Autotrophic and Heterotrophic Metabolism of Hydrogenomonas: Regulation of Autotrophic Growth by Organic Substrates

The effects of a number of organic substrates on the autotrophic metabolism of Hydrogenomonas eutropha were examined. Dual substrate (mixotrophic) cultivation in the presence of hydrogen plus either fructose or alanine allowed autotrophic growth to begin immediately after the exhaustion of the organic substrate. On the other hand, the presence of acetate, pyruvate, or glutamate caused a lengthy lag to occur before autotrophic growth commenced. With acetate or pyruvate this lag (plateau) in the dicyclic growth curve was due to the repression of ribulose diphosphate carboxylase (RDPC) synthesis during mixotrophic growth. During heterotrophic growth with glutamate, RDPC was partially repressed; however, during mixotrophic growth, RDPC activity was high. Thus the delay of autotrophic growth was not due to a repression of RDPC by glutamate. The data suggest that glutamate interferes with autotrophic metabolism by repressing the incorporation of inorganic nitrogen. The repression of these vital autotrophic functions by acetate, pyruvate, and glutamate occurred both in the presence and absence of hydrogen, i.e., during both heterotrophic and mixotrophic cultivation. The derepression of the affected systems during the plateau phase of the dicyclic growth curves was demonstrated. Carbon dioxide assimilation by whole cells agreed well with the RDPC activity of extracts from cells grown under similar conditions.     

Changes of ribulose 1,5-diphosphate carboxylase level during the processes of degeneration and regeneration of chloroplasts in Chlorella protothecoides

RuDP carboxylase was active mainly in chloroplasts and PEP carboxylaseactive principally outside of chloroplasts in Chlorella protothecoides. During the process of chloroplast degeneration in algal cellsinduced by addition of glucose, the activity of RuDP carboxylasesignificantly decreased, whereas the activities of PEP-carboxylaseand -carboxykinase markedly increased. During the process of chloroplast regeneration in "glucose-bleached"algal cells, which contained no detectable amounts of FractionI protein and showed only traces of RuDP carboxylase activity,a light-dependent development of RuDP carboxylase proceededalmost in parallel with the light-induced formation of chlorophyll.The activities of PEP-carboxylase and -carboxykinase, whichwere negligibly low in glucose-bleached cells, developed independentlyof light. Both chloramphenicol and cycloheximide severely inhibited thedevelopment of RuDP carboxylase activity. A relatively low concentrationof glucose also caused a significant suppression. Under theseconditions, chlorophyll formation was inhibited only slightlyby chloramphenicol and very strongly by cycloheximide and glucose. ¹ Deceased, 11 June, 1972. (Received April 25, 1972; )     

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.     

Growth and physiology of Thiobacillus novellus under nutrient-limited mixotrophic conditions.

Thiobacillus novellus was cultivated in a chemostate under the individual limitations of thiosulfate, glucose, and thiosulfate plus glucose. At dilution rate (D) of 0.05 h-1 or lower, the steady-state biomass concentration in mixotrophic medium was additive of the heterotrophic and autotrophic biomass at corresponding D values. The ambient concentrations of thiosulfate, glucose, or both in the various cultures were low and were very similar in mixotrophic, heterotrophic, and autotrophic environments at a given D value. At D = 0.05 h-1, mixotrophic cells possessed higher activities of sulfite oxidase and thiosulfate oxidation compared to autotrophic cells, as well as higher activities of glucose enzymes and glucose oxidation than heterotrophic cells. Thus, in contrast to nutrient-excess conditions, in nutrient-limited mixotrophic environments at these D values, T. novellus did not exhibit characteristics of uncoupled substrate oxidation, inhibition of substrate utilization, and repression of enzymes of energy metabolism. It is concluded that T. novellus responds to mixotrophic growth conditions differently in environments of different nutritional status, and the ecological and physiological significance of this finding is discussed.     

Hydrogenase and ribulose diphosphate carboxylase during autotrophic, heterotrophic, and mixotrophic growth of scotochromogenic mycobacteria.

