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     

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     

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.     

Yield Coefficients of Thiobacillus neapolitanus in Continuous Culture

Thiobacillus neapolitanus, when grown in continuous culture with thiosulfate limiting growth, possessed an apparent maximal molar growth yield of 8.0 g (dry weight) per mole of thiosulfate. The substrate requirement for energy of maintenance was the highest yet reported, amounting to 21.8 mmoles of thiosulfate per g per hr. The molar growth yield, corrected for this maintenance energy requirement, was 13.9 g (dry weight) per mole of thiosulfate. It was concluded that substrate-level phosphorylation during sulfite oxidation accounted for about 45% of the adenosine triphosphate (ATP) requirement for CO₂ assimilation and maintenance during growth on limiting thiosulfate, that three sites of energy conservation exist in the electron-transport chain terminating in oxygen, and that 7.8 moles of ATP are required to fix and assimilate 1 mole of CO₂ into cell material.     

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.     

Relations between d-ribulose-1,5-bisphosphate carboxylase,carboxysomes and CO2 fixing capacity in the obligate chemolithotroph Thiobacillus neapolitanus grown under different limitations in the chemostat

An adaptation of the d-ribulose-1,5-bisphosphate carboxylase (RuBPCase) activity to changing CO₂ concentrations in the growth medium in the chemostat was observed in the obligate chemolithotroph Thiobacillus neapolitanus. RuBPCase activity has been separated in a soluble and particulate fraction. The activity of the particulate fraction appeared to be associated with the carboxysomes.The total activity of RuBPCase of CO₂ limited cultures was about 5-fold higher than the activity of thiosulphate limited cultures grown in the presence of 5% CO₂ whilst the particulate activity and the soluble activity were about 8- and 1.5-fold higher, respectively. The fluctuation of the total and particulate RuBPCase activity correlated with the changes in volume density of carboxysomes in the cell.An inverse correlation between maximal CO₂ fixing capacity by whole cells and the volume density of carboxysomes was observed. The change in ratio of soluble RuBPCase activity to particulate RuBPCase activity paralleled the change in maximal CO₂ fixation by whole cells during the different growth conditions.     

The major soluble cytochromes of the obligately aerobic sulfur bacterium,Thiobacillus neapolitanus

Four cytochromes were isolated from soluble extracts of the aerobic sulfur bacterium, Thiobacillus neapolitanus. The two most abundant proteins were purified to homogeneity and thoroughly characterized. Cytochrome c-554 (547) is a monomeric, small molecular weight protein which is unusual in having two well-resolved alpha peaks in UV-visible absorption spectra. The redox potential is 208 mV. Native cytochrome c-549 is oligometric, but has a subunit size of about 26.000. The yield of this protein could be improved dramatically by washing membranes with 30% ammonium sulfate, but the material solubilized by this method had a larger native molecular weight than that in the initial 0.1 M Tris-Cl extract and behaved differently on chromatography. The properties of cytochrome c-549 including subunit size and UV-visible absorption spectra are similar to mitochondrial cytochrome c ₁ and chloroplast cytochrome f, which suggests that it may be a modified form of the predominant membrane cytochrome. Based on cytochrome content, it is suggested that T. neapolitanus is not closely related to other thiobacilli.Dedicated to Prof. Dr. G. Drews on the occasion of his sixtieth birthday     

Influence of amino acids and organic antimetabolites on growth and biosynthesis of the chemoautotroph Thiobacillus neapolitanus strain C

