Mixotrophic growth of Thiobacillus A2 on acetate and thiosulfate as growth limiting substrates in the chemostat

During heterotrophic growth on acetate, in batch culture, the autotrophic growth potential of Thiobacillus A2, i.e. the capacity to oxidize thiosulfate and to fix carbon dioxide via the Calvin cycle, was completely repressed. The presence of thiosulfate in a batch culture with acetate as the organic substrate partly released the repression of the thiosulfate oxidizing system. Cultivation of the organism in continuous culture at a dilution rate of 0.05 h^-1 with different concentration ratios of thiosulfate and acetate in the reservoir medium led to mixotrophic growth under dual substrate limitation. Growth on the different mixtures of acetate and thiosulfate yielded upto 30% more cell dry weight than predicted from the growth yields on comparable amounts of these substrates separately. The extent to which the carbon dioxide fixation capacity and the maximum thiosulfate and acetate oxidation capacity are repressed appeared to be a function of the thiosulfate to acetate concentration ratio in the reservoir medium. The results of ¹⁴C-acetate assimilation experiments and of gas-analysis demonstrated that the extent to which acetate was assimilated depended also on the substrate ratio in the inflowing medium. Under the different growth conditions surprisingly little variation was found in some tri-carboxylic acid cycle enzyme activities. Cultivation of T. A2 at different growth rates with a fixed mixture of thiosulfate (18 mM) and acetate (11 mM) in the medium, showed that dual substrate limitation occured at dilution rates ranging from 0.03–0.20 h^-1.Abbreviations PPO 2,5-diphenoloxazol - RubPCase Ribulose-1,5-bisphophate carboxylase - Tris tris (hydroxymethyl) aminomethane - EDTA ethylenediaminetetra-acetic acid     

Reactivity versus flexibility in thiobacilli

The results of ecophysiological studies on obligately and facultatively chemolithotrophic thiobacilli performed over the past years clearly show that the two types of organisms occupy different ecological niches. Chemostat experiments with cultures of the obligate chemolithotroph Thiobacillus neapolitanus and the facultative chemolithotroph Thiobacillus A2 have been carried out to explain the competitiveness of T. neapolitanus under conditions of strongly fluctuating substrate supply. Thiobacillus neapolitanus appeared to be very resistant to starvation periods whereafter it could oxidize sulfide (or thiosulfate) almost instantaneously at the original rate. Under alternate supply of 4 h sulfide and 4 h sulfate (or acetate which does not support growth of the organism either) to a chemostat culture of T. neapolitanus (D=0.05 h^–1) the sulfide concentration in the growth vessel never reached levels higher than 4m. This strategy is aimed at maximal reactivity. In contrast to T. neapolitanus the facultative chemolithotroph T.A2 appeared to be very flexible with respect to its energy generation. Under alternate supply of 4 h sulfide and 4 h acetate (D=0.05 h^–1) T.A2 was able to grow continuously since it directed its metabolism to either heterotrophy or autotrophy by rapid induction-repression mechanisms. This flexible strategy seems to be incompatible with a reactive strategy within one organism, since the oxidation capacity for sulfide decreased during the acetate period resulting in accumulation of sulfide during the sulfide period. It is concluded that T.A2 needs a continuous supply of an inorganic and an organic substrate to thrive whereas T. neapolitanus needs only a continuous supply of a reduced inorganic sulfur source but also will persist in environments with interrupted addition of sulfide provided that the starvation period does not last too long.     

Influence of the glucose input concentration on the kinetics of metabolite production by Klebsiella aerogenes NCTC 418: Growing in chemostat culture in potassium- or ammonia-limited environments

