U-2131 Beach Hall, University of Connecticut, Storrs, CT 06269, USA
Correspondence
Walter Godchaux
Godchaux{at}uconn.edu
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ABSTRACT |
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INTRODUCTION |
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The purple nonsulfur bacteria (PNS) are exceptionally versatile in the range of organic compounds that they can utilize (Dutton & Evans, 1969, 1978
; Harwood & Gibson, 1986
, 1988
; Imhoff & Trüper, 1992
; Madigan & Gest, 1979
; Pfennig, 1978
). These bacteria are unique in the ability to grow by a variety of modes of metabolism. However, it is their versatile photometabolism that distinguishes them from other phototrophic bacteria. PNS are able to use a variety of organic acids, alcohols, sugars and aromatic compounds as carbon sources and/or electron donors during photoheterotrophic growth (Harwood & Gibson, 1986
). Their metabolic and physiological diversity makes them an important component of wastewater treatment, photoassimilating and mineralizing a variety of organic compounds (Kobayashi & Nakanishi, 1971
).
The ability of the PNS to use carbon-linked sulfur compounds as sources of sulfur and as a source of carbon and electron donors was first demonstrated with thiols (Visscher & Taylor, 1993). In that study, isolates of Rhodopseudomonas were able to use these compounds in a more conventional way, cleaving the thiol group of mercaptomalate and using it as a source of electrons for the reduction of CO2. One isolate was also able to grow photoorganotrophically using the thiol sulfur and assimilating the residual carbon for biosynthesis. The utilization of thiol sulfur is consistent with the reported ability of the PNS to use low levels of H2S as a source of electrons (Imhoff & Trüper, 1992
). Recently, it was demonstrated that Rhodobacter capsulatus was able to grow phototrophically with taurine as sulfur source (Masepohl et al., 2001
), but this work did not address the possible utilization of taurine either as a carbon source or as an electron donor; no mention was made of the desulfonative abilities of other PNS.
Previous reports have demonstrated the anaerobic desulfonation of taurine (Chien et al., 1995; Denger et al., 1996
, 1997a
). Subsequent research has established the importance of inducible enzyme activities for the anaerobic metabolism of this compound (Chien et al., 1997
; Laue & Cook, 2000
; R. T. Novak and others, unpublished results). Also, the utilization of taurine as a source of carbon and electrons under anoxic, nitrate- and sulfate-respiratory growth conditions has been established (Denger et al., 1997a
; Lie et al., 1999
). We now report a new aspect of anaerobic taurine metabolism: two strains of PNS utilize the sulfonate taurine as an electron donor for phototrophic growth. In addition, we will show that these organisms can utilize the sulfonate as a sole source of nitrogen and sulfur and, in one strain, as a partial source of carbon.
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METHODS |
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Enrichment cultures were incubated at 27 °C under incandescent light. Cultures deemed to be phototrophic bacteria (light dependence, development of pigments) were subcultured twice in the above media. Pure cultures were obtained following growth on agar media at 27 °C in glass Brewer jars under incandescent light. GasPak H2 and CO2 gas generators (BBL) with palladium catalyst were used to produce anoxic conditions. Culture purity was checked microscopically and by growth on complex medium (1 %, w/v, yeast extract, 1 %, w/v, Bacto Agar).
Phototrophic growth experiments with the pure cultures were done with 20 mM taurine (unless otherwise indicated for individual experiments) in minimal salts medium (9·5 ml) added to modified Hungate tubes (Miller & Wolin, 1974); the headspace of the tubes was initially 100 % argon. Following autoclaving, filter-sterilized vitamin mix (described above) was added (2 ml l1). Filter-sterilized, anoxic sodium bicarbonate (concentration dependent on individual experiment) was then added to each tube. In some experiments, cells were grown in a non-sulfonate medium [0·1 M potassium phosphate buffer, pH 6·8, 30 mM sodium bicarbonate, 20 mM sodium acetate, 10 mM ammonium chloride, 100 µM sodium sulfate, mineral base and vitamin mix, with a head space of CO2/argon (20 : 80, v/v)]. Aerobic (dark) growth studies were conducted using a similar medium without added CO2, with 30 mM succinate as a carbon and energy source and taurine as a sole nitrogen and sulfur source, and cultures were incubated at 27 °C in Erlenmeyer flasks on an orbital shaker (New Brunswick Scientific) at 140 r.p.m. Growth of cultures was followed by measuring OD650 (Cary 50 UV/vis spectrophotometer; Varian) in modified Hungate tubes, and readings were converted to 1 cm path-length values. All growth experiments were repeated at least three times; typical results are shown.
