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Department of Biology, The University, D-78457 Konstanz, Germany
Correspondence
Alasdair M. Cook
alasdair.cook{at}uni-konstanz.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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(Microbiology 150, 805–816) on the basis of a partial genome sequence. In the present study, R. sphaeroides 2.4.1 was found to grow exponentially with taurine as the sole source of carbon and energy for growth. When taurine was the sole source of nitrogen in succinate-salts medium, the taurine was rapidly degraded, and most of the organic nitrogen was excreted as the ammonium ion, which was then utilized for growth. Most of the enzymes involved in dissimilation, taurine dehydrogenase (TDH), sulfoacetaldehyde acetyltransferase (Xsc) and phosphate acetyltransferase (Pta), were found to be inducible, and evidence for transcription of the corresponding genes (tauXY, xsc and pta), as well as of tauKLM, encoding the postulated TRAP transporter for taurine, and of tauZ, encoding the sulfate exporter, was obtained by reverse-transcription PCR. An additional branch of the pathway, observed by Novak et al. (2004) (Microbiology 150, 1881–1891) in R. sphaeroides TAU3, involves taurine : pyruvate aminotransferase (Tpa) and a presumptive ABC transporter (NsbABC). No evidence for a significant role of this pathway, or of the corresponding alanine dehydrogenase (Ald), was obtained for R. sphaeroides 2.4.1. The anaplerotic pathway needed under these conditions in R. sphaeroides 2.4.1 seems to involve malyl-CoA lyase, which was synthesized inducibly, and not malate synthase (GlcB), whose presumed gene was not transcribed under these conditions.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) is ‘a phylogenetically ancient compound with a disjunct distribution in the biosphere’ (Huxtable, 1992). Absent in plants, it is a major organic solute in mammals [1 g (kg wet wt)–1], which excrete it (largely in urine), and it is found free or derivatized in all vertebrates, many marine creatures, algae and bacteria (e.g. Allen & Garrett, 1971; Vollrath et al., 1990; Huxtable, 1992; Yin et al., 2000; Abraham et al., 2004). Spiders secrete molar concentrations of N-acetyltaurine on their webs (Vollrath et al., 1990). The widespread supply of extracellular (and intracellular) taurine is presumed to explain the large number of terrestrial and marine bacteria able to utilize the compound as sole source of carbon for aerobic growth, as a source or sink of electrons in respirations, and in fermentations or in photoheterotrophic growth (e.g. Lie et al., 1998; Cook & Denger, 2002; Denger et al., 2004a; Novak et al., 2004). The compound can also serve as a sole source of sulfur for growth (e.g. Kertesz, 2000), and a range of phenomena can be involved when organisms utilize the compound as a sole source of nitrogen; these include deamination without desulfonation, not always concomitant with substrate utilization (Denger et al., 2004b; Styp von Rekowski et al., 2005; Weinitschke et al., 2005), and utilization concomitant with taurine carbon (Denger et al., 2004a). Two general pathways for the dissimilation of taurine have been hypothesized (Cook & Denger, 2006), one of which has received significant experimental support in Silicibacter pomeroyi DSS-3, especially for the genes encoding an ATP-binding cassette transporter (TauABC), taurine : pyruvate aminotransferase (Tpa) and alanine dehydrogenase (Ald) (analogous to Fig. 1a, bottom left), as well as sulfoacetaldehyde acetyltransferase (Xsc) and phosphate acetyltransferase (Pta) (Gorzynska et al., 2006).
