Comparative aspects of utilization of sulfonate and other sulfur sources by Escherichia coli K12

Selected biochemical features of sulfonate assimilation in Escherichia coli K-12 were studied in detail. Competition between sulfonate-sulfur and sulfur sources with different oxidation states, such as cysteine, sulfite and sulfate, was examined. The ability of the enzyme sulfite reductase to attack the C-S linkage of sulfonates was directly examined. Intact cells formed sulfite from sulfonate-sulfur. In cysteine-grown cells, when cysteine was present with either cysteate or sulfate, assimilation of both of the more oxidized sulfur sources was substantially inhibited. In contrast, none of three sulfonates had a competitive effect on sulfate assimilation. In studies of competition between different sulfonates, the presence of taurine resulted in a decrease in cysteate uptake by one-half, while in the presence of isethionate, cysteate uptake was almost completely inhibited. In sulfite-grown cells, sulfonates had no competitive effect on sulfite utilization. An E. coli mutant lacking sulfite reductase and unable to utilize isethionate as the sole source of sulfur formed significant amounts of sulfite from isethionate. In cell extracts, sulfite reductase itself did not utilize sulfonate-sulfur as an electron acceptor. These findings indicate that sulfonate utilization may share some intermediates (e.g. sulfite) and regulatory features (repression by cysteine) of the assimilatory sulfate reductive pathway, but sulfonates do not exert regulatory effects on sulfate utilization. Other results suggest that unrecognized aspects of sulfonate metabolism, such as specific transport mechanisms for sulfonates and different regulatory features, may exist.     

The ssu locus plays a key role in organosulfur metabolism in Pseudomonas putida S-313

Pseudomonas putida S-313 can utilize a broad range of aromatic sulfonates as sulfur sources for growth in sulfate-free minimal medium. The sulfonates are cleaved monooxygenolytically to yield the corresponding phenols. miniTn5 mutants of strain S-313 which were no longer able to desulfurize arylsulfonates were isolated and were found to carry transposon insertions in the ssuEADCBF operon, which contained genes for an ATP-binding cassette-type transporter (ssuABC), a two-component reduced flavin mononucleotide-dependent monooxygenase (ssuED) closely related to the Escherichia coli alkanesulfonatase, and a protein related to clostridial molybdopterin-binding proteins (ssuF). These mutants were also deficient in growth with a variety of other organosulfur sources, including aromatic and aliphatic sulfate esters, methionine, and aliphatic sulfonates other than the natural sulfonates taurine and cysteate. This pleiotropic phenotype was complemented by the ssu operon, confirming its key role in organosulfur metabolism in this species. Further complementation analysis revealed that the ssuF gene product was required for growth with all of the tested substrates except methionine and that the oxygenase encoded by ssuD was required for growth with sulfonates or methionine. The flavin reductase SsuE was not required for growth with aliphatic sulfonates or methionine but was needed for growth with arylsulfonates, suggesting that an alternative isozyme exists for the former compounds that is not active in transformation of the latter substrates. Aryl sulfate ester utilization was catalyzed by an arylsulfotransferase, and not by an arylsulfatase as in the related species Pseudomonas aeruginosa.     

Expression and functional roles of Bradyrhizobium japonicum genes involved in the utilization of inorganic and organic sulfur compounds in free-living and symbiotic conditions

Strains of Bradyrhizobium spp. form nitrogen-fixing symbioses with many legumes, including soybean. Although inorganic sulfur is preferred by bacteria in laboratory conditions, sulfur in agricultural soil is mainly present as sulfonates and sulfur esters. Here, we show that Bradyrhizobium japonicum and B. elkanii strains were able to utilize sulfate, cysteine, sulfonates, and sulfur-ester compounds as sole sulfur sources for growth. Expression and functional analysis revealed that two sets of gene clusters (bll6449 to bll6455 or bll7007 to bll7011) are important for utilization of sulfonates sulfur source. The bll6451 or bll7010 genes are also expressed in the symbiotic nodules. However, B. japonicum mutants defective in either of the sulfonate utilization operons were not affected for symbiosis with soybean, indicating the functional redundancy or availability of other sulfur sources in planta. In accordance, B. japonicum bacteroids possessed significant sulfatase activity. These results indicate that strains of Bradyrhizobium spp. likely use organosulfur compounds for growth and survival in soils, as well as for legume nodulation and nitrogen fixation.     