Two key autotrophic enzyme systems, hydrogenase and ribulose diphosphate carboxylase, were examined in Mycobacterium gordonae and two other chemolithotrophic, scotochromogenic mycobacteria under different cultural conditions. In all three organisms both enzymes were inducible and were produced in significant levels only in the presence of the specific substrate, hydrogen or carbon dioxide. M. gordonae exhibited increased growth rates and yields, indicating mixotrophic growth, in the presence of a number of single organic substrates, including acetate, pyruvate, glucose, fructose, and glycerol. In contrast to other aerobic hydrogen autotrophs, the presence of either acetate or pyruvate did not repress ribulose diphosphate carboxylase, and mixotrophic growth was rapid with these substrates. In the absence of carbon dioxide, growth in glycerol medium under an atmosphere of hydrogen and oxygen was severely inhibited, even with cells preadapted to heterotrophic growth on glycerol. Cyclic adenosine monophosphate was not effective in inducing hydrogenase or carboxylase in heterotrophic, mixotrophic, or hydrogen-inhibited cultures.     

Regulation of autotrophic and heterotrophic metabolism in Pseudomonas oxalaticus OX1: Growth on mixtures of oxalate and formate in continuous culture

Pseudomonas oxalaticus was grown in carbon- and energy-limited continuous cultures either with oxalte or formate or with mixtures of these substrates. During growth on the mixtures, simultaneous utilization of the two substrates occurred at all dilution rates tested. Under these conditions oxalate repressed the synthesis of ribulosebisphosphate carboxylase. The degree of this repression was dependent on the dilution rate and the ratio of oxalate and formate in the medium reservoir. At a fixed oxalate/formate ratio repression was greatest at intermediate dilution rates, whereas derepression occurred at both low and high dilution rates. Progressive depression of ribulosebisphosphate carboxylase synthesis and of autotrophic CO₂ fixation at low dilution rates was attributed to the decreasing concentration of intracellular repressor molecule(s), parallel to the decreasing concentration of the growth-limiting substrates in the culture. To account for the derepression at higher dilution rates, it is proposed that the rate of oxalyl-CoA production from oxalate limits the supply of metabolic intermediates and that additional energy and reducing power generated from formate drains the pools of metabolic intermediates sufficiently to lower the intracellular concentration of the repressor(s). During growth of Pseudomonas oxalaticus on the heterotrophic substrate oxalate alone, at dilution rates below 10% of the maximum specific growth rate, derepression of ribulosebisphosphate carboxylase synthesis and of autotrophic CO₂ fixation was observed to a level which was 50% of that observed during growth on formate alone at the same dilution rate. It is concluded that in Pseudomonas oxalaticus the synthesis of enzymes involved in autotrophic CO₂ fixation via the Calvin cycle is regulated by a repression/derepression mechanism and that the contribution of autotrophic CO₂ fixation to the biosynthesis of cell material in this organism is mainly controlled via the synthesis of these enzymes.Abbreviations RuBPCase ribulosebisphosphate carboxylase - PMS phenazine methosulphate - DCPIP 2,6-dichlorophenolindophenol - FDH formate dehydrogenase - S_R concentration of growth-limiting substrate in reservoir     

Regulation of autotrophic and heterotrophic metabolism in Pseudomonas oxalaticus OX1: Growth on mixtures of acetate and formate in continuous culture