Summary The growth of Thiobacillus neapolitanus strain C in liquid cultures was depressed by phenylalanine, p-fluorophenylalanine, cysteine, methionine, nor-leucine, azetidine-2-carboxylic acid, and chloramphenicol, but was little affected by glutamic acid, glycine, proline, azathymine, or oligomycin.Growing cultures assimilated ¹⁴C-labelled glycine, glutamic acid, phenylalanine, and tyrosine into protein. Tyrosine and phenylalamine were incorporated unchanged, but glutamate was used also for synthesis of arginine and proline. Glycine-¹⁴C contributed also to adenine and guanine synthesis. The extremely large amounts of phenylalanine incorporated into protein could indicate its toxicity to depend on its producing abnormal protein synthesis. Azetidine-2-carboxylic acid appeared to lower the amount of proline in the protein.Assimilation of glutamate and glycine by non-growing organisms was almost entirely dependent on energy from thiosulphate oxidation, thus suggesting a cause of obligate chemoautotrophy. Chloramphenicol specifically inhibited this thiosulphate-dependent incorporation of glutamate, glycine or CO₂ into protein at concentrations which did not affect total CO₂-fixation. Provided that energy is available from thiosulphate-oxidation this Thiobacillus is thus able to (a) activate exogenous amino acids; (b) incorporate them and CO₂ into protein by a chloramphenicol sensitive mechanism; (c) synthesise proline and arginine from glutamate; or adenine and guanine from glycine. Its biosynthesis thus depends on mechanisms like those of heterotrophs but requires to be driven by a chemolithotrophic energy supply.     

Energetic aspects of CO2 uptake in Thiobacillus neapolitanus

Uptake of inorganic carbon (C_i) in the form of CO₂ and/or HCO ₃ ^- was studied in the chemolithoautotroph Thiobacillus neapolitanus under energy (thiosulphate) or carbon (CO₂) limitation. Uptake of C₁ was found to be a metabolic energy dependent process since in the presence of uncouplers no uptake was observed. The accumulation level of C_i was higher in the CO₂-limited cells (1000-to 1500-fold) in comparison to the thiosulphate-limited cells (500-to 800-fold). The process of uptake could be influenced by addition of ionophores. Inhibition of uptake and accumulation of C_i was found after addition of valinomycin which completely dissipated the electrical potential (). After addition of nigericin an increase in the uptake and accumulation of C_i was observed with a concomitant increase of the . These results suggest that the is the main driving force for uptake of C_i. However, uptake of C_i could never be found in the absence of electron transfer, or in cells in which the electron transfer chain was blocked by potassium cyanide. Electron transfer therefore appears to be an additional requirement for C_i uptake. Kinetic experiment on the uptake of inorganic carbon at different pH values suggest that CO₂ is the carbon species taken up by T. neapolitanus.Abbreviations RuBisCO ribulose-1,5-bisphosphate carboxylase - DCCD N,N¹-dicyclohexylcarbodiimide - CCCP carbonyl cyanide m-chlorophenyl hydrazone - FCCP carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone - EDTA sodium ethylene diamine tetraacetate     

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.     

Competition between the facultatively chemolithotrophic Thiobacillus A2, an obligately chemolithotrophic Thiobacillus and a heterotrophic spirillum for inorganic and organic substrates

Competition in a chemostat between the versatile Thiobacillus A2 and the specialized T. neapolitanus for thiosulfate as the sole growth-limiting substrate, led to dominance of the specialized over the versatile organism, at dilution rates 0.025 h^-1. Increasing concentrations of acetate or glycollate in the thiosulfate medium caused increased relative numbers of T. A2 in steady states at D=0.07 h^-1. Eventually, with 10–12 mmol of organic substrate per litre, complete dominance of T. A2 over T. neapolitanus occurred.Mixed cultures of T. A2 and a specialized spirillumshaped heterotroph, competing for acetate as sole growth-limiting substrate resulted in complete dominance of the heterotroph at dilution rates of 0.07 and 0.15 h^-1. In this case increasing concentrations of thiosulfate in the acetate medium, up to 10 mM, eventually led to the elimination of the heterotroph.These results have been interpreted as evidence that T. A2 was growing mixotrophically. As the concentration of the second substrate was raised, the number of T. A2 cells increased and as a result T. A2 consumed an increasing portion of the common substrate.In mixed chemostat cultures containing all three organisms, T. A2 could maintain itself with all tested ratios of acetate and thiosulfate in the inflowing medium. The heterotroph was excluded from the culture below a relatively low acetate to thiosulfate ratio, whilst above a relatively high acetate to thiosulfate ratio T. neapolitanus was completely eliminated.These results were discussed in relation to the ecological niche of Thiobacillus A2-type organisms.     

