs the characterized SQ Embden-Meyerhof-Parnas pathway, as confirmed by mutational and proteomic analyses. For the second tier, a DHPS-degrading Desulfovibrio sp. isolate from anaerobic sewage sludge was used, strain DF1. It catalyzes another novel fermentation, of the DHPS to acetate and H2S. Its DHPS desulfonation pathway was identified by differential proteomics and demonstrated by heterologously produced enzymes: DHPS is oxidized via 3-sulfolactaldehyde to 3-sulfolactate (SL) by two NAD+-dependent dehydrogenases (DhpA, SlaB); the SL is cleaved by an SL sulfite-lyase known from aerobic bacteria (SuyAB) to pyruvate and sulfite. The pyruvate is oxidized to acetate, while the sulfite is used as electron acceptor in respiration and reduced to H2S. In conclusion, anaerobic sulfidogenic SQ degradation was demonstrated as a novel link in the biogeochemical sulfur cycle. SQ is also a constituent of the green-vegetable diet of herbivores and omnivores and H2S production in the intestinal microbiome has many recognized and potential contributions to human health and disease. Hence, it is important to examine bacterial SQ degradation also in the human intestinal microbiome, in relation to H2S production, dietary conditions and human health.

Sulphoglycolysis in Escherichia coli K-12 closes a gap in the biogeochemical sulphur cycle.

Sulphoquinovose (SQ, 6-deoxy-6-sulphoglucose) has been known for 50 years as the polar headgroup of the plant sulpholipid in the photosynthetic membranes of all higher plants, mosses, ferns, algae and most photosynthetic bacteria. It is also found in some non-photosynthetic bacteria, and SQ is part of the surface layer of some Archaea. The estimated annual production of SQ is 10,000,000,000 tonnes (10 petagrams), thus it comprises a major portion of the organo-sulphur in nature, where SQ is degraded by bacteria. However, despite evidence for at least three different degradative pathways in bacteria, no enzymic reaction or gene in any pathway has been defined, although a sulphoglycolytic pathway has been proposed. Here we show that Escherichia coli K-12, the most widely studied prokaryotic model organism, performs sulphoglycolysis, in addition to standard glycolysis. SQ is catabolised through four newly discovered reactions that we established using purified, heterologously expressed enzymes: SQ isomerase, 6-deoxy-6-sulphofructose (SF) kinase, 6-deoxy-6-sulphofructose-1-phosphate (SFP) aldolase, and 3-sulpholactaldehyde (SLA) reductase. The enzymes are encoded in a ten-gene cluster, which probably also encodes regulation, transport and degradation of the whole sulpholipid; the gene cluster is present in almost all (>91%) available E. coli genomes, and is widespread in Enterobacteriaceae. The pathway yields dihydroxyacetone phosphate (DHAP), which powers energy conservation and growth of E. coli, and the sulphonate product 2,3-dihydroxypropane-1-sulphonate (DHPS), which is excreted. DHPS is mineralized by other bacteria, thus closing the sulphur cycle within a bacterial community.

Paracoccus denitrificans PD1222 utilizes hypotaurine via transamination followed by spontaneous desulfination to yield acetaldehyde and, finally, acetate for growth.

Hypotaurine (HT; 2-aminoethane-sulfinate) is known to be utilized by bacteria as a sole source of carbon, nitrogen, and energy for growth, as is taurine (2-aminoethane-sulfonate); however, the corresponding HT degradation pathway has remained undefined. Genome-sequenced Paracoccus denitrificans PD1222 utilized HT (and taurine) quantitatively for heterotrophic growth and released the HT sulfur as sulfite (and sulfate) and HT nitrogen as ammonium. Enzyme assays with cell extracts suggested that an HT-inducible HT:pyruvate aminotransferase (Hpa) catalyzes the deamination of HT in an initial reaction step. Partial purification of the Hpa activity and peptide fingerprinting-mass spectrometry (PF-MS) identified the Hpa candidate gene; it encoded an archetypal taurine:pyruvate aminotransferase (Tpa). The same gene product was identified via differential PAGE and PF-MS, as was the gene of a strongly HT-inducible aldehyde dehydrogenase (Adh). Both genes were overexpressed in Escherichia coli. The overexpressed, purified Hpa/Tpa showed HT:pyruvate-aminotransferase activity. Alanine, acetaldehyde, and sulfite were identified as the reaction products but not sulfinoacetaldehyde; the reaction of Hpa/Tpa with taurine yielded sulfoacetaldehyde, which is stable. The overexpressed, purified Adh oxidized the acetaldehyde generated during the Hpa reaction to acetate in an NAD(+)-dependent reaction. Based on these results, the following degradation pathway for HT in strain PD1222 can be depicted. The identified aminotransferase converts HT to sulfinoacetaldehyde, which desulfinates spontaneously to acetaldehyde and sulfite; the inducible aldehyde dehydrogenase oxidizes acetaldehyde to yield acetate, which is metabolized, and sulfite, which is excreted.

