Sulfonates: novel electron acceptors in anaerobic respiration

The enrichment and isolation in pure culture of a bacterium, identified as a strain of Desulfovibrio, able to release and reduce the sulfur of isethionate (2-hydroxyethanesulfonate) and other sulfonates to support anaerobic respiratory growth, is described. The sulfonate moiety was the source of sulfur that served as the terminal electron acceptor, while the carbon skeleton of isethionate functioned as an accessory electron donor for the reduction of sulfite. Cysteate (alanine-3-sulfonate) and sulfoacetaldehyde (acetaldehyde-2-sulfonate) could also be used for anaerobic respiration, but many other sulfonates could not. A survey of known sulfate-reducing bacteria revealed that some, but not all, strains tested could utilize the sulfur of some sulfonates as terminal electron acceptor. Isethionate-grown cells of Desulfovibrio strain IC1 reduced sulfonate-sulfur in preference to that of sulfate; however, sulfate-grown cells reduced sulfate-sulfur in preference to that of sulfonate. Received: 2 May 1996 / Accepted: 8 June 1996     

Sulfonate-sulfur can be assimilated for fermentative growth

Abstract Bacterial assimilation of sulfonate-sulfur under anaerobic conditions has been demonstrated. Two different bacteria able to grow fermentatively using sulfonate-sulfur as sole sulfur source were isolated by enrichment culture; neither were able to utilize sulfonates as sole source of carbon and energy for growth. The isolate of Clostridium pasteurianum assimilated the sulfur of isethionate (2-hydroxyethanesulfonate), taurine (2-aminoethanesulfonate), or p -toluenesulfonate. A facultatively fermentative Klebsiella strain did not utilize the sulfur of any of these sulfonates, but assimilated cysteate-sulfur; in contrast, when growing by aerobic respiration, the range of sulfonates able to serve as sulfur source was greater. Both bacteria displayed a preferential utilization of sulfate-sulfur to that of the sulfonates tested. Thus, bacterial assimilation of sulfonate-sulfur during anaerobic growth has direct parallels with features until now recognized only for aerobic assimilatory processes.     

Utilization of sulfonates as sole sulfur source by soil bacteria including Comamonas acidovorans

Bacteria able to use cysteate, taurine or isethionate as sole source of carbon and energy were isolated from the soil. Tests of sulfur assimilation showed that sulfonate sulfur and sulfate sulfur supported comparable cell yields. Methanesulfonate, 1-dodecanesulfonate and p-toluenesulfonate also served as sole source of sulfur for strain I91, identified as Comamonas (Pseudomonas) acidovorans. Competition studies with strain I91 showed that the presence of sulfate inhibits cysteate, isethionate or taurine incorporation. Pseudomonas aeruginosa PAO1, Comamonas acidovorans 14 and 105, and Acidovorax (Pseudomonas) facilis 332 used cysteate, isethionate, or taurine as sole source of sulfur while P. aeruginosa PAO716 and PAO718 used only taurine.     

Sulfonate-sulfur assimilation by yeasts resembles that of bacteria

Abstract Three sulfonates were tested for their ability to serve as nutrients for Hansenula wingei, Rhodotorula glutinis, Trigonopsis variabilis and Saccharomyces cerevisiae . Cysteate, taurine and isethionate, under aerobic conditions, could be utilized as sources of sulfur, although in some instances final cell yields were less than those obtained with an equimolar amount of sulfate-sulfur. Sulfonate assimilation by S. cerevisiae resembled that of bacteria (reported earlier by us) in several aspects: first, sulfate-S was used in preference to that of sulfonate, when both were present; second, mutants unable to use a source of sulfur because of deficiencies in ATP sulfurylase, adenylylsulfate kinase (APS kinase) or PAPS reductase were able to utilize sulfonates; and third, mutants deficient in sulfite reductase were unable to utilize sulfonates.     

Sulfonates as terminal electron acceptors for growth of sulfite-reducing bacteria (Desulfitobacterium spp.) and sulfate-reducing bacteria: effects of inhibitors of sulfidogenesis.