For physiological comparison, type cultures of Rp. palustris ATCC 17001T and Rb. sphaeroides (2.4.1) ATCC 17023T were kindly provided by Professors Caroline Harwood of the University of Iowa, Iowa City, and Samuel Kaplan of the University of Texas Medical School, Houston.
Taxonomic status of strains Tau1 and Tau3.
Cell lysis, amplification of 16S rRNA genes and 16S rRNA gene sequencing procedures are described in detail elsewhere (Dewhirst et al., 1999). For identification of closest relatives, the sequences from strains Tau1 and Tau3 were compared to the 16S rRNA gene sequences of over 4000 microorganisms in the Forsyth Institute's database, 16 000 sequences in the Ribosomal Database Project (RDP; Maidak et al., 2000
), and GenBank.
Chemicals.
All of the chemicals used in this study were of the highest purity from Fisher Scientific, Sigma/Aldrich Chemical Co. (Taurine 99 %, no. T 0625), Difco and Roche-Boehringer. Sulfoacetaldehyde bisulfite adduct (SABA) was synthesized as described by Kondo et al. (1971)
. The structure was confirmed by electrospray mass spectrometry (Gritzer et al., 2003
). Gas (argon and CO2/argon) was ultrahigh purity grade supplied by Airgas East.
Phototrophic growth physiology and analytical methods.
The consumption of substrate (taurine) and the formation of one product (sulfate) were routinely assayed by HPLC, using refractometric methods following chromatography on an ion-exclusion and partition/adsorption column (Shodex KC811), with a conductivity detector (Waters) used in conjunction with a Shodex KC-810P precolumn; the mobile phase was 0·1 % phosphoric acid at 70 °C, 1 ml min1 flow rate, and 400 p.s.i. (2760 kPa). Products were identified by comparison to the elution profiles of authentic reference compounds and quantified using standard curves. Sulfate ion was assayed using a modification of the BaCl2 technique (Schauder et al., 1986). Because of interference by some low-molecular-mass species that were potential metabolites, we were unable to identify definitively one of the products of taurine metabolism (which we suspected to be sulfate) simply by a comparison to a reference compound. Cultures were grown using media with 0·1 M Tris buffer (pH 6·8) replacing phosphate buffer, which, like sulfate and sulfite, is precipitated by barium and hence interferes with this determination. The culture supernatants were assayed by HPLC following treatment with BaCl2 and centrifugation; where this caused disappearance of a peak with an elution time essentially that of sulfate (which was well separated from sulfite) we identified that peak as sulfate.
Cell extracts and enzymology.
Cells were grown in MSV medium with 20 mM taurine and 20 mM bicarbonate in 500 ml screw-capped bottles with butyl rubber septa (which allowed introduction of substrates or withdrawal of samples with syringes) and an initial headspace of 100 % argon. Mid-exponential-phase cells were harvested by centrifugation and washed with breaking buffer (50 mM potassium phosphate, 100 µM pyridoxal 5'-phosphate, pH 7·5) three times. After resuspension in a small volume of buffer (0·4 g wet weight cells ml1), the cells were broken using a French pressure cell at 15 000 p.s.i (104 MPa). The preparation was then centrifuged (12 000 g, 20 min, 4 °C) to remove whole cells and cell debris, and the supernatant was subsequently ultracentrifuged (63 000 g, 60 min, 4 °C) to sediment cell membranes. Crude cell extracts were stored on ice and used for enzyme assays on the same day. Protein concentrations were measured by the bicinchoninic acid method (Smith et al., 1985), with BSA as a protein standard.
