Structural studies on the Escherichia coli O101 lipopolysaccharide found in association with F41 and K99 fimbriae

Abstract Pseudomonas aeruginosa PAO1 used a broad range of alkanesulfonic acids as sole sulfur source for growth, with molar growth yields of 2.2 to 2.9 kg protein per mol sulfur. 4-Phenylbutane-1-sulfonate was desulfonated in vivo to yield 4-pheny 1-1-butyric acid quantitatively as the sole product, suggesting that the desulfonation mechanism is the same as when alkanesulfonates serve as a carbon source for growth. This contrasts with aromatic sulfonate utilization in other organisms, where different desulfonation reactions are used to provide carbon and sulfur. Desulfonation of alkanesulfonates to provide sulfur was repressed by sulfate or thiocyanate, and derepressed in their absence. The alkanesulfonatase system is hence controlled as part of the sulfate starvation-induced stimulon.     

Taurine-Sulfur Assimilation and Taurine-Pyruvate Aminotransferase Activity in Anaerobic Bacteria

We demonstrated the ability of strictly fermentative, as well as facultatively fermentative, bacteria to assimilate sulfonate sulfur for growth. Taurine (2-aminoethanesulfonate) can be utilized by Clostridium pasteurianum C1 but does not support fermentative growth of two Klebsiella spp. and two different Clostridium spp. However, the latter are able to assimilate the sulfur of a variety of other sulfonates (e.g., cysteate, 3-sulfopyruvate, and 3-sulfolactate) anaerobically. A novel taurine-pyruvate aminotransferase activity was detected in cell extracts of C. pasteurianum C1 grown with taurine as the sole sulfur source. This activity was not detected in extracts of other bacteria examined, in C. pasteurianum C1 grown with sulfate or sulfite as the sulfur source, or in a Klebsiella isolate assimilating taurine-sulfur by aerobic respiration. More common aminotransferase activities (e.g., with aspartate or glutamate as the amino donor and pyruvate, oxalacetate, or (alpha)-ketoglutarate as the amino acceptor) were present, no matter what sulfur source was used for growth. Partial characterization of the taurine-pyruvate aminotransferase revealed an optimal temperature of 37(deg)C and a broad optimal pH range of 7.5 to 9.5.     

Utilisation of aminomethane sulfonate by Chromohalobacter marismortui VH1

Chromohalobacter marismortui VH1 was screened for its ability to utilise organosulfonate compounds at a range of NaCl concentrations. Only aminomethane sulfonate, of seven sulfonates tested, was utilised. Length of lag phase during growth on aminomethane sulfonate, as either nitrogen and/or sulfur source, increased with increasing NaCl concentration. Cell yields increased linearly with increasing aminomethane sulfonate concentration up to 5 mM. Resting cells pregrown on aminomethane sulfonate as sole nitrogen source exhibited carbon-sulfur bond cleaving [0.123 nmol sulfate accumulated h(-1) (mg cells)(-1)] and sulfite-oxidising [0.185 nmol sulfate accumulated h(-1) (mg cells)(-1)] activities. C. marismortui VH1 is capable of sulfur-starvation deregulated metabolism of aminomethane sulfonate under high salt conditions.     

Utilization of sulfonic acids as the only sulfur source for growth of photosynthetic organisms

Growth on ethanesulfonic acid as the only sulfur source was found to occur in ten of the 14 green algae tested and in three of the ten cyanobacteria analyzed. Similar growth could not be demonstrated in the higher plant Lemna minor, or in tissue cultures of anise, sunflower and tobacco. Organisms growing on sulfonic acids as the only sulfur source developed an uptake system for ethanesulfonate found neither in algae growing on sulfate nor in algae unable to utilize sulfonic acids for growth. The development of sulfonate transport was not caused by substrate induction, but by conditions of sulfate starvation. The presence of this uptake system was always correlated with an increased sulfate-uptake capacity. Enhanced sulfate uptake was found in all S-deficient and sulfonate-grown cultures tested, indicating sulfate limitation as the regulatory signal. A lag period of 2–2.5 h after transfer to sulfate deprivation was needed for expression of both enhanced sulfate uptake and ethanesulfonate uptake in case of the green alga Chlorella fusca. It is speculated that the availability of sulfate (pool size) or a metabolic product in equilibrium with oxidized sulfur compounds (sulfate ester? sulfolipids?) controls sulfate and sulfonate uptake systems. The principle of (coordinated) derepression by starvation is discussed as a general strategy in photosynthetic organisms.     