Growth of Pseudomonas oxalaticus in carbon- and energy-limited continuous cultures with mixtures of acetate and formate resulted in the simultaneous utilization of both substrates at all dilution rates tested. During growth on these mixtures, acetate repressed the synthesis of ribulosebisphosphate carboxylase. The degree of this repression was dependent on the dilution rate and on the ratio of acetate and formate in the medium reservoir. At fixed acetate and formate concentrations in the inflowing medium of 30 and 100 mM, respectively, and dilution rates above 0.10h^-1, the severe repression of autotrophic enzymes resulted in a marked increase in bacterial dry weight compared to the growth yield of the organisms on the two substrates separately. Also, at these dilution rates a significant increase in isocitrate lyase activity was observed in the cells as compared to growth on acetate alone. This indicated that under these conditions more acetate was assimilated and less dissimilated since acetate was partly replaced by formate as the energy source. When formate was added to the reservoir of an acetate-limited culture (S_R=30 mM), derepression of RuBPCase synthesis was observed at formate concentrations of 50 mM and above. Below this concentration formate only served as an energy source for acetate assimilation; when its concentration was increased above 50 mM a progressively increasing contribution of carbon dioxide fixation to the total carbon assimilation was observed as the activity of RuBPCase in the cells increased. It is concluded that in Pseudomonas oxalaticus the synthesis of enzymes involved in autotrophic carbon dioxide fixation via the Calvin cycle is regulated by a repression/derepression mechanism.Abbreviations RuBPCase ribulosebisphosphate carboxylase - PMS phenazine methosulphate - DCPIP 2,6-dichlorophenol-indophenol - FDH formate dehydrogenase - S_R concentration of growth-limiting substrate in reservoir     

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     

Effects of organic matter on the growth of Thiobacillus intermedius

London, Jack (University of California, Los Angeles), and Sydney C. Rittenberg. Effects of organic matter on the growth of Thiobacillus intermedius. J. Bacteriol. 91:1062-1069. 1966.-Yeast extract, glucose, glutamate, and other organic materials stimulate the rate and extent of growth of Thiobacillus intermedius in thiosulfate broth. Growth did not occur in glucose or glutamate mineral salts medium in the absence of thiosulfate, although a stable variant was obtained which grows on yeast extract alone. Cells harvested from media supplemented with organic matter have a reduced rate of thiosulfate oxidation (20 to 30% of autotrophic), oxidize the organic supplement, and have an additive rate of oxidation in the presence of both the organic substrate and thiosulfate. Carboxydismutase synthesis is repressed, and the incorporation of bicarbonate carbon into cell material is almost completely eliminated by the presence of organic matter in the growth medium. It is concluded that the availability of organic matter eliminates the autotrophic assimilatory mechanisms of T. intermedius but not its autotrophic energy-generating system. The data are discussed in relation to the existence of "obligate" chemoautotrophic bacteria.     

Chemoautotrophic growth of hydrogen-uptake-positive strains of Rhizobium japonicum.

Recently reported research from this laboratory has demonstrated the autotrophic growth of certain hydrogen-uptake-positive strains of Rhizobium japonicum and defined minimal conditions for such growth. Ribulose 1,5-bisphosphate carboxylase has been detected in autotrophically growing cells, but at low specific activity. Moreover, growth rates were low, and growth ceased at low cell densities. We report here improved autotrophic growth rates of R. japonicum SR through the use of a modified mineral salts/vitamins medium and a programmed increase in oxygen tension as autotrophic growth proceeds. Under these conditions, ribulose, 1,5-biphosphate carboxylase activity increased greater than 10-fold and crude-extract-uptake-hydrogenase activities were from 20 to 47 times those heretofore reported for free-living R. japonicum. It is likely that previous assays for these enzymes were done on preparations of cells in which their synthesis had been partially repressed. The contribution of CO2 fixation to organic carbon accumulation in autotrophic cells was assessed as sufficient to support observed growth. Enzymological determination of the product of carbon fixation has established a stoichiometric ratio of 1.9 mol of 3-phosphoglycerate per mol of CO2 fixed and unequivocally assigns the role of carbon fixation catalysis to ribulose 1,5-bisphosphate carboxylase. Ammonium served best as a nitrogen source, nitrate was less effective, and gaseous nitrogen would not support autotrophic growth. Ecological, evolutionary, and practical considerations of autotrophy in the rhizobia are briefly discussed in the light of our findings.     

