Ann P. Wood

Chemolithotrophic metabolism of the newly-isolated moderately thermophilic, obligately autotrophic Thiobacillus tepidarius

Thiobacillus tepidarius, isolated from the hot springs at Bath, Avon, UK, grew optimally at 43–45°C and pH 6.0–7.5 on thiosulphate or tetrathionate. In batch culture, thiosulphate was oxidized stoichiometrically to tetrathionate, with a rise in pH. The tetrathionate was then oxidized to sulphate, supporting growth and producing a fall in pH to a minimum of ph 4.8. The organism contained high levels of thiosulphate-oxidizing enzyme, rhodanese and ribulose bisphosphate carboxylase. It was obligately chemolithotrophic and autotrophic. In chemostat culture, T. tepidarius grew autotrophically with the following sole energy-substrates: sulphide, thiosulphate, trithionate, tetrathionate, hexathionate or heptathionate. Thiocyanate, dithionate and sulphite were not used as sole substrates, although sulphite enhanced growth yields in the presence of thiosulphate. Maximum specific growth rate on tetrathionate was 0.44 h-1. ‘True growth yields’ (Ymax) and maintenance coefficients (m) were calculated for sulphide, thiosulphate, trithionate and tetrathionate and observed yields at a single fixed dilution rate compared with those on hexathionate and heptathionate. Mean values for Ymax, determined from measurements of absorbance, dry wt, total organic carbon and cell protein, were similar for sulphide, thiosulphate and trithionate (10.9 g dry wt/mol substrate) as expected from their equivalent oxygen consumption for oxidation. Ymax for tetrathionate (20.5) and the relative Yo values (as g dry wt/g atom oxygen consumed) for thiosulphate and all four polythionates indicated that substrate level phosphorylation did not contribute significantly to energy conservation. These Ymax values were 40–70% higher than any of those previously reported for obligately aerobic thiobacilli. Mean values for m were 6.7 mmol substrate oxidized/g dry wt·h for sulphide, thiosulphate and trithionate, and 2.6 for tetrathionate.

Autotrophic growth of four Sulfolobus strains on tetrathionate and the effect of organic nutrients

Autotrophic growth yields of four strains of Sulfolobus using tetrathionate as sole energy substrate fell in the range 6.2–7.8 g dry weight (mol tetrathionate oxidized)-1. Autotrophic organisms lacked ribulose 1,5-bis-phosphate carboxylase, but contained pyruvate and phosphoenolpyruvate carboxylases. S. brierleyi and strains B6-2 and LM exhibited mixotrophic growth, with tetrathionate oxidation, CO2-fixation and organic substrate assimilation occurring concurrently, using media containing glucose or acetate. Yeast extract or succinate supported heterotrophic growth and showed strain-dependent repression of one or both of tetrathionate oxidation and CO2-fixation resulting in biphasic growth. All four carbon atoms of succinate were assimilated to cell-carbon during growth. Acetate was the major source of cell-carbon during mixotrophic growth. These observations are not inconsistent with the possibility of a reductive carboxylic acid cycle in these organisms. Radiorespirometric analysis of glucose oxidation indicated CO2 release to occur by means of an Entner-Doudoroff pathway (followed by pyruvate decarboxylation) and oxidative pentose phosphate pathway reactions. There was little evidence from the glucose radiorespirometry of the large-scale use of an oxidative tricarboxylic acid cycle for terminal oxidation of acetate derived from pyruvate. These results demonstrate the considerable metabolic versatility of Sulfolobus strains and show that there is significant variation among them.

Metabolism of Thiobacillus A2 Grown Under Autotrophic, Mixotrophic and Heterotrophic Conditions in Chemostat Culture

Thiobacillus A2 was grown in chemostat culture under four distinct types of substrate limitation: chemolithoautotrophically with limitation by thiosulphate or CO2; heterotrophically with limitation by glucose; and mixotrophically with dual limitation by both thiosulphate and glucose. Under mixotrophic conditions energy was obtained from the oxidation of both thiosulphate and glucose, and carbon was derived both from CO2 fixation by the Calvin cycle and from glucose. Ribulosebisphosphate carboxylase (RuBP carboxylase) activity was negligible and chemolithotrophic thiosulphate oxidation and autotrophic CO2 fixation were apparently repressed in bacteria which had been grown heterotrophically. Conversely, under autotrophic conditions the ability to oxidize glucose was repressed. Growth yields from mixotrophic cultures were the sum of those obtained under single substrate limitation. Intermediate activities of RuBP carboxylase were detected in mixotrophic cultures, but more glucose was assimilated mixotrophically than heterotrophically. Glucose was metabolized by the Entner-Doudoroff (85 to 90%) and pentose phosphate (10 to 15%) pathways under both heterotrophic and mixotrophic conditions, with slight involvement also of the Embden-Meyerhof pathway (<9%) heterotrophically. RuBP carboxylase activity in autotrophic cultures was enhanced four- or fivefold by CO2 limitation. Repression of RuBP carboxylase activity and thiosulphate-oxidizing ability during the transition from autotrophy to heterotrophy and the activities of carbohydrate-metabolizing enzymes in autotrophic, heterotrophic and mixotrophic cultures are described.