This study demonstrates the ability of Desulfitobacterium spp. to utilize aliphatic sulfonates as terminal electron acceptors (TEA) for growth. Isethionate (2-hydroxyethanesulfonate) reduction by Desulfitobacterium hafniense resulted in acetate as well as sulfide accumulation in accordance with the expectation that the carbon portion of isethionate was oxidized to acetate and the sulfur was reduced to sulfide. The presence of a polypeptide, approximately 97 kDa, was evident in isethionate-grown cells of Desulfitobacterium hafniense, Desulfitobacterium sp. strain PCE 1, and the two sulfate-reducing bacteria (SRB)-Desulfovibrio desulfuricans IC1 (T. J. Lie, J. R. Leadbetter, and E. R. Leadbetter, Geomicrobiol. J. 15:135-149, 1998) and Desulfomicrobium norvegicum; this polypeptide was not detected when these bacteria were grown on TEA other than isethionate, suggesting involvement in its metabolism. The sulfate analogs molybdate and tungstate, effective in inhibiting sulfate reduction by SRB, were examined for their effects on sulfonate reduction. Molybdate effectively inhibited sulfonate reduction by strain IC1 and selectively inhibited isethionate (but not cysteate) reduction by Desulfitobacterium dehalogenans and Desulfitobacterium sp. strain PCE 1. Desulfitobacterium hafniense, however, grew with both isethionate and cysteate in the presence of molybdate. In contrast, tungstate only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium spp. Similarly, another inhibitor of sulfate reduction, 1,8-dihydroxyanthraquinone, effectively inhibited sulfate reduction by SRB but only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium hafniense.     

Sulfonates as Terminal Electron Acceptors for Growth of Sulfite-Reducing Bacteria (Desulfitobacterium spp.) and Sulfate-Reducing Bacteria: Effects of Inhibitors of Sulfidogenesis

This study demonstrates the ability of Desulfitobacterium spp. to utilize aliphatic sulfonates as terminal electron acceptors (TEA) for growth. Isethionate (2-hydroxyethanesulfonate) reduction by Desulfitobacterium hafniense resulted in acetate as well as sulfide accumulation in accordance with the expectation that the carbon portion of isethionate was oxidized to acetate and the sulfur was reduced to sulfide. The presence of a polypeptide, approximately 97 kDa, was evident in isethionate-grown cells of Desulfitobacterium hafniense, Desulfitobacterium sp. strain PCE 1, and the two sulfate-reducing bacteria (SRB)—Desulfovibrio desulfuricans IC1 (T. J. Lie, J. R. Leadbetter, and E. R. Leadbetter, Geomicrobiol. J. 15:135–149, 1998) and Desulfomicrobium norvegicum; this polypeptide was not detected when these bacteria were grown on TEA other than isethionate, suggesting involvement in its metabolism. The sulfate analogs molybdate and tungstate, effective in inhibiting sulfate reduction by SRB, were examined for their effects on sulfonate reduction. Molybdate effectively inhibited sulfonate reduction by strain IC1 and selectively inhibited isethionate (but not cysteate) reduction by Desulfitobacterium dehalogenans and Desulfitobacterium sp. strain PCE 1. Desulfitobacterium hafniense, however, grew with both isethionate and cysteate in the presence of molybdate. In contrast, tungstate only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium spp. Similarly, another inhibitor of sulfate reduction, 1,8-dihydroxyanthraquinone, effectively inhibited sulfate reduction by SRB but only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium hafniense.     

Taurine reduction in anaerobic respiration of Bilophila wadsworthia RZATAU.

Organosulfonates are important natural and man-made compounds, but until recently (T. J. Lie, T. Pitta, E. R. Leadbetter, W. Godchaux III, and J. R. Leadbetter. Arch. Microbiol. 166:204-210, 1996), they were not believed to be dissimilated under anoxic conditions. We also chose to test whether alkane- and arenesulfonates could serve as electron sinks in respiratory metabolism. We generated 60 anoxic enrichment cultures in mineral salts medium which included several potential electron donors and a single organic sulfonate as an electron sink, and we used material from anaerobic digestors in communal sewage works as inocula. None of the four aromatic sulfonates, the three unsubstituted alkanesulfonates, or the N-sulfonate tested gave positive enrichment cultures requiring both the electron donor and electron sink for growth. Nine cultures utilizing the natural products taurine, cysteate, or isethionate were considered positive for growth, and all formed sulfide. Two clearly different pure cultures were examined. Putative Desulfovibrio sp. strain RZACYSA, with lactate as the electron donor, utilized sulfate, aminomethanesulfonate, taurine, isethionate, and cysteate, converting the latter to ammonia, acetate, and sulfide. Strain RZATAU was identified by 16S rDNA analysis as Bilophila wadsworthia. In the presence of, e.g., formate as the electron donor, it utilized, e.g., cysteate and isethionate and converted taurine quantitatively to cell material and products identified as ammonia, acetate, and sulfide. Sulfite and thiosulfate, but not sulfate, were utilized as electron sinks, as was nitrate, when lactate was provided as the electron donor and carbon source. A growth requirement for 1,4-naphthoquinone indicates a menaquinone electron carrier, and the presence of cytochrome c supports the presence of an electron transport chain. Pyruvate-dependent disappearance of taurine from cell extracts, as well as formation of alanine and release of ammonia and acetate, was detected. We suspected that sulfite is an intermediate, and we detected desulfoviridin (sulfite reductase). We thus believe that sulfonate reduction is one aspect of a respiratory system transferring electrons from, e.g., formate to sulfite reductase via an electron transport system which presumably generates a proton gradient across the cell membrane.     