ADAPTIVE PHOTOSYNTHESIS RESPONSES TO TEMPERATURE EXTREMES BY THE THERMOPHILIC CYANOPHYTE SYNECHOCOCCUS LIVIDUS1

The physiological and biochemical changes associated with and resulting in adaptation to both sub- and supra-optimal temperatures are presented for the thermophilic cyanophyte Synechococcus lividus Copeland. The optimum temperature for growth was 45 C. An increase in the optimum temperature of photosynthesis from 50 to 55 C was shown for cells grown at the supra-optimum temperature of 57 C; whereas, cells grown at the sub-optimal temperature of 35 C exhibited a decrease in the optimal temperature from 50 to 45 C for ¹⁴CO₂ uptake. These changes in optimal temperatures are interpreted as adaptive. Associated with the 5 C increase in optimal temperature for photosynthesis was an increase in chlorophyll a, plastoquinone A, and activity of ribulose-1,5-diphosphate carboxylase (RuDP carboxylase). However, the increase in the temperature optimum for 57 C grown cells was associated with a reduced O₂ yield correlated with a reduced ferricyanide photoreduction capacity. RuDP carboxylase activity decreased rapidly above 55 C. Therefore reduced rates above 55 C resulted from damage to ferricyanide reducing systems and reduced RuDP-carboxylase activity, whereas low photosynthesis rates at sub-optimal temperatures were probably due to rate limiting effect of low temperatures on RuDP carboxylase activity with no evidence of damage to ferricyanide photoreducing systems.     

Regulation of Autotrophic and Heterotrophic Carbon Dioxide Fixation in Hydrogenomonas facilis

After growth on various carbon sources, sonic extracts of Hydrogenomonas facilis contained ribulosediphosphate (RuDP) carboxylase and phosphoribulokinase (Ru5-P kinase). After very short sonic treatment, a reductive adenosine triphosphate (ATP)-dependent incorporation of (14)CO(2) was also detectable. Reduced nicotinamide adenine dinucleotide (NADH(2)) served as reductant 30-fold more effectively than reduced nicotinamide adenine dinucleotide phosphate (NADPH(2)). Adenosine 5'-phosphate (AMP) and adenosine 5'-pyrophosphate (ADP) inhibited Ru5-P kinase and NADH(2)-, ATP-dependent CO(2) fixation. The levels and duration of CO(2) fixation suggested that it is a cyclic process. The requirement of reduced pyridine nucleotide and ATP and the sensitivity of fixation to AMP and ADP support the conjecture that it occurs via the Calvin cycle. After thorough study of variables affecting catalysis, specific activities (millimicromoles of substrate disappearing per milligram of protein) at 30 C were determined for RuDP carboxylase (C), Ru5-P kinase (K) and ATP-, NADH(2)- dependent CO(2) fixation (CO(2) F) after growth autotrophically on fructose, glucose, ribose, glutamate, lactate, succinate, and acetate. Values for these growth modes were, respectively-for C: 67.3, 51.1, 51.4, 24.6, 2.05, 10.2, 2.25, 1.4; for K: 24.7, 24.0, 23.2, 14.2, 12.8, 12.9, 13.4, 2.8; and for CO(2) F: 4.54, 4.83, 3.10, 2.87, 0.85, 1.51, 0.24, 0.41. The qualitative parallel between values for RuDP carboxylase and CO(2) fixation suggests that one major control point in fixation is the step catalyzed by RuDP carboxylase.     

Microbial sulfide oxidation in the oxic–anoxic transition zone of freshwater sediment: involvement of lithoautotrophic Magnetospirillum strain J10

The oxic–anoxic transition zone (OATZ) of freshwater sediments, where opposing gradients exist of reduced iron and sulfide with oxygen, creates a suitable environment for microorganisms that derive energy from the oxidation of iron or sulfide. Gradient microcosms incubated with freshwater sediment showed rapid microbial turnover of sulfide and oxygen compared with sterile systems. Microcosms with FeS as a substrate also showed growth at the OATZ and subsequent dilution series resulted in the isolation of three novel strains, of which strain J10 grows chemolithoautotrophically with reduced sulfur compounds under microaerobic conditions. All three strains are motile spirilla with bipolar flagella, related to the genera Magnetospirillum and Dechlorospirillum within the Alphaproteobacteria . Strain J10 is closely related to Magnetospirillum gryphiswaldense and is the first strain in this genus found to be capable of autotrophic growth. Thiosulfate was oxidized completely to sulfate, with a yield of 4 g protein mol⁻¹ thiosulfate, and autotrophic growth was evidenced by incorporation of ¹³C derived from bicarbonate into biomass. A putative gene encoding ribulose 1,5-bisphosphate carboxylase/oxygenase type II was identified in strain J10, suggesting that the Calvin–Benson–Bassham cycle is used for autotrophic growth. Analogous genes are also present in other magnetospirilla, and in the autotrophically growing alphaproteobacterium magnetic vibrio MV-1.     