Isolation and physiological characterisation of Thiobacillus thyasiris sp. nov., a novel marine facultative autotroph and the putative symbiont of Thyasira flexuosa

A novel facultatively chemolithoautotropic Thiobacillus, isolated from the gill tissue of the marine bivalve Thyasira flexuosa, is described. It is believed to be the symbiont from this animal, providing the animal with carbon fixed by the Calvin cycle. The organism grows lithoautotrophically on thiosulphate, tetrathionate and elemental sulphur, which are oxidised to sulphate. It oxidizes sulphide, thiosulphate, trithionate, tetrathionate and hexathionate, but not thiocyanate. Kinetic constants for these substrates are presented. In autotrophic batch culture it produces yields that are among the lowest reported for thiosulphate or tetrathionate as energy substrates (1.25 and 2.5 g cell-carbon per mol substrate, respectively). Autotrophic cultures contain ribulose bisphosphate carboxylase and excreted 20% of their fixed carbon into the medium during growth. Mixotrophic growth on acetate and thiosulphate resulted in partial repression of the carboxylase. The organism is slightly halophilic and markedly halotolerant, showing optimum growth at about pH 7.5 and maximum growth rate at 37° C. It contains ubiquinone Q-10 and its DNA contains 52 mol % G+C. These characteristics distinguish it from any other Thiobacillus or Thiomicrospira species previously described. The organism is formally described and named as Thiobacillus thyasiris.

Simultaneous operation of three catabolic pathways in the metabolism of glucose by Thiobacillus A2

Enzymes essential to the operation of the Embden-Meyerhof glycolytic pathway, the Entner-Doudoroff pathway and oxidative pentose phosphate pathway were present in Thiobacillus A2 grown on glucose and other sugars. Radiorespirometry under various conditions with Thiobacillus A2 oxidising glucose specifically labelled with 14C in carbon atoms 1, 2, 3, 3+4, 6 or universally labelled demonstrated the simultaneous operation of the Embden-Meyerhof (48%), Entner-Doudoroff (28%), and pentose phosphate (24%) pathways in release of carbon dioxide from glucose. Growth on succinate, or autotrophically on formate or thiosulphate resulted in repression of most enzymes of the pathways, but high aldolase levels were retained indicating its role in gluconeogenesis and the Calvin cycle. Different fructose diphosphatase activities were found in succinate- and thiosulphate-grown organisms. The results indicate that all three major catabolic pathways for glucose function in Thiobacillus A2 grown on sugars. Thiobacillus acidophilus showed a different radiorespirometric pattern and apparently used the Entner-Doudoroff (64.5%) and pentose phosphate (35.5%) pathways, but showed unusually high release of carbon atom 6, as was also found for T. ferrooxidans.

Isolation and physiological characterisation of Thiobacillus aquaesulis sp. nov., a novel facultatively autotrophic moderate thermophile

A moderately thermophilic, facultatively chemolithoautotrophic thiobacillus isolated from a thermal sulphur spring is described. It differs from all other species currently known to be in culture. It grows lithoautotrophically on thiosulphate, trithionate or tetrathionate, which are oxidized to sulphate. Batch cultures on thiosulphate do not produce tetrathionate, but do precipitate elemental sulphur during growth. In autotrophic chemostat cultures the organism produces yields on thiosulphate, trithionate and tetrathionate that are among the highest observed for a Thiobacillus. Autotrophic cultures contain ribulose bisphosphate carboxylase. Heterotrophic growth has been observed only on complex media such as yeast extract and nutrient broth. It is capable of autotrophic growth and denitrification under anaerobic conditions with thiosulphate and nitrate. It grows between 30 to 55° C, and pH 7 to 9, with best growth at about 43°C and pH 7.6. It contains ubiquinone Q-8, and its DNA contains 65.7 mol% G+C. The organism is formally described and named as Thiobacillus aquaesulis.

Reclassification of Thiobacillus thyasiris as Thiomicrospira thyasirae comb. nov., an organism exhibiting pleomorphism in response to environmental conditions

The halotolerant, facultatively chemolithoautotrophic, gramnegative sulphur-oxidizing organism isolated from the gills of Thyasira flexuosa is shown to exhibit pleomorphism consistent with its being a member of the genus Thiomicrospira and not a Thiobacillus as originally classified. Its distinctive morphology, DNA base composition, and similarity of 16S rRNA sequences to that of Thiomicrospira sp. strain L-12 (which differs only in detail from the type species Thiomicrospira pelophila), lead us to reclassify this strain as Thiomicrospira thyasirae DSM5322.