Thiobacillus neapolitanus grown in minerals medium in a thiosulfate-limited chemostat excreted 15% of all the carbon dioxide fixed as ¹⁴C-organic compounds at a dilution rate (D) of 0.03 h^-1. At D=0.36 h^-1 this excretion was 8.5%. Up to a D of 0.2h^-1 glycolate was the major excretion product. Glycolate excretion was maximal at a pO₂ of 100% air saturation (a.s.) and not detectable at a pO₂ of 5% (a.s.). Increasing the pCO₂ of the gassing mixture to 5% (v/v), at a pO₂ of 50% a.s. resulted in a lowering of the glycolate excretion from 3.5% of the total CO₂ fixed to 1.8%. These results indicate that glycolate excretion in T. neapolitanus is due to oxygenase activity of D-ribulose-1,5-bisphosphate carboxylase. HPMS (2-pyridylhydroxymethanesulfonate), an inhibitor of glycolate metabolism, did not stimulate the glycolate production in T. neapolitanus. Glycolate excretion was not observed in thiosulfate-limited chemostat cultures of the obligately chemolithotrophic Thiomicrospira pelophila or in thiosulfate- or formate-grown cultures of the facultatively chemolithotrophic Thiobacillus A2.Abbreviation HPMS 2-pyridylhydroxymethanesulfonate     

Thiobacillus strain Q,a chemolithoheterotrophic sulphur bacterium

The physiological properties of an organism isolated from a selective chemostat enrichment using acetate and thiosulphate as the limiting substrates, provisionally called Thiobacillus Q, were investigated. Although the organism made up 85% of the community in the enrichment culture, its expected chemolithotrophic nature was not apparent in batch experiments. The growth yield was not enhanced by the addition of thiosulphate to an acetate containing mineral medium, even though up to 50% of the thiosulphate was oxidized. Under acetate limitation in the chemostat, there was a linear increase in yield with thiosulphate addition up to a concentration of 7 mM. Higher thiosulphate concentrations resulted in loss of thiosulphate oxidizing capacity and a decrease in the biomass to the level obtained with acetate alone. This loss may be due to the presence of inhibitory (50–100 M) levels of sulphite which is probably produced as an intermediate of the biological thiosulphate oxidation. Experiments with sulphide showed that Thiobacillus Q could also use it as an additional energy source. The complete lack of autotrophic growth, both in batch and chemostat experiments, together with the absence of even very low amounts of the key enzymes of the Calvin cycle demonstrated that this organism is a typical chemolithoheterotroph. Although this organism has provisionally been placed in the genus Thiobacillus, standard taxonomic procedures showed a close relationship with Pseudomonas alcaligenes. This study stresses the importance of quantitative chemostat studies in establishing the role of inorganic oxidations in energy metabolism and in the understanding of the role of heterotrophic sulphur oxidation in natural environments.     

Occurrence,structure and function of intracellular polyglucose in the obligate chemolithotroph Thiobacillus neapolitanus

Nitrogen-limited cells of the obligate chemolithotroph Thiobacillus neapolitanus formed an intracellular polymer during growth in the chemostat. This polymer was isolated and characterized as a branched polyglucose composed of units joined by -14 and -16 linkages. Polyglucose in T. neapolitanus can be considered a storage compound since formation of this compound took place during excess of energy and CO₂ whilst shortage of CO₂ resulted in rapid breakdown of polyglucose. Moreover the breakdown of polyglucose generated metabolically useful energy as could be demonstrated by polyglucose-dependent protein synthesis. Possession of polyglucose did not influence the viability of T. neapolitanus during prolonged periods of energy starvation. Activities of key enzymes of the oxidative pentose phosphate cycle, glucose-6-phosphatedehydrogenase and 6-phospho-gluconate-dehydrogenase, were demonstrated in cell free extracts of T. neapolitanus and appeared to increase 5- and 3-fold, respectively, during growth on NO ₃ ^- instead of NH ₄ ⁺ as a nitrogen source.     

Quantification and intracellular distribution of ribulose-1,5-bisphosphate carboxylase in Thiobacillus neapolitanus,as related to possible functions of carboxysomes