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, Table 1) was first formulated from data in the partial genome sequence of the facultative phototroph Rhodobacter sphaeroides 2.4.1 (Brüggemann et al., 2004), which now serves as a model organism that represents several genomes or sequences of clusters of taurine-related genes. The hypothesis involves in part a gene cluster now known to be encoded on plasmid B of R. sphaeroides 2.4.1 (Fig. 1b). The cluster contains the genes for a putative regulator (tauR) and a tripartite ATP-independent (TRAP) transport system (tauKLM), for which there is currently no experimental support. The tauXY genes are excellent candidates to encode taurine dehydrogenase (TDH), but this is still unproven (Weinitschke et al., 2006). The xsc gene of R. sphaeroides 2.4.1 has a high level (87 %) of identity to the orthologue encoding a purified Xsc in the phylogenetically related Paracoccus denitrificans NKNIS (Brüggemann et al., 2004). The tauZ gene is presumed to encode a sulfate exporter (Rein et al., 2005). The group of pta genes is well known, but the enzyme is seldom characterized (Lawrence et al., 2006). In addition to the functions ascribed to the gene products from the cluster, an unknown exporter of the ammonium ion released by TDH is needed, and an ammonium/methylammonium transporter, possibly AmtB, has been proposed (Cook & Denger, 2006; Gorzynska et al., 2006). The oxidation of sulfite to sulfate is also postulated, but no genes (sorAB) encoding the known sulfite dehydrogenase (Kappler et al., 2000) are present on the genome of R. sphaeroides 2.4.1. Whereas the Krebs cycle is known in the organism, the anaplerotic sequence is not (Filatova et al., 2005; Meister et al., 2005). The hypothesis (Fig. 1a) is further complicated by the fact that the genome of R. sphaeroides 2.4.1 includes a putative tpa gene (Fig. 1b), whose derived product shares 75 % identity of position with a confirmed Tpa in the partially sequenced genome of Rhodobacter capsulatus SB1003, in which the enzyme is involved in the assimilation of taurine sulfur (Masepohl et al., 2001; see also Laue et al., 2006). The genes located upstream of the putative tpa (Fig. 1b) represent a putative nitrate/sulfonate/bicarbonate ABC transporter (NsbABC; NsbA has <20 % identity with any known TauA). A different isolate of R. sphaeroides, strain Tau3, is reported to express both Xsc and Tpa under conditions of photoheterotrophic utilization of taurine (Novak et al., 2004); the genome of R. sphaeroides 2.4.1 potentially encodes, besides the candidate Tpa, a candidate Ald (Table 1), which would allow the regeneration of pyruvate (Fig. 1a) if the Tpa were active. This latter situation is standard in organisms which degrade taurine via Tpa (Laue & Cook, 2000a, b; Denger et al., 2004a; Gorzynska et al., 2006). The multiple metabolic possibilities involving sequenced genes can be readily studied with a combination of analyses of transcription by reverse-transcription (RT-) PCR and enzymic analyses.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) minimal medium. It contained 50 mM potassium phosphate buffer, pH 7.2, 0.25 mM MgSO4, 9 mM NaCl, 1.2 mM MgCl2, 0.2 mM CaCl2, trace elements and a vitamin solution (Thurnheer et al., 1986). Different combinations of sources of carbon and nitrogen for growth were used: 10 mM taurine as sole and limiting source of carbon with 2 mM NH4Cl [taurine(carbon)-ammonium medium]; 2 mM taurine as nitrogen source (a growth-limiting concentration) with 9 mM succinate [taurine(nitrogen)-succinate medium]; 2 mM NH4Cl with 9 mM succinate (succinate-ammonium medium). Purity was checked by streaking on Luria–Bertani agar plates and observing homogeneous, red-coloured colonies.
Precultures (5 ml) were grown in 30 ml screw-cap tubes in a roller. Growth experiments, or cultures for harvesting and preliminary enzyme assays, were done with 50 ml cultures in 300 ml Erlenmeyer flasks on a rotary shaker. Samples were taken in intervals to measure OD580, to quantify protein (OD580 1
205 mg protein l–1) and to determine the concentrations of substrate and products. Cells for the preparation of total RNA were grown in the required selective medium and harvested in the mid-exponential phase of growth (OD580 0.3–0.7) by centrifugation at 5000 g; RNA was extracted immediately. Storage of intact cells at –70 °C before RNA extraction resulted in complete loss of mRNA.
Preparation of cells grown with taurine as a nitrogen source for RNA extraction or enzyme determination involved a growth phase in taurine(nitrogen)-succinate-salts medium followed by renewed induction by addition of taurine (to 2 mM) 3–10 h before harvesting. Cells for enzyme purification were grown with taurine as a carbon source at the 1-litre scale in 5-litre Erlenmeyer flasks. Disruption of cells and the preparation of crude extracts, a membrane fraction and a soluble fraction were as described elsewhere (Weinitschke et al., 2006).
Enzyme assays and protein separation.
