Assimilation of Homotaurine-Nitrogen by Burkholderia Sp. and Excretion of Sulfopropanoate Jutta Mayer1, Karin Denger1, Katrin Kaspar2, Klaus Hollemeyer3, Theo H

RESEARCH LETTER Assimilation of homotaurine-nitrogen by Burkholderia sp. and excretion of sulfopropanoate Jutta Mayer1, Karin Denger1, Katrin Kaspar2, Klaus Hollemeyer3, Theo H. M. Smits1, Thomas Huhn2 & Alasdair M. Cook1

1Department of Biology University of Konstanz, Konstanz, Germany; 2Department of Chemistry, University of Konstanz, Konstanz, Germany; and 3Institute of Biochemical Engineering, University of the Saarland, Saarbr ¨ucken,Germany

Correspondence: Alasdair M. Cook, Abstract Department of Biology University of Konstanz, D-78457 Konstanz, Germany. Tel.: Homotaurine (3-aminopropanesulfonate), free or derivatized, is in widespread 149 7531 88 4247; fax: 149 7531 88 2966; pharmaceutical and laboratory use. Studies with enrichment cultures indicated E-mail: [email protected] 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 The sequence reported in this paper has been assimilated the amino group from homotaurine in a glucose–salts medium, and deposited in the GenBank database which released an organosulfonate, 3-sulfopropanoate, into the medium stoichio- (accession no. EU035638). metrically. The deamination involved an inducible 2-oxoglutarate-dependent aminotransferase to yield glutamate, and 3-sulfopropanal. Release of the amino Present address: Theo H.M. Smits, Agroscope Changins-Wadenswil¨ ACW, Swiss group was attributed to the measured NADP-coupled glutamate dehydrogenase. Federal Research Station, Schloss, Postfach 185, CH-8820 Wadenswil,¨ Switzerland.

First published online December 2007.

Editor: Christiane Dahl

Keywords assimilation of homotaurine-nitrogen; Burkholderia sp.; excretion of sulfopropanoate; homotaurine:2-oxoglutarate aminotransferase.

assimilation of taurine-nitrogen was seldom accompanied Introduction by cleavage of the carbon–sulfonate bond, and organosulfo- Taurine (2-aminoethanesulfonate) is a major organic solute nates were excreted quantitatively into the growth medium in mammals, and with its derivatives, it is found in all (Denger et al., 2004a, b; Styp von Rekowski et al., 2005; vertebrates and a wide range of marine invertebrates (Allen Weinitschke et al., 2005). & Garrett, 1971; Huxtable, 1992). Major roles have been The many functions of taurine in mammals led pharma- proposed for taurine in brain and muscle, and they have led cologists to examine the properties of homologues of to prolonged interest in the compound (e.g. Oja & Saransaari, taurine. Aminomethanesulfonate decays spontaneously in 2006). Corresponding to the widespread occurrence of mineral medium for bacterial growth (K. Denger, unpub- taurine and derivatives in eukaryotic organisms, the com- lished data), but homotaurine (3-aminopropanesulfonate, pound has been found to serve as a sole source of sulfur, APS) (Fig. 1) is stable and is a drug candidate (AlzhemedTM, nitrogen or carbon for a range of aerobic bacteria (Eichhorn CerebrilTM) for two major conditions (Alzheimer and et al., 1997, 2000; Cook & Denger, 2006; Mayer et al., 2006; stroke). The N-acetyl derivative of homotaurine Weinitschke et al., 2006; see also Lie et al., 1998). The (Camprals) is used to treat alcoholics (Scott et al., 2005). 78

3-Sulfopropanoate Homotaurine O− + H3N − SO3 − O SO3

I membrane V

− H2O 3-Sulfopropanal O II IV +H N − − − 3 SO3 O SO3 O SO3

2-OG Glu acceptorox acceptorred + H2O + III NADP H H O NADPH 2 + NH4 Cell polymers