Studies on the growth of Thiobacillus ferrooxidans

Summary A method for enumeration of viable numbers of Thiobacillus ferrooxidans using membrane filters on ferrous-iron agar is presented. Factors affecting colony production were the concentration and brand of agar, pH of the medium, and type of membrane filter. The results suggest that inhibition of T. ferrooxidans by agar is a result of the acid hydrolysis of agar, the main product of which is d-galactose. Colony development was suppressed by aged medium, by acid-hydrolysed agar and by 0.1% galactose. Sartorius and Millipore membrane filters were suitable for the experiments, whereas Oxoid MF-50 membranes virtually suppressed the production of colonies. The method was employed to follow growth of T. ferrooxidans in pH 1.3 medium. The viable cell numbers were correlated with ¹⁴CO₂-fixation and ferrous iron oxidation. Generation time was 6 h 22 min with a yield of 2.2×10¹² organisms/g atom Fe²⁺ oxidized. Growth of T. neapolitanus on thiosulphate medium was not affected by agar-type or membrane filters and yield of the organism was 1.5×10¹³ organisms/g molecule Na₂S₂O₃ oxidized.     

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.     

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)     

Mixotrophic and Autotrophic Growth of Thiobacillus acidophilus on Glucose and Thiosulfate

Mixotrophic growth of the facultatively autotrophic acidophile Thiobacillus acidophilus on mixtures of glucose and thiosulfate or tetrathionate was studied in substrate-limited chemostat cultures. Growth yields in mixotrophic cultures were higher than the sum of the heterotrophic and autotrophic growth yields. Pulse experiments with thiosulfate indicated that tetrathionate is an intermediate during thiosulfate oxidation by cell suspensions of T. acidophilus. From mixotrophic growth studies, the energetic value of thiosulfate and tetrathionate redox equivalents was estimated to be 50% of that of redox equivalents derived from glucose oxidation. Ribulose 1,5-bisphosphate carboxylase (RuBPCase) activities in cell extracts and rates of sulfur compound oxidation by cell suspensions increased with increasing thiosulfate/glucose ratios in the influent medium of the mixotrophic cultures. Significant RuBPCase and sulfur compound-oxidizing activities were detected in heterotrophically grown T. acidophilus. Polyhedral inclusion bodies (carboxysomes) could be observed at low frequencies in thin sections of cells grown in heterotrophic, glucose-limited chemostat cultures. Highest RuBPCase activities and carboxysome abundancy were observed in cells from autotrophic, CO₂-limited chemostat cultures. The maximum growth rate at which thiosulfate was still completely oxidized was increased when glucose was utilized simultaneously. This, together with the fact that even during heterotrophic growth the organism exhibited significant activities of enzymes involved in autotrophic metabolism, indicates that T. acidophilus is well adapted to a mixotrophic lifestyle. In this respect, T. acidophilus may have a competitive advantage over autotrophic acidophiles with respect to the sulfur compound oxidation in environments in which organic compounds are present.     

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.     

Combined degradation of covellite by Thiobacillus thiooxidans and Thiobacillus ferrooxidans

Summary In the presence of iron, which is always associated with natural sulphide ores, the percentages of copper dissolution in the bioleaching of covellite were 34 and 45 % when Thiobacillus thiooxidans and Thiobacillus ferrooxidans were used together and when an indirect bioleaching with attached bacteria was performed respectively. In the latter, the percentage of copper dissolution was still higher than the percentages obtained with pure cultures (36 % with a T. thiooxidans culture and 40 % with a T. ferrooxidans culture).     

