Sulphoquinovose (SQ, 6-deoxy-6-sulphoglucose) has been known for 50 years as the polar headgroup of the plant sulpholipid in the photosynthetic membranes of all higher plants, mosses, ferns, algae and most photosynthetic bacteria. It is also found in some non-photosynthetic bacteria, and SQ is part of the surface layer of some Archaea. The estimated annual production of SQ is 10,000,000,000 tonnes (10 petagrams), thus it comprises a major portion of the organo-sulphur in nature, where SQ is degraded by bacteria. However, despite evidence for at least three different degradative pathways in bacteria, no enzymic reaction or gene in any pathway has been defined, although a sulphoglycolytic pathway has been proposed. Here we show that Escherichia coli K-12, the most widely studied prokaryotic model organism, performs sulphoglycolysis, in addition to standard glycolysis. SQ is catabolised through four newly discovered reactions that we established using purified, heterologously expressed enzymes: SQ isomerase, 6-deoxy-6-sulphofructose (SF) kinase, 6-deoxy-6-sulphofructose-1-phosphate (SFP) aldolase, and 3-sulpholactaldehyde (SLA) reductase. The enzymes are encoded in a ten-gene cluster, which probably also encodes regulation, transport and degradation of the whole sulpholipid; the gene cluster is present in almost all (>91%) available E. coli genomes, and is widespread in Enterobacteriaceae. The pathway yields dihydroxyacetone phosphate (DHAP), which powers energy conservation and growth of E. coli, and the sulphonate product 2,3-dihydroxypropane-1-sulphonate (DHPS), which is excreted. DHPS is mineralized by other bacteria, thus closing the sulphur cycle within a bacterial community.
Isolation of bacteria able to utilize s-triazines as the sole and limiting nitrogen sources for growth is described. Three strains of Pseudomonas (A, D, and F) and two strains of Klebsiella pneumoniae (90 and 99) were examined. Strains D and F utilized N-ethylammelide, N-isopropylammelide, ammeline, ammelide, cyanuric acid, and ammonium ion as nitrogen sources. Strain A utilized melamine, ammeline, ammelide, cyanuric acid, ammonium ion, and deaminated N-ethylammeline and N-isopropylammeline. Strains 90 and 99 utilized ammelide, cyanuric acid, and ammonium ion. Growth yields of strains were independent of the nitrogen source, and specific growth rates with s-triazines were similar to those with ammonium ion as the nitrogen source (-0.3-0.6 h-l). Suspensions of nongrowing cells generally gave quantitative yields of ammonium ion from s-triazines, and ring carbon atoms were released as carbon dioxide. N-Alkylammelines in mixtures of strains A and D were quantitatively degraded to ammonium ion.Papers claiming microbial degradation of s-triazines, usually herbicides, are widespread, but reviewers have various interpretations of the rates of s-triazine degradation. Thus Alexander (1979) labels s-triazines recalcitrant, Cripps and Roberts (1978) imply ready degradability, whereas other reviewers refrain from comment (Esser et al., 1975;Kaufman and Kearney, 1970; Knuesli et al., 1969; Mikrobiologisches Institut, Eidgenossische Technische Hochschule, ETH-Zentnun, CH-8092 Zurich, Switzerland. Harris et al., 1968). Jordan et al. (1970) complement these data by citing extensive nonbiological degradation of striazines, usually on clay mineral surfaces. s-Triazines do not accumulate in the soils studied by Ramsteiner et al. (1972).Metabolites from s-triazines in experiments with animals, plants, and microorganisms have been reviewed by Fishbein (1975). Definitive proof of s-triazine metabolism by microorganisms in pure culture has been provided
Five anaerobic bacteria were tested for their abilities to transform tetrachloromethane so that information about enzymes involved in reductive dehalogenations of polychloromethanes could be obtained. Cultures of the sulfate reducer Desulfobactenium autotrophicum transformed some 80 ,uM tetrachloromethane to trichloromethane and a small amount of dichloromethane in 18 days under conditions of heterotrophic growth. The acetogens Acetobacterium woodii and Clostridium thermoaceticum in fructose-salts and glucose-salts media, respectively, degraded some 80 ,M tetrachloromethane completely within 3 days. Trichloromethane accumulated as a transient intermediate, but the only chlorinated methanes recovered at the end of the incubation were 8 ,uM dichloromethane and traces of chloromethane. Desulfobacter hydrogenophilus and an autotrophic, nitrate-reducing bacterium were unable to transform tetrachloromethane. Reduction of chlorinated methanes was thus observed only in the organisms with the acetyl-coenzyme A pathway. Experiments with ['4C]tetrachloromethane were done to determine the fate of this compound in the acetogen A. woodii. Radioactivity in an 11-day heterotrophic culture was largely (67%) recovered in C02, acetate, pyruvate, and cell material. In experiments with cell suspensions to which [14C]tetrachloromethane was added, 14Co2 appeared within 20 s as the major transformation product. A. woodii thus catalyzes reductive dechlorinations and transforms tetrachloromethane to CO2 by a series of unknown reactions. * Corresponding author. MATERIALS AND METHODS Materials. [U-'4C]acetate (2.07 TBq/mol), [2-'4C]acetate (1.96 TBq/mol), and ['4C]tetrachloromethane (2.3 TBq/mol;
, 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.