Fig. 1. Presumed pathway of assimilatory deamination of homotaurine-nitrogen by Burkholderia sp. strain N-APS2. It is axiomatic that sulfonates require transport into the cell (Graham et al., 2002), and it was presumed that either an ATP-binding cassette transporter or a tripartite ATP-independent transporter (I) is involved in transporting homotaurine into the cell, analogous to the transport of other sulfonates (Kertesz, 2001; Denger et al., 2006; Gorzynska et al., 2006). Evidence is provided in the text for the presence of homotaurine:2-oxoglutarate aminotransferase (EC 2.6.1.-) (II) and glutamate dehydrogenase (EC 1.4.1.4) (III). The authors postulate a sulfopropanal dehydrogenase (EC 1.2.1.-) (IV) to generate the sulfopropanoate, which was detected in the medium (Fig. 2), and an exporter (V), because the cell membrane is impermeable to sulfonates. Glu, L-glutamate; 2-OG, 2- oxoglutarate.

Other derivatives of homotaurine are the amphoteric the enrichment and isolation procedures were described surfactant 3-[(3-cholamidopropyl)-dimethylammonio]-1- previously (Mayer et al., 2006). The isolate Burkholderia sp. propanesulfonate (CHAPS) and the buffers cyclohexylami- strain N-APS2 was deposited with the German Culture nopropanesulfonate and 3-[N-morpholino]propanesulfonate Collection DSMZ, Braunschweig, Germany, under the (MOPS). Homotaurine and derivatives can, thus, be ex- accession number DSM 19529. pected in communal sewage works. Growth of Burkholderia sp. strain N-APS2 with 2 mM This study reports that homotaurine can be utilized by homotaurine as sole source of nitrogen (carbon source: bacteria as a sole source of carbon or of nitrogen for growth, 5 mM glucose) was followed in 100-mL cultures in 1-L as indicated in a review (Cook et al., 2006). The assimilation Erlenmeyer flasks shaken at 30 1C in a water bath. Samples of nitrogen was readily studied in pure culture. It involved a were taken at intervals to measure turbidity and to deter- homotaurine:2-oxoglutarate aminotransferase and the mine the concentrations of protein, homotaurine, 3-sulfo- excretion of 3-sulfopropanoate. propanoate, and ammonium and sulphate ions. Crude cell extracts for enzyme assays were prepared from Materials and methods cultures (50 mL) grown with homotaurine or ammonium chloride as sole nitrogen source. Harvesting and cell disrup- Enrichment cultures, isolations, growth media, tion with a French pressure cell were done as described growth conditions and cell extracts elsewhere (Denger et al., 2004a). Enrichment cultures (3 mL in 30-mL tubes) were grown at Analytical methods 30 1C in a 50 mM potassium phosphate-buffered salts medium, pH 7.2, which contained 0.25 mM magnesium sulfate Growth was followed as turbidity at 580 nm or quantified as and trace elements (Thurnheer et al., 1986). Carbon-limited protein in a Lowry-type reaction (Cook & Hutter,¨ 1981). enrichments contained 20 mM ammonium chloride and Sulfate was quantified turbidimetrically as a suspension of

7.5 mM homotaurine; nitrogen-limited medium contained BaSO4 (Sorbo,¨ 1987). Sulﬁte was determined photometri- 2 mM homotaurine and 5 mM glucose, 7 mM succinate and cally as the fuchsin-derivative (Denger et al., 2001). Ammo- 10 mM glycerol. Negative controls were done by omitting nium ion was assayed colorimetrically by the Berthelot homotaurine, positive controls by replacing the compound reaction (Gesellschaft Deutscher Chemiker, 1996). Homo- with glucose, succinate and glycerol (carbon-limited taurine and glutamate were determined after derivatization cultures) or with ammonium chloride (nitrogen-limited with 2,4-dinitroﬂuorobenzene (DNFB) (Sanger, 1945) and cultures). The inocula were from garden soil, forest soil or separation by HPLC. Reactions in samples (50 mL; activated sludge from the wastewater treatment plant in 0–100 nmol amine) from growth experiments or enzyme