Dissimilation of C3-sulfonates

Cysteate and sulfolactate are widespread natural products in the environment, while propanesulfonate, 3-aminopropanesulfonate and propane-1,3-disulfonate are xenobiotics. While some understanding of the bacterial assimilation of cysteate sulfur has been achieved, details of the dissimilation of cysteate and sulfolactate by microbes together with information on the degradation of the xenobiotics have only recently become available. This minireview centres on bacterial catabolism of the carbon moiety in these C₃-sulfonates and on the fate of the sulfonate group. Three mechanisms of desulfonation have been established. Firstly, cysteate is converted via sulfopyruvate to sulfolactate, which is desulfonated to pyruvate and sulfite; the latter is oxidized to sulfate by a sulfite dehydrogenase and excreted as sulfate in Paracoccus pantotrophus NKNCYSA. Secondly, sulfolactate can be converted to cysteate, which is cleaved in a pyridoxal 5′-phosphate-coupled reaction to pyruvate, sulfite and ammonium ions; in Silicibacter pomeroyi DSS-3, the sulfite is excreted largely as sulfite. Both desulfonation reactions seem to be widespread. The third desulfonation mechanism is oxygenolysis of, e.g. propanesulfonate(s), about which less is known.     

Sulfidogenesis from 2-aminoethanesulfonate (taurine) fermentation by a morphologically unusual sulfate-reducing bacterium, Desulforhopalus singaporensis sp. nov.

A pure culture of an obligately anaerobic marine bacterium was obtained from an anaerobic enrichment culture in which taurine (2-aminoethanesulfonate) was the sole source of carbon, energy, and nitrogen. Taurine fermentation resulted in acetate, ammonia, and sulfide as end products. Other sulfonates, including 2-hydroxyethanesulfonate (isethionate) and cysteate (alanine-3-sulfonate), were not fermented. When malate was the sole source of carbon and energy, the bacterium reduced sulfate, sulfite, thiosulfate, or nitrate (reduced to ammonia) but did not use fumarate or dimethyl sulfoxide as a terminal electron acceptor for growth. Taurine-grown cells had significantly lower adenylylphosphosulfate reductase activities than sulfate-grown cells had, which was consistent with the notion that sulfate was not released as a result of oxidative C-S bond cleavage and then assimilated. The name Desulforhopalus singaporensis is proposed for this sulfate-reducing bacterium, which is morphologically unusual compared to the previously described sulfate-reducing bacteria by virtue of the spinae present on the rod-shaped, gram-negative, nonmotile cells; endospore formation was not discerned, nor was desulfoviridin detected. Granules of poly-beta-hydroxybutyrate were abundant in taurine-grown cells. This organism shares with the other member of the genus Desulforhopalus which has been described a unique 13-base deletion in the 16S ribosomal DNA. It differs in several ways from a recently described endospore-forming anaerobe (K. Denger, H. Laue, and A. M. Cook, Arch. Microbiol. 168:297-301, 1997) that reportedly produces thiosulfate but not sulfide from taurine fermentation. D. singaporensis thus appears to be the first example of an organism which exhibits sulfidogenesis during taurine fermentation. Implications for sulfonate sulfur in the sulfur cycle are discussed.     

Sulfidogenesis from 2-Aminoethanesulfonate (Taurine) Fermentation by a Morphologically Unusual Sulfate-Reducing Bacterium, Desulforhopalus singaporensis sp. nov.