Two enzymes of a complete degradation pathway for linear alkylbenzenesulfonate (LAS) surfactants: 4-sulfoacetophenone Baeyer-Villiger monooxygenase and 4-sulfophenylacetate esterase in Comamonas testosteroni KF-1.

Complete biodegradation of the surfactant linear alkylbenzenesulfonate (LAS) is accomplished by complex bacterial communities in two steps. First, all LAS congeners are degraded into about 50 sulfophenylcarboxylates (SPC), one of which is 3-(4-sulfophenyl)butyrate (3-C(4)-SPC). Second, these SPCs are mineralized. 3-C(4)-SPC is mineralized by Comamonas testosteroni KF-1 in a process involving 4-sulfoacetophenone (SAP) as a metabolite and an unknown inducible Baeyer-Villiger monooxygenase (BVMO) to yield 4-sulfophenyl acetate (SPAc) from SAP (SAPMO enzyme); hydrolysis of SPAc to 4-sulfophenol and acetate is catalyzed by an unknown inducible esterase (SPAc esterase). Transcriptional analysis showed that one of four candidate genes for BVMOs in the genome of strain KF-1, as well as an SPAc esterase candidate gene directly upstream, was inducibly transcribed during growth with 3-C(4)-SPC. The same genes were identified by enzyme purification and peptide fingerprinting-mass spectrometry when SAPMO was enriched and SPAc esterase purified to homogeneity by protein chromatography. Heterologously overproduced pure SAPMO converted SAP to SPAc and was active with phenylacetone and 4-hydroxyacetophenone but not with cyclohexanone and progesterone. SAPMO showed the highest sequence homology to the archetypal phenylacetone BVMO (57%), followed by steroid BVMO (55%) and 4-hydroxyacetophenone BVMO (30%). Finally, the two pure enzymes added sequentially, SAPMO with NADPH and SAP, and then SPAc esterase, catalyzed the conversion of SAP via SPAc to 4-sulfophenol and acetate in a 1:1:1:1 molar ratio. Hence, the first two enzymes of a complete LAS degradation pathway were identified, giving evidence for the recruitment of members of the very versatile type I BVMO and carboxylester hydrolase enzyme families for the utilization of a xenobiotic compound by bacteria.

(R)-Cysteate-nitrogen assimilation by Cupriavidus necator H16 with excretion of 3-sulfolactate: a patchwork pathway.

Cupriavidus necator H16 grew exponentially with (R)-cysteate, a structural analogue of aspartate, as sole source of nitrogen in succinate-salts medium. Utilization of cysteate was quantitative and concomitant with growth and with the excretion of the deaminated product (R)-sulfolactate, which was identified thoroughly. The deaminative pathway started with transport of (R)-cysteate into the cell, which we attributed to an aspartate transporter. Transamination to sulfopyruvate involved an aspartate/(R)-cysteate:2-oxoglutarate aminotransferase (Aoa/Coa) and regeneration of the amino group acceptor by NADP⁺-coupled glutamate dehydrogenase. Reduction of sulfopyruvate to (R)-sulfolactate was catalyzed by a (S)-malate/(R)-sulfolactate dehydrogenase (Mdh/Sdh). Excretion of the sulfolactate could be attributed to the sulfite/organosulfonate exporter TauE, which was co-encoded and co-expressed, with sulfoacetaldehyde acetyltransferase (Xsc), though Xsc was irrelevant to the current pathway. The metabolic enzymes could be assayed biochemically. Aoa/Coa and Mdh/Sdh were highly enriched by protein separation, partly characterized, and the relevant locus-tags identified by peptide-mass fingerprinting. Finally, RT-PCR was used to confirm the transcription of all appropriate genes. We thus demonstrated that Cupriavidus necator H16 uses a patchwork pathway by recruitment of 'housekeeping' genes and sulfoacetaldehyde-degradative genes to scavenge for (R)-cysteate-nitrogen.