Organosulfonates are widespread compounds, be they natural products of low or high molecular weight, or xenobiotics. Many commonly found compounds are subject to desulfonation, even if it is not certain whether all the corresponding enzymes are widely expressed in nature. Sulfonates require transport systems to cross the cell membrane, but few physiological data and no biochemical data on this topic are available, though the sequences of some of the appropriate genes are known. Desulfonative enzymes in aerobic bacteria are generally regulated by induction, if the sulfonate is serving as a carbon and energy source, or by a global network for sulfur scavenging (sulfate-starvation-induced (SSI) stimulon) if the sulfonate is serving as a source of sulfur. It is unclear whether an SSI regulation is found in anaerobes. The anaerobic bacteria examined can express the degradative enzymes constitutively, if the sulfonate is being utilized as a carbon source, but enzyme induction has also been observed. At least three general mechanisms of desulfonation are recognisable or postulated in the aerobic catabolism of sulfonates: (1) activate the carbon neighboring the C-SO3- bond and release of sulfite assisted by a thiamine pyrophosphate cofactor; (2) destabilize the C-SO3- bond by addition of an oxygen atom to the same carbon, usually directly by oxygenation, and loss of the good leaving group, sulfite; (3) an unidentified, formally reductive reaction. Under SSIS control, different variants of mechanism (2) can be seen. Catabolism of sulfonates by anaerobes was discovered recently, and the degradation of taurine involves mechanism (1). When anaerobes assimilate sulfonate sulfur, there is one common, unknown mechanism to desulfonate the inert aromatic compounds and another to desulfonate inert aliphatic compounds; taurine seems to be desulfonated by mechanism (1).
The facultatively anaerobic bacterium Alcaligenes defragrans NKNTAU was found to oxidize taurine (2-aminoethanesulphonate) with nitrate as the terminal electron acceptor. Taurine was transaminated to 2-sulphoacetaldehyde. This was not converted into sulphite and acetate by a "sulphoacetaldehyde sulpho-lyase" (EC 4.4.1.12), but into sulphite and acetyl phosphate, which was identified by three methods. The enzyme, which required the addition of phosphate, thiamin diphosphate and Mg(2+) ions for activity, was renamed sulphoacetaldehyde acetyltransferase (Xsc; EC 2.3.1.-). Inducible Xsc was expressed at high levels, and a three-step 11-fold purification yielded an essentially homogeneous soluble protein, which was a homotetramer in its native form; the molecular mass of the subunit was found to be between about 63 kDa (SDS/PAGE) and 65.3 kDa (matrix-assisted laser-desorption ionization-time-of-flight MS). The N-terminal and two internal amino acid sequences were determined, and PCR primers were generated. The xsc gene was amplified and sequenced; the derived molecular mass of the processed protein was 65.0 kDa. The downstream gene presumably encoded the inducible phosphate acetyltransferase (Pta) found in crude extracts. The desulphonative enzymes ("EC 4.4.1.12") from Achromobacter xylosoxidans NCIMB 10751 and Desulfonispora thiosulfatigenes GKNTAU were shown to be Xscs. We detected at least three subclasses of xsc in Proteobacteria and in Gram-positive bacteria, and they comprised a distinct group within the acetohydroxyacid synthase supergene family. Genome sequencing data revealed xsc genes in Burkholderia fungorum (80% sequence identity) and Sinorhizobium meliloti (61%) with closely linked pta genes. Different patterns of regulation for the transport and dissimilation of taurine were hypothesized for S. meliloti and B. fungorum.
Two-dimensional gel electrophoresis of proteins from Escherichia coli, Pseudomonas putida, and Staphylococcus aureus, grown with methionine or one of a variety of organosulfates and organosulfonates as the sole source of sulfur, showed expression of specific sets of 7 to 14 proteins which were not observed during growth with sulfate or cysteine for all three species or with thiocyanate for P. putida and S. aureus. Under the same conditions, arylsulfatase activity in P. putida and S. aureus was seen to increase by up to 140-fold, suggesting that the proteins induced under these conditions may be involved in sulfur metabolism. We propose that these proteins are members of a sulfate starvation-induced stimulon.
1. The degradative pathway of melamine (1,3,5-triazine-2,4,6-triamine) was examined in Pseudomonas sp. strain A. 2. The bacterium grew with melamine, ammeline, ammelide, cyanuric acid or NH+4 as sole source of nitrogen, and each substrate was entirely metabolized. Utilization of ammeline, ammelide, cyanuric acid or NH+4 was concomitant with growth. But with melamine as substrate, a transient intermediate was detected, which was identified as ammeline by three methods. 3. Enzymes from strain A were separated by chromatography on DEAE-cellulose, and four activities were examined. 4. Melamine was converted stoichiometrically into equimolar amounts of ammeline and NH+4. 5. Ammeline was converted stoichiometrically into equimolar amounts of ammelide and NH+4; ammelide was identified by four methods. 6. Ammelide was converted stoichiometrically into equimolar amounts of cyanuric acid and NH+4; cyanuric acid was identified by four methods. 7. Cyanuric acid was converted by an enzyme preparation into an unidentified product with negligible release of NH+4. 8. The specific activities of the degradative enzymes (greater than or equal to 0.3 mkat/kg of protein) were high enough to explain the growth rate of the organism. 9. The bacterium converted 0.4 mM-melamine anaerobically into 2.3 mM-NH+4. 10. Two other pseudomonads and two strains of Klebsiella pneumoniae were also examined, with similar results. 11. The degradative pathway of melamine appears to be hydrolytic, and proceeds by three successive deaminations to cyanuric acid, which is further metabolized.
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