Role of Reduced Exogenous Organic Compounds in the Physiology of the Blue-Green Bacteria (Algae): Photoheterotrophic Growth of a “Heterotrophic” Blue-Green Bacterium

Nostoc sp. (strain Mac) was shown to be capable of using glucose, fructose, or sucrose as a sole source of carbon and energy in the dark. In the light in the absence of exogenously supplied CO(2), this strain exhibited a more versatile metabolism. In addition to the three sugars above, glycerol and acetate served as sole sources of carbon. This photoheterotrophic growth in the absence of exogenously supplied CO(2) appears to involve O(2)-evolving photosynthesis. The action spectrum for photoheterotrophic growth on acetate closely resembles the action spectrum for photosynthesis. The physiology of photoheterotrophic growth was further investigated through determinations of stable carbon isotope ratios and measurements of gas exchanges. These investigations suggest that respired CO(2) from substrate oxidation is assimilated by the photosynthetic machinery.     

Photosynthetic intermediates, the warburg effect, and glycolate synthesis in isolated spinach chloroplasts

Increasing levels of CO(2) have been shown to stimulate the rate of photosynthesis, eliminate the oxygen inhibition of photosynthesis (Warburg effect), and decrease glycolate formation in isolated spinach chloroplasts. Ribose 5-phosphate and fructose 1,6-diphosphate at concentrations of 5 to 10 mum also stimulate the rate of plastid photosynthesis and eliminate the Warburg effect. In contrast to the effect of high CO(2) levels, these sugar phosphates have little effect on glycolate formation. Evidence is presented to show that the level of intermediates of the photosynthetic carbon reduction cycle may influence the Warburg effect in vivo. It is postulated that the formation of glycolate is not the causal factor of the Warburg effect.     

Phototrophic growth of a Rubisco-deficient mesophilic purple nonsulfur bacterium harboring a Type III Rubisco from a hyperthermophilic archaeon

The hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1 harbors a structurally novel, Type III Rubisco (Rbc(Tk)). In terms of protein engineering of Rubiscos, the enzyme may provide an alternative target to the conventional Type I and Type II enzymes. With a future aim to improve the catalytic properties of Rbc(Tk), here we examined whether or not the enzyme could support growth of a mesophilic organism dependent on CO2 fixation. Via double-crossover homologous recombination, we first deleted three Rubisco genes present on the chromosome of the photosynthetic mesophile Rhodopseudomonas palustris No. 7. The mutant strain (delta3) could neither grow under photoautotrophic nor photoheterotrophic conditions. We introduced the rbc(Tk) gene into strain delta3 either on a plasmid, or by integrating the gene onto the chromosome. The two transformant strains harboring rbc(Tk) displayed growth under photoautotrophic and photoheterotrophic conditions, both dependent on CO2 fixation. Specific growth rates and Rubisco activity levels were compared under photoheterotrophic conditions among the two transformants and the wild-type strain. We observed that the levels of Rubisco activity in the respective cell-free extracts correlated well with the specific growth rates. Immunoprecipitation experiments revealed that Rubisco activity detected in the transformants was derived solely from Rbc(Tk). These results demonstrated that the Type III Rbc(Tk) from a hyperthermophile could support CO2 fixation in a mesophilic organism, and that the specific growth rate of the transformant can be used as a convenient parameter for selection of engineered proteins with improved Rubisco activity.     

Redox homeostasis phenotypes in RubisCO-deficient Rhodobacter sphaeroides via ensemble modeling

Photosynthetic bacteria are capable of carrying out the fundamental biological processes of carbon dioxide assimilation and photosynthesis. In this work, ensemble modeling (EM) was used to examine the behavior of mutant strains of the nonsulfur purple photosynthetic bacterium Rhodobacter sphaeroides containing a blockage in the primary CO(2) assimilatory pathway, which is responsible for cellular redox balance. When the Calvin-Benson-Bassham (CBB) pathway is nonfunctional, spontaneous adaptive mutations have evolved allowing for the use of at least two separate alternative redox balancing routes enabling photoheterotrophic growth to occur. The first of these routes expresses the nitrogenase complex, even in the presence of normal repressing ammonia levels, dissipating excess reducing power via its inherent hydrogenase activity to produce large quantities of hydrogen gas. The second of these routes may dissipate excess reducing power through reduction of sulfate by the formation of hydrogen sulfide. EM was used here to investigate metabolism of R. sphaeroides and clearly shows that inactivation of the CBB pathway affects the organism's ability to achieve redox balance, which can be restored via the above-mentioned alternative redox routes. This work demonstrates that R. sphaeroides is capable of adapting alternative ways via mutation to dissipate excess reducing power when the CBB pathway is inactive, and that EM is successful in describing this behavior.     