Sulfoquinovose degraded by pure cultures of bacteria with release of C3-organosulfonates: complete degradation in two-member communities.

Sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) was synthesized chemically. An HPLC-ELSD method to separate SQ and other chromophore-free sulfonates, e.g. 2,3-dihydroxypropane-1-sulfonate (DHPS), was developed. A set of 10 genome-sequenced, sulfonate-utilizing bacteria did not utilize SQ, but an isolate, Pseudomonas putida SQ1, from an enrichment culture did so. The molar growth yield with SQ was half of that with glucose, and 1 mol 3-sulfolactate (mol SQ)(-1) was formed during growth. The 3-sulfolactate was degraded by the addition of Paracoccus pantotrophus NKNCYSA, and the sulfonate sulfur was recovered quantitatively as sulfate. Another isolate, Klebsiella oxytoca TauN1, could utilize SQ, forming 1 mol DHPS (mol SQ)(-1) ; the molar growth yield with SQ was half of that with glucose. This DHPS could be degraded by Cupriavidus pinatubonensis JMP134, with quantitative recovery of the sulfonate sulfur as sulfate. We presume that SQ can be degraded by communities in the environment.

Molecular genetics and biochemistry of N-acetyltaurine degradation by Cupriavidus necator H16.

Cupriavidus necator H16 (DSM 428), whose genome has been sequenced, was found to degrade N-acetyltaurine as a sole source of carbon and energy for growth. Utilization of the compound was quantitative. The degradative pathway involved an inducible N-acetyltaurine amidohydrolase (NaaS), which catalysed the cleavage of N-acetyltaurine to acetate and taurine. The degradation of the latter compound is via an inducible, degradative pathway that involves taurine dehydrogenase [EC 1.4.2.-], sulfoacetaldehyde acetyltransferase [EC 2.3.3.15], phosphotransacetylase [EC 2.4.1.8], a sulfite exporter [TC 9.A.29.2.1] and sulfite dehydrogenase [EC 1.8.2.1]. Induction of the expression of representative gene products, encoded by at least four gene clusters, was confirmed biochemically. The acetate released by NaaS was activated to acetyl-CoA by an inducible acetate-CoA ligase [EC 6.2.1.1]. NaaS was purified to homogeneity; it had a K(m) value of 9.4 mM for N-acetyltaurine, and it contained tightly bound Zn and Fe atoms. The denatured enzyme has a molecular mass of about 61 kDa (determined by SDS-PAGE) and the native enzyme was apparently monomeric. Peptide-mass fingerprinting identified the locus tag as H16_B0868 in a five-gene cluster, naaROPST (H16_B0865-H16_B0869). The cluster presumably encodes a LysR-type transcriptional regulator (NaaR), a membrane protein (NaaO), a solute : sodium symporter-family permease [TC 2.A.21] (NaaP), the metal-dependent amidohydrolase (NaaS) and a putative metallochaperone (COG0523) (NaaT). Reverse-transcription PCR indicated that naaOPST were inducibly transcribed.

2,3-Dihydroxypropane-1-sulfonate degraded by Cupriavidus pinatubonensis JMP134: purification of dihydroxypropanesulfonate 3-dehydrogenase.