Ribulose-1,5-bisphosphate carboxylase (RuBPCase) has been quantified by immunological methods in Thiobacillus neapolitanus cultivated under various growth conditions in the chemostat at a fixed dilution rate of 0.07 h^-1. RuBPCase was a major protein in T. neapolitanus accounting for a maximum of 17% of the total protein during CO₂ limitation and for a minimum of 4% during either ammonium- or thiosulfate limitation in the presence of 5% CO₂ (v/v) in the gasphase. The soluble RuBPCase (i.e. in the cytosol) and the particulate RuBPCase (i.e. in the carboxysomes) were shown to be immunologically identical. The intracellular distribution of RuBPCase protein between carboxysomes and cytosol was quantified by rocket immunoelectrophoresis. The particulate RuBPCase content, which correlated with the volume density of carboxysomes, was minimal during ammonium limitation (1.3% of the total protein) and maximal during CO₂ limitation (6.8% of the total protein). A protein storage function of carboxysomes is doubtful since nitrogen starvation did not result in degradation of particulate RuBPCase within 24 h. Proteolysis of RuBPCase was not detected. Carboxysomes, on the other hand, were degraded rapidly (50% within 1 h) after change-over from CO₂ limitation to thiosulfate limitation with excess CO₂. Particulate RuBPCase protein became soluble during this degradation of carboxysomes, but this did not result in an increase in soluble RuBPCase activity. Modification of RuBPCase resulting in a lower true specific activity was suggested to explain this phenomenon. The true specific activity was very similar for soluble and particulate RuBPCase during various steady state growth conditions (about 700 nmol/min·mg RuBPCase protein), with the exception of CO₂-limited growth when the true specific activity of the soluble RuBPCase was extremely low (260 nmol/min ·mg protein). When chemostat cultures of T. neapolitanus were exposed to different oxygen tensions, neither the intracellular distribution of RuBPCase nor the content of RuBPCase were affected. Short-term labelling experiments showed that during CO₂ limitation, when carboxysomes were most abundant, CO₂ is fixed via the Calvin cycle. The data are assessed in terms of possible functions of carboxysomes.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase - PEP phosphoenolpyruvate - RIE rocket immunoelectrophoresis - CIE crossed immunoelectrophoresis     

Metabolic flexibility of Thiobacillus A 2 during substrate transitions in the chemostat

During autotrophic growth, cells of Thiobacillus A 2 retained a considerable capacity to oxidize various organic energy sources. Heterotrophically grown cultures, on the other hand, were completely devoid of the capacity to fix CO₂ via the Calvin cycle and to generate energy from thiosulfate. During transitions from organic media to inorganic thiosulfate-containing media in the chemostat, a long lag-phase was observed before energy generation, CO₂ fixation and, consequenctly, measurable growth occurred. This lag-phase was practically abolished if substrates were presentm at very low concentrations in the thiosulfate mineral medium which could be used as an energy source. The same result was obtained when the cells contained reserve material at the moment of the transition. During transitions from thiosulfate-limited growth to starvation, the -thiosulfate and the capacity to fix CO₂ decreased very slowly, after an initial short (± 4 h) increase of both enzyme systems. In contrast, these two metabolic functions were inactivated relatively rapidly in the presence of an oxidizable organic carbon and energy source. This process of inactivation was instantaneously stopped and reversed into rapid enzyme synthesis upon replacement of the organic substrate by thiosulfate.     

Isolation and characterization of Thiobacillus hydrothermalis sp. nov., a mesophilic obligately chemolithotrophic bacterium isolated from a deep-sea hydrothermal vent in Fiji Basin

A novel obligately chemolithotrophic Thiobacillus species isolated from a deep-sea hydrothermal vent is described. This organism grows lithoautotrophically on thiosulphate, tetrathionate, sulphide and sulphur which are oxidized to sulphate. The isolate is slightly halophilic and markedly halotolerant, showing optimum growth at pH 7.5 and at 35°C. The G+C content of the DNA is 67.1 mol%. The 16S rRNA sequence is distinct from any other Thiobacilli sequences. Phylogenetic analysis shows the organism to be a representative of the -group of proteobacteria and a specific relative of Thiobacillus neapolitanus. The ubiquinone is ubiquinone-8. These characters distinguish the isolate from any other Thiobacillus or Thiomicrospira species previously reported and is a new species described as Thiobacillus hydrothermalis. The type strain is isolate R3, DSM7121.     