Chemical inhibition of the glycolate pathway in soybean leaf cells

Isolated soybean (Glycine max [L.] Merr.) leaf cells were treated with three inhibitors of the glycolate pathway in order to evaluate the potential of such inhibitors for increasing photosynthetic efficiency. Preincubation of cells under acid conditions in α-hydroxypyridinemethanesulfonic acid increased ¹⁴CO₂ incorporation into glycolate, but severely inhibited photosynthesis. Isonicotinic acid hydrazide (INH) increased the incorporation of ¹⁴CO₂ into glycine and reduced label in serine, glycerate, and starch. Butyl 2-hydroxy-3-butynoate (BHB) completely and irreversibly inhibited glycolate oxidase and increased the accumulation of ¹⁴C into glycolate. Concomitant with glycolate accumulation was the reduction of label in serine, glycerate, and starch, and the elimination of label in glycine. The inhibitors INH and BHB did not eliminate serine synthesis, suggesting that some serine is synthesized by an alternate pathway. The per cent incorporation of ¹⁴CO₂ into glycolate by BHB-treated cells or glycine by INH-treated cells was determined by the O₂/CO₂ ratio present during assay. Photosynthesis rate was not affected by INH or BHB in the absence of O₂, but these compounds increased the O₂ inhibition of photosynthesis. This finding suggests that the function of the photorespiratory pathway is to recycle glycolate carbon back into the Calvin cycle, so if glycolate metabolism is inhibited, Calvin cycle intermediates become depleted and photosynthesis is decreased. Thus, chemicals which inhibit glycolate metabolism do not reduce photorespiration and increase photosynthetic efficiency, but rather exacerbate the problem of photorespiration.     

Dimethyl sulfoxide reduction by a hyperhermophilic archaeon Thermococcus onnurineus NA1 via a cysteine-cystine redox shuttle

A variety of microbes grow by respiration with dimethyl sulfoxide (DMSO) as an electron acceptor, and several distinct DMSO respiratory systems, consisting of electron carriers and a terminal DMSO reductase, have been characterized. The heterotrophic growth of a hyperthermophilic archaeon Thermococcus onnurineus NA1 was enhanced by the addition of DMSO, but the archaeon was not capable of reducing DMSO to DMS directly using a DMSO reductase. Instead, the archaeon reduced DMSO via a cysteine-cystine redox shuttle through a mechanism whereby cystine is microbially reduced to cysteine, which is then reoxidized by DMSO reduction. A thioredoxin reductase-protein disulfide oxidoreductase redox couple was identified to have intracellular cystine-reducing activity, permitting recycle of cysteine. This study presents the first example of DMSO reduction via an electron shuttle. Several Thermococcales species also exhibited enhanced growth coupled with DMSO reduction, probably by disposing of excess reducing power rather than conserving energy.     

Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana

We introduced the Escherichia coli glycolate catabolic pathway into Arabidopsis thaliana chloroplasts to reduce the loss of fixed carbon and nitrogen that occurs in C(3) plants when phosphoglycolate, an inevitable by-product of photosynthesis, is recycled by photorespiration. Using step-wise nuclear transformation with five chloroplast-targeted bacterial genes encoding glycolate dehydrogenase, glyoxylate carboligase and tartronic semialdehyde reductase, we generated plants in which chloroplastic glycolate is converted directly to glycerate. This reduces, but does not eliminate, flux of photorespiratory metabolites through peroxisomes and mitochondria. Transgenic plants grew faster, produced more shoot and root biomass, and contained more soluble sugars, reflecting reduced photorespiration and enhanced photosynthesis that correlated with an increased chloroplastic CO(2) concentration in the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase. These effects are evident after overexpression of the three subunits of glycolate dehydrogenase, but enhanced by introducing the complete bacterial glycolate catabolic pathway. Diverting chloroplastic glycolate from photorespiration may improve the productivity of crops with C(3) photosynthesis.     