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.     

Dimethyl sulfoxide reductase is not required for trimethylamine N-oxide reduction in Escherichia chcoli

Deletion mutants of Escherichia coli lacking dimethyl sulfoxide (DMSO) reductase activity and consequently unable to utilize DMSO as an electron acceptor for anaerobic growth have been isolated. These mutants retained the ability to use trimethylamine N-oxide (TMAO) as an electron acceptor and the TMAO reductase activity was found to be unaltered. Heating the cell-free extract of the wild-type strain at 70 degrees C for 15 min selectively inactivated the DMSO reductase activity while the TMAO reductase activity remained unchanged for at least 1 h.     

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     

Nitrate reduction mutants of fusarium moniliforme (gibberella fujikuroi)

Twelve strains of Fusarium moniliforme were examined for their ability to sector spontaneously on toxic chlorate medium. All strains sectored frequently; 91% of over 1200 colonies examined formed chlorate-resistant, mutant sectors. Most of these mutants had lesions in the nitrate reduction pathway and were unable to utilize nitrate (nit mutants). nit mutations occurred in seven loci: a structural gene for nitrate reductase (nit1), a regulatory gene specific for the nitrate reduction pathway (nit3), and five genes controlling the production of a molybdenum-containing cofactor that is necessary for nitrate reductase activity (nit2, nit4, nit5, nit6, nit7). No mutations affecting nitrite reductase or a major nitrogen regulatory locus were found among over 1000 nit mutants. Mutations of nit1 were recovered most frequently (39-66%, depending on the strain) followed by nit3 mutations (23-42%). The frequency of isolation of each mutant type could be altered, however, by changing the source of nitrogen in the chlorate medium. We concluded that genetic control of nitrate reduction in F. moniliforme is similar to that in Aspergillus and Neurospora, but that the overall regulation of nitrogen metabolism may be different.     

Competitiveness of different polysaccharide utilization mutants of Bacteroides thetaiotaomicron in the intestinal tracts of germfree mice.

Bacteroides thetaiotaomicron, an obligate anaerobe found in high numbers in human colons, can utilize a variety of polysaccharides. To determine which type of polysaccharide contributes most to the nutrition of B. thetaiotaomicron in vivo, we isolated and characterized transposon-generated mutants deficient in the ability to use different polysaccharides. Some mutants were deficient in polysaccharide utilization because of the inability to utilize a component monosaccharide. These mutants included a mutant that was unable to utilize L-fucose (a component of goblet cell mucin), a mutant that was unable to utilize D-galactose (a component of raffinose, stachyose, arabinogalactan, and goblet cell mucin), and a mutant that was unable to utilize either glucuronic acid (a component of mucopolysaccharides) or galacturonic acid (a component of polygalacturonic acid or pectin). Other mutants were unable to use the polysaccharide but could use the component sugars. These included four mutants that were unable to utilize starch and one mutant that was unable to utilize polygalacturonic acid. The mutants were tested for the ability to compete with the wild type for colonization of the intestinal tracts of germfree mice. The only mutants against which the wild type competed successfully in the intestinal tracts of germfree mice were a galactose-negative mutant and a uronic acid-negative mutant. These mutations differed from the others tested in that they affected utilization of more than one type of polysaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Competitiveness of different polysaccharide utilization mutants of Bacteroides thetaiotaomicron in the intestinal tracts of germfree mice