Heterotrophic growth of Thiobacillus acidophilus in batch and chemostat cultures

Heterotrophic growth of the facultatively chemolithoautotrophic acidophile Thiobacillus acidophilus was studied in batch cultures and in carbon-limited chemostat cultures. The spectrum of carbon sources supporting heterotrophic growth in batch cultures was limited to a number of sugars and some other simple organic compounds. In addition to ammonium salts and urea, a number of amino acids could be used as nitrogen sources. Pyruvate served as a sole source of carbon and energy in chemostat cultures, but not in batch cultures. Apparently the low residual concentrations in the steady-state chemostat cultures prevented substrate inhibition that already was observed at 150 M pyruvate. Molar growth yields of T. acidophilus in heterotrophic chemostat cultures were low. The Y _max and maintenance coefficient of T. acidophilus grown under glucose limitation were 69 g biomass · mol^–1 and 0.10 mmol · g^–1 · h^–1, respectively. Neither the Y _max nor the maintenance coefficient of glucose-limited chemostat cultures changed when the culture pH was increased from 3.0 to 4.3. This indicates that in T. acidophilus the maintenance of a large pH gradient is not a major energy-requiring process. Significant activities of ribulose-1,5-bisphosphate carboxylase were retained during heterotrophic growth on a variety of carbon sources, even under conditions of substrate excess. Also thiosulphate- and tetrathionate-oxidising activities were expressed under heterotrophic growth conditions.     

Effects of organic compounds on growth of chemostat cultures of Thiomicrospira pelophila,Thiobacillus thioparus and Thiobacillus neapolitanus

Summary Cultures of Thiomicrospira pelophila, Thiobacillus thioparus and Thiobacillus neapolitanus were grown in thiosulfate-limited chemostats in a mineralsthiosulfate medium with and without organic supplements. Acetate, succinate and mixtures of amino acids increased the dry weight by 12–24% and the protein by 11–38%. Addition of both acetate and succinate had a cumulative effect. Saccharose, glucose, fructose, ribose, glycerol, glycerate, pyruvate, lactate or malate were without effect. The increase in dry weight of T. neapolitanus by ¹⁴C-acetate was directly related to the relative contribution of this compound to the total cell carbon.In CO₂-limited cultures of T. neapolitanus the effects of acetate on dry weight and protein were similar to those found in thiosulfate-limited cultures. In CO₂-limited cultures of T. pelophila a combination of acetate and succinate caused an increase in dry weight of 27% and of 50% in protein, the increase in protein being twice as high as in thiosulfate-limited cultures.There were no measurable differences in the activities of ribulosediphosphate carboxylase (RudPcase) in cell free extracts obtained from thiosulfate- or CO₂-limited cultures of T. pelophila or T. neapolitanus grown in the presence or absence of organic compounds. In T. pelophila the RudPcase activity was almost constant at all growth rates tested, and independent of the type of growth-limitation. For T. neapolitanus the specific RudPcase activity varied slightly with the growth rate. In CO₂-limited cultures the activity was three times that found in thiosulfate-limited cultures, thus showing that the RudPcase activity can be influenced by nutritional conditions.     

Energetics of mixotrophic and autotrophic C1-metabolism by Thiobacillus acidophilus

Although the facultatively autotrophic acidophile Thiobacillus acidophilus is unable to grow on formate and formaldehyde in batch cultures, cells from glucose-limited chemostat cultures exhibited substrate-dependent oxygen uptake with these C₁-compounds. Oxidation of formate and formaldehyde was uncoupler-sensitive, suggesting that active transport was involved in the metabolism of these compounds. Formate- and formaldehyde-dependent oxygen uptake was strongly inhibited at substrate concentrations above 150 and 400 M, respectively. However, autotrophic formate-limited chemostat cultures were obtained by carefully increasing the formate to glucose ratio in the reservoir medium of mixotrophic chemostat cultures. The molar growth yield on formate (Y=2.5 g ·mol^-1 at a dilution rate of 0.05 h^-1) and RuBPCase activities in cell-free extracts suggested that T. acidophilus employs the Calvin cycle for carbon assimilation during growth on formate. T. acidophilus was unable to utilize the C₁-compounds methanol and methylamine. Formate-dependent oxygen uptake was expressed constitutively under a variety of growth conditions. Cell-free extracts contained both dye-linked and NAD-dependent formate dehydrogenase activities. NAD-dependent oxidation of formaldehyde required reduced glutathione. In addition, cell-free extracts contained a dye-linked formaldehyde dehydrogenase activity. Mixotrophic growth yields were higher than the sum of the heterotrophic and autotrophic yields. A quantitative analysis of the mixotrophic growth studies revealed that formaldehyde was a more effective energy source than formate.     