Influence of glycerate on photosynthesis by wheat chloroplasts

Glycerate was found to effect photosynthetic O2 evolution in wheat chloroplasts by its conversion to triose phosphate and by influencing the rate of photosynthesis through the reductive pentose phosphate pathway. In the absence of bicarbonate, the photosynthetic O2 evolution with glycerate was low (10 to 25 mumol mg chlorophyll-1 h-1), and only about 15% of the rate of bicarbonate-dependent O2 evolution under optimum conditions. This corresponds to a rate of glycerate conversion to triose phosphate of 20 to 50 mumol mg chlorophyll-1 h-1, which appears sufficient to accommodate flux through the glycolate pathway in vivo. Pi was required for this glycerate-dependent O2 evolution; rates remained relatively constant between 0.1 and 40 mM Pi, and proceeded with little lag upon illumination (less than 0.5 min). Evidence for O2 evolution due to glycerate conversion to triose phosphate could be conclusively demonstrated by addition of glycolaldehyde, an inhibitor of the regenerative phase of photosynthesis, which prevents CO2 fixation. The effect of glycerate on photosynthesis in the presence of bicarbonate was determined by measuring both photosynthetic O2 evolution and 14CO2 fixation at varying Pi concentrations. Low concentrations of glycerate (micro- to millimolar levels) prevented inhibition of photosynthesis by Pi. With 1 mM bicarbonate and pH 8.2, which is favorable for glycolate synthesis, maximum rates of photosynthesis were obtained at low Pi (25 microM), whereas strong inhibition of photosynthesis occurred at only 0.2 mM Pi. Addition of glycerate relieved the inhibition of photosynthesis by Pi, indicating the possible importance of glycerate metabolism in the chloroplast under photorespiratory conditions. The initiation of photosynthesis by glycerate at inhibitory Pi levels occurred with little reduction in the ratio of CO2 fixed/O2 evolved, and the main effect of glycerate was on carbon assimilation. While the basis for the beneficial effect of glycerate on CO2 assimilation under moderate to high Pi levels is uncertain, it may increase the concentration of 3-phosphoglycerate (PGA) in the chloroplast, and thus make conditions more favorable for induction of photosynthesis and reduction of PGA to triose phosphate.     

The Absence of Alternative Oxidase AOX1A Results in Altered Response of Photosynthetic Carbon Assimilation to Increasing CO2 in Arabidopsis thaliana

In higher plants, the mitochondrial electron transport chain has non-phosphorylating alternative pathways that include the alternative terminal oxidase (AOX). This alternative pathway has been suggested to act as a sink for dissipating excess reducing power, minimizing oxidative stress and possibly optimizing photosynthesis in response to changing conditions. The expression patterns of the AOX genes have been well characterized under different growth conditions, particularly in response to light and temperature stress. Additionally, it has been suggested that mitochondrial electron transport is important for avoiding chloroplast over-reduction and balancing energy partitioning among photosynthesis, photorespiration and respiration. Nonetheless, the role AOX plays in optimizing photosynthetic carbon metabolism is unclear. Therefore, the response of photosynthesis to the disruption of AOX was investigated in the Arabidopsis thaliana T-DNA mutant aox1a (SALK_084897). Gas exchange analysis revealed a lower net CO(2) assimilation rate (A) at high CO(2) concentrations in the aox1a mutant compared to wild type. This decrease in A was accompanied by a lower maximum electron transport rate and quantum yield of PSII, and higher excitation pressure on PSII and non-photochemical quenching. The aox1a mutant also exhibited a lower estimated rate of ribulose 1,5-bisphosphate regeneration, and the ribulose 1,5-bisphosphate content was lower at high CO(2) concentrations, suggesting an ATP limitation of the Calvin-Benson cycle. Additionally, the activity of the malate-oxaloacetate shuttle was lower in the mutant compared to wild type. These results indicate that AOX is important for optimizing rates of photosynthetic CO(2) assimilation in response to rising CO(2) concentration by balancing the NAD(P)H/ATP ratio and rates of ribulose 1,5-bisphosphate regeneration within the chloroplast.     

Carbonic anhydrase activity and photosynthetic rate in the tree species Paulownia tomentosa Steud. Effect of dimethylsulfoxide treatment and zinc accumulation in leaves

The enzyme carbonic anhydrase (CA) (EC 4.2.1.1) catalyzes the reversible conversion of CO2 to HCO3- and has been shown to be involved in photosynthesis. The enzyme has been shown in animals, plants, eubacteria and viruses, but similar reports on the evidence for CA activity in tree plants does not be appear to be available. In the preliminary analyses of the work, the CA activity in leaf extracts from the tree species Paulownia tomentosa Steud. (introduced in Bulgaria) is described. A connection between CA activity and the rate of photosynthetic CO2 fixation is shown. In the second portion of the work, the effect of 10(-4) mol/L and 10(-2) mol/L dimethylsulfoxide (DMSO) on the zinc accumulation in leaves is demonstrated. It is suggested that CA activity is an indicator of the level of physiologically active zinc in leaves of P. tomentosa Steud. A connection between the process of zinc accumulation in leaves and the activity of the enzymes CA and glycolate oxidase (GO) (EC 1.1.3.1) is established.     