2,3-Dihydroxypropane-1-sulfonate (DHPS) is a widespread intermediate in plant and algal transformations of sulfoquinovose (SQ) from the plant sulfolipid sulfoquinovosyl diacylglycerol. Further, DHPS is recovered quantitatively during bacterial degradation of SQ by Klebsiella sp. strain ABR11. DHPS is also a putative precursor of sulfolactate in e.g. Ruegeria pomeroyi DSS-3. A bioinformatic approach indicated that some 28 organisms with sequenced genomes might degrade DHPS inducibly via sulfolactate, with three different desulfonative enzymes involved in its degradation in different organisms. The hypothesis for Cupriavidus pinatubonensis JMP134 (formerly Ralstonia eutropha) involved a seven-gene cluster (Reut_C6093-C6087) comprising a LacI-type transcriptional regulator, HpsR, a major facilitator superfamily uptake system, HpsU, three NAD(P)(+)-coupled DHPS dehydrogenases, HpsNOP, and (R)-sulfolactate sulfo-lyase (SuyAB) [EC 4.4.1.24]. HpsOP effected a DHPS-racemase activity, and HpsN oxidized (R)-DHPS to (R)-sulfolactate. The hypothesis for Roseovarius nubinhibens ISM was similar, but involved a tripartite ATP-independent transport system for DHPS, HpsKLM, and two different desulfonative enzymes, (S)-cysteate sulfo-lyase [EC 4.4.1.25] and sulfoacetaldehyde acetyltransferase (Xsc) [EC 2.3.3.15]. Representative organisms were found to grow with DHPS and release sulfate. C. pinatubonensis JMP134 was found to express at least one NAD(P)(+)-coupled DHPS dehydrogenase inducibly, and three different peaks of activity were separated by anion-exchange chromatography. Protein bands (SDS-PAGE) were subjected to peptide-mass fingerprinting, which identified the corresponding genes (hpsNOP). Purified HpsN converted DHPS to sulfolactate. Reverse-transcription PCR confirmed that hpsNOUP were transcribed inducibly in strain JMP134, and that hpsKLM and hpsNOP were transcribed in strain ISM. DHPS degradation is widespread and diverse, implying that DHPS is common in marine and terrestrial environments.

Racemase activity effected by two dehydrogenases in sulfolactate degradation by Chromohalobacter salexigens: purification of (S)-sulfolactate dehydrogenase.

Chromohalobacter salexigens DSM 3043, whose genome has been sequenced, is known to degrade (R,S)-sulfolactate as a sole carbon and energy source for growth. Utilization of the compound(s) was shown to be quantitative, and an eight-gene cluster (Csal_1764-Csal_1771) was hypothesized to encode the enzymes in the degradative pathway. It comprised a transcriptional regulator (SuyR), a Tripartite Tricarboxylate Transporter-family uptake system for sulfolactate (SlcHFG), two sulfolactate dehydrogenases of opposite sulfonate stereochemistry, namely novel SlcC and ComC [(R)-sulfolactate dehydrogenase] [EC 1.1.1.272] and desulfonative sulfolactate sulfo-lyase (SuyAB) [EC 4.4.1.24]. Inducible reduction of 3-sulfopyruvate, inducible SuyAB activity and induction of an unknown protein were detected. Separation of the soluble proteins from induced cells on an anion-exchange column yielded four relevant fractions. Two different fractions reduced sulfopyruvate with NAD(P)H, a third yielded SuyAB activity, and the fourth contained the unknown protein. The latter was identified by peptide-mass fingerprinting as SlcH, the candidate periplasmic binding protein of the transport system. Separated SuyB was also identified by peptide-mass fingerprinting. ComC was partially purified and identified by peptide-mass fingerprinting. The (R)-sulfolactate that ComC produced from sulfopyruvate was a substrate for SuyAB, which showed that SuyAB is (R)-sulfolactate sulfo-lyase. SlcC was purified to homogeneity. This enzyme also formed sulfolactate from sulfopyruvate, but the latter enantiomer was not a substrate for SuyAB. SlcC was obviously ( S)-sulfolactate dehydrogenase.

Bifurcated degradative pathway of 3-sulfolactate in Roseovarius nubinhibens ISM via sulfoacetaldehyde acetyltransferase and (S)-cysteate sulfolyase.