Bacteroides thetaiotaomicron, an obligate anaerobe found in high numbers in human colons, can utilize a variety of polysaccharides. To determine which type of polysaccharide contributes most to the nutrition of B. thetaiotaomicron in vivo, we isolated and characterized transposon-generated mutants deficient in the ability to use different polysaccharides. Some mutants were deficient in polysaccharide utilization because of the inability to utilize a component monosaccharide. These mutants included a mutant that was unable to utilize L-fucose (a component of goblet cell mucin), a mutant that was unable to utilize D-galactose (a component of raffinose, stachyose, arabinogalactan, and goblet cell mucin), and a mutant that was unable to utilize either glucuronic acid (a component of mucopolysaccharides) or galacturonic acid (a component of polygalacturonic acid or pectin). Other mutants were unable to use the polysaccharide but could use the component sugars. These included four mutants that were unable to utilize starch and one mutant that was unable to utilize polygalacturonic acid. The mutants were tested for the ability to compete with the wild type for colonization of the intestinal tracts of germfree mice. The only mutants against which the wild type competed successfully in the intestinal tracts of germfree mice were a galactose-negative mutant and a uronic acid-negative mutant. These mutations differed from the others tested in that they affected utilization of more than one type of polysaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cystathionine Metabolism in Methionine Auxotrophic and Wild-Type Strains of Saccharomyces cerevisiae

The role of cystathionine in methionine biosynthesis in wild-type and auxotrophic strains of Saccharomyces cerevisiae was studied. Homocysteine and cysteinerequiring mutants were selected for detailed study. Exogenously supplied cystathionine, although actively transported by all strains tested, could not satisfy the organic sulfur requirements of the mutants. Cell-free extracts of the wild-type, homocysteine, and cysteine auxotrophs were shown to cleave cystathionine. Pyruvic acid and homocysteine were identified as teh products of this cleavage. A mutant containing an enzyme which could cleave cystathionine to homocysteine in cell-free experiments was unable to use cystathionine as a methionine precursor in the intact organisms. The significance of this finding is discussed.     

Genetic evidence for the physiological significance of the D-tagatose 6-phosphate pathway of lactose and D-galactose degradation in staphylococcus aureus

Mutants of Staphylococcus aureus were isolated which were unable to utilize d-galactose or lactose, but which were able to utilize all other carbohydrates tested. Growth of the mutants on a peptone-containing medium was inhibited by d-galactose. Of those mutants selected for further study, one (tagI2) was missing d-galactose 6-phosphate isomerase, one (tagK3) was missing d-tagatose 6-phosphate kinase, and one (tagA4) was missing d-tagatose 1, 6-diphosphate aldolase. Each of these mutants accumulated the substrate of the missing enzyme intracellularly. Spontaneous revertants of each of the mutants simultaneously regained their ability to utilize d-galactose and lactose, lost their sensitivity to d-galactose, regained the missing enzymatic activities, and no longer accumulated intermediates of the d-tagatose 6-phosphate pathway. These data support our previous contention that the physiologically significant route for the metabolism of d-galactose and the d-galactosyl moiety of lactose in S. aureus is the d-tagatose 6-phosphate pathway. Furthermore, a mutant constitutive for all three enzymes of this pathway was isolated, indicating that the products of the tagI, tagK, and tagA genes are under common genetic control. This conclusion was supported by the demonstration that d-galactose 6-phosphate isomerase, d-tagatose 6-phosphate kinase, and d-tagatose 1, 6-diphosphate aldolase are coordinately induced in the parental strain.     