Anaerobic Desulfonation of 4-Tolylsulfonate and 2-(4-Sulfophenyl) Butyrate by a Clostridium sp

Alkyl- and arylsulfonates were tested as sole added sources of sulfur for the growth of enrichment cultures under strictly anaerobic denitrifying or fermentative conditions. Cultures that utilized taurine, ethylsulfonate, the dyestuffs orange II and acid red I, tolylsulfonate, 2-(4-sulfophenyl)butyrate (SPB), a dialkyltetralinesulfonate, and 1-(4-sulfophenyl)octane were readily obtained. We chose to work with the simple aromatic compounds and isolated a fermentative bacterium, strain EV4, which utilized SPB as the sole added source of sulfur in glucose-mineral medium. The organism was identified as a Clostridium sp. related to Clostridium beijerinckii. Clostridium sp. strain EV4 utilized seven of seven tested arylsulfonates quantitatively. The growth yield was about 3 kg of protein per mol of sulfur, whether sulfonate or sulfate was utilized. A major product specific to each sulfonate could be observed. Although no product was identified, the existence of anaerobic desulfonation has been established.     

Saccharin as a sole source of carbon and energy for<Emphasis Type="Italic"> Sphingomonas xenophaga</Emphasis> SKN

A bacterium, strain SKN, that was able to utilize saccharin as the sole source of carbon and energy for aerobic growth, was enriched and isolated from communal sewage. The isolate was identified as a strain of Sphingomonas xenophaga. Saccharin was quantitatively converted to cell material, sulfate, ammonium and, presumably, CO(2). The specific rate of saccharin-dependent oxygen uptake during growth reached a maximum before the culture entered the stationary phase and then fell to undetectable levels. Saccharin was degraded only in the presence of molecular oxygen. Catechol was detected as an intermediate during degradation of saccharin in whole cells and catechol 1,2-dioxygenase was expressed inducibly during growth with saccharin. There was an apparent requirement of 2 mol O(2)/mol saccharin to remove the substituents on the ring and to cleave the ring. We presume that S. xenophaga SKN synthesizes a multi-component saccharin dioxygenase that simultaneously cleaves off both vicinal substituents from the aromatic ring to yield catechol and the undefined precursor of CO(2) as well as sulfate and ammonium ions.     

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.     

Characterization of main sulfur source of wood-degrading basidiomycetes by S K-edge X-ray absorption near edge spectroscopy (XANES)

The main wood degraders in aerobic terrestrial ecosystems belong to the white- and brown-rot fungi, where their biomass can be created on wood decay only. However, total sulfur (S) concentration in wood is very low and only little is known about the different sulfur compounds in wood today. Sulfur-starved brown-rot fungi Gloeophyllum trabeum and Oligoporus placenta were incubated on sterilized pine wood blocks whereas Lentinus cyathiformis and the white-rot fungi Trametes versicolor were incubated on sterilized beech wood blocks. After 19 weeks of incubation, the S oxidation status was analyzed in wood, in degraded wood, and in biomass of wood-degrading fungi by synchrotron based S K-edge XANES, and total S and sulfate were quantified. Total sulfur and sulfate content in pine wood blocks were approximately 50 and 1 ??g g⁻¹, respectively, while in beech wood approximately 100 and 20 ??g g⁻¹ were found, respectively. Sulfur in beech was dominated by sulfate-esters. In contrast, pine wood also contained larger amounts of reduced S. Three out of four selected fungi caused a reduction of the S oxidation state in wood from oxidized S (sulfate-ester, sulfate) to intermediate S (sulfonate, sulfoxide) or reduced S (thiols, e.g., proteins, peptides, enzyme cofactors). Only O. placenta shifted thiol to sulfonate. Growth experiments of these fungi on selective minimal media showed that in particular cysteine (thiol), sulfonates, and sulfate enhanced total mycelium growth. Consequently, wood-degrading fungi were able to utilize a large variety of different wood S sources for growth but preferentially transformed in vivo sulfate-esters and thiol into biomass structures.     