Taurine dehydrogenase (TDH) was measured photometrically with dichlorophenol indophenol as the electron acceptor as described previously (Brüggemann et al., 2004). Taurine : pyruvate aminotransferase (Tpa) was assayed discontinuously as the pyruvate-dependent disappearance of taurine concomitant with the formation of alanine (Brüggemann et al., 2004). Alanine dehydrogenase (Ald) was routinely measured photometrically as the reduction of NAD+ (Laue & Cook, 2000a); S. pomeroyi DSS-3 (Denger et al., 2006) served as a positive control. Sulfoacetaldehyde acetyltransferase (Xsc) was assayed by GC as the ThDP- and phosphate-dependent release of acetate after acidification to hydrolyse the acetyl phosphate formed, or assayed as the formation of sulfite (Ruff et al., 2003). Phosphate acetyltransferase (Pta) was assayed photometrically as the HS-CoA-dependent formation of acetyl-CoA (Bergmeyer et al., 1983). Sulfite dehydrogenase was assayed with potassium ferricyanide or beef cytochrome c (Reichenbecher et al., 1999) as the electron acceptor, and Sinorhizobium meliloti Rm1021 (Brüggemann et al., 2004) and Delftia acidivorans NAT (Mayer et al., 2006) were used as positive controls. Other putative electron acceptors, O2, NAD(P), FAD and nitrate, were tested, as was the possibility of an AMP- or ADP-dependent oxidation with ferricyanide or cytochrome c (Hagen & Nelson, 1997). Isocitrate lyase and malate synthase were assayed as described by Dixon & Kornberg (1959), and D. acidovorans NAT (Mayer et al., 2006) served as positive control. Malyl-CoA lyase was assayed as described elsewhere (Meister et al., 2005). Anion-exchange and hydrophobic interaction chromatography were done with soluble fractions free of nucleic acids as described elsewhere (Ruff et al., 2003; Rein et al., 2005).
Analytical methods.
Growth was followed as turbidity (OD580) and quantified as protein in a Lowry-type reaction (Cook & Hütter, 1981). Taurine and alanine were derivatized with dinitrofluorobenzene and subjected to separation by HPLC (Denger et al., 1997). Sulfite was quantified as the fuchsin derivative as described elsewhere (Denger et al., 2001). Sulfate was determined turbidimetrically as a suspension of BaSO4 (Sörbo, 1987). Ammonium ion was assayed colorimetrically by the Berthelot reaction (Gesellschaft Deutscher Chemiker, 1996). Protein in extracts was assayed by protein-dye binding (Bradford, 1976). Denatured proteins were separated in 12 % SDS-PAGE gels and stained with Coomassie Brilliant Blue R250 (Laemmli, 1970).
Molecular methods.
Oligonucleotides were synthesized by Microsynth (Balgach, Switzerland). Taq DNA polymerase and M-MuLV reverse transcriptase were from MBI Fermentas and they were used as specified by the supplier. Chromosomal DNA was isolated from bacteria as described by Desomer et al. (1991). Total RNA was isolated using the EZNA bacterial RNA kit (Peqlab Biotechnologie), and contaminant DNA was removed with RNase-free DNase (Qiagen). The RNA was tested for residual DNA before reverse transcription (RT) by PCR using the primer set RsXscF-RsXscR. The reverse PCR primers listed in Table 2 were used for RT-PCR reactions. Subsequent PCR reactions were done as described previously (Innis et al., 1990) using chromosomal DNA of R. sphaeroides 2.4.1 as a positive control. PCR products were visualized on 1 % or 1.5 % agarose gels according to standard methods (Sambrook et al., 1989). The GeneRuler DNA ladder mix (MBI Fermentas) was used as molecular marker.
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) at NCBI. The Lasergene package (DNAStar) was used for routine DNA analyses in silico. Primers for RT and PCR were designed using the program Amplify (version 1.2). |
RESULTS |
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). The utilization of taurine and the excretion of both ammonium and sulfate ions were concomitant with growth (Fig. 2b). The yield of sulfate at the end of growth was 11 mM. No sulfite was detected. A net increase of about 9 mM ammonium ion was found in the medium at the end of growth, and we calculated there to be about 2 mM combined nitrogen in cell material in the medium. The molar growth yield, 6 g protein (mol C)–1, represented quantitative utilization of the carbon source (Cook, 1987). The data were interpreted to represent a complete mass balance for the carbon, nitrogen and sulfur moieties in taurine.