Glycollate production and excretion byAlcaligenes eutrophus

Autotrophic cultures of the facultative chemolithotrophAlcaligenes eutrophus have been found to excrete glycollate. This excretion was greatly stimulated by the incorporation of up to 20% (v/v) oxygen in the hydrogen used for gassing. The stimulatory effect of oxygen was prevented by the addition of 10% (v/v) CO₂ to the gassing mixture. Glycollate excretion only in the presence of oxygen was increased by the addition of 2-pyridyl-hydroxymethane sulphonic acid (HPMS), an inhibitor of glycollate oxidation, indicating that glycollate formation itself was stimulated by oxygen. No glycollate excretion by cultures grown heterotrophically on pyruvate was detected, either in the absence or presence of HPMS, under heterotrophic or autotrophic conditions.Extracts from autotrophic cells showed phosphoglycollate phosphatase and glycollate oxidoreductase activities, which were considerably lower in extracts prepared from pyruvate- or fructose-grown (heterotrophic) cells. The increase in activity of both enzymes upon cell transfer from heterotrophic to autotrophic growth was prevented by chloramphenicol and resembled the induction ofd0ribulose 1,5-diphosphate carboxylase under the same conditions.Abbreviations DTE dithioerythritol - EDTA ethylenediamine tetraacetate - HPMS 2-pyridyl-hydroxymethane sulphonie acid - RuDP d-ribulose 1,5-diphosphate     

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.     

Induced formation of ribulose 1,5-diphosphate carboxylase in Rhodopseudomonas spheroides with particular concern to its relation with chromatophore formation

A comparative study was made on features of the induced synthesisof RuDP carboxylase in three strains of R. spheroides with differentbiochemical properties. In strains Sb and Sa, which were able to grow under either light-anaerobicor dark-aerobic conditions, activities of RuDP carboxylase inthe light-grown cells were much higher than those in dark-growncells. The level of RuDP carboxylase activity in dark-growncells of the Sb strain (wild type strain) increased two to threetimes in the dark by incubating the heavy cell suspension underlow aeration, but, for a further increase in enzyme activity,a light-anaerobic condition was required. This is in contrastto the induced formation of bacteriochlorophyll which has beenshown to proceed actively in the dark as well as in the light.On the other hand, with dark-grown cells of the Sa strain, whichhad possible partial defects in the chlorophyll synthesis system,the induced synthesis of RuDP carboxylase under the light-anaerobiccondition was markedly retarded as compared to that with theSb strain. RuDP carboxylase formation was not induced in L-57(a colorless mutant) under any of these conditions. The induced formation of RuDP carboxylase, as well as of bacteriochlorophyll,under the light-anaerobic condition was considerably suppressedby hydroxyurea and mitomycin C. This suggests that the geneticcontrol systems of RuDP carboxylase synthesis may be closelyrelated with those for the formation of the photosynthetic apparatus. ¹This work was supported in part by Public Health Research GrantAM 08016 from the National Institute of Arthritis and MetabolicDiseases, U.S.A. (G. K.). ²Present address: Laboratory of Radioisotope Experiment, TohokuUniversity School of Medicine, Sendai, Japan. (Received September 6, 1968; )     