AUTOTROPHIC METABOLISM OF FORMATE BY THIOBACILLUS STRAIN-A2

SUMMARY: ThiobacillusA2 grew on formate as its sole source of carbon and energy. Growth was autotrophic: formate and carbon dioxide were equivalent as carbon sources during growth on formate. Carbon dioxide was fixed by the Calvin cycle in formate- or thiosulphate-grown bacteria which contained comparable high specific activities of ribulosebisphosphate carboxylase. Formate incorporated by bacteria growing heterotrophically or on thio- sulphate showed more restricted metabolism, particularly providing carbon for purines. Bacteria growing on formate or thiosulphate assimilated acetate but showed disproportionately high incorporation into glutamate, proline, arginine, leucine, pyrimidines and lipid. Growth kinetics on formate were studied using extended cultures held at constant formate concentrations at pH 7.8. Yield was a function of substrate concentration and growth rate, which were linearly related in the range 4 to 40 mM-formate. Formate was an inhibitory substrate at higher concentrations. A computer analysis of the inhibited growth kinetics indicated K s and K i values of 26.5 and 187 mM-formate, respectively, and a true μ max of 0.299 h−1.

Mixotrophic Growth of Thiobacillus A2 in Chemostat Culture on Formate and Glucose

In aerobic chemostat culture Thiobacillus A2-GFI grew autotrophically on formate and heterotrophically on glucose with maximum specific growth rates (μmax) of 021 and 033 h-1, respectively. At dilution rates of 01 and 018 h-1, it grew mixotrophically on formate + glucose mixtures, completely consuming both substrates. Ribulose-1,5-bisphosphate carboxylase and formate dehydrogenase were present at high specific activity in autotrophic and mixotrophic cultures, but were repressed in cultures on glucose alone. A greater proportion of added glucose was assimilated in mixotrophic culture than in heterotrophic culture. Raising the dilution rate of a mixotrophic culture from 01 or 018 to 03 h-1 resulted in washout (with an apparent μmax for mixotrophic growth of 025 h-1) and the establishment of a culture dependent on glucose for growth. Growth yields on formate and glucose were, respectively, 33 and 100 g dry wt (mol substrate consumed)-1. Steady state biomass production in mixotrophic culture indicated additive growth yields. The biomass produced in cultures on formate + glucose at a dilution rate of 03 h-1 suggested that growth only occurred on glucose, but organisms still contained high activities of ribulose-1,5-bisphosphate carboxylase and formate dehydrogenase. At a formate: glucose ratio (mm) of 100:1, some formate was oxidized and CO2 was fixed, but formate was not used when this ratio was 50:5. Formate-glucose mixotrophy benefits Thiobacillus A2-GFI when substrates are limited at low growth rates (< μmax for formate), but is characterized by a μmax below that possible on glucose. Physiological behaviour at high growth rates was influenced by the formate:glucose ratio, resulting under some conditions, at least, in loss of mixotrophy and the establishment of heterotrophic growth.

Methylotrophic and autotrophic bacteria isolated from lucinid and thyasirid bivalves containing symbiotic bacteria in their gills

Bacteria have been isolated into pure culture from the gills of Lucinoma borealis, Myrtea spinifera, Thyasira flexuosa and T. sarsi collected from several British and Scandinavian coastal sediments. It is proposed that these bacteria are symbiotic with the animals. The bacteria were either autotrophs, obtaining energy from oxidation of inorganic sulphur compounds, or methylotrophs using methanol or methylamine as the source of carbon and energy. The latter could also oxidize thiosulphate to tetrathionate and precipitate elemental sulphur. The pure cultures have been characterised in some detail, showing that (1) their DNA base composition falls in the range 47–58% guanine + cytosine; (2) although all grow well at the natural environment temperatures of 8–15°C, their optima lie between 30 and 35°C; (3) all produce copious amounts of extracellular slime when grown at low temperatures; (4) the autotrophic isolate is facultatively heterotrophic, containing a repressible ribulose bisphosphate carboxylase, and excreting up to 30% of its carbon dioxide fixation into the culture medium; (5) the methylotrophs include some containing high activities of hexulose phosphate synthase for assimilation of formaldehyde by the ribulose monophosphate cycle; others may have novel assimilatory metabolism. The possibility is discussed that environmental factors may determine whether a methylotroph-animal or autotroph-animal symbiosis is established rather than the symbiont type being an invariable species-specific character.