Energy requirement for maintenance of the transmembrane potassium gradient in Klebsiella aerogenes NCTC 418: A continuous culture study

Molar growth yield studies on chemostat cultures of Thiobacillus neapolitanus grown in thiosulfate-minerals medium have confirmed earlier observations that the dry weight increased linearly with the dilution rate. The observed increase can be explained neither by a change in cell composition nor by the observed excretion of organic compounds. The increase of the molar growth yield over the full range of growth rates, that is also observed in other obligate chemolithotrophs, was not found in the facultatively chemolithotrophic Thiobacillus A2, grown on thiosulfate or formate. The interpretation of the results in terms of maintenance energy requirement is discussed. It is concluded that these results do not allow a mathematical treatment according to the empirical formula of Pirt.Abbreviation APS Adenosine-5-phosphosulfate     

Salinity requirements of a marine Thiobacillus intermedius

Thiobacillus intermedius was isolated from a salt marsh sediment with an interstitial water salinity of 30. This bacterium was cultured in a chemostat for 9 months. The optimum salinity for CO₂ fixation by this Thiobacillus was 10, much less than the salinity of its natural environment. Respiration of cultures increased at high salinities and the pathway of thiosulfate oxidation was altered so that polythionates accumulated rapidly. One ecological conclusion from these results is that in nature this bacterium probably grows at its maximum possible rate only rarely.     

Assimilation and Metabolism of Exogenous Organic Compounds by the Strict Autotrophs Thiobacillus thioparus and Thiobacillus neapolitanus

The assimilation and utilization of the individual carbon atoms of pyruvate and acetate by cells of Thiobacillus thioparus and T. neapolitanus, in the presence and absence of an energy source, were studied by use of radioactive substrates. Both organisms produced ¹⁴CO₂ from ¹⁴C-labeled pyruvate, but more came from carbon 1 than from carbons 2 or 3. The conversion of the carbons of acetate to CO₂ by both organisms was much less than that from any of the pyruvate carbons. When labeled pyruvate and acetate were incubated with these organisms, small amounts of radioactivity were found in the tricholoacetic acid-soluble material, nucleic acids, and lipids, and larger amounts were found in the protein fraction. The composition of the incubation medium affected the amount of utilization and incorporation of labeled substrates by both organisms. The presence of an exogenous energy source (Na₂S₂O₃) suppressed incorporation of the labeled substrates into various cellular components by T. thioparus, but enhanced incorporation by T. neapolitanus. When ¹⁴C-pyruvate was used as a substrate, as many as 12 radioactive compounds were found in the water-soluble fraction in the experiments with T. neapolitanus, whereas no more than three radioactive compounds were detected in this fraction in the experiments with T. thioparus. Of the total ¹⁴C activity found in the water-soluble fractions, malic acid contained the highest percentage. These findings are discussed in light of the overall metabolism of these two sulfur-oxidizing obligate chemoautotrophs, as well as in relation to the biochemical basis of chemoautotrophy.     

Characterization of Thiobacillus species by gas-liquid chromatography of cellular fatty acids

Summary Fatty acids of 18 strains representing 10 species of Thiobacillus were extracted from whole cells and examined as methyl esters by gas-liquid chromatography. Both visual and quantitative comparison of the resulting chromatograms for the presence and relative amounts of major peaks allowed rapid differentiation between such closely related species as Thiobacillus neapolitanus and T. thioparus and of eight other species. Except for a feature common to all thiobacilli tested, T. thiooxidans, T. neapolitanus and T. thioparus each possessed a characteristic fatty acid methyl ester profile that was exhibited by all the strains of that species. Hence, the thiobacilli could be divided into three distinct groups. It was possible to use the gas-liquid chromatographic patterns of the cellular fatty acids for rapid identification or grouping of these microorganisms since the fatty acid composition of the genus Thiobacillus thus appeared to be of taxonomic significance.Non-standard abbreviations GLC Gas-liquid chromatography - FAME Fatty acid methyl ester(s)     

Tricarboxylic acid and glyoxylate cycle enzyme activities in Thiobacillus versutus,an isocitrate lyase negative organism