Konstanz, Germany. The preparation of inocula as well as assays were stopped by addition to 1 mL of 0.1 M NaHCO3, 79 and derivatization mixtures were shaken at 30 1C for 2 h Chemicals after addition of 0.2 mL 3% (v/v) DNFB in ethanol. Deriva- The sodium salt of 3-sulfopropanoate was synthesized in a tization was stopped by acidification with 50 mL of concen- modification of the published method (Kharasch & Brown, trated HCl, and after centrifugation (1 min, 11 000 g at room 1940). The key alteration was the addition of the radical starter temperature) the supernatant fluid was subject to analysis by azoisobutyronitrile [2,20-azobis(2-methylpropionitrile)] to reversed-phase HPLC (0–80% methanol gradient in 50 mM the mixture of sulfonyl chloride (0.25 mol) in an excess of potassium phosphate buffer, pH 2.2 over 17 min: the propanoic acid (0.5 mol) under reflux and illumination from detector was set at 360 nm). Aldehydes were deriva- a 200 W tungsten lamp. Residual propanoic acid was removed tized with 2-(diphenylacetyl)indane-1,3-dione-1-hydrazone under vacuum. Further unreacted material and putative by- (DIH; 0.3 mg mL1) in acetonitrile (100 mL sample plus products were removed by extraction with 400 mL light 300 mL DIH reagent) and then separated by reversed-phase petroleum. The white crystals of 3-sulfopropanoic acid (yield HPLC (Cunningham et al., 1998). A gradient of acetonitrile 16%; lit.: 37%) were identified by nuclear magnetic resonance (30–80%) in 0.11 M NaClO was applied over 17 min and 4 (NMR) and shown to contain o 1% impurity with the the detection wavelength was 400 nm. A Eurospher 100–5 educts. Analysis by 1H-NMR (400 MHz, D6–DMSO) gave C18 column (125 mm 3 mm; Knauer, Berlin, Germany) 3 1 the following data: d [ppm] = 2.49 (2H; t, J=8.2Hz, was used at a flow of 0.5 mL min . Confirmation of the 3 –CH2–COOH), 2.75 (2H; t, J = 8.0 Hz, CH2–SO3H), 12.78 formation of an o-aldehyde in enzyme reactions was 13 (2H; s, –COOH, –SO H). Analysis by C-NMR (100 MHz, obtained colorimetrically by reaction with o-aminobenzal- 3 D6–DMSO) gave the following data:[ppm] = 30.4 (–CH –), dehyde, which does not react with oxo-groups (Toyama 2 46.9 (–CH –SO H), 173.2 (–COOH). This product, however, et al., 1974). Matrix-assisted laser-desorption ionization 2 3 was oxygen-sensitive and had to be stored under nitrogen, so time-of-flight MS (MALDI-TOF-MS) in the negative-ion no melting point was determined. Neutralization of the free mode was used to confirm the identity of homotaurine (m/z acid with NaOH resulted in the disodium salt that was stable = 138 = [M-H]1), assay its presence, and identify the inter- in air; this compound decomposed at about 298 1C. Elemental mediate, 3-sulfopropanal, and the excretion product 3-sulfo- analysis of the disodium salt gave data, from which the propanoate (Tholey et al., 2002). 3-Sulfopropanoate was composition of the crystals could be concluded as quantified by ion chromatography with the conditions de- C H Na O S 1/2 H O. scribed for sulfoacetate (Denger et al., 2004b). Standard 3 4 2 5 2 Commercial chemicals were of the highest purity avail- methods were used for the Gram reaction, and to assay catalase able, and they were purchased from Fluka, Merck, Roth, or cytochrome c-oxidase activity (Gerhardt et al., 1994). Serva or Sigma. Chromosomal DNA was isolated as described elsewhere (Desomer et al., 1991). Amplification of the 16S rRNA gene, Results and discussion sequencing and sequence analysis were done as reported previously (Weinitschke et al., 2006). Enrichment cultures and the identification of Burkholderia sp. strain N-APS2 Enzyme assays Enrichment cultures from forest soil and sewage sludge Homotaurine:2-oxoglutarate aminotransferase was assayed reproducibly yielded cultures, which grew slowly with discontinuously at 30 1C as the 2-oxoglutarate-dependent homotaurine as the sole source of carbon and energy for formation of glutamate and the concomitant depletion of growth, and which were prokaryotes. Cultures reproducibly homotaurine. The reaction mixture contained (in a final required 4–5 days to convert 7.5 mM homotaurine quanti- volume of 1.0 mL): 40 mmol Tris/HCl, pH 9.0, 1–10 mmol tatively to cell material, and the ammonium and sulfate ions. homotaurine, 10 mmol 2-oxoglutarate, 0.1 mmol pyridoxal-5- This slow growth made the authors to concentrate on the phosphate and 0.25 mg protein with which the reaction was assimilation of homotaurine-nitrogen. started. Samples were taken at intervals, as described above. Enrichment cultures from soil and sewage sludge yielded The possible presence of a pyruvate-coupled aminotrans- cultures of prokaryotes whose subcultures grew in 2 days ferase was tested by replacing 2-oxoglutarate with pyruvate. with homotaurine as the sole source of combined nitrogen. The colorimetric assay for a homotaurine-deaminating There was no growth (within 4 days) in the absence of dehydrogenase with dichlorophenol indophenol (DCPIP) homotaurine. Three pure cultures of bacteria were obtained, as electron acceptor was done as reported previously for and the most convenient isolate, strain N-APS2, was exam- taurine dehydrogenase (Bruggemann¨ et al., 2004), replacing ined in more detail. It was a motile, Gram-negative, non- taurine with homotaurine. Glutamate dehydrogenase (EC spore-forming rod, which was oxidase-positive and catalase- 1.4.1.3 and EC 1.4.1.4) was assayed photometrically positive and did not grow at 41 1C. A 1498-bp fragment of (Schmidt, 1974). the 16S rRNA gene was sequenced and found to share 99.5% 80