Ribulose-1, 5-diphosphate carboxylase of Chromatium strain D

RuDP carboxylase isolated from autotrophically grown cells ofphotosynthetic sulfur bacterium, Chromatium strain D, was partiallypurified by (NH₄)₂SO₄ precipitation and Sephadex G-200 gel filtration.The molecular size of the bacterial RuDP carboxylase was foundto be large, analogous to that of the plant enzyme, in agreementwith results of previous workers. Sucrose density gradient centrifugationshowed the S_rel to be approximately 18; the omission of Mg⁺⁺caused no dissociation of the enzyme molecule in its subunits.Chromatium RuDP carboxylase showed similarities to the plantenzyme in some of its kinetic properties; (a) a shift of pHoptimum to the neutral side from the alkaline side on the additionof Mg⁺⁺, (b) deviation of the substrate concentration (NaHCO₃)-activityrelationship from the MICHAELIS formula and (c) a marked stimulativeeffect of Mg⁺⁺. A unique sigmoidal saturation curve of the enzymeto RuDP, which had been detected in Rhodospirillum rubrum andRhodopseudomonas spheroides RuDP carboxylase in the absenceof Mg⁺⁺, was not found. Another characteristic feature of ChromatiumRuDP carboxylase is its partial immunological response to therabbit anti-spinach RuDP carboxylase serum as detected by theinhibition of the carboxylation reaction due to the antibody-antigenreaction. ¹Part X, Structure and Function of Chloroplast Proteins. Supportedin part by research grants from the Ministry of Education ofJapan (No. 8719) and USPHS (AM-10792-03) (Received July 4, 1969; )     

Carbon metabolism in Sulfobacillus thermosulfidooxidans subsp. asporogenes, strain 41

The activities of carbon metabolism enzymes were determined in cellular extracts of the moderately thermophilic, chemolithotrophic, acidophilic bacterium Sulfobacillus thermosulfidooxidans subsp. asporogenes, strain 41, grown either at an atmospheric content of CO2 in the gas phase (autotrophically, heterotrophically, or mixotrophically) or autotrophically at a CO2 content increased to 5-10%. Regardless of the growth conditions, all TCA cycle enzymes (except for 2-oxoglutarate dehydrogenase), one glyoxylate cycle enzyme (malate synthase), and some carboxylases (ribulose bisphosphate carboxylase, pyruvate carboxylase, and phosphoenolpyruvate carboxylase) were detected in the cellular extracts of strain 41. During autotrophic cultivation of strains 41 and 1269, the increase in the CO2 content of the supplied air to 5-10% resulted in the activation of growth and iron oxidation, a 20-30% increase in the cellular content of protein, enhanced activity of the key TCA enzymes (citrate synthase and aconitase), and, in strain 41, a decrease in the activity of carboxylases.     

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.Abbreviations PIPES Piperazine-N,N-bis (2-ethane sulphonic acid)     

Ribulose 1,5-diphosphate carboxylase and Chlorobium thiosulfatophilum

1. Cell-free extracts of the photosynthetic bacterium Chlorobium thiosulfatophilum, strains 8327 and Tassajara, were assayed for ribulose 1,5-diphosphate (RuDP) carboxylase and phosphoribulokinase-the two enzymes peculiar to the reductive pentose phosphate cycle. 2. RuDP carboxylase was consistently absent in strain 8327. The Tassajara strain showed a low RuDP-dependent CO₂ fixation activity that was somewhat higher in cells following transatlantic air shipment than in freshly grown cells. The stability and behaviour of this activity in sucrose density gradients were similar to those described by other workers. 3. The radioactive carboxylation products formed in the presence of RuDP by enzyme preparations from the Tassajara strain did not include 3-phosphoglycerate-the known product of the RuDP carboxylase reaction, but instead consisted of the unrelated acids glutamate, aspartate and malate. 4. Phosphoribulokinase was absent in all preparations of the two Chlorobium strains tested. By contrast, phosphoribulokinase as well as RuDP carboxylase were readily demonstrated in preparations from pea chloroplasts and the photosynthetic bacterium Rhodospirillum rubrum. 5. It is concluded that C. thiosulfatophilum appears to lack RuDP carboxylase, phosphoribulokinase, and hence, the reductive pentose phosphate cycle.Support of a J. S. Guggenheim Fellowship is gratefully acknowledged