An analysis was made of the specific enzyme activities of the TCA and glyoxylate cycle in Thiobacillus versutus cells grown in a thiosulphate- or acetate-limited chemostat. Activities of all enzymes of the TCA cycle were detected, irrespective of the growth substrate and they were invariably lower in the thiosulphate-grown cells. Of the glyoxylate cycle enzymes, isocitrate lyase was absent but malate synthase activity was increased from 15 nmol·min^-1·mg^-1 protein in thiosulphate-grown cells to 58 nmol·min^-1·mg^-1 protein in acetate-grown cells. Suspensions of cells grown on thiosulphate were able to oxidize acetate, although the rate was 3 times lower than that observed with acetate-grown cells. The respiration of acetate was completely inhibited by 10 mM fluoroacetate or 5 mM arsenite. Partially purified citrate synthase from both thiosulphate- and acetate-grown cells was completely inhibited by 0.5 mM NADH and was insensitive to inhibition by 1 mM 2-oxoglutarate or 1 mM ATP. The specific enzyme activities of the TCA and glyoxylate cycle in T. versutus were compared with those of Pseudomonas fluorescens, an isocitrate lyase positive organism, after growth in a chemostat limited by acetate, glutarate, succinate or glutamate. The response of the various enzyme activities to a change in substrate was similar in both organisms, with the exception of isocitrate lyase.Abbreviations TCA tricarboxylic acid - DNTB 2,2-dinitro-5,5-dithiobenzoic acid - APAD acetylpyridine adenine dinucleotide - PMS phenazine methosulphate - DCPIP 2,6-dichlorophenol-indophenol - DOC dissolved organic carbon     

Metabolism of organic acids by Thiobacillus neapolitanus

Summary A comparative study has been made of the metabolism in several strains of Thiobacillus neapolitanus of formate, acetate, propionate, butyrate, valerate and pyruvate. Conflicting reports in the literature concerning the mechanism of pyruvate assimilation in thiobacilli have been resolved. Pyruvate undergoes decarboxylation to yield acetyl coenzyme A, which is converted to glutamate, proline and arginine via reactions of the incomplete Krebs' cycle of this organism. Pyruvate is converted also to alanine, valine, isoleucine, leucine and lysine by mechanisms like those in heterotrophs. No aspartate is formed from the C-3 of pyruvate. Removal of the C-1 of pyruvate yields carbon dioxide, which is refixed into all cell constituents. Formate is not produced by this scission reaction, as formate itself is incorporated almost exclusively into purines. Aspartate can be synthesized by the activities of phosphoenolpyruvate carboxylase and oxaloacetate-glutamate transamination. Carbon from propionate is converted principally to lipids, although some amino acid production occurs with the same distinctive labelling pattern as is found after acetate assimilation by T. neapolitanus strains C and X. Butyrate and valerate also showed some distinctive patterns of incorporation into cell constituents. Fluoropyruvate and fluoropropionate inhibited the growth of T. neapolitanus and the mechanisms of this poisoning are discussed.Generally these compounds contributed only small proportions of the total cell carbon and tended to be converted to limited numbers of cell components. The thiobacilli thus tend to conserve carbon from these compounds and not to degrade them to carbon dioxide on a large scale when growing in an otherwise autotrophic medium.     

Characterization of a new metal-mobilizing Thiobacillus isolate

A new acidophilic, mineral sulphide oreoxidizing bacterium was isolated from a uranium mine near Salamanca, Spain. Cells were rod-shaped, motile and gram-negative. They were aerobes, could grow on pyrite and use sulphur or thiosulphate as sole energy source, suggesting this new isolate belongs to the genus Thiobacillus. It could grow neither with glucose nor with yeast extract as sole substrates. It could not grow on ferrous sulphate as the only energy source, although it grew in the same medium supplemented with glucose, yeast extract or thiosulphate. It was a mesophilic and extremely acidophilic Thiobacillus, with an optimal pH of 1.5 2. The G+C content of the DNA was 58%. The new isolate could grow in cultures on pyrite where electrophoretic pattern was clearly different from those of other thiobacilli, such as T. ferrooxidans.Abbreviations G+C Guanine + Cytosine     