Data from the genome sequence of the aerobic, marine bacterium Roseovarius nubinhibens ISM were interpreted such that 3-sulfolactate would be degraded as a sole source of carbon and energy for growth via a novel bifurcated pathway including two known desulfonative enzymes, sulfoacetaldehyde acetyltransferase (EC 2.3.3.15) (Xsc) and cysteate sulfo-lyase (EC 4.4.1.25) (CuyA). Strain ISM utilized sulfolactate quantitatively with stoichiometric excretion of the sulfonate sulfur as sulfate. A combination of enzyme assays, analytical chemistry, enzyme purification, peptide mass fingerprinting, and reverse transcription-PCR data supported the presence of an inducible, tripartite sulfolactate uptake system (SlcHFG), and a membrane-bound sulfolactate dehydrogenase (SlcD) which generated 3-sulfopyruvate, the point of bifurcation. 3-Sulfopyruvate was in part decarboxylated by 3-sulfopyruvate decarboxylase (EC 4.1.1.79) (ComDE), which was purified. The sulfoacetaldehyde that was formed was desulfonated by Xsc, which was identified, and the acetyl phosphate was converted to acetyl-coenzyme A by phosphate acetyltransferase (Pta). The other portion of the 3-sulfopyruvate was transaminated to (S)-cysteate, which was desulfonated by CuyA, which was identified. The sulfite that was formed was presumably exported by CuyZ (TC 9.B.7.1.1 in the transport classification system), and a periplasmic sulfite dehydrogenase is presumed. Bioinformatic analyses indicated that transporter SlcHFG is rare but that SlcD is involved in three different combinations of pathways, the bifurcated pathway shown here, via CuyA alone, and via Xsc alone. This novel pathway involves ComDE in biodegradation, whereas it was discovered in the biosynthesis of coenzyme M. The different pathways of desulfonation of sulfolactate presumably represent final steps in the biodegradation of sulfoquinovose (and exudates derived from it) in marine and aquatic environments.

Amphoteric surfactant N-oleoyl-N-methyltaurine utilized by Pseudomonas alcaligenes with excretion of N-methyltaurine.

The amphoteric surfactant N-oleoyl-N-methyltaurine, which is in use in skin-care products, was utilized by aerobic bacteria as the sole source of carbon or of nitrogen in enrichment cultures. One isolate, which was identified as Pseudomonas alcaligenes, grew with the xenobiotic compound as the sole source of carbon and energy. The sulfonate moiety, N-methyltaurine, was excreted quantitatively during growth, while the fatty acid was dissimilated. The initial degradative reaction was shown to be hydrolytic and inducible. This amidase reaction could be demonstrated with crude cell extracts. The excreted N-methyltaurine could be utilized by other bacteria in cocultures. Complete degradation of similar natural compounds in bacterial communities seems likely.

Sulfoacetate released during the assimilation of taurine-nitrogen by Neptuniibacter caesariensis: purification of sulfoacetaldehyde dehydrogenase.

Taurine (2-aminoethanesulfonate) is a widespread natural product whose nitrogen moiety was recently shown to be assimilated by bacteria, usually with excretion of an organosulfonate via undefined novel pathways; other data involve transcriptional regulator TauR in taurine metabolism. A screen of genome sequences for TauR with the BLAST algorithm allowed the hypothesis that the marine gammaproteobacterium Neptuniibacter caesariensis MED92 would inducibly assimilate taurine-nitrogen and excrete sulfoacetate. The pathway involved an ABC transporter (TauABC), taurine:pyruvate aminotransferase (Tpa), a novel sulfoacetaldehyde dehydrogenase (SafD) and exporter(s) of sulfoacetate (SafE) (DUF81). Ten candidate genes in two clusters involved three sets of paralogues (for TauR, Tpa and SafE). Inducible Tpa and SafD were detected in cell extracts. SafD was purified 600-fold to homogeneity in two steps. The monomer had a molecular mass of 50 kDa (SDS-PAGE); data from gel filtration chromatography indicated a tetrameric native protein. SafD was specific for sulfoacetaldehyde with a K (m)-value of 0.12 mM. The N-terminal amino acid sequence of SafD confirmed the identity of the safD gene. The eight pathway genes were transcribed inducibly, which indicated expression of the whole hypothetical pathway. We presume that this pathway is one source of sulfoacetate in nature, where this compound is dissimilated by many bacteria.

Different bacterial strategies to degrade taurocholate.

Aerobic enrichment cultures with taurocholate or alkanesulfonates as sole sources of carbon and energy for growth were successful and yielded nine bacterial isolates, all of which utilized taurocholate. Growth was complex and involved not only many, usually transient, excretion products but also sorption of taurocholate and cholate to cells. Three metabolic strategies to dissimilate taurocholate were elucidated, all of which involved bile salt hydrolase cleaving taurocholate to cholate and taurine. Comamonas testosteroni KF-1 utilized both the taurine and the cholate moieties for growth. Pseudomonas spp., e.g. strain TAC-K3 and Rhodococcus equi TAC-A1 grew with the cholate moiety and released taurine quantitatively. Delftia acidovorans SPH-1 utilized the taurine moiety and released cholate.

