Autotrophic Cells Research Articles

Cytokinin autotrophy and differentiation in tissue cultures of haploid Nicotiana tabacum L.

Explants from the apical region (10 cm from the tip) of haploid Nicotiana tabacum cv. Wisconsin-38 were cultured on media with and without kinetin. Cell lines were selected in the dark and in the light. Cytokinins were extracted from the apical region of haploid plants and from callus tissues after 84 days of growth (third transfer culture). Chlorophyll was extracted from callus grown under light after 21 days of growth at each of the four cell line selection steps. Kinetin (+) cell lines and cytokinin autotrophic tissues grown in the light showed a compact growth pattern. Microscopic examination of these callus showed the presence of large numbers of nodules consisting of tracheary elements, parenchymatic cells, sieve elements and meristematic cells. Cytokinin-autotrophic callus grown in the dark showed an irregular growth pattern presenting regions of compact tissue and friable tissue. The compact tissue contained large amounts of nodules similar to those of kinetin (+) tissues and of cytokinin autotrophic tissues grown in the light. Extraction of the compact and the friable callus components showed high cytokinin activity in the compact region and low activity in the friable portion. It is suggested that cytokinin synthesis is related to the differentiation of the nodular structures. The amount of chlorophyll increased during the process of cytokinin autotrophic cell line selection.

Control of autotrophic carbon assimilation in Alcaligenes eutrophus by inactivation and reactivation of phosphoribulokinase.

Phosphoribulokinase in Alcaligenes eutrophus was partially inactivated when an autotrophic culture was shifted to heterotrophic growth with pyruvate as the sole source of carbon and energy. A similar response was observed on addition of various organic substrates to autotrophic cultures during the transition to mixotrophic growth. The extent of inactivation depended on the added substrate. Pyruvate or lactate caused the strongest inactivation among the tested substrates. Up to 75% of the phosphoribulokinase activity found in the autotrophic cells was lost within 30 min after supplementation of the cultures with either of these two substrates. This loss of enzyme activity was not the result of degradation of enzyme protein. Inactivation of phosphoribulokinase was accompanied by a decrease in the CO2 fixation rate of the cells. Reactivation of the enzyme occurred after exhaustion of pyruvate from the medium. Neither inactivation nor reactivation required de novo protein synthesis; however, continued energy conversion was necessary for the inactivation to occur. We suggest that the pyruvate metabolism of A. eutrophus is involved in these regulatory processes which act on phosphoribulokinase. They appear to contribute to the control of autotrophic CO2 assimilation in this organism.

The complexity of poly(A)+ RNA in the alga Chlorella fusca.

RNA excess hybridization with radioactively labelled complementary DNA (cDNA) was used to reveal the complexity of poly(A)+ RNA from Chlorella fusca var. vacuolata. RNA was tested from cells during photosynthetic exponential growth and during adaptation to heterotrophic growth in the dark on acetate. Both RNA populations were resolved into abundant, intermediate and rare sequence classes. Abundant sequences (200-400 copies per cell) constituted a significantly larger proportion of total poly(A)+ RNA in acetate-adapting cells than in exponentially growing autotrophic cells. Both types of RNA contained a rare sequence class of complexity consistent with a composition of about 20 000 different sequences. This indicated a substantially greater complexity of genes expressed in this alga and Euglena than in fungi and slime moulds. Heterologous hybridization, and hybridization with fractionated cDNA, showed that the majority of differences between RNA populations from exponentially growing and adapting cells were changes in relative abundance of groups of sequences, rather than presence of different sets of sequences in the two populations.