Assimilation of sulfur from alkyl- and arylsulfonates by Clostridium spp.

Organisms able to utilize one of several alkyl- and arylsulfonates as sole source of sulfur under anoxic conditions were enriched. Three fermenting bacteria, all putative Clostridium spp., were isolated in pure culture. All three organisms had wide substrate ranges for alkylsulfonates, taurine and arylsulfonates, presumably due to three different enzyme systems. One organism, strain KNNDS (DSM 10612) was selected for further characterization. The organism was possibly a new Clostridium sp., with Clostidium intestinalis as its nearest neighbor (97.6% similarity of rDNA). Strain KNNDS catalyzed complete sulfonate utilization concomitant with growth. Growth yields of approximtely 3 kg protein/mol sulfur were observed, independent of the sulfur source [e.g. sulfate, sulfide, 4-(phenyl)butyl-1-sulfonate, 2,6-naphthyldisulfonate or 4-nitrocatechol sulfate]. We failed to detect significant amounts of either an arylsulfonatase or an arylsulfatase, and we hypothesize different arylsulfatases [EC 3.1.6.1] in aerobes and in Clostridium spp. Received: 15 October / Accepted: 29 November 1996     

Bacterial cleavage of nitrogen to sulfone bonds in sulfamide and 1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide: formation of 2-nitrobenzamide by Gordonia sp.

Pure cultures of aerobic bacteria were isolated which could utilize sulfamate, sulfamide or 1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (BTDD) as sole source of sulfur for growth and thus cleave a N–S(O)_x bond. The molar growth yields indicated that each source of sulfur was utilized quantitatively. This was confirmed directly for Gordonia sp. strain BT2 utilizing BTDD, which was converted quantitatively via an unidentified intermediate to 2-nitrobenzamide. Another isolate, strain BT1, could utilize saccharin to yield salicylamide, thus cleaving both the N–S(O)_x and C–S(O)_x bonds.     

Anaerobic degradation of citrate under sulfate reducing and methanogenic conditions

Citrate is an important component of metal processing effluents such as chemical mechanical planarization wastewaters of the semiconductor industry. Citrate can serve as an electron donor for sulfate reduction applied to promote the removal of metals, and it can also potentially be used by methanogens that coexist in anaerobic biofilms. The objective of this study was to evaluate the degradation of citrate with sulfate-reducing and methanogenic biofilms. During batch bioassays, the citrate, acetate, methane and sulfide concentrations were monitored. The results indicate that independent of the biofilm or incubation conditions used, citrate was rapidly fermented with specific rates ranging from 566 to 720 mg chemical oxygen demand (COD) consumed per gram volatile suspended solids per day. Acetate was found to be the main fermentation product of citrate degradation, which was later degraded completely under either methanogenic or sulfate reducing conditions. However, if either sulfate reduction or methanogenesis was infeasible due to specific inhibitors (2-bromoethane sulfonate), absence of sulfate or lack of adequate microorganisms in the biofilm, acetate accumulated to levels accounting for 90–100% of the citrate-COD consumed. Based on carbon balances measured in phosphate buffered bioassays, acetate, CO₂ and hydrogen are the main products of citrate fermentation, with a molar ratio of 2:2:1 per mol of citrate, respectively. In bicarbonate buffered bioassays, acetogenesis of H₂ and CO₂ increased the yield of acetate. The results taken as a whole suggest that in anaerobic biofilm systems, citrate is metabolized via the formation of acetate as the main metabolic intermediate prior to methanogenesis or sulfate reduction. Sulfate reducing consortia must be enriched to utilize acetate as an electron donor in order to utilize the majority of the electron-equivalents in citrate.     