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). There was no growth in the absence of combined nitrogen. Taurine was rapidly metabolized, and it disappeared before 25 % of the total growth occurred (Fig. 3b). Over the same period, 2 mM sulfate was formed, so there was mass balance for sulfur; no sulfite was detected (Fig. 3b). Taurine nitrogen was released as the ammonium ion, and the organism then grew with this ammonium; growth was then concomitant with ammonium utilization (Fig. 3b). The overall molar growth yield was 70 g protein (mol taurine-N)–1. The same growth yield [about 70 g protein (mol N)–1] was observed in succinate-ammonium medium (not shown), which indicated quantitative assimilation of nitrogen into cell material (e.g. Cook, 1987). The specific growth rate in the presence of taurine was 0.08 h–1; a lower growth rate (maximally 0.03 h–1) was observed when the excreted ammonium ion was the sole source of nitrogen (Fig. 3).
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). No transcript of any of the corresponding genes was detected in succinate-ammonium-grown cells (Table 3). An amplicon of each of these genes, but especially of tauL and tauM, was obtained from taurine(carbon)-ammonium-grown cells (Table 3). Amplicons of tauK, tauL and tauM could also be detected in taurine(nitrogen)-succinate-grown cells (Table 3). This is the first direct evidence to show that the tauKLM genes are inducibly transcribed, and that their gene products are presumably relevant to taurine metabolism.
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) but not in succinate-ammonium-grown cells, so these enzymes were considered to be inducible. Inducible transcription of xsc and pta was detected in these cells (Table 3); inducible synthesis of TDH, Xsc and Pta, with the corresponding transcripts, was observed in taurine(nitrogen)-succinate-grown cells (Table 3). As in previous work (Brüggemann et al., 2004; Weinitschke et al., 2006), TDH was found to be associated with the particulate fraction of cell extracts, whereas Xsc and Pta were found in the soluble fraction. A transcript from the tauXY genes was detected in taurine(carbon)-ammonium-grown cells and in the taurine(nitrogen)-succinate-grown cells, but not in succinate-ammonium-grown cells (Table 3); the amplicon overlapped the tauX and tauY genes, so the signal indicates that these genes are co-transcribed. These data were considered to be further support for the postulate that tauXY encodes TDH (Brüggemann et al., 2004; Weinitschke et al., 2006).
Transamination of taurine was detected, but activity was higher in extracts of succinate-ammonium-grown cells than in extracts of taurine(carbon)-ammonium-grown or taurine(nitrogen)-succinate-grown cells (Table 3). Correspondingly, the transcript from the putative tpa gene was detected in cells from all cultures (Table 3). Further, no Ald activity was detected in extracts of cells grown in the presence of taurine (Table 2), so negligible amounts of pyruvate could be regenerated from alanine. It was concluded that the putative Tpa and Ald made a negligible contribution to taurine dissimilation.
Sulfite dehydrogenase must have been present, because no sulfite was observed during growth (Figs 2b and 3b), but no enzyme activity was detected in cell extracts. The acceptors tested for the direct oxidation (Kappler & Dahl, 2001) were cytochrome c and ferricyanide, so neither the known sulfite dehydrogenase, SorAB (Kappler et al., 2000), nor the cytochrome c-independent enzyme (Reichenbecher et al., 1999; Gorzynska et al., 2006; Weinitschke et al., 2006), was detected. The acceptors tested for the AMP-dependent (indirect) pathway of sulfite oxidation (Hagen & Nelson, 1997; Kappler & Dahl, 2001) were ferricyanide and cytochrome c. The absence of SorAB (and SoxCD) could be anticipated from the genome sequence, and the indirect pathway is oxygen-sensitive (Schiffer et al., 2006), so it is unclear whether the undefined ferricyanide-coupled enzyme (Reichenbecher et al., 1999), or a novel enzyme, catalyses the oxidation of sulfite.
The product of sulfite dehydrogenase is sulfate, which is believed to be exported from the cell by TauZ (Rein et al., 2005). A strong transcript of the tauZ gene was detected in taurine(carbon)-ammonium-grown cells, but not in succinate-ammonium-grown cells (Table 3). An amplicon overlapping the tauZ and pta genes was also obtained when using primer RsPtaR for RT (Fig. 4), so these neighbouring genes were co-transcribed. No amplicon was obtained for a transcript representing tauX to xsc (using primers RsTauXF-RsXscR), or of a transcript representing xsc to tauZ (using primers RsXscF-RsTauZR); this suggests separate transcripts for tauXY, for xsc and for tauZ-pta in R. sphaeroides 2.4.1.