A pure culture of an obligately anaerobic marine bacterium was obtained from an anaerobic enrichment culture in which taurine (2-aminoethanesulfonate) was the sole source of carbon, energy, and nitrogen. Taurine fermentation resulted in acetate, ammonia, and sulfide as end products. Other sulfonates, including 2-hydroxyethanesulfonate (isethionate) and cysteate (alanine-3-sulfonate), were not fermented. When malate was the sole source of carbon and energy, the bacterium reduced sulfate, sulfite, thiosulfate, or nitrate (reduced to ammonia) but did not use fumarate or dimethyl sulfoxide as a terminal electron acceptor for growth. Taurine-grown cells had significantly lower adenylylphosphosulfate reductase activities than sulfate-grown cells had, which was consistent with the notion that sulfate was not released as a result of oxidative C-S bond cleavage and then assimilated. The name Desulforhopalus singaporensis is proposed for this sulfate-reducing bacterium, which is morphologically unusual compared to the previously described sulfate-reducing bacteria by virtue of the spinae present on the rod-shaped, gram-negative, nonmotile cells; endospore formation was not discerned, nor was desulfoviridin detected. Granules of poly-β-hydroxybutyrate were abundant in taurine-grown cells. This organism shares with the other member of the genus Desulforhopalus which has been described a unique 13-base deletion in the 16S ribosomal DNA. It differs in several ways from a recently described endospore-forming anaerobe (K. Denger, H. Laue, and A. M. Cook, Arch. Microbiol. 168:297–301, 1997) that reportedly produces thiosulfate but not sulfide from taurine fermentation. D. singaporensis thus appears to be the first example of an organism which exhibits sulfidogenesis during taurine fermentation. Implications for sulfonate sulfur in the sulfur cycle are discussed.     

Regulation of sulfate assimilation by nitrogen and sulfur nutrition in poplar trees

Plants cover their need for sulfur by taking up inorganic sulfate, reducing it to sulfide, and incorporating it into the amino acid cysteine. In herbaceous plants the pathway of assimilatory sulfate reduction is highly regulated by the availability of the nutrients sulfate and nitrate. To investigate the regulation of sulfate assimilation in deciduous trees we used the poplar hybrid Populus tremula × P. alba as a model. The enzymes of the pathway are present in several isoforms, except for sulfite reductase and -glutamylcysteine synthetase; the genomic organization of the pathway is thus similar to herbaceous plants. The mRNA level of APS reductase, the key enzyme of the pathway, was induced by 3 days of sulfur deficiency and reduced by nitrogen deficiency in the roots, whereas in the leaves it was affected only by the withdrawal of nitrogen. When both nutrients were absent, the mRNA levels did not differ from those in control plants. Four weeks of sulfur deficiency did not affect growth of the poplar plants, but the content of glutathione, the most abundant low molecular thiol, was reduced compared to control plants. Sulfur limitation resulted in an increase in mRNA levels of ATP sulfurylase, APS reductase, and sulfite reductase, probably as an adaptation mechanism to increase the efficiency of the sulfate assimilation pathway. Altogether, although distinct differences were found, e.g. no effect of sulfate deficiency on APR in poplar leaves, the regulation of sulfate assimilation by nutrient availability observed in poplar was similar to the regulation described for herbaceous plants.     

The role of the novel adenosine 5′-phosphosulfate reductase in regulation of sulfate assimilation of <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Sulfate assimilation provides reduced sulfur for the synthesis of the amino acids cysteine and methionine and for a range of other metabolites. The key step in control of plant sulfate assimilation is the reduction of adenosine 5′-phosphosulfate to sulfite. The enzyme catalyzing this reaction, adenosine 5′phosphosulfate reductase (APR), is found as an iron sulfur protein in plants, algae, and many bacteria. In the moss Physcomitrella patens, however, a novel isoform of the enzyme, APR-B, has recently been discovered lacking the co-factor. To assess the function of the novel APR-B we used homologous recombination to disrupt the corresponding gene in P. patens. The knock-out plants were able to grow on sulfate as a sole sulfur source and the content of low molecular weight thiols was not different from wild type plants or plants where APR was disrupted. However, when treated with low concentrations of cadmium the APR-B knockout plants were more sensitive than both wild type and APR knockouts. In wild type P. patens, the two APR isoforms were not affected by treatments that strongly regulate this enzyme in flowering plants. The data thus suggest that in P. patens APS reduction is not the major control step of sulfate assimilation.     

Regulation of sulfate assimilation in Cytophaga johnsonae

We have studied the regulation of sulfate assimilation by the gliding bacterium Cytophaga johnsonae in which 20% of the total sulfur is in the sulfornate moiety of sulfonolipid. Added cystine inhibited sulfate uptake and growth with cystine as sulfur source resulted in a repression of sulfate uptake. However, low concentrations of cystine preferentially repressed the terminal reactions of sulfate assimilation responsible for cysteine synthesis while allowing the transport and activation of sulfate for sulfonolipid synthesis. The significance of this novel pattern of regulation in bacteria is discussed.     