Regulation of glucose catabolism in Thiobacillus A2 grown in the chemostat under dual limitation by succinate and glucose

In contrast to its diauxic behaviour in batch culture, Thiobacillus A2 grew in chemostat culture using glucose and succinate as dual limiting substrates. Biomass production under dual limitations was the sum of that on single substrates with each substrate being oxidized and assimilated to similar extents in single and dual substrate-limited cultures. In glucose and glucose + succinate-limited cultures glucose was oxidized largely by the Entner-Doudoroff and pentose phosphate pathways, but other mechanisms also contributed and the ratios of pathways depended on substrate ratios and the previous substrate-history of the culture. Variations in specific activities of enzymes of carbohydrate metabolism following switches from single to mixed substrates were considerable, ranging from fourfold for fructose diphosphate aldolase to more than 200-fold for hexokinase, fructose diphosphatase, glucose 6-phosphate and 6-phosphogluconate dehydrogenases. Changes in specific activities occurred only over prolonged time periods in the chemostat, probably reflecting low concentrations of free substrates in carbon-limited cultures and consequent low levels of catabolite repression.     

Chemolithoautotrophy and mixotrophy in the thiophene-2-carboxylic acid-utilizing Xanthobacter tagetidis

Xanthobacter tagetidis grew as a chemolithotrophic autotroph on thiosulfate and other inorganic sulfur compounds, as a heterotroph on thiophene-2-carboxylic acid, acetic acid and α-ketoglutaric acid, and as a mixotroph on thiosulfate in combination with thiophene-2-carboxylic acid and/or acetic acid. Autotrophic growth on one-carbon organosulfur compounds, and intermediates in their oxidation are also reported. Thiosulfate enhanced the growth yields in mixotrophic cultures, presumably by acting as a supplementary energy source, since ribulose bisphosphate carboxylase was only active in thiosulfate-grown cells and was not detected in mixotrophic cultures using thiosulfate with thiophene-2-carboxylic acid. Bacteria grown on thiophene-2-carboxylic acid also oxidized sulfide, thiosulfate and tetrathionate, indicating these as possible sulfur intermediates in thiophene-2-carboxylic acid degradation. Thiosulfate and tetrathionate were oxidized completely to sulfate and, consequently, did not accumulate as products of thiophene-2-carboxylic acid oxidation in growing cultures. K _m and V _max values for the oxidation of thiosulfate, tetrathionate or sulfide were 13 μM and 83 nmol O₂ min^–1 (mg dry wt.)^–1, respectively; thiosulfate and tetrathionate became autoinhibitory at concentrations above 100 μM. The true growth yield (Y_max) on thiophene-2-carboxylic acid was estimated from chemostat cultures (at dilution rates of 0.034–0.094 h^–1) to be 112.2 g mol^–1, with a maintenance coefficient (m) of 0.3 mmol thiophene-2-carboxylic acid (g dry wt.)^–1 h^–1, and the maximum specific growth rate (μ_max) was 0.116 h^–1. Growth in chemostat culture at a dilution rate of 0.041 h^–1 indicated growth yields [g dry wt. (mol substrate)^–1] of 8.1 g (mol thiosulfate)^–1, 60.9 g (mol thiophene-2-carboxylic acid)^–1, and 17.5 g (mol acetic acid)^–1, with additive yields for growth on mixtures of these substrates. At a dilution rate of 0.034 h^–1, yields of 57.8 g (mol α-ketoglutaric acid)^–1 and 60.7 g (mol thiophene-2-carboxylic acid)^–1 indicated some additional energy conservation from oxidation of the thiophene-sulfur. SDS-PAGE of cell-free preparations indicated a polypeptide (M _r, 21.0 kDa) specific to growth on thiophene-2-carboxylic acid for which no function can yet be ascribed: no metabolism of thiophene-2-carboxylic acid by cell-free extracts was detected. It was shown that X. tagetidis exhibits a remarkable degree of metabolic versatility and is representative of facultatively methylotrophic and chemolithotrophic autotrophs that contribute significantly to the turnover of simple inorganic and organic sulfur compounds (including substituted thiophenes) in the natural environment. Received: 1 July 1997 / Accepted: 3 November 1997