Fermentation and Sulfur Reduction in the Mat-Building Cyanobacterium Microcoleus chthonoplastes

The mat-building cyanobacterium Microcoleus chthonoplastes carried out a mixed-acid fermentation when incubated under anoxic conditions in the dark. Endogenous storage carbohydrate was fermented to acetate, ethanol, formate, lactate, H(inf2), and CO(inf2). Cells with a low glycogen content (about 0.3 (mu)mol of glucose per mg of protein) produced acetate and ethanol in equimolar amounts. In addition to glycogen, part of the osmoprotectant, glucosyl-glycerol, was degraded. The glucose component of glucosyl-glycerol was fermented, whereas glycerol was released into the medium. Cells with a high content of glycogen (about 2 (mu)mol of glucose per mg of protein) did not utilize glucosyl-glycerol. These cells produced more acetate than ethanol. M. chthonoplastes was also capable of using elemental sulfur as the electron acceptor during fermentation, resulting in the production of sulfide. With sulfur present, acetate production increased whereas ethanol production decreased. Also, less formate was produced and the evolution of hydrogen ceased completely. In general, the carbon recoveries were satisfactory but the oxidation-reduction balances were too high. The latter could be explained by assuming the reduction of ferric iron, which is associated with the cells, mediated by the oxidation of formate. The switch from photoautotrophic to fermentative metabolism did not require de novo protein synthesis, and fermentation started immediately upon transfer to dark anoxic conditions. From the molar ratios of the fermentation products and from measurement of enzyme activities in cell extracts, we concluded that glucose derived from glycogen and glucosyl-glycerol is degraded via the Embden-Meyerhof-Parnas pathway.     

Aerobic biodegradation of 2,2'-dithiodibenzoic acid produced from dibenzothiophene metabolites

Dibenzothiophene is a sulfur heterocycle found in crude oils and coal. The biodegradation of dibenzothiophene through the Kodama pathway by Pseudomonas sp. strain BT1d leads to the formation of three disulfides: 2-oxo-2-(2-thiophenyl)ethanoic acid disulfide, 2-oxo-2-(2-thiophenyl)ethanoic acid-2-benzoic acid disulfide, and 2,2'-dithiodibenzoic acid. When provided as the carbon and sulfur source in liquid medium, 2,2'-dithiodibenzoic acid was degraded by soil enrichment cultures. Two bacterial isolates, designated strains RM1 and RM6, degraded 2,2'-dithiodibenzoic acid when combined in the medium. Isolate RM6 was found to have an absolute requirement for vitamin B12, and it degraded 2,2'-dithiodibenzoic acid in pure culture when the medium was supplemented with this vitamin. Isolate RM6 also degraded 2,2'-dithiodibenzoic acid in medium containing sterilized supernatants from cultures of isolate RM1 grown on glucose or benzoate. Isolate RM6 was identified as a member of the genus Variovorax using the Biolog system and 16S rRNA gene analysis. Although the mechanism of disulfide metabolism could not be determined, benzoic acid was detected as a transient metabolite of 2,2'-dithiodibenzoic acid biodegradation by Variovorax sp. strain RM6. In pure culture, this isolate mineralized 2,2'-dithiodibenzoic acid, releasing 59% of the carbon as carbon dioxide and 88% of the sulfur as sulfate.     

Fructose degradation byDesulfovibrio sp. in pure culture and in coculture withMethanospirillum hungatei

In a mineral medium containing sulfate, the sulfate-reducing bacteriumDesulfovibrio sp. strain JJ degraded 1 mol of fructose stoichiometrically to 1 mol of H₂S, 2 mol of acetate, and presumably 2 mol of CO₂. The doubling time was 10 h, and the yield was 41.6 g dry weight/mol fructose degraded. In the absence of sulfate, the hydrogenophilic methanogenMethanospirillum hungatei replaced sulfate as hydrogen sink. In such cocultures, 1 mol of fructose was converted to acetate, methane, succinate, and presumably CO₂ in varying concentrations. The growth yield of the H₂-transferring association was 33 g dry weight/mol fructose. In the absence of sulfate,Desulfovibrio strain JJ slowly fermented 1 mol of fructose to 1 mol of succinate, 0.5 mol of acetate, and 0.5 mol of ethanol. The results are compared with those of other anaerobic hexose-degrading bacteria.     

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.     