Sulfate assimilation in Rhodopseudomonas globiformis

Rhodopseudomonas globiformis is able to grow on sulfate as sole source of sulfur, but only at concentrations below 1 mM. Good growth was observed with thiosulfate, cysteine or methionine as sulfur sources. Tetrathionate supported slow growth. Sulfide and sulfite were growth inhibitory. Growth inhibition by higher sulfate concentrations was overcome by the addition of O-acetylserine, which is known as derepressor of sulfate-assimilating enzymes, and by reduced glutathione. All enzymes of the sulfate assimilation pathway. ATP-sulfurylase, adenylylphosphate-sulfotransferase, thiosulfonate reductase and O-acetylserine sulfhydrylase are present in R. globiformis. Sulfate was taken up by the cells and the sulfur incorporated into the amino acids cysteine, methionine and homocysteine. It is concluded, that the failure of R. globiformis to grow on higher concentrations of sulfate is caused by disregulation of the sulfate assimilation pathway. Some preliminary evidence for this view is given in comparing the activities of some of the involved enzymes after growth on different sulfur sources and by examining the effect of O-acetylserine on these activities.Abbreviations DTE dl-dithioerythritol - APS adenosine 5-phosphosulfate, adenylyl sulfate - PAPS 3-phosphoadenosine 5-phosphosulfate, 3-phosphoadenylylsulfate     

Synthesis of the sulfur amino acids: cysteine and methionine

This review will assess new features reported for the molecular and biochemical aspects of cysteine and methionine biosynthesis in Arabidopsis thaliana with regards to early published data from other taxa including crop plants and bacteria (Escherichia coli as a model). By contrast to bacteria and fungi, plant cells present a complex organization, in which the sulfur network takes place in multiple sites. Particularly, the impact of sulfur amino-acid biosynthesis compartmentalization will be addressed in respect to localization of sulfur reduction. To this end, the review will focus on regulation of sulfate reduction by synthesis of cysteine through the cysteine synthase complex and the synthesis of methionine and its derivatives. Finally, regulatory aspects of sulfur amino-acid biosynthesis will be explored with regards to interlacing processes such as photosynthesis, carbon and nitrogen assimilation.     

Sulfite formation by wine yeasts

Four strains of wine yeasts of two different species (Saccharomyces cerevisiae var. ellipsoideus and S. bayanus) were investigated with respect to regulation of NADPH- and benzyl viologen-dependent sulfite reductases by various sulfur sources. The enzyme activity was followed over a growth period of 96 h. The low sulfite-producing strains showed an increased biosynthesis of NADPH-dependent sulfite reductase during the exponential growth phase in the presence of sulfate, sulfite and djencolic-acid. This increase was not observed in the high sulfite-producing strains. Methionine and cysteine prevented this derepression. At the end of the exponential growth phase, enzyme biosynthesis was repressed again, presumably by sulfur-containing amino acids which were produced during growth. The regulatory influence of the various sulfur sources on benzyl viologen dependent sulfitereductase activity is obviously much weaker.Abbreviation BV benzyl viologen     

Cysteine is not an obligatory intermediate in the biosynthesis of cysteate by Cytophaga johnsonae

A cysteine auxotroph of Cytophaga johnsonae was able to incorporate sulfur from sulfate into cysteate, and thus into sulfonolipid, in the absence of cysteine synthesis. This indicates that cysteine is not an obligatory intermediate of the cysteate biosynthetic pathway even though cysteine sulfur can be utilized for cysteate synthesis.     

Interaction of sulfate assimilation with nitrate assimilation as a function of nutrient status and enzymatic co-regulation inbrassica juncea roots

The interaction of sulfate assimilation with nitrate assimilation inBrassica juncea roots was analyzed by monitoring the regulation of ATP sulfurylase (AS), adenosine-5’-phosphosulfate reductase (AR), sulfite reductase (SiR), and nitrite reductase (NiR). Depending on the status of sulfur and nitrogen nutrition, AS and AR activities and mRNA levels were increased by sulfate starvation but decreased by nitrate starvation. The activation of AS and AR by sulfate starvation was inhibited by sulfate/nitrate starvation. However, the rise in steady-state mRNA levels for AS and AR by sulfate starvation was not affected by sulfate/nitrate starvation. SiR gene expression was slightly activated by both sulfate starvation and sulfate/nitrate starvation, but was decreased by nitrate starvation. Although NiR gene expression was little affected by sulfate starvation, it was diminished significantly by either nitrate or nitrate/sulfate starvation. Cysteine (Cys) also decreased AS and AR activities and mRNA levels even when plants were simultaneously starved for sulfate; in contrast, both SiR and NiR gene expressions were only slightly, if at all, affected under the same conditions. This supports our conclusion that Cys, the end-product of sulfate assimilation, is the key regulatory signal. Moreover, SiR and NiR apparently are not the linking step in the co-regulation of sulfate and nitrate assimilation in plants.