R E S E A R C H L E T T E R
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, Department of Biology University of Konstanz, D-78457 Konstanz, Germany. Tel.:
149 7531 88 4247; fax:149 7531 88 2966;
E-mail: alasdair.cook@uni-konstanz.de
The sequence reported in this paper has been deposited in the GenBank database (accession no. EU035638).
Present address:Theo H.M. Smits, Agroscope Changins-W ¨adenswil ACW, Swiss Federal Research Station, Schloss, Postfach 185, CH-8820 W ¨adenswil, Switzerland.
First published online December 2007.
Editor: Christiane Dahl
Keywords
assimilation of homotaurine-nitrogen;
Burkholderia sp.; excretion of sulfopropanoate;
homotaurine:2-oxoglutarate aminotransferase.
Abstract
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 ofBurkholderiasp. was isolated which assimilated the amino group from homotaurine in a glucose–salts medium, and which released an organosulfonate, 3-sulfopropanoate, into the medium stoichio- metrically. 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.
Introduction
Taurine (2-aminoethanesulfonate) is a major organic solute in mammals, and with its derivatives, it is found in all vertebrates and a wide range of marine invertebrates (Allen
& Garrett, 1971; Huxtable, 1992). Major roles have been proposed for taurine in brain and muscle, and they have led to prolonged interest in the compound (e.g. Oja & Saransaari, 2006). Corresponding to the widespread occurrence of taurine and derivatives in eukaryotic organisms, the com- pound has been found to serve as a sole source of sulfur, nitrogen or carbon for a range of aerobic bacteria (Eichhorn et al., 1997, 2000; Cook & Denger, 2006; Mayeret al., 2006;
Weinitschke et al., 2006; see also Lie et al., 1998). The
assimilation of taurine-nitrogen was seldom accompanied by cleavage of the carbon–sulfonate bond, and organosulfo- nates were excreted quantitatively into the growth medium (Denger et al., 2004a, b; Styp von Rekowski et al., 2005;
Weinitschkeet al., 2005).
The many functions of taurine in mammals led pharma- cologists to examine the properties of homologues of taurine. Aminomethanesulfonate decays spontaneously in mineral medium for bacterial growth (K. Denger, unpub- lished data), but homotaurine (3-aminopropanesulfonate, APS) (Fig. 1) is stable and is a drug candidate (AlzhemedTM, CerebrilTM) for two major conditions (Alzheimer and stroke). The N-acetyl derivative of homotaurine (Camprals) is used to treat alcoholics (Scottet al., 2005).
Konstanzer Online-Publikations-System (KOPS) URL: http://www.ub.uni-konstanz.de/kops/volltexte/2008/6764/
URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-67645
Other derivatives of homotaurine are the amphoteric surfactant 3-[(3-cholamidopropyl)-dimethylammonio]-1- propanesulfonate (CHAPS) and the buffers cyclohexylami- nopropanesulfonate and 3-[N-morpholino]propanesulfonate (MOPS). Homotaurine and derivatives can, thus, be ex- pected in communal sewage works.
This study reports that homotaurine can be utilized by bacteria as a sole source of carbon or of nitrogen for growth, as indicated in a review (Cooket al., 2006). The assimilation of nitrogen was readily studied in pure culture. It involved a homotaurine:2-oxoglutarate aminotransferase and the excretion of 3-sulfopropanoate.
Materials and methods
Enrichment cultures, isolations, growth media, growth conditions and cell extracts
Enrichment cultures (3 mL in 30-mL tubes) were grown at 301C in a 50 mM potassium phosphate-buffered salts med- ium, pH 7.2, which contained 0.25 mM magnesium sulfate and trace elements (Thurnheeret al., 1986). Carbon-limited enrichments contained 20 mM ammonium chloride and 7.5 mM homotaurine; nitrogen-limited medium contained 2 mM homotaurine and 5 mM glucose, 7 mM succinate and 10 mM glycerol. Negative controls were done by omitting homotaurine, positive controls by replacing the compound with glucose, succinate and glycerol (carbon-limited cultures) or with ammonium chloride (nitrogen-limited cultures). The inocula were from garden soil, forest soil or activated sludge from the wastewater treatment plant in Konstanz, Germany. The preparation of inocula as well as
the enrichment and isolation procedures were described previously (Mayeret al., 2006). The isolateBurkholderiasp.
strain N-APS2 was deposited with the German Culture Collection DSMZ, Braunschweig, Germany, under the accession number DSM 19529.
Growth of Burkholderia sp. strain N-APS2 with 2 mM homotaurine as sole source of nitrogen (carbon source:
5 mM glucose) was followed in 100-mL cultures in 1-L Erlenmeyer flasks shaken at 301C in a water bath. Samples were taken at intervals to measure turbidity and to deter- mine the concentrations of protein, homotaurine, 3-sulfo- propanoate, and ammonium and sulphate ions.