Ametryne and Prometryne as Sulfur Sources for Bacteria

Bacteria were isolated that could utilize quantitatively the s-triazine herbicide prometryne [N,N′ -bis(1-methylethyl)-6-(methylthio)-1,3,5-triazine-2,4-diamine] or ametryne [N-ethyl-N′-(1-methylethyl)-6-(methylthio)-1,3,5-triazine- 2,4-diamine], or both, as a sole source of sulfur for growth. The success of enrichments depended on previous exposure of the soil inoculum to s-triazine herbicides. Deaminoethylametryne [4-(1-methylethyl)amino-6-(methylthio)-1,3,5-triazine-2-(1H)-one], methylsulfonic acid, and sodium sulfate could also be used as sulfur sources. Utilization of a compound was quantified as the growth yield per mole of sulfur supplied. Yields were about 6 kg of protein per mol of sulfur. The product of the desulfuration of an s-triazine was identified as the corresponding hydroxy-derivative. This is the first substantiated report of the utilization of these s-triazines as sulfur sources by bacteria.     

Energetics of Microbacterium thermosphactum in glucose-limited continuous culture.

Microbacterium thermosphactum was grown at 25 degrees C in glucose-limited continuous culture under aerobic (greater than 120 microM oxygen) and anaerobic (less than 0.2 microM oxygen) conditions. The end products of the anaerobic metabolism of glucose were identified as L-lactate and ethanol. Together these compounds accounted for between 85 and 90% of the glucose utilized over the full range of growth rates studied. In addition, 4% of the glucose utilized was incorporated into cellular material. Under anaerobic conditions the molar growth yield was 40 g (dry weight) of cells per mol of glucose utilized, and the maintenance energy coefficient was 0.4 mmol of glucose utilized per g (dry weight) of cells per h. For cells grown under aerobic conditions in the corresponding values were 73 g/mol and 0.2 mmol/g per h, respectively. The molar growth yield with respect to adenosine 5'-triphosphate varied with the growth rate of the culture, and the true molar growth yield with respect to adenosine 5'-triphosphate was found to be 20 g/mol of adenosine 5'-triphosphate.     

Energetics of Microbacterium thermosphactum in glucose-limited continuous culture.

Microbacterium thermosphactum was grown at 25 degrees C in glucose-limited continuous culture under aerobic (greater than 120 microM oxygen) and anaerobic (less than 0.2 microM oxygen) conditions. The end products of the anaerobic metabolism of glucose were identified as L-lactate and ethanol. Together these compounds accounted for between 85 and 90% of the glucose utilized over the full range of growth rates studied. In addition, 4% of the glucose utilized was incorporated into cellular material. Under anaerobic conditions the molar growth yield was 40 g (dry weight) of cells per mol of glucose utilized, and the maintenance energy coefficient was 0.4 mmol of glucose utilized per g (dry weight) of cells per h. For cells grown under aerobic conditions in the corresponding values were 73 g/mol and 0.2 mmol/g per h, respectively. The molar growth yield with respect to adenosine 5'-triphosphate varied with the growth rate of the culture, and the true molar growth yield with respect to adenosine 5'-triphosphate was found to be 20 g/mol of adenosine 5'-triphosphate.     

Lithoautotrophic growth of the freshwater strain Beggiatoa D-402 and energy conservation in a homogeneous culture under microoxic conditions

The freshwater filamentous bacterium Beggiatoa D-402 was shown to grow lithoautotrophically in a homogeneous culture under microoxic conditions only, the growth yield being the highest at 0.1 mg O(2) l(-1). High activities of the Calvin cycle key enzymes and of the dissimilatory path thiosulfate oxidation enzymes were found in the bacterial cells. The rate of CO(2) fixation above 112 nmol min(-1) (mg protein)(-1), an about 90% increase in the protein carbon at the expense of CO(2) carbon and an increase in the molar yield up to 12 mg dry weight (mmol oxidized thiosulfate)(-1) indicate the bacterial growth was autotrophic. Thiosulfate was oxidized by the strain almost completely into sulfate. The metabolically useful energy was conserved by oxidative phosphorylation that was coupled to oxidation of sulfur compounds. The bacterial membranes were found to contain CO-binding cytochromes b and two cytochromes c with M(r) 23 and 26 kDa, the terminal part of the respiratory chain containing presumably a cbb(3)-type oxidase. A cytochrome c with M(r) 12 kDa was detected in the soluble fraction.