In vitro assay of aminotransferase activity.
The enzyme assay system contained 10 µmol of an amino donor (taurine, -alanine, 2-aminoethanephosphonate, 1-butylamine, 1-amylamine, 1,4-butanediamine, spermidine, spermine, 1,6-hexanediamine or 1,7-heptanediamine), 20 µmol of an amino acceptor (pyruvate, oxalacetate or 2-oxoglutarate), 100 µmol of potassium phosphate (pH 8·5), 4 µg of pyridoxal 5'-phosphate and 100 µl cell extract (0·10·2 mg protein) in a final volume of 400 µl. The reaction mixture was incubated at 37 °C for 60 min, and then inactivated by heat (90 °C, 10 min). Following heating, denatured proteins were removed by centrifugation (10 000 g, 5 min, 25 °C). In some experiments, amino acids formed in the reaction mixture were separated by TLC on cellulose by isobutyric acid/water (4 : 1, v/v), along with the amino acid standards alanine, aspartate and glutamate. The amino acids were detected using ninhydrin spray (0·5 % ninhydrin in 95 % ethanol).
Characterization and quantitative determination of taurinepyruvate aminotransferase.
The calculated activity of taurinepyruvate aminotransferase (EC 2.6.1.77) was based on a two-part fixed-time assay, as described by Chien et al. (1997). In the first part of the assay, alanine was formed from the transamination of taurine and pyruvate. In the second part of the assay, the alanine formed was quantitatively determined using commercial alanine dehydrogenase (Sigma) (Yoshida & Freese, 1965
). Various amino donors and amino acceptors (listed above) were tested as replacements for taurine and pyruvate.
In vitro assay of sulfoacetaldehyde desulfonation.
Desulfonation of sulfoacetaldehyde by sulfoacetaldehyde acetyltransferase (EC 2.3.3.15), forming bisulfite and acetyl phosphate, by cell extracts, was examined. The enzyme system contained 5 mM taurine and 5 mM pyruvate, or 5 mM SABA, plus 0·1 M potassium phosphate buffer, pH 7·5, 0·1 mM pyridoxal 5'-phosphate (omitted when SABA was the substrate), 1 mM thiamin pyrophosphate, 5 mM MgCl2 and 1 mg protein ml1. The reaction mixture was incubated at 30 °C for 60 min, and the enzymes then chilled in an ice bath. Controls contained boiled extract (100 °C, 15 min), or were without substrate. The presence of desulfonation activity was based on the measurement of bisulfite and acetyl phosphate produced (liberated from sulfoacetaldehyde, the transamination product of taurine, or SABA) during the fixed-time assay. Sulfoacetaldehyde is unstable in solution, but SABA (the stable bisulfite adduct) functions as a substrate in this assay (Gritzer et al., 2003).
Determination of bisulfite.
Assays for the determination of bisulfite formed in the desulfonation assay contained 10 µg of Ellman's reagent [5,5'-dithiobis-(2-nitrobenzoic acid)], and 10 µl of assay mixture containing reaction product formed in the complete standard assay system (described above), in a final volume of 1 ml. In this latter assay system, bisulfite formed from desulfonation activity was determined by measuring the A415 at t0 and at 60 min. Controls showed that loss of bisulfite due to oxidation to sulfate was negligible during the assay period. In controls where boiled extracts were used, small amounts of bisulfite were produced chemically, and values for other samples were corrected accordingly. When SABA was used as an assay substrate, the reagent reacted with the adduct bisulfite, yielding a background that was subtracted from experimental values (for assays in which desulfuration of the sulfonates produced additional Ellman reactivity).
Determination of acetyl phosphate.
One of the products of sulfoacetaldehyde acetyltransferase has been identified previously as acetyl phosphate (Ruff et al., 2003). This product was carefully preserved and identified in our extracts of taurine-grown cells by the procedure (reaction with hydroxylamine to form hydroxamic acids which are then complexed with ferric ion to form coloured products) of Stadtman (1957)
. The assay volume was reduced to 1·5 ml, and succinic anhydride was used as standard.