Importance of organosulfur utilization for survival of Pseudomonas putida in soil and rhizosphere

The sulfur present in both agricultural and uncultivated soils is largely in the form of sulfonates and sulfate esters and not as free, bioavailable inorganic sulfate. Desulfurization of the former compounds in vitro has previously been studied in Pseudomonas putida, a common rhizosphere inhabitant. Survival of P. putida strains was now investigated in three sulfur-deficient Danish soils which were found to contain 60 to 70% of their sulfur in sulfonate or sulfate ester form, as determined by X-ray near-edge spectroscopy. The soil fitness of P. putida S-313 was compared with that of isogenic strains with mutations in the sftR and asfA genes (required for in vitro desulfurization of sulfate esters and arylsulfonates, respectively) and in the ssu locus (required in vitro for the desulfurization of both sulfonates and sulfate esters). asfA or sftR mutants showed significantly reduced survival compared to the parent strain in bulk soil that had been enriched with carbon and nitrogen to mimic rhizosphere conditions, but this reduced survival was not observed in the absence of these additives. In a tomato rhizosphere grown in compost, survival of sftR and ssu mutants was reduced relative to the parent strain. The results demonstrate that the ability to desulfurize sulfonates and sulfate esters is critical for survival of bacteria in the rhizosphere but less so in bulk soils outside the influence of plant roots, where carbon is the limiting nutrient for growth.     

alpha-Aminoadipate as a primary nitrogen source for Saccharomyces cerevisiae mutants.

In contrast to wild-type strains of the yeast Saccharomyces cerevisiae, lys2 and lys5 mutants are able to utilize alpha-aminoadipate as a primary source of nitrogen. Chattoo et al. (B. B. Chattoo, F. Sherman, D. A. Azubalis, T. A. Fjellstedt, D. Mehnert, and M. Ogur, Genetics 93:51-65, 1979) relied on this difference in the effective utilization of alpha-aminoadipate to develop a procedure for directly selecting lys2 and lys5 mutants. In this study we used a range of mutant strains and various media to determine why normal strains are unable to utilize alpha-aminoadipate as a nitrogen source. Our results demonstrate that the anabolism of high levels of alpha-aminoadipate through the biosynthetic pathway of lysine results in the accumulation of a toxic intermediate and, furthermore, that lys2 and lys5 mutants contain blocks leading to the formation of this intermediate.     

Conversion of Pipecolic Acid into Lysine in Penicillium chrysogenum Requires Pipecolate Oxidase and Saccharopine Reductase: Characterization of the lys7 Gene Encoding Saccharopine Reductase

Pipecolic acid is a component of several secondary metabolites in plants and fungi. This compound is useful as a precursor of nonribosomal peptides with novel pharmacological activities. In Penicillium chrysogenum pipecolic acid is converted into lysine and complements the lysine requirement of three different lysine auxotrophs with mutations in the lys1, lys2, or lys3 genes allowing a slow growth of these auxotrophs. We have isolated two P. chrysogenum mutants, named 7.2 and 10.25, that are unable to convert pipecolic acid into lysine. These mutants lacked, respectively, the pipecolate oxidase that converts pipecolic acid into piperideine-6-carboxylic acid and the saccharopine reductase that catalyzes the transformation of piperideine-6-carboxylic acid into saccharopine. The 10.25 mutant was unable to grow in Czapek medium supplemented with alpha-aminoadipic acid. A DNA fragment complementing the 10.25 mutation has been cloned; sequence analysis of the cloned gene (named lys7) revealed that it encoded a protein with high similarity to the saccharopine reductase from Neurospora crassa, Magnaporthe grisea, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. Complementation of the 10.25 mutant with the cloned gene restored saccharopine reductase activity, confirming that lys7 encodes a functional saccharopine reductase. Our data suggest that in P. chrysogenum the conversion of pipecolic acid into lysine proceeds through the transformation of pipecolic acid into piperideine-6-carboxylic acid, saccharopine, and lysine by the consecutive action of pipecolate oxidase, saccharopine reductase, and saccharopine dehydrogenase.     