Thylakoid morphology of the cyanobacteria Anabaena variabilis and Nostoc MAC grown under light and dark conditions

Anabaena variabilis (ATCC 29413) and Nostoc sp. strain MAC were grown photoautotrophically and chemoheterotrophically. Thylakoid morphology of cells from both types of culture was investigated by freeze-etching electron microscopy. Peripheral and concentric arrays of thylakoid membranes seen in autotrophic cells were replaced, in heterotrophic cells, by numerous vesicular or tubular structures; the changes were reversible. Heterotrophic cells showed a reduced overall content of chlorophyll but started photosynthetic oxygen evolution immediately upon illumination. The results apparently show that in Anabaena and Nostoc the gross morphology of the intracytoplasmic membranes (thylakoid membranes) may depend on growth conditions similarly as is known for the photosynthetic bacteria sensu stricto (Rhodospirillales).

Autotrophic Picoplankton in the Tropical Ocean

In phytoplankton of the eastern tropical Pacific Ocean from 25 to 90 percent of the biomass (measured as chlorophyll a) and 20 to 80 percent of the inorganic carbon fixation were attributable to particles that could pass a screen with a 1-micrometer pore diameter. Evidence is presented that these are indeed autotrophic cells and not cell fragments.

Effect of enclosure in large plastic bags on diurnal change in oxygen concentration in tropical ocean water

From November 1980 to February 1981 the concentration of oxygen dissolved in the surface mixed layer of the oligotrophic Caribbean Sea off Curacao was quite constant (420.77±1.98 μg at l-1). However, immediately following enclosure in 4500-1 plastic bags reaching to a depth of 5 m the oxygen concentration began to decrease, down to values below saturation (405 μg at l-1) within 48 h. Autotrophic and heterotrophic nanoplankton cell numbers and algal pigments in bags remained constant or increased slightly during the first 24 h of enclosure. The rate of decrease in oxygen concentration in bags was significantly higher during daylight hours than in the night, which suggests that photo-oxidative processes were involved in the additional daytime loss of oxygen. The dramatic “enclosure effect” on the oxygen content of the water in the bags can be taken as evidence of the dependence of the oxygen concentration near the tropical ocean's surface on supply from below: in water freely circulating in the euphotic zone deviations from the mean oxygen concentration during a diurnal cycle were 0.47% at most, differential losses near the surface being counteracted through vertical exchange; while in water separated from the rest of the mixed layer in the plastic bags losses due to respiration of the enclosed plankton community plus an even greater loss, assigned to non-biological, photosensitized oxidation processes, were up to 10 μg at O2 l-1 in 24 h. Although photo-oxidation is confined to the very surface the oxygen flux involved may be important enough to necessitate consideration of a photochemically induced loss factor in oxygen budget calculations, e.g. when primary production is to be estimated from diurnal oxygen curves.

Heterotrophic and photoheterotrophic utilization of lactate by the diatom,Cylindrotheca fusiformis

The rates of lactate utilization, evaluated in terms of uptake, incorporation into acid insoluble products and respiration, were measured in the light and dark in cells which had been (1) growing autotrophically without lactate in the medium, (2) growing heterotrophically on lactate or (3) growing autotrophically and were then starved in the dark without lactate for 2–4 days. Autotrophic cells have low but significant lactate utilization rates which increase in dark-starved cells and increase further after adaptation to heterotrophic growth. Incubation in the light dramatically increases incorporation rates in dark-starved and heterotrophic cells, an effect that is not due solely to the refixation of respired lactate carbon. Assimilation efficiencies were influenced by the light or dark incubation conditions during the experiment rather than by previous growth conditions of the cells. Our results, including experiments with a photosynthetic inhibitor, support the hypothesis that the induction of lactate utilization systems involves incorporation and respiration pathways as well as lactate uptake systems.