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), as observed and cited by, for example, Filatova et al. (2005) and anticipated from the absence of the corresponding gene in the genome. The apparent activity of malate synthase was detected at a high level in taurine(carbon)-ammonium-grown cells, but at much lower levels in taurine(nitrogen)-succinate grown cells and in succinate-ammonium grown cells (Table 3). Malate synthase is one enzyme in Ivanovsky's anaplerotic pathway (e.g. Filatova et al., 2005), but Meister et al. (2005) have shown this to result from the combined activity of malyl-CoA lyase (EC 4.1.3.24) and of a putative malyl-CoA hydrolase. We could confirm the presence of malyl-CoA lyase in crude extracts (Table 3) and detect a putative malyl-CoA hydrolase activity in fractions of the extract (not shown), analogous to the data of Meister et al. (2005). Correspondingly, the gene annotated to encode malate synthase, RSP_1980, was not transcribed under the conditions tested (Table 3). Proteins in the extracts of cells grown in succinate-ammonium medium or in taurine(carbon)-ammonium medium were separated by SDS-PAGE (not shown). One inducible protein only could be observed in extracts of taurine(carbon)-ammonium-grown cells and taurine(nitrogen)-succinate-grown cells. The protein had a molecular mass of about 63 kDa, i.e that of Xsc. Soluble extract of taurine(carbon)-ammonium-grown cells was subjected to anion-exchange chromatography and hydrophobic interaction chromatography, and the fraction containing Xsc activity, which was ThDP-dependent, was the essentially pure 63 kDa protein. The activity of Pta was lost rapidly and traces were found in many fractions from the anion exchanger. A single fraction with Pta activity was obtained from soluble extract on the hydrophobic interaction column, but the losses in activity prevented both further chromatography, and useful analyses by SDS-PAGE.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) has been confirmed, and growth was concomitant with substrate utilization (Fig. 2a, b). Similarly, the idea was confirmed that the organism would utilize taurine as a sole source of nitrogen for growth (Fig. 3a, b). The assimilation of taurine-nitrogen sometimes involves utilization of the compound concomitant with growth, as found in Acinetobacter calcoaceticus SW1, Alcaligenes faecalis MT1 and Rhodococcus opacus ISO-5 (Denger et al., 2004a; Weinitschke et al., 2005, 2006), and sometimes with excretion and subsequent utilization of the ammonium ion as in Rhodopseudomonas palustris CGA009 (Denger et al., 2004b), as shown in Fig. 3(b). When the organism can utilize taurine as sole source of both carbon and nitrogen, the same pathway is obviously involved, via Tpa (and Ald) in R. opacus ISO-5 (Denger et al., 2004a) and via TDH in R. sphaeroides 2.4.1 (Table 3). We have no explanation for the excretion or non-excretion of the ammonium ion during growth of different organisms with limiting amounts of taurine.
One key, novel aspect of the degradative pathway proposed by Brüggemann et al. (2004) is the TRAP transporter, TauKLM. We now present the first experimental evidence (Table 3) that the genes are transcribed, and presumably translated. The assumption of function is the sequence similarity to TRAP transporters for dicarboxylates (DctPQM) from Rhodobacter capsulatus (e.g. Forward et al., 1997): TauK and DctP share 22 % identity. Gene orthologues of tauKLM have now been found in the genomes of several organisms which contain the xsc gene and do, or can be expected to, dissimilate taurine [two other members of the Rhodobacterales (e.g. Brüggemann et al., 2004), Desulfotalea psychrophila LSv54 (R. Rabus, personal communication) and three untested 
-Proteobacteria (Vibrionales)]. Transport of taurine via a TRAP transporter thus seems to be widespread.
The degradative pathway for taurine via TDH (Brüggemann et al., 2004) was detected (Table 3). There was no direct proof that tauXY was involved in the pathway, but the inducible co-transcription of the two genes supports a role for the gene products, which we consider to be TDH. Heterologous expression of the tauXY genes has not yet been successful (T. H. M. Smits, unpublished), and solublization of the membrane fraction led to loss of enzyme activity (K. Denger, unpublished). The identity of the cytochrome c which accepts electrons from TDH, is also unknown.