The incorporation of acetate by the chemoautotroph Thiobacillus neapolitanus strain C

Summary Cultures of Thiobacillus neapolitanus strain C assimilate ¹⁴C-labelled acetate and aspartate. Both carbon atoms of acetate are incorporated, and 25% of the cell carbon can arise from acetate. Aspartate-¹⁴C contributes 4–5% of the cell carbon, and is found in pyrimidines and in protein as aspartate and its related amino acids. Acetate-¹⁴C contributes to lipid, glutamate, arginine, proline and leucine, but not to aspartate. Acetate assimilation by washed organisms requires carbon dioxide and energy from thiosulphate oxidation. Degradation of ¹⁴C-glutamic acid from acetate-¹⁴C-labelled bacteria; the accumulation of ¹⁴C-citrate in the presence of fluoroacetate and [¹⁴C] acetate; short-term kinetic experiments on acetate-¹⁴C turnover; and the demonstration of citrate synthesis by cell-free extracts all indicate glutamate synthesis from -ketoglutarate formed by reactions of the tricarboxylic acid cycle. The cycle is believed to be incomplete, probably not proceeding further than -ketoglutarate, and functions as a glutamate-synthesising system, using oxaloacetate derived solely from carbon dioxide fixation. Malate synthase (and the glyoxylate cycle) appear to be insignificant in the metabolism, but extracts did form citramalate from acetate and pyruvate.     

Mixotrophic and autotrophic growth of Thiobacillus acidophilus on tetrathionate

Thiobacillus acidophilus can grow in batch and chemostat culture as a heterotroph on glucose, a chemolithoautotroph on tetrathionate and CO₂, or as a mixotroph. Mixotrophically it obtains energy from the simultaneous oxidation of tetrathionate and glucose, and carbon from both glucose and CO₂. Mixotrophic cultures contain lower activities of ribulose 1,5-bisphosphate carboxylase and exhibit lower specific rates of tetrathionate oxidation than do autotrophic cultures. Mixotrophic cultures with low concentrations of glucose have growth rates that are intermediate between slow autotrophic growth and fast heterotrophic growth. Slightly more glucose-carbon is assimilated by mixotrophic cultures than by heterotrophic ones provided with the same concentrations of glucose. Mixotrophic yield in the chemostat is also slightly greater than predicted from autotrophic and heterotrophic yields. These observations indicate that there is preferential assimilation of glucose, at the expense of energy from tetrathionate oxidation, during mixotrophy, resulting in an overall energy saving that produces enhanced growth yield. These observations are relevant to understanding the regulatory behaviour of T. acidophilus in its acidic, mineral-leaching habitats.     

Transpositional Mutagenesis of Thiobacillus novellus and Thiobacillus versutus

Transpositional mutagenesis of Thiobacillus novellus by Tn501 was achieved by means of the incompatibility of IncP plasmids. Tn501 insertion caused three types of mutant phenotypes: isoleucine auxotrophy, lysine auxotrophy, and a reduced ability to oxidize reduced sulfur compounds and to fix CO₂. Oxidation rates for elemental sulfur (S⁰), thiosulfate (S₂O₃²⁻), and tetrathionate (S₄O₆²⁻) in mutants of the latter type were reduced relative to those of the nonmutant control strain. Incorporation of labeled bicarbonate (H¹⁴CO₃⁻) was also significantly impaired. Although suicide vehicles were not useful for the introduction of transposons into T. novellus, this method was effective for the Tn1721-induced mutagenesis of Thiobacillus versutus. Tn1721 insertions resulted in the loss of the natural resistance of T. versutus to arsenate and gentamicin and in auxotrophies for isoleucine-valine, arginine, phenylalanine, valine, and panthothenate. Transpositional mutagenesis by either method should prove to be a useful tool for further study of these and other members of the genus Thiobacillus.