Regulation of the Synthesis of Ribulose-l,5-bisphosphate Carboxylase and Its Subunits in the Flagellate Chlorogonium elongatum. II. Coordinated Synthesis of the Large and Small Subunits

Abstract Transfer of heterotrophically grown cells of the unicellular green alga Chlorogonium elongatum to autotrophic growth conditions causes a 10 -15 fold increase in the amount of the chloroplastic enzyme ribulose-1,5-bisphosphate carboxylase. This increase was found to be due to de novo synthesis. The relative proportions of large and small subunits of the enzyme do not change. Their ratio is close to 3.4, the proportions in weight of the two subunits in the holoenzyme. Continous labelling with [35S]sulfate reveals that the ratios of incorporation into large and small subunits are essentially the same in autotrophic and heterotrophic cells. Pulse-chase experiments show that the subunits are degraded synchronously. The coordinated subunit synthesis cannot be uncoupled using inhibitors of protein and RNA synthesis or high temperature of cultivation of the alga. The results suggests a very tightly coordinated synthesis of the large and small subunits of ribulosebisphosphate carboxylase.

Influence of light on plastocyanin formation in the alga Scenedesmus acutus

Scenedesmus acutus, pregrown autotrophically, forms high levels of plastocyanin during an initiation period of 24 hr in the light, after cupric ions have been added to depleted cells. In the dark, no plastocyanin formation is observed. This is in contrast to heterotrophic cells which, over the same period and under identical conditions, yield about half the plastocyanin level regardless of whether they are incubated in the dark or in the light. The ATP level is high in all cases when substantial formation of plastocyanin takes place, while the (35)S-sulfolipid level, the marker of thylakoid formation, is at variance. After the 24-hr plastocyanin formation period, the (35)S-sulfolipid level is high in illuminated autotrophic cells as well as in dark-incubated heterotrophic ones, but low in illuminated heterotrophic cells, all of them having substantial plastocyanin contents. Additional experiments on light dependence of copper uptake, influence of light quality, occurrence of apoplastocyanins, provide a set of data to assume that the role of light in plastocyanin formation is primarily through photophosphorylation, the ATP presumably being necessary for processing of plastocyanin precursors to holoprotein.

Complexity of polyadenylated RNA in autotrophic and acetate-adapted cells of Chlorella fusca var, vacuolata.

Complexity of polyadenylated RNA in autotrophic and acetate-adapted cells of Chlorella fusca var, vacuolata.

Regulation of glucose transport and metabolism in Thiobacillus novellus.

To investigate the physiological basis of decreased rate of glucose utilization by Thiobacillus novellus in a mixotrophic environment (R. C. Perez and A. Matin, J. Bacteriol. 142:633-638, 1980), its glucose transport system was characterized and the modulation of this system as well as enzymes of glucose metabolism by the growth environment was examined. Uptake of 2-deoxy-d-glucose by cell suspensions was almost abolished by respiratory chain inhibitors, and the sugar accumulated unchanged inside the cells against a concentration gradient: its transport is probably linked to the proton electrochemical gradient. The glucose transport system, as well as several enzymes of glucose metabolism, had a high specific activity in heterotrophic cells, intermediate activity in mixotrophic cells, and low activity in autotrophic cells; thus, they are induced by glucose but repressed by thiosulfate, its metabolites, or both. Thiosulfate and sulfite inhibited the glucose transport system uncompetitively and noncompetitively, respectively (apparent K(i) = 3.1 x 10(-2) M and 3.3 x 10(-7) M, respectively) and also inhibited glucose-6-phosphate dehydrogenase activity. Thus, the rate of glucose utilization in mixotrophic environments decreased because thiosulfate and its metabolites repress as well as inhibit the glucose transport system and enzymes of glucose metabolism. The significance of this and other regulatory phenomena that come into play in such environments is discussed.

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.

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.