(a) identity of position with the gene in Stanier strain 383 (LMG 1 22485), in the multi-species Burkholderia cepacia complex. This tentative identiﬁcation was consistent with the taxo- nomic tests (above), and with the pattern of usage of carbon sources (Holt et al., 1994) (below), so the organism was termed Burkholderia sp. strain N-APS2. The organism 0.1 utilized glucose, glycerol, succinate, acetate, tartrate, 580 nm b-alanine, g-aminobutyrate, taurine, N-acetyltaurine and OD isethionate as sole sources of carbon and energy for growth, whereas homotaurine, N-methyltaurine, taurocholate, sulfoacetate and L-cysteate were not utilized. The organism 0.01 utilized acetamide, b-alanine, g-aminobutyrate, taurine, N-acetyltaurine and L-cysteate as sole sources of nitrogen 01020304050 for growth, but not N-methyltaurine or taurocholate. Time (h)

Formation of 3-sulfopropanoate from (b) 2.5 homotaurine during growth

Burkholderia sp. strain N-APS2 grew exponentially with 2.0 2 mM homotaurine in glucose–salts medium (Fig. 2a) with a maximum specific growth rate (m) of 0.15 h1. Utilization 1.5 of homotaurine was concomitant with growth (Fig. 2b) and the overall molar growth yield was 49 g protein (mol N)1. 1.0 This value was essentially the same as for growth with the ammonium ion [46 g protein (mol N)1] and indicates 0.5 Concentration (mM) quantitative utilization of homotaurine-nitrogen (Cook, 1987). The specific degradation rate of homotaurine was 0.0 calculated to be 0.9 mkat (kg protein)1. No detectable am- 0 20406080100 monium ion was found in the medium during growth (Fig. 2b). Protein (µg mL−1) Further, no additional sulfate (Fig. 2b) [and no sulfite (not shown)] was detected during growth, so it was presumed Fig. 2. A representative set of data on the growth of Burkholderia sp. that an organosulfonate was formed from homotaurine, as strain N-APS2 with 2 mM homotaurine as the sole source of combined observed in taurine metabolism (e.g. Weinitschke et al., nitrogen in 5-mM glucose-salts medium. Growth was assayed as turbidity (a) and quantified as protein (b). , turbidity; ’, homotaurine; 2005). Anion-chromatography of the culture medium m, 3-sulfopropanoate; , ammonium ion;H, sulfate. showed the formation of an unknown compound (Fig. 2b), which was not formed during growth with the ammonium detected in the presence of DCPIP as an electron acceptor. ion as source of combined nitrogen. Homotaurine was not a substrate for any putative pyruvate- The unknown product was identified initially by MALDI- coupled aminotransferase [e.g. (EC 2.6.1.77) or (EC TOF-MS: m/z = 153 = [M-H]1, which is consistent with 2.6.1.18)]. In contrast, activity of a homotaurine:2-oxoglu- M = 154 = C H O S for protonated sulfopropanoic acid. 3 6 5 tarate aminotransferase was detected in extracts of homo- The identification was confirmed when the putative 3- taurine-grown cells in the form of generation of glutamate sulfopropanoate was found to cochromatograph with and consumption of homotaurine. The formation of an authentic material on the anion-exchange column. Quanti- unknown aldehyde was also