The Cryptococcus neoformans STE11alpha gene is similar to other fungal mitogen-activated protein kinase kinase kinase (MAPKKK) genes but is mating type specific

Partial sequence analysis of the Cryptococcus neoformans MATalpha mating type locus revealed the presence of a gene with substantial sequence similarity to other fungal mitogen-activated protein (MAP) kinase kinase kinase (MAPKKK) genes. The C. neoformans gene, designated STE11alpha, showed the highest degree of similarity to the Neurospora crassa nrc-1, Schizosaccharomyces pombe byr2 and Saccharomyces cerevisiae STE11 genes. A polymerase chain reaction-mediated sib-selection technique was successfully adapted for the purpose of disrupting STE11alpha. C. neoformans ste11alphaDelta mutants were found to be sterile, consistent with the phenotypes of ste11 and byr2 mutants in S. cerevisiae and S. pombe respectively. Haploid ste11alphaDelta mutants were also found to be unable to produce hyphae, suggesting that the C. neoformans gene is functionally conserved when compared with its S. cerevisiae MAPKKK counterpart. Comparison of the wild-type STE11alpha strain with a ste11alphaDelta disruptant for virulence using the mouse model showed that the ste11alphaDelta strain was less virulent, but the difference was only minor. In spite of some of the conserved functions of STE11alpha, linkage analysis showed that STE11alpha is only found in mating type alpha strains. These results demonstrate that, although functionally conserved, the mating pathway in C. neoformans has a unique organization.     

Desulfurization and desulfonation: applications of sulfur-controlled gene expression in bacteria

Inorganic sulfate is the preferred sulfur source for the growth of most microorganisms but, in its absence, many organosulfur compounds can be degraded microbially to provide sulfur. Desulfurization of dibenzothiophene (DBT) by Rhodococcus sp. and of aromatic sulfonates by Pseudomonas sp. has considerable biotechnological potential. Both these pathways require non-flavin-containing FMNH2-dependent monoxygenases (DszC/DszA and SsuD, respectively). FMNH2 is provided from the freely diffusible FMNH2 pool in the cell, and is replenished by specific NAD(P)H:FMN oxidoreductases (DszD and SsuE). Overexpression of the DszD FMN reductase in a heterologous system increases the efficiency of DBT desulfurization but is detrimental to cell growth at high levels. Expression of the sulfonatase that cleaves aromatic sulfonates (surfactants, dyes) is accompanied by synthesis of a thiol-specific antioxidant protein, which may protect the cell from superoxide radicals generated by autoxidation of the reduced flavin. Effective application of DBT desulfurization in the biodesulfurization of crude oil, and of arylsulfonate desulfonation in bioremediation, may require optimization of both flavin reductase levels and antioxidant protection systems within the cell.     

Importance of Organosulfur Utilization for Survival of Pseudomonas putida in Soil and Rhizosphere

The sulfur present in both agricultural and uncultivated soils is largely in the form of sulfonates and sulfate esters and not as free, bioavailable inorganic sulfate. Desulfurization of the former compounds in vitro has previously been studied in Pseudomonas putida, a common rhizosphere inhabitant. Survival of P. putida strains was now investigated in three sulfur-deficient Danish soils which were found to contain 60 to 70% of their sulfur in sulfonate or sulfate ester form, as determined by X-ray near-edge spectroscopy. The soil fitness of P. putida S-313 was compared with that of isogenic strains with mutations in the sftR and asfA genes (required for in vitro desulfurization of sulfate esters and arylsulfonates, respectively) and in the ssu locus (required in vitro for the desulfurization of both sulfonates and sulfate esters). asfA or sftR mutants showed significantly reduced survival compared to the parent strain in bulk soil that had been enriched with carbon and nitrogen to mimic rhizosphere conditions, but this reduced survival was not observed in the absence of these additives. In a tomato rhizosphere grown in compost, survival of sftR and ssu mutants was reduced relative to the parent strain. The results demonstrate that the ability to desulfurize sulfonates and sulfate esters is critical for survival of bacteria in the rhizosphere but less so in bulk soils outside the influence of plant roots, where carbon is the limiting nutrient for growth.