Determination and Characterization of the Effects of Fluometuron and MSMA onChlorella

Studies were conducted on the effects of fluometuron [1,1-dimethyl-3-(α,α,α-trifluoro-m-tolyl)urea] on autotrophic growth (no sugar), heterotrophic growth (with sugar), and oxygen evolution and uptake and on the effects of MSMA (monosodium methanearsonate) on autotrophic growth and oxygen evolution and uptake by the unicellular green algaChlorella pyrenoidosaEmerson strain (University of Texas). Two methods of analysis (probit and kinetic) were used to characterize the dosage-response ofChlorellato these herbicides. All measurements ofChlorellaresponse to fluometuron were satisfactorily characterized by probit analysis, but only autotrophic cell number was adequately characterized by kinetic analysis. The fluometuron concentrations giving 50% inhibition on the probit plot were as follows: autotrophic cell number, 2.4 × 10−6M; chlorophyll at 400 nm, 3.3 × 10−6M; heterotrophic cell number, 2.4 × 10−6M; oxygen evolution, 20 × 10−6M. The response of the alga to MSMA was stimulatory instead of inhibitory. Since this stimulation did not consistently range between 10 and 90% of the control, it was not possible to characterize the MSMA dosage-Chlorellaresponse using either type of analysis.

Siroheme sulfite reductase isolated from Chromatium vinosum. Purification and investigation of some of its molecular and catalytic properties

Cells of the phototrophic bacterium Chromatium vinosum strain D were shown to contain a siroheme sulfite reductase after autotrophic growth in a sulfide/bicarbonate medium. The enzyme could not be detected in cells grown heterotrophically in a malate/sulfate medium. Siroheme sulfite reductase was isolated from autotrophic cells and obtained in an about 80% pure preparation which was used to investigate some molecular and catalytic properties of the enzyme. It was shown to consist of two different types of subunits with molecular weights of 37,000 and 42,000, most probably arranged in an α4β4-structure. The molecular weight of the native enzyme was determined to 280,000, 51 atoms of iron and 47 atoms of acid-labile sulfur were found per enzyme molecule. The absorption spectrum indicated siroheme as prosthetic group; it had maxima at 280 nm, 392 nm, 595 nm, and 724 nm. The molar extinction coefficients were determined as 302×103 cm2xmmol-1 at 392 nm, 98×103 cm2 xmmol-1 at 595 nm and 22×103 cm2x-mmol-1 at 724 nm. With reduced viologen dyes as electron donor the enzyme reduced sulfite to sulfide, thiosulfate, and trithionate. The turnover number with 59 (2 e-/enzyme moleculexmin) was low. The pH-optimum was at 6.0. C. vinosum sulfite reductase closely resembled the corresponding enzyme from Thiobacillus denitrificans and also desulfoviridin, the dismilatory sulfite reductase from Desulfovibrio species. It is proposed that C. vinosum catalyses anaerobic oxidation of sulfide and/or elemental sulfur to sulfite in the course of dissimilatory oxidation of reduced sulfur compounds to sulfate.

Eukaryotic [formula omitted] transformylase : Some properties of the Euglena gracilis enzyme

The N 10- formyl-H 4 folate:methionyl-tRNA fMet transformylase was extracted from Euglena cells, and the level of enzyme activity found to be of 0.15 pmol/min per 10 6 cells for autotrophic and 0.016 pmol/min per 10 6 cells for streptomycin-bleached cells. The enzyme has been purified by DEAE-cellulose and CM-Sephadex chromatography about 1000-fold from autotrophic cells and about 650-fold from chloroplasts obtained by the non-aqueous technique. A methionyl-tRNA synthetase with apparently high specificity for the initiator tRNA Met has also been found which allowed the estimation of enzyme activity even with unfractionated tRNAs from different sources, since only formylatable species were present in methionylated form. As judged from the initial velocity the Euglena transformylase showed the same kinetic behaviour towards Met-tRNA fMet from E. gracilis, Escherichia coli and yeast. The chloroplast enzyme showed different properties from those of the bacterial enzyme. It appears to have a partial requirement for K +, a higher molecular weight, and a lower Mg 2+ optimum. Moreover, the chloroplast transformylase showed sigmoidal rather than hyperbolic kinetics respect to Met-tRNA fMet ( n H = 1.81 ; K′ = 4 · 10 −14 ) as well as N 10- formyl-H 4 folate ( n H = 2.46 ; K′ = 5.2 · 10 −16 ), thus indicating a strong positive cooperativity for both substrates.

