Effect of Osmotic Stress on Carbon Metabolism in Chlamydomonas reinhardtii: Accumulation of Glycerol as an Osmoregulatory Solute

NaCl, KCl, and sucrose at equiosmolar concentrations had similar inhibitory effects on photosynthetic carbon metabolism by the freshwater green alga, Chlamydomonas reinhardtii. Inhibitory concentrations of these solutes altered the products of photosynthetic ¹⁴CO₂ incorporation, resulting in reduced incorporation into starch, sugar phosphates, lactate, and glycolate, but caused an accumulation of glycerol both intracellularly and in the medium.     

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.     

H(2) and CO(2) Evolution by Anaerobically Adapted Chlamydomonas reinhardtii F-60

Using manometric and enzymic techniques, H₂ and CO₂ evolution in darkness and light has been studied in the green alga Chlamydomonas reinhardtii F-60. F-60 is a mutant strain characterized by an incomplete photosynthetic carbon reduction cycle but an intact electron transport chain.     

Glyoxylate and glutamate effects on photosynthetic carbon metabolism in isolated chloroplasts and mesophyll cells of spinach

Addition of millimolar sodium glyoxylate to spinach (Spinacia oleracea) chloroplasts was inhibitory to photosynthetic incorporation of ¹⁴CO₂ under conditions of both low (0.2 millimolar or air levels) and high (9 millimolar) CO₂ concentrations. Incorporation of ¹⁴C into most metabolites decreased. Labeling of 6-P-gluconate and fructose-1,6-bis-P increased. This suggested that glyoxylate inhibited photosynthetic carbon metabolism indirectly by decreasing the reducing potential of chloroplasts through reduction of glyoxylate to glycolate. This hypothesis was supported by measuring the reduction of [¹⁴C]glyoxylate by chloroplasts. Incubation of isolated mesophyll cells with glyoxylate had no effect on net photosynthetic CO₂ uptake, but increased labeling was observed in 6-P-gluconate, a key indicator of decreased reducing potential. The possibility that glyoxylate was affecting photosynthetic metabolism by decreasing chloroplast pH cannot be excluded. Increased ¹⁴C-labeling of ribulose-1,5-bis-P and decreased 3-P-glyceric acid and glycolate labeling upon addition of glyoxylate to chloroplasts suggested that ribulose-bis-P carboxylase and oxygenase might be inhibited either indirectly or directly by glyoxylate. Glyoxylate addition decreased ¹⁴CO₂ labeling into glycolate and glycine by isolated mesophyll cells but had no effect on net ¹⁴CO₂ fixation. Glutamate had little effect on net photosynthetic metabolism in chloroplast preparations but did increase ¹⁴CO₂ incorporation by 15% in isolated mesophyll cells under air levels of CO₂.     

Photoassimilation of Glycolate, Glycine and Serine by Euglena gracilis

SYNOPSIS. Glycolate was readily utilized for growth by Euglena gracilis , strain Z, in the light at pH 3.8 under a variety of atmospheric conditions, including CO₂-free air and nitrogen. Glycolate did not support growth in the dark as sole carbon source; no significant uptake of glycolate was observed under these conditions. However, cells grown in the light with glycolate as sole carbon source were still capable of glycolate uptake for up to 3 hr after transfer to darkness, and glycolate was taken up by cells utilizing glucose in the dark. The energy requirement for glycolate utilization could thus be met either by light, or by the aerobic metabolism of glucose in the dark. DCMU, an inhibitor of photosystem II, inhibited photoassimilation of glycolate. In the light, but again not in the dark, glycine and serine also served as sole source of carbon under CO₂-free air, but not under nitrogen. Net release of ammonia to the medium accompanied the photoassimilation of glycine and serine. Of the several metabolicallyrelated compounds tested, only glycolate was utilized as sole carbon source in the light under "anaerobic" conditions. A lag in net chlorophyll synthesis occurred during the photoassimilation of glycolate glycine or serine. Determination of rates of photosynthetic ¹⁴CO₂ fixation confirmed that some inhibition of photosynthetic capacity had occurred in response to utilization of glycolate and related compounds.     

Involvement of glycolate metabolism in acclimation of Chlorella vulgaris cultures to low phosphate supply

Chlorella vulgaris (Beijer.) was grown for 8 d under air in cultures with complete (Control) or with phosphorus-deficient (–P) medium limiting culture growth. The cells assimilated only 5–17 % of orthophosphate supplied from the complete medium, whereas from medium of –P cultures, orthophosphate was almost totally exhausted. Despite limited phosphorus availability, cells in the oldest –P cultures contained the same amount of inorganic orthophosphate as the control cells and only slightly less organic phosphates. The –P cells showed normal chlorophyll concentration and increased V_max and 1/K_0.5 dissolved inorganic carbon (DIC) of photosynthetic O₂ evolution. Phosphorus deficiency enhanced production, excretion and metabolism of glycolate during the whole investigated period. In the initial phase of –P culture growth, medium acidification and low DIC concentration were conducive to glycolate production. With subsequent medium alkalization, DIC content and cell carbonic anhydrase activity increased the photosynthetic O₂ evolution of –P cells two-fold. At that period, the elevated intrachloroplast O₂ concentration might be the main reason of enhancement of glycolate metabolism. The results support the suggestion that involvement of glycolate metabolism in acclimation to low phosphorus supply improves regeneration of inorganic orthophosphate and protects chloroplasts against photoinhibitory damage by consumption of excess of absorbed light energy.     

Impairment of Photorespiratory Carbon Flow into Rubber by the Inhibition of the Glycolate Pathway in Guayule (Parthenium argentatum Gray)

Cut shoots of guayule (Parthenium argentatum Gray) were treated with four inhibitors of the glycolate pathway (α-hydroxypyridinemethanesulfonic acid; isonicotinic acid hydrazide, glycine hydroxamate, and amino-oxyacetate, AOA) in order to evaluate the role of photorespiratory intermediates in providing precursors for the biosynthesis of rubber. Photorespiratory CO₂ evolution in guayule leaves was severely inhibited by AOA. Application of each of the four inhibitors has resulted in a significantly decreased incorporation of ¹⁴C into rubber fractions suggesting that the glycolate pathway is involved in the biosynthesis of rubber