detected, firstly as a DIH- fication of the sulfopropanoate formed during growth derivative with retention-time (10.8 min) similar to that of showed its stoichiometric formation concomitant with derivatized sulfoacetaldehyde (10.7 min), and secondly as a growth and concomitant with disappearance of homotaur- derivative of o-aminobenzaldehyde. This compound was ine (Fig. 2b). presumed to be 3-sulfopropanal, which was confirmed by MALDI-TOF-MS in the negative-ion mode: m/z Homotaurine:2-oxoglutarate aminotransferase = 137 = [M-H]1,soM = 138, which is consistent with and the derived degradative pathway C3H6O4S, and the aldehyde with this formula is presumably The deamination of homotaurine could involve a dehydro- HC(O)–CH2–CH2–SO3H. The observed specific activity of genase, analogous to taurine dehydrogenase (EC 1.4.2.-) the transaminase [1.5 mkat (kg protein)1] is higher than (Bruggemann¨ et al., 2004), but no redox reaction was the minimum specific degradation rate calculated for 81 growing cells, and is sufficient to explain the growth rate. No Acknowledgements homotaurine transaminase activity was detected in extracts The authors are grateful to Marijke I. Baldock for data of ammonium-grown cells. generated in a practical course for advanced students and to Activity [0.6 mkat (kg protein)1] of NADP-coupled glu- Ulrich Groth, in whose laboratories the synthesis was under- tamate dehydrogenase (EC 1.4.1.4) was detected in extracts taken. The project was funded by the University of Konstanz of homotaurine-grown cells; there was negligible activity and the Deutsche Forschungsgemeinschaft (Co 206/7-1). with NAD1 (EC 1.4.1.2 and EC 1.4.1.3). Presumably, the amino group from homotaurine is released by glutamate dehydrogenase and assimilated into cell material. What is unusual in this context is that no detectable ammonium ion References is observed in the growth medium (Fig. 2b). Experience with Allen JA & Garrett MR (1971) Taurine in marine invertebrates. utilization of taurine-nitrogen showed that oxidative dea- Adv Mar Biol 9: 205–253. mination of an amino acid, directly or after transamination, Bruggemann¨ C, Denger K, Cook AM & Ruff J (2004) Enzymes was usually accompanied by transient excretion of the and genes of taurine and isethionate dissimilation in ammonium ion (Denger et al., 2004b; Weinitschke et al., Paracoccus denitrificans. Microbiology (Reading, UK) 150: 2005); strain N-APS2 obviously maintains a low intracellu- 805–816. lar concentration of free ammonium ion. Cook AM (1987) Biodegradation of s-triazine xenobiotics. FEMS The sole excretion product from homotaurine is 3-sulfo- Microbiol Rev 46: 93–116. propanoate (Fig. 1). The enzymic formation of sulfopropanal Cook AM & Denger K (2006) Metabolism of taurine in was shown above, so the formation of the 3-sulfopropanoate microorganisms: a primer in molecular diversity? Adv Exp is envisaged as a simple oxidation step, whose electron Med Biol 583: 3–13. acceptor has not been established, as yet (Fig. 1). Cook AM & Hutter¨ R (1981) s-Triazines as nitrogen sources for bacteria. J Agric Food Chem 29: 1135–1143.