Crude cell extracts for enzyme assays were prepared from cultures (50 mL) grown with homotaurine or ammonium chloride as sole nitrogen source. Harvesting and cell disrup- tion with a French pressure cell were done as described elsewhere (Dengeret al., 2004a).
Analytical methods
Growth was followed as turbidity at 580 nm or quantified as protein in a Lowry-type reaction (Cook & H¨utter, 1981).
Sulfate was quantified turbidimetrically as a suspension of BaSO4(S¨orbo, 1987). Sulfite was determined photometri- cally as the fuchsin-derivative (Dengeret al., 2001). Ammo- nium ion was assayed colorimetrically by the Berthelot reaction (Gesellschaft Deutscher Chemiker, 1996). Homo- taurine and glutamate were determined after derivatization with 2,4-dinitrofluorobenzene (DNFB) (Sanger, 1945) and separation by HPLC. Reactions in samples (50mL;
0–100 nmol amine) from growth experiments or enzyme assays were stopped by addition to 1 mL of 0.1 M NaHCO3,
Cell polymers
+H3N
SO3−
I
+H3N SO3− O SO3− O SO3−
O−
2-OG Glu H2O
H+ NADPH NH4+
NADP+ H2O
acceptorox acceptorred H2O
II
III
IV
O SO3−
O−
membrane V
3-Sulfopropanal
Homotaurine 3-Sulfopropanoate
Fig. 1. Presumed pathway of assimilatory deamination of homotaurine-nitrogen byBurkholderiasp. strain N-APS2. It is axiomatic that sulfonates require transport into the cell (Grahamet 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; Dengeret al., 2006;
Gorzynskaet 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.
and derivatization mixtures were shaken at 301C for 2 h after addition of 0.2 mL 3% (v/v) DNFB in ethanol. Deriva- tization was stopped by acidification with 50mL of concen- trated HCl, and after centrifugation (1 min, 11 000gat room temperature) the supernatant fluid was subject to analysis by reversed-phase HPLC (0–80% methanol gradient in 50 mM potassium phosphate buffer, pH 2.2 over 17 min: the detector was set at 360 nm). Aldehydes were deriva- tized with 2-(diphenylacetyl)indane-1,3-dione-1-hydrazone (DIH; 0.3 mg mL1) in acetonitrile (100mL sample plus 300mL DIH reagent) and then separated by reversed-phase HPLC (Cunninghamet al., 1998). A gradient of acetonitrile (30–80%) in 0.11 M NaClO4was applied over 17 min and the detection wavelength was 400 nm. A Eurospher 100–5 C18 column (125 mm3 mm; Knauer, Berlin, Germany) was used at a flow of 0.5 mL min1. Confirmation of the formation of an o-aldehyde in enzyme reactions was obtained colorimetrically by reaction witho-aminobenzal- dehyde, which does not react with oxo-groups (Toyama et al., 1974). Matrix-assisted laser-desorption ionization time-of-flight MS (MALDI-TOF-MS) in the negative-ion mode was used to confirm the identity of homotaurine (m/z
= 138 = [M-H]1), assay its presence, and identify the inter- mediate, 3-sulfopropanal, and the excretion product 3-sulfo- propanoate (Tholey et al., 2002). 3-Sulfopropanoate was quantified by ion chromatography with the conditions de- scribed for sulfoacetate (Denger et al., 2004b). Standard methods were used for the Gram reaction, and to assay catalase or cytochromec-oxidase activity (Gerhardtet al., 1994).
Chromosomal DNA was isolated as described elsewhere (Desomeret al., 1991). Amplification of the 16S rRNA gene, sequencing and sequence analysis were done as reported previously (Weinitschkeet al., 2006).
Enzyme assays
Homotaurine:2-oxoglutarate aminotransferase was assayed discontinuously at 301C as the 2-oxoglutarate-dependent formation of glutamate and the concomitant depletion of homotaurine. The reaction mixture contained (in a final volume of 1.0 mL): 40mmol Tris/HCl, pH 9.0, 1–10mmol homotaurine, 10mmol 2-oxoglutarate, 0.1mmol pyridoxal-5- phosphate and 0.25 mg protein with which the reaction was started. Samples were taken at intervals, as described above.
The possible presence of a pyruvate-coupled aminotrans- ferase was tested by replacing 2-oxoglutarate with pyruvate.
The colorimetric assay for a homotaurine-deaminating dehydrogenase with dichlorophenol indophenol (DCPIP) as electron acceptor was done as reported previously for taurine dehydrogenase (Br¨uggemannet al., 2004), replacing taurine with homotaurine. Glutamate dehydrogenase (EC 1.4.1.3 and EC 1.4.1.4) was assayed photometrically (Schmidt, 1974).