Photosynthetic electron transport and anaerobic metabolism in purple non-sulfur phototrophic bacteria

Purple non-sulfur phototrophic bacteria, exemplifed byRhodobacter capsulatus andRhodobacter sphaeroides, exhibit a remarkable versatility in their anaerobic metabolism. In these bacteria the photosynthetic apparatus, enzymes involved in CO₂ fixation and pathways of anaerobic respiration are all induced upon a reduction in oxygen tension. Recently, there have been significant advances in the understanding of molecular properties of the photosynthetic apparatus and the control of the expression of genes involved in photosynthesis and CO₂ fixation. In addition, anaerobic respiratory pathways have been characterised and their interaction with photosynthetic electron transport has been described. This review will survey these advances and will discuss the ways in which photosynthetic electron transport and oxidation-reduction processes are integrated during photoautotrophic and photoheterotrophic growth.     

Effect of Aminoacetonitrile on the CO2 Exchange Rate in Rice Leaves

Aminoacetonitrile (AAN), a specific inhibitor of glycine oxidation in the photorespiratory glycolate pathway, did not inhibit photosynthetic CO₂ fixation, but inhibited the apparent photosynthesis of rice leaves under high photosynthetic conditions. However, under such low photosynthetic conditions as low light intensity or senescent leaves, the apparent photosynthesis was not inhibited by AAN. The application of AAN to the leaves led to a greater accumulation of glycine under a high photosynthetic condition like strong light intensity.

From these results, it can be postulated that the inhibition of apparent photosynthesis by AAN was due to the accumulation of intermediate metabolites in the photorespiratory glycolate pathway which was induced by AAN treatment.     

Effects of acetate on facultative autotrophy in Chlamydomonas reinhardtii assessed by photosynthetic measurements and stable isotope analyses

The green alga Chlamydomonas reinhardtii can grow photoautotrophically utilizing CO(2), heterotrophically utilizing acetate, and mixotrophically utilizing both carbon sources. Growth of cells in increasing concentrations of acetate plus 5% CO(2) in liquid culture progressively reduced photosynthetic CO(2) fixation and net O(2) evolution without effects on respiration, photosystem II efficiency (as measured by chlorophyll fluorescence), or growth. Using the technique of on-line oxygen isotope ratio mass spectrometry, we found that mixotrophic growth in acetate is not associated with activation of the cyanide-insensitive alternative oxidase pathway. The fraction of carbon biomass resulting from photosynthesis, determined by stable carbon isotope ratio mass spectrometry, declined dramatically (about 50%) in cells grown in acetate with saturating light and CO(2). Under these conditions, photosynthetic CO(2) fixation and O(2) evolution were also reduced by about 50%. Some growth conditions (e.g. limiting light, high acetate, solid medium in air) virtually abolished photosynthetic carbon gain. These effects of acetate were exacerbated in mutants with slowed electron transfer through the D1 reaction center protein of photosystem II or impaired chloroplast protein synthesis. Therefore, in mixotrophically grown cells of C. reinhardtii, interpretations of the effects of environmental or genetic manipulations of photosynthesis are likely to be confounded by acetate in the medium.     

Molecular analysis of dimethyl sulphide dehydrogenase from Rhodovulum sulfidophilum: its place in the dimethyl sulphoxide reductase family of microbial molybdopterin-containing enzymes

Dimethyl sulphide dehydrogenase catalyses the oxidation of dimethyl sulphide to dimethyl sulphoxide (DMSO) during photoautotrophic growth of Rhodovulum sulfidophilum. Dimethyl sulphide dehydrogenase was shown to contain bis(molybdopterin guanine dinucleotide)Mo, the form of the pterin molybdenum cofactor unique to enzymes of the DMSO reductase family. Sequence analysis of the ddh gene cluster showed that the ddhA gene encodes a polypeptide with highest sequence similarity to the molybdopterin-containing subunits of selenate reductase, ethylbenzene dehydrogenase. These polypeptides form a distinct clade within the DMSO reductase family. Further sequence analysis of the ddh gene cluster identified three genes, ddhB, ddhD and ddhC. DdhB showed sequence homology to NarH, suggesting that it contains multiple iron-sulphur clusters. Analysis of the N-terminal signal sequence of DdhA suggests that it is secreted via the Tat secretory system in complex with DdhB, whereas DdhC is probably secreted via a Sec-dependent mechanism. Analysis of a ddhA mutant showed that dimethyl sulphide dehydrogenase was essential for photolithotrophic growth of Rv. sulfidophilum on dimethyl sulphide but not for chemo-trophic growth on the same substrate. Mutational analysis showed that cytochrome c2 mediated photosynthetic electron transfer from dimethyl sulphide dehydrogenase to the photochemical reaction centre, although this cytochrome was not essential for photoheterotrophic growth of the bacterium.     