Glycollate production and excretion byAlcaligenes eutrophus

Autotrophic cultures of the facultative chemolithotroph Alcaligenes 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 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 of D-ribulose 1,5-diphosphate carboxylase under the same conditions.

Lactate dehydrogenase from autotrophic and heterotrophic cells of the marine diatom Cylindrotheca fusiformis Reimann & Lewin

Cultures of Cylindrotheca furisormis grown either autotrohpically or heterotrophically on lactate contained significant amounts of NAD-dependent L(+)-lactate dehydrogenase (EC 1.1.1.27). Polyacylamide gel electrophoresis of crude enzyme extracts revealed a single band which was indistinguishable between autotrohpic and heterotrohpic cells. The Km for lactate of partially purified preparations was lower under heterotrophic conditions. The specific activity in crude extracts was higher under autotrophic than heterotrophic conditions; it dropped precipitously when autotrophic cells were transferred to the dark, increasing again only in the presence of lactate. These and related observations suggest that this enzyme has at most only a minor role in the assimilation of lactate during heterotrophic growth on lactate.

Ribulose Diphosphate Carboxylase from Autotrophic Euglena gracilis

Ribulose 1,5-diphosphate carboxylase (RUDPcase) from autotrophically grown Euglena gracilis was purified to homogeneity as measured by analytical ultracentrifugation, polyacrylamide gel electrophoresis, and immunoprecipitation reactions. The enzyme represented about 9% of total protein and 24% of soluble protein in the autotrophic cell. Light-grown, heterotrophic cells seemed to contain considerably less RUDPcase. Native carboxylase from autotrophic Euglena showed an s(20, w) at low protein concentrations of 17 to 17.5, suggesting a molecular weight of >500,000 daltons. Upon denaturation, the enzyme dissociated into two subunits having different amino acid compositions and molecular weights of 59,000 and 12,000 daltons. Based upon the amino acid mass ratios, a quaternary organization of 7 to 8 large and 8 to 10 small subunits per native enzyme molecule was indicated.The phylogenetic relationship of carboxylase from Euglena and from three higher plants was investigated. In general, the size, subunit formation, and quaternary structure of RUDPcase from the various sources seemed to be similar. A partial immunochemical reaction between anti-RUDPcase serum from Euglena and the enzymes from lettuce, cucumber, and New Zealand spinach suggested that the algal and higher plant carboxylases were related but not identical. This was borne out by amino acid analyses which showed a close correspondence between the large, but not the small, subunits of Euglena and lettuce.

Localization of Enzymes of Glycolate Metabolism in the Alga Chlorogonium elongatum

Organelles in homogenates from autotrophic cells of Chlorogonium elongatum were separated on linear sucrose gradients. The distribution of enzymes typical of leaf peroxisomes was determined.Whereas more than 60% of the catalase activity was particulate and recovered in microbodies at a mean density of 1.225 g/cm(3) within the gradient, in most experiments only 5 to 10% (as a maximum 30%) of the NAD-dependent hydroxypyruvate reductase was particulate, and this was recovered principally at density 1.19 g/cm(3). This distribution coincides with that of cytochrome oxidase, malate dehydrogenase, and isocitrate dehydrogenase, the mitochondrial markers. Glyoxylate-glutamate aminotransferase and glycolate dehydrogenase showed a similar distribution pattern to that of NAD-dependent hydroxypyruvate reductase. Thus in Chlorogonium the enzymes of the glycolate pathway are not associated with the microbodies that are recovered at density 1.225 g/cm(3).The single large chloroplasts of the Chlorogonium cells are broken during grinding, and this probably accounts for the finding that NADP-glyoxylate reductase was recovered only in the soluble fractions of the gradient.