Cook AM, Denger K & Smits THM (2006) Dissimilation of C3- Significance of homotaurine biotransformation sulfonates. Arch Microbiol 185: 83–90. Cunningham C, Tipton KF & Dixon HBF (1998) Conversion of The buffers mentioned in the Introduction are probably an taurine into N-chlorotaurine (taurine chloramine) and insignificant portion of the carbon flux through a sewage sulphoacetaldehyde in response to oxidative stress. Biochem J works, but with about 4% of the population suffering from 330: 939–945. alcohol dependence, the relevance of Camprals is signifi- 1 Denger K, Ruff J, Rein U & Cook AM (2001) Sulfoacetaldehyde cant, because it is dosed at 2 g day (Scott et al., 2005). The sulfo-lyase [EC 4.4.1.12] from Desulfonispora thiosulfatigenes: same order of magnitude is, apparently, usual for the dosage purification, properties and primary sequence. Biochem J 357: of other pharmaceutical sulfonates (‘Introduction’), so it is 581–586. important that these pharmaceuticals be degraded during Denger K, Ruff J, Schleheck D & Cook AM (2004a) Rhodococcus wastewater treatment, or at least inactivated. Complete opacus expresses the xsc gene to utilize taurine as a carbon degradation as a carbon source is obviously possible, source or as a nitrogen source but not as a sulfur source. but it seems to be slower than the process that is sketched Microbiology (Reading, UK) 150: 1859–1867. in Fig. 1. Denger K, Weinitschke S, Hollemeyer K & Cook AM (2004b) Dissimilation of organosulfonates with recovery of the Sulfoacetate generated by Rhodopseudomonas palustris from sulfonate moiety as sulfate (and transient sulfite) in the taurine. Arch Microbiol 182: 254–258. medium has a long history, but identification of the Denger K, Smits THM & Cook AM (2006) Genome-enabled exporters is only now becoming available (Denger et al., analysis of the utilization of taurine as sole source of carbon or 2006; Weinitschke et al., 2007). The transformation of nitrogen by Rhodobacter sphaeroides 2.4.1. Microbiology organosulfonates with the excretion of other organosulfo- (Reading, UK) 152: 3167–3174. Desomer J, Crespi M & Van Montagu M (1991) Illegitimate nates has also been observed for many years (e.g. Schleheck integration of non-replicative vectors in the genome of et al., 2004), but only recent work on the biotransformation Rhodococcus fascians upon electrotransformation as an of taurine (Denger et al., 2004b; Styp von Rekowski et al., insertional mutagenesis system. Mol Microbiol 5: 2115–2124. 2005; Weinitschke et al., 2005) gave access to readily Eichhorn E, van der Ploeg JR, Kertesz MA & Leisinger T (1997) quantifiable processes. The current work is the first example Characterization of a-ketoglutarate-dependent taurine of the quantitative transformation of a single xenobiotic dioxygenase from Escherichia coli. J Biol Chem 272: sulfonate to a single sulfonated product in a simple pathway. 23031–23036. the authors hope that it will lead to the identification of not Eichhorn E, van der Ploeg JR & Leisinger T (2000) Deletion only the genes encoding the enzymes but also to the nature analysis of the Escherichia coli taurine and alkanesulfonate of the transporters involved (Fig. 1). transport systems. J Bacteriol 182: 2687–2795. 82