Metabolic shifts in hypersaline microbial mats upon addition of organic substrates

The responses of hypersaline microbial mats to the addition of acetate, glycolate or glucose were investigated using oxygen, pH and sulphide microsensors. Changes in community structure were investigated with molecular techniques. Acetate addition inhibited respiration in the photic zone, stimulated respiration in the aphotic zone and had no effect on gross photosynthesis. Glycolate addition strongly increased both respiration and gross photosynthesis in the photic zone. Thus, glycolate and acetate were probably consumed in those regions of the mat where these substrates are usually formed. Moreover, photosynthesis was only stimulated by increased respiration and concomitant CO2 production in the photic zone which indicates that the photosynthetic and respiratory populations must be present in close proximity to each other. Glucose addition had an unexpected negative effect on the microbial population, strongly inhibiting both respiration and gross photosynthesis within hours. After four days, oxygen profiles in the light were equal to those measured in the dark. After replacing the water phase with unamended water, photosynthesis and respiration recovered within a week. None of the physiological changes were accompanied by detectable shifts in the cyanobacterial or the overall microbial community. The mechanism of inhibition of photosynthesis by glucose requires further investigation.     

果糖-1,6-二磷酸醛缩酶和丙糖磷酸异构酶共表达对蓝藻光合作用效率的影响

以转高等植物ALD和TPI基因的鱼腥藻 7120为对象 ,研究了ALD和TPI两个酶表达量对细胞光合固碳效率的影响。考察了初始pH、NaHCO₃浓度和CO₂浓度对转基因藻和野生藻生长、光合活性及无机碳亲和力的影响。结果表明 ,转基因藻在较高碳源浓度下 ,其生长速率和光合放氧活性比野生藻有显著的提高 ,并且可以比野生藻耐受更高的pH。在含有2%CO₂的空气中 ,转基因藻对外源无机碳的亲和力比野生藻提高了4.06倍.     

Maintenance and control of redox poise in Rhodobacter capsulatus strains deficient in the Calvin-Benson-Bassham pathway

Carbon dioxide serves as the preferred electron acceptor during photoheterotrophic growth of nonsulfur purple photosynthetic bacteria such as Rhodobacter capsulatus and Rhodobacter sphaeroides. This CO2, produced as a result of the oxidation of preferred organic carbon sources, is reduced through reactions of the Calvin-Benson-Bassham reductive pentose phosphate pathway. This pathway is thus crucial to maintain a balanced intracellular oxidation-reduction potential (or redox poise) under photoheterotrophic growth conditions. In the absence of a functional Calvin-Benson-Bassham pathway, either an exogenous electron acceptor, such as dimethylsulfoxide, must be supplied or the organism must somehow develop alternative electron acceptor pathways to preserve the intracellular redox state of the cell. Spontaneous variants of Rba. capsulatus strains deficient in the Calvin-Benson-Bassham pathway that have become photoheterotrophically competent (in the absence of an exogenous electron acceptor) were isolated. These strains (SBP-PHC and RCNd1, RCNd3, and RCNd4) were shown to obviate normal ammonia control and derepress synthesis of the dinitrogenase enzyme complex for the dissipation of excess reducing equivalents and generation of H2 gas via proton reduction. In contrast to previous studies with other organisms, the dinitrogenase reductase polypeptides were maintained in an active and unmodified form in strain SBP-PHC and the respective RCNd strains. Unlike the situation in Rba. sphaeroides, the Rba. capsulatus strains did not regain full ammonia control when complemented with plasmids that reconstituted a functional Calvin-Benson-Bassham pathway. Moreover, dinitrogenase derepression in Rba. capsulatas was responsive to the addition of the auxiliary electron acceptor dimethylsulfoxide. These results indicated a hierarchical control over the removal of reducing equivalents during photoheterotrophic growth that differs from strains of Rba. sphaeroides and Rhodospirillum rubrum deficient in the Calvin-Benson-Bassham pathway.