Assay of glyoxylate cycle enzymes.
Cell extracts were also assayed for the presence of (inducible) glyoxylate cycle enzymes (malate synthase, isocitrate lyase), following the procedure of Dixon & Kornberg (1959).
Incorporation of taurine carbon into cell material: radiolabelling experiments.
Rp. palustris Tau1 and Rb. sphaeroides Tau3 were grown in 20 mM taurine (taurine-limiting)/20 mM CO2/MSV medium to an OD650 of 1·0. Two separate sets of tubes were labelled with either [U-14C]taurine or 14CO2 at a specific activity of 600 c.p.m. (µg-atom carbon)1. Samples (1 ml) were removed and collected on filters (25 mm diameterx0·45 µm pore size; Millipore) and washed three times with 5 ml portions of distilled, deionized water. The filters were placed in 5 ml scintillation cocktail (Optifluor, Packard Instruments). The radioactivity incorporated into cells was quantified with an ISOCAP/300 liquid scintillation counter (Searle) and expressed as µg-atom carbon (OD650 unit) of culture1. The number of OD650 units was obtained by multiplying the OD at 650 nm by the sample volume in ml.
The same cultures (tubes) were then acidified with concentrated HCl to release dissolved CO2. The culture was bubbled with N2 for 20 min (10 ml min1) into 14CO2 traps consisting of two 7 ml scintillation vials in series filled with 0·5 ml -phenethylamine in 4 ml scintillation cocktail (Brune et al., 1995
).
Phototrophic growth requirement for CO2.
These organisms would not grow phototrophically using taurine in the absence of CO2. To assess the proportion of cellular carbon derived from each source, a medium was used that contained [U-14C]taurine (20 mM) at a specific activity of 3000 c.p.m. (µg-atom carbon)1 and variable amounts of non-radioactive bicarbonate (10200 mM). Cells were harvested, and the CO2 from the supernatant was trapped and counted as described above. Incorporation of radioactivity into cell material was quantified as described in the previous section.
In order to assess whether the CO2 fixation during phototrophic growth of Rb. sphaeroides Tau3 occurred over the entire course of growth or only at a particular growth phase, a medium was constructed as above with 20 mM taurine and 60 mM bicarbonate, the latter labelled with 14CO2 at a specific activity of 600 c.p.m. (µg-atom carbon)1, in a total volume of 400 ml in 500 ml screw-capped serum bottles with butyl rubber septa. Samples were removed (0·5 OD650 units of cells, e.g. 5 ml of a culture of OD650 0·1) at various times during growth (OD650 0·1, 0·2, 0·3, 0·4, 0·6, 1·0 and 1·2) and counted; the supernatant was acidified and CO2 trapped as described above. Radioactivity incorporated into cell material was quantified as described above.
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RESULTS |
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Phototrophic growth of organisms with taurine
Rb. sphaeroides ATCC 17023T and isolate Tau3, and Rp. palustris ATCC 17001T and isolate Tau1 were examined for their ability to grow phototrophically using taurine. None of these organisms demonstrated significant growth with taurine as sole carbon, nitrogen and sulfur source and electron donor. Only the environmental isolates Tau1 and Tau3 were able to grow (cf. Fig. 1 for Tau1: the results for Tau3 were virtually identical) with taurine as sole electron donor, sulfur and nitrogen source, and only when CO2 was provided as well. Neither organism was able to grow with sulfite as sole electron donor, but both were able to utilize acetate as electron donor.
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Enzymology of growth with taurine
Extracts of Rp. palustris Tau1 and Rb. sphaeroides Tau3 grown phototrophically using taurine as sole electron donor and sulfur and nitrogen source, and also of cells grown aerobically in the dark using succinate as a carbon and energy source and taurine as sole sulfur and nitrogen source, were assayed for the presence of enzyme activities (below) known to be involved in taurine metabolism (Chien et al., 1997; Denger et al., 2001
; Laue & Cook, 2000
; Ruff et al., 2003
).