Bacterial sulfite dehydrogenases in organotrophic metabolism: separation and identification in Cupriavidus necator H16 and in Delftia acidovorans SPH-1.

The utilization of organosulfonates as carbon sources by aerobic or nitrate-reducing bacteria usually involves a measurable, uncharacterized sulfite dehydrogenase. This is tacitly assumed to be sulfite : ferricytochrome-c oxidoreductase [EC 1.8.2.1], despite negligible interaction with (eukaryotic) cytochrome c: the enzyme is assayed at high specific activity with ferricyanide as electron acceptor. Purified periplasmic sulfite dehydrogenases (SorAB, SoxCD) are known from chemoautotrophic growth and are termed 'sulfite oxidases' by bioinformatic services. The catalytic unit (SorA, SoxC; termed 'sulfite oxidases' cd02114 and cd02113, respectively) binds a molybdenum-cofactor (Moco), and involves a cytochrome c (SorB, SoxD) as electron acceptor. The genomes of several bacteria that express a sulfite dehydrogenase during heterotrophic growth contain neither sorAB nor soxCD genes; others contain at least four paralogues, for example Cupriavidus necator H16, which is known to express an inducible sulfite dehydrogenase during growth with taurine (2-aminoethanesulfonate). This soluble enzyme was enriched 320-fold in four steps. The 40 kDa protein (denatured) had an N-terminal amino acid sequence which started at position 42 of the deduced sequence of H16_B0860 (termed 'sulfite oxidase' cd02114), which we named SorA. The neighbouring gene is an orthologue of sorB, and the sorAB genes were co-transcribed. Cell fractionation showed SorA to be periplasmic. The corresponding enzyme in Delftia acidovorans SPH-1 was enriched 270-fold, identified as Daci_0055 (termed 'sulfite oxidase' cd02110) and has a cytochrome c encoded downstream. We presume, from genomic data for bacteria and archaea, that there are several subgroups of sulfite dehydrogenases, which all contain a Moco, and transfer electrons to a specific cytochrome c.

Assimilation of homotaurine-nitrogen by Burkholderia sp. and excretion of sulfopropanoate.

Homotaurine (3-aminopropanesulfonate), free or derivatized, is in widespread pharmaceutical and laboratory use. Studies with enrichment cultures indicated that the compound is degradable as a sole source of carbon or as a sole source of nitrogen for bacterial growth. A pure culture of Burkholderia sp. was isolated which assimilated the amino group from homotaurine in a glucose-salts medium, and which released an organosulfonate, 3-sulfopropanoate, into the medium stoichiometrically. The deamination involved an inducible 2-oxoglutarate-dependent aminotransferase to yield glutamate, and 3-sulfopropanal. Release of the amino group was attributed to the measured NADP-coupled glutamate dehydrogenase.

The DUF81 protein TauE in Cupriavidus necator H16, a sulfite exporter in the metabolism of C2 sulfonates.

The degradation of taurine, isethionate and sulfoacetate in Cupriavidus necator (Ralstonia eutropha) H16 was shown by enzyme assays to be inducible, and each pathway involved sulfoacetaldehyde, which was subject to phosphatolysis by a common sulfoacetaldehyde acetyltransferase (Xsc, H16_B1870) to yield acetyl phosphate and sulfite. The neighbouring genes encoded phosphate acetyltransferase (Pta, H16_B1871) and a hypothetical protein [domain of unknown function (DUF)81, H16_B1872], with eight derived transmembrane helices. RT-PCR showed inducible transcription of these three genes, and led to the hypothesis that H16_B1872 and orthologous proteins represent a sulfite exporter, which was named TauE.

Roseovarius sp. strain 217: aerobic taurine dissimilation via acetate kinase and acetate-CoA ligase.

The genome sequence of Roseovarius sp. strain 217 indicated that many pathway enzymes found in other organisms for the degradation of taurine are represented, but that a novel, apparently energy-dependent pathway is involved in the conversion of acetyl phosphate to acetyl CoA. Thus, an ABC transporter for taurine could be postulated, while inducible taurine: pyruvate aminotransferase, alanine dehydrogenase, sulfoacetaldehyde acetyltransferase and sulfite dehydrogenase could be assayed. Whereas phosphate acetyltransferase has been found in other organisms, none was indicated in the genome sequence and no activity was found in cell-free extracts. Instead, acetate kinase was active as was acetate-CoA ligase.