Chemicals
The sodium salt of 3-sulfopropanoate was synthesized in a modification of the published method (Kharasch & Brown, 1940). The key alteration was the addition of the radical starter azoisobutyronitrile [2,20-azobis(2-methylpropionitrile)] to the mixture of sulfonyl chloride (0.25 mol) in an excess of propanoic acid (0.5 mol) under reflux and illumination from a 200 W tungsten lamp. Residual propanoic acid was removed under vacuum. Further unreacted material and putative by- products were removed by extraction with 400 mL light petroleum. The white crystals of 3-sulfopropanoic acid (yield 16%; lit.: 37%) were identified by nuclear magnetic resonance (NMR) and shown to contain o1% impurity with the educts. Analysis by 1H-NMR (400 MHz, D6–DMSO) gave the following data: d [ppm] = 2.49 (2H; t, 3J = 8.2 Hz, –CH2–COOH), 2.75 (2H;t, 3J = 8.0 Hz, CH2–SO3H), 12.78 (2H; s, –COOH, –SO3H). Analysis by13C-NMR (100 MHz, D6–DMSO) gave the following data:[ppm] = 30.4 (–CH2–), 46.9 (–CH2–SO3H), 173.2 (–COOH). This product, however, was oxygen-sensitive and had to be stored under nitrogen, so no melting point was determined. Neutralization of the free acid with NaOH resulted in the disodium salt that was stable in air; this compound decomposed at about 2981C. Elemental analysis of the disodium salt gave data, from which the composition of the crystals could be concluded as C3H4Na2O5S1/2 H2O.
Commercial chemicals were of the highest purity avail- able, and they were purchased from Fluka, Merck, Roth, Serva or Sigma.
Results and discussion
Enrichment cultures and the identification of Burkholderia sp. strain N-APS2
Enrichment cultures from forest soil and sewage sludge reproducibly yielded cultures, which grew slowly with homotaurine as the sole source of carbon and energy for growth, and which were prokaryotes. Cultures reproducibly required 4–5 days to convert 7.5 mM homotaurine quanti- tatively to cell material, and the ammonium and sulfate ions.
This slow growth made the authors to concentrate on the assimilation of homotaurine-nitrogen.
Enrichment cultures from soil and sewage sludge yielded cultures of prokaryotes whose subcultures grew in 2 days with homotaurine as the sole source of combined nitrogen.
There was no growth (within 4 days) in the absence of homotaurine. Three pure cultures of bacteria were obtained, and the most convenient isolate, strain N-APS2, was exam- ined in more detail. It was a motile, Gram-negative, non- spore-forming rod, which was oxidase-positive and catalase- positive and did not grow at 411C. A 1498-bp fragment of the 16S rRNA gene was sequenced and found to share 99.5%
identity of position with the gene in Stanier strain 383 (LMG 22485), in the multi-speciesBurkholderia cepaciacomplex.
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 utilized glucose, glycerol, succinate, acetate, tartrate, b-alanine, g-aminobutyrate, taurine, N-acetyltaurine and isethionate as sole sources of carbon and energy for growth, whereas homotaurine,N-methyltaurine, taurocholate, sul- foacetate and L-cysteate were not utilized. The organism utilized acetamide, b-alanine, g-aminobutyrate, taurine, N-acetyltaurine and L-cysteate as sole sources of nitrogen for growth, but notN-methyltaurine or taurocholate.
Formation of 3- sulfopropanoate from homotaurine during growth
Burkholderia sp. strain N-APS2 grew exponentially with 2 mM homotaurine in glucose–salts medium (Fig. 2a) with a maximum specific growth rate (m) of 0.15 h1. Utilization of homotaurine was concomitant with growth (Fig. 2b) and the overall molar growth yield was 49 g protein (mol N)1. This value was essentially the same as for growth with the ammonium ion [46 g protein (mol N)1] and indicates quantitative utilization of homotaurine-nitrogen (Cook, 1987). The specific degradation rate of homotaurine was calculated to be 0.9 mkat (kg protein)1. No detectable am- monium ion was found in the medium during growth (Fig. 2b).
Further, no additional sulfate (Fig. 2b) [and no sulfite (not shown)] was detected during growth, so it was presumed that an organosulfonate was formed from homotaurine, as observed in taurine metabolism (e.g. Weinitschke et al., 2005). Anion-chromatography of the culture medium showed the formation of an unknown compound (Fig. 2b), which was not formed during growth with the ammonium ion as source of combined nitrogen.
The unknown product was identified initially by MALDI- TOF-MS: m/z= 153 = [M-H]1, which is consistent with M= 154 = C3H6O5S for protonated sulfopropanoic acid.