Taurinepyruvate aminotransferase
Those organisms grown phototrophically using taurine contained inducible aminotransferase activity; this was specific for the transamination of taurine or its carboxyl analogue, -alanine, with pyruvate as amino acceptor (Table 1
; only data for Rb. sphaeroides aminotransferase are shown; results were essentially identical for Rp. palustris cells). Growth with
-alanine+sulfate also induced an aminotransferase activity in a similar amount and with similar substrate specifity. This induction was also observed in Clostridium pasteurianum C1 grown in the presence of
-alanine, and may reflect the structural similarity of
-alanine to the sulfonate (unpublished data). As we describe below, the acetyltransferase was induced by
-alanine as if it and the aminotransferase were co-induced by the amino donor. These observations are consistent with the idea that the aminotransferase activities induced by either compound represent the same enzyme, but more detailed studies are needed to establish this point. In contrast, cells grown aerobically in the dark, with taurine as sole sulfur and nitrogen source, contained an aminotransferase that was not specific for taurine (results not shown). Activity was not detected when either taurine or pyruvate were left out of the assay, and was also absent when heat-inactivated extract was substituted in the assays. The absence of pyridoxal 5'-phosphate from the assay conditions did not affect the activity of the enzyme. This cofactor was added to the extraction buffer to maximize stability of the enzyme over time, and thus may remain tightly bound to the enzyme (Chien et al., 1997
).
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[U-14C]Taurine incorporation versus 14CO2 incorporation during phototrophic growth.
In order to determine whether taurine was used as a carbon source, as opposed to merely as an electron donor to provide the reducing power for CO2 reduction, separate tubes containing 20 mM taurine and 60 mM bicarbonate were labelled with either [U-14C]taurine or [14C]bicarbonate to a specific activity of 3000 c.p.m. (µg-atom carbon)1. Rb. sphaeroides Tau3 and Rp. palustris Tau1 were inoculated and harvested (OD650 1·2), and the cellular radioactivity was quantified (Table 3). In Rp. palustris Tau1, almost all of the cellular carbon was derived from CO2. Rb. sphaeroides Tau3, on the other hand, derived a majority of its cellular carbon from taurine, but also incorporated some CO2. As shown earlier, in both organisms, taurine is completely consumed, and there were no detectable carbon products of its metabolism excreted into the supernatant. Thus, we surmised that any carbon was either incorporated into cell material or completely oxidized to CO2. The supernatants from cultures grown with [14C]taurine were examined for the presence of either labelled taurine or CO2 (Table 3
). Almost all of the remaining labelled taurine not found in cellular material could be accounted for in the form of CO2 (Table 3
). Following acidification of the media and measurement of the trapped 14CO2, the culture supernatant contained negligible label. Thus, in both organisms, all of the taurine carbon was either incorporated into cell material or oxidized to CO2.
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DISCUSSION |
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Both Rp. palustris and Rb. sphaeroides are known to be able to grow using acetate either as a source of carbon (photoheterotrophic) or only as an electron donor (photoautotrophic) (Albers & Gottschalk, 1976; Barbosa et al., 2001
; Berg et al., 2002
; Schauder et al., 1986
; Vigenschow et al., 1986
; Yang et al., 2002
). Both isolates Tau1 and Tau3 grown with taurine+CO2 contained the core enzyme activities of anaerobic taurine metabolism: taurinepyruvate aminotransferase and sulfoacetaldehyde acetyltransferase. Interestingly, our own (unpublished) searches of available sequence databases reveal an ORF with high sequence similarity to a known taurinepyruvate aminotransferase in Rb. sphaeroides, and one with high similarity to omega amino acid aminotransferase in Rp. palustris. The products of this taurine metabolism are bisulfite and acetyl phosphate. Acetate (derived from acetyl phosphate) is the most likely candidate for an intermediate electron donor for photometabolism, since sulfite does not serve as sole electron donor in either organism (data not shown). However, the pathway between acetyl phosphate and cell material apparently is complicated and has not been completely elucidated either by us or by others (Albers & Gottschalk, 1976
; Berg et al., 2002
), although a citramalate cycle has been proposed. In this cycle, citramalate would be formed by condensation of pyruvate and acetyl-CoA and ultimately converted to glyoxyate and regenerated pyruvate; the glyoxalate could be converted to malate by malate synthase and thus be assimilated into cellular metabolites without involvement of either isocitrate or its lyase.