Gerhardt P, Murray RGE, Wood WA & Krieg NR (1994) Methods alkylbenzenesulfonate (LAS) by defined pairs of heterotrophic for General and Molecular Bacteriology. American Society for bacteria. Appl Environ Microbiol 70: 4053–4063. Microbiology, Washington, DC. Schmidt E (1974) Glutamat-dehydrogenase UV-test. Methoden Gesellschaft Deutscher Chemiker (1996) German Standard der enzymatischen Analyse (Bergmeyer HU, ed), pp. 689–696. Methods for the Laboratory Examination of Water, Waste Water Verlag Chemie, Weinheim. and Sludge. Verlag Chemie, Weinheim. Scott LJ, Figgitt DP, Keam SJ & Waugh J (2005) Acamprosate: a Gorzynska AK, Denger K, Cook AM & Smits THM (2006) review of its use in the maintenance of abstinence in patients Inducible transcription of genes involved in taurine uptake with alcohol dependence. CNS Drugs 19: 445–464. and dissimilation by Silicibacter pomeroyi DSS-3T. Arch Sorbo¨ B (1987) Sulfate: turbidimetric and nephelometric Microbiol 185: 402–606. methods. Methods Enzymol 143: 3–6. Graham DE, Xu H & White RH (2002) Identification of Styp von Rekowski K, Denger K & Cook AM (2005) Isethionate as coenzyme M biosynthetic phosphosulfolactate synthase: a new a product from taurine during nitrogen-limited growth of family of sulfonate biosynthesizing enzymes. J Biol Chem 277: Klebsiella oxytoca Tau-N1. Arch Microbiol 183: 325–330. Tholey A, Wittmann C, Kang MJ, Bungert D, Hollemeyer K & 13421–13429. Heinzle E (2002) Derivatization of small biomolecules for Holt JG, Krieg NR, Sneath PHA, Staley JT & Williams ST (1994) optimized matrix-assisted laser desorption/ionization mass Bergey’s Manual of Determinative Bacteriology. Williams & spectrometry. J Mass Spectrom 37: 963–973. Wilkins, Baltimore. Thurnheer T, Kohler¨ T, Cook AM & Leisinger T (1986) Orthanilic Huxtable RJ (1992) Physiological actions of taurine. Physiol Rev acid and analogues as carbon sources for bacteria: growth 72: 101–163. physiology and enzymic desulphonation. J Gen Microbiol 132: Kertesz MA (2001) Bacterial transporters for sulfate and 1215–1220. organosulfur compounds. Res Microbiol 152: 279–290. Toyama S, Yasuda M, Miyasato K, Hirasawa T & Soda K (1974) A Kharasch MS & Brown HC (1940) Chlorinations with sulfuryl new assay method of o-amino acid aminotransferase with o- chloride. III (a) The peroxide-catalyzed chlorination of aminobenzaldehyde. Agric Biol Chem 38: 2263–2264. aliphatic and acid chlorides. (b) The photochemical Weinitschke S, Styp von Rekowski K, Denger K & Cook AM sulfonation of aliphatic acids. J Am Chem Soc 62: (2005) Sulfoacetaldehyde is excreted quantitatively by 925–929. Acinetobacter calcoaceticus SW1 during growth with taurine as Lie TL, Leadbetter JR & Leadbetter ER (1998) Metabolism of sole source of nitrogen. Microbiology (Reading, UK) 151: sulfonic acids and other organosulfur compounds by sulfate- 1285–1290. reducing bacteria. Geomicrobiol J 15: 135–149. Weinitschke S, Denger K, Smits THM, Hollemeyer K & Cook AM Mayer J, Denger K, Smits THM, Hollemeyer K, Groth U & Cook (2006) The sulfonated osmolyte N-methyltaurine is AM (2006) N-Acetyltaurine dissimilated via taurine by Delftia dissimilated by Alcaligenes faecalis and by Paracoccus versutus acidovorans NAT. Arch Microbiol 186: 61–67. with release of methylamine. Microbiology (Reading, UK) 152: Oja SS & Saransaari P (2006) Taurine 6. Springer, New York. 1179–1186. Sanger F (1945) The free amino groups of insulin. Biochem J 39: Weinitschke S, Denger K, Cook AM & Smits THM (2007) The 507–515. DUF81 protein TauE in Cupriavidus necator H16, a sulfite

Schleheck D, Knepper TP, Fischer K & Cook AM (2004) exporter in the metabolism of C2-sulfonates. Microbiology Mineralization of individual congeners of linear (Reading, UK) 153: 3055–3060.