The identification was confirmed when the putative 3- sulfopropanoate was found to cochromatograph with authentic material on the anion-exchange column. Quanti- fication of the sulfopropanoate formed during growth showed its stoichiometric formation concomitant with growth and concomitant with disappearance of homotaur- ine (Fig. 2b).
Homotaurine:2-oxoglutarate aminotransferase and the derived degradative pathway
The deamination of homotaurine could involve a dehydro- genase, analogous to taurine dehydrogenase (EC 1.4.2.-) (Br¨uggemann et al., 2004), but no redox reaction was
detected in the presence of DCPIP as an electron acceptor.
Homotaurine was not a substrate for any putative pyruvate- coupled aminotransferase [e.g. (EC 2.6.1.77) or (EC 2.6.1.18)]. In contrast, activity of a homotaurine:2-oxoglu- tarate aminotransferase was detected in extracts of homo- taurine-grown cells in the form of generation of glutamate and consumption of homotaurine. The formation of an unknown aldehyde was also detected, firstly as a DIH- derivative with retention-time (10.8 min) similar to that of derivatized sulfoacetaldehyde (10.7 min), and secondly as a derivative of o-aminobenzaldehyde. This compound was presumed to be 3-sulfopropanal, which was confirmed by MALDI-TOF-MS in the negative-ion mode: m/z
= 137 = [M-H]1, so M= 138, which is consistent with C3H6O4S, and the aldehyde with this formula is presumably HC(O)–CH2–CH2–SO3H. The observed specific activity of the transaminase [1.5 mkat (kg protein)1] is higher than the minimum specific degradation rate calculated for
0 20 40 60 80 100
0.0 0.5 1.0 1.5 2.0 2.5
Concentration (mM)
Protein (µg mL−1)
0 10 20 30 40 50
0.01 0.1 (a) 1
(b) OD580 nm
Time (h)
Fig. 2.A representative set of data on the growth ofBurkholderiasp.
strain N-APS2 with 2 mM homotaurine as the sole source of combined nitrogen in 5-mM glucose-salts medium. Growth was assayed as turbidity (a) and quantified as protein (b)., turbidity;’, homotaurine;
m, 3-sulfopropanoate;, ammonium ion;H, sulfate.
growing cells, and is sufficient to explain the growth rate. No homotaurine transaminase activity was detected in extracts of ammonium-grown cells.
Activity [0.6 mkat (kg protein)1] of NADP-coupled glu- tamate dehydrogenase (EC 1.4.1.4) was detected in extracts of homotaurine-grown cells; there was negligible activity 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 is observed in the growth medium (Fig. 2b). Experience with utilization of taurine-nitrogen showed that oxidative dea- mination of an amino acid, directly or after transamination, was usually accompanied by transient excretion of the ammonium ion (Denger et al., 2004b; Weinitschke et al., 2005); strain N-APS2 obviously maintains a low intracellu- lar concentration of free ammonium ion.
The sole excretion product from homotaurine is 3-sulfo- propanoate (Fig. 1). The enzymic formation of sulfopropanal was shown above, so the formation of the 3-sulfopropanoate is envisaged as a simple oxidation step, whose electron acceptor has not been established, as yet (Fig. 1).
Significance of homotaurine biotransformation The buffers mentioned in the Introduction are probably an insignificant portion of the carbon flux through a sewage works, but with about 4% of the population suffering from alcohol dependence, the relevance of Campralsis signifi- cant, because it is dosed at 2 g day1(Scottet al., 2005). The same order of magnitude is, apparently, usual for the dosage of other pharmaceutical sulfonates (‘Introduction’), so it is important that these pharmaceuticals be degraded during wastewater treatment, or at least inactivated. Complete degradation as a carbon source is obviously possible, but it seems to be slower than the process that is sketched in Fig. 1.
Dissimilation of organosulfonates with recovery of the sulfonate moiety as sulfate (and transient sulfite) in the medium has a long history, but identification of the exporters is only now becoming available (Denger et al., 2006; Weinitschke et al., 2007). The transformation of organosulfonates with the excretion of other organosulfo- nates has also been observed for many years (e.g. Schleheck et al., 2004), but only recent work on the biotransformation of taurine (Dengeret al., 2004b; Styp von Rekowskiet al., 2005; Weinitschke et al., 2005) gave access to readily quantifiable processes. The current work is the first example of the quantitative transformation of a single xenobiotic sulfonate to a single sulfonated product in a simple pathway.
the authors hope that it will lead to the identification of not only the genes encoding the enzymes but also to the nature of the transporters involved (Fig. 1).
Acknowledgements
The authors are grateful to Marijke I. Baldock for data generated in a practical course for advanced students and to Ulrich Groth, in whose laboratories the synthesis was under- taken. The project was funded by the University of Konstanz and the Deutsche Forschungsgemeinschaft (Co 206/7-1).
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