Rp. palustris strain Tau1 grew with taurine as sole electron donor, sulfur and nitrogen source in the presence of CO2 (Fig. 4a). Studies of doseresponse, with varying amounts of non-radioactive bicarbonate, that demonstrated linear growth yield over the range 120 mM (data not shown), suggested that the limiting nutrient for growth was CO2; hence growth is photoautotrophic (data not shown). Experiments where either the taurine or the CO2 carbon was labelled in taurine+CO2 medium demonstrated that the carbon in cell mass was derived entirely from CO2.
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The two isolates differed in their ability to assimilate taurine carbon, even though some similar taurine metabolic enzymes and (presumably) transport mechanisms are present. Why then is only Rb. sphaeroides Tau3 capable of assimilating taurine carbon? In Alcaligenes defragrans the actual carbon-containing product of sulfoacetaldehyde acetyltransferase is acetyl phosphate (Ruff et al., 2003). We assume that acetyl phosphate is converted to acetyl-CoA, and subsequently acetate, in Rb. sphaeroides. Therefore, the answer to the question posed may relate to the apparent induction of the glyoxylate-cycle enzyme activities. Rp. palustris Tau1 grown photoheterotrophically with acetate contained both glyoxylate cycle enzymes. However, growth with taurine+CO2 resulted in induction of neither enzyme. Taurine (20 mM) was completely metabolized in cultures of this organism. Therefore, it may be that the acetate generated from taurine is readily consumed for the reduction of CO2 to cell material, and that acetate does not accumulate in the cell to a level sufficient to induce the glyoxylate cycle. It is somewhat surprising that taurine is not used as a substrate for photoheterotrophic growth by Rp. palustris Tau1 in the absence of CO2; this may be a reflection of transport efficiency.
On the other hand, Rb. sphaeroides Tau3 assimilates taurine carbon for a majority of cell mass. The metabolism of acetate in this organism is complex, and to date has not been completely elucidated (Birgit Albers, personal communication; Albers & Gottschalk, 1976; Berg et al., 2002
). Our isolate contained an inducible malate synthase in both acetate- and taurine-grown cells, but no isocitrate lyase was detected; this is consistent with a genomic analysis of this organism (Joint Genome Institute, 2003
). There have been suggestions that pyruvate carboxylase is involved in the synthesis of C4-dicarboxylic acids from acetate and CO2, but a mutant strain of Rb. sphaeroides devoid of pyruvate carboxylase was still able to grow with acetate and CO2 (Payne & Morris, 1969
), so there must be additional mechanisms for acetate assimilation in the presence of CO2 which do not involve pyruvate carboxylase. Regardless of the mechanism involved, the data presented here indicate that taurine-carbon assimilation during phototrophic growth of Rb. sphaeroides Tau3 requires CO2; nonetheless, the phototrophic utilization of taurine carbon suggests a new aspect of sulfonate carbon cycling.
This work describes the first example of a sulfonate utilized as a sole electron donor, sulfur and nitrogen source by members of the purple nonsulfur bacteria, but this occurs only under phototrophic conditions. In addition, it also demonstrates that Rb. sphaeroides Tau3 can derive a majority of its cellular carbon directly from the sulfonate taurine. The difference in sulfonate metabolism between Rp. palustris Tau1 and Rb. sphaeroides Tau3 demonstrates another aspect of the metabolic diversity within the purple nonsulfur bacteria.
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ACKNOWLEDGEMENTS |
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Received 8 January 2003;
revised 5 February 2004;
accepted 11 February 2004.
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