The prediction of a role for Tpa in taurine dissimilation by R. sphaeroides 2.4.1, from both sequence analysis (see Introduction) and enzyme assays in R. sphaeroides Tau3 (Novak et al., 2004), was not confirmed. Some aminotransferase activity could, indeed, be detected, but at lower levels than in succinate-ammonium-grown cells (Table 3). We presume the taurine : pyruvate aminotransferase activity to represent the side reaction of a different aminotransferase. Work with the Tpa from Bilophila wadsworthia RZATAU showed that the sequences of some Tpa orthologues cluster together, whereas others are spread amongst class III aminotransferases of different or unknown functions (Laue et al., 2006). The hypothetical ABC transporter (‘NsbABC’; Fig. 1b, Table 3) encoded upstream of this transaminase is a member of a large group of uncharacterized ABC transporters closely related to TC 3.A.1.12/16/17.– which are unfortunately termed TauABC, identical to the TauABC transporters involved in taurine metabolism (TC 3.A.1.17.1) (Eichhorn et al., 2000; Gorzynska et al., 2006). TauA and NsbA share <20 % sequence identity. It is unlikely that ‘NsbABC’ (Table 3) plays any role in taurine metabolism in R. sphaeroides 2.4.1.
Xsc is the only enzyme in the pathway which has been regularly purified (Denger et al., 2001, 2004a; Ruff et al., 2003; Brüggemann et al., 2004). In the present paper, the enzyme could be purified to near homogeneity in two steps. Indeed, the xsc gene is our marker for the presence of the degradation of a C2-sulfonate in a genome (see Cook & Denger, 2002), and some 25 organisms have been confirmed to express the enzyme (e.g. Gorzynska et al., 2006), or at least grow with a C2-sulfonate (K. Denger, unpublished), and the number is steadily increasing.
Pta, in contrast, frequently presents a problem, because the enzyme is labile under our standard conditions (e.g. Weinitschke et al., 2006), a phenomenon which seems to be widespread (Lawrence et al., 2006). In the present study, the enzyme rapidly lost activity and could not be satisfactorily separated by anion-exchange chromatography. Higher stability was attained for hydrophobic interaction chromatography, but the very low amount of partially purified protein did not allow an estimation of the molecular mass of the enzyme.
There will be several fates for the acetyl-CoA formed by Pta. A minor portion will flow to fatty acid synthesis. A major portion will be oxidized via the Krebs cycle, and a significant portion will flow through an anaplerotic sequence to maintain the supply of oxaloacetate for the Krebs cycle. The nature of this anaplerotic pathway is unclear, because R. sphaeroides has been known for some 40 years to lack one key enzyme of the glyoxylate shunt, isocitrate lyase. Ivanovsky's group, working in crude extracts, has postulated a citramalate cycle, which involves malate synthase (e.g. Filatova et al., 2005). Fuchs' group has started to clarify this pathway by purifying enzymes, and a recent article shows that the apparent measurement of malate synthase represents the combined activity of malyl-CoA lyase and a malyl-CoA hydrolase (Meister et al., 2005). Our data (e.g. Table 3) support this latter observation. Whatever the nature of the complete anaplerotic pathway (see Alber et al., 2006), the apparent malate synthase in crude extract shows the cycle to be present at high levels in the taurine(carbon)-ammonium-grown cells, but only in traces in taurine(nitrogen)-succinate-grown cells (Table 3). Presumably, when taurine is supplying nitrogen in the presence of a C4 carbon source in excess (in our case succinate), the anaplerotic sequence is not necessary, and is regulated independently of the operation of the taurine degradative pathway. The gene RSP_1980, annotated as malate synthase (glcB), is not transcribed under the tested conditions (Table 3), so it plays no role in the anaplerotic sequence; an alternative function for an orthologue in Mycobacterium tuberculosis has been demonstrated to be that of an adhesin (Kinhikar et al., 2006).
During the dissimilation of taurine, not only the flow of carbon but also the fluxes of ammonia/ammonium ion and sulfite/sulfate ions must be contained. It seems logical to consider that the single copy of the amtB gene will be involved (e.g. Khademi et al., 2004; Gorzynska et al., 2006), but we obtained no reproducible data. The nature of the oxidation of sulfite to sulfate is also unknown. The sulfate is presumably excreted via the inducible TauZ (Fig. 1a, b, Table 3).
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ACKNOWLEDGEMENTS |
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Received 6 June 2006;
revised 25 July 2006;
accepted 26 July 2006.
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, taurine; 
, ammonium ion; 
, sulfate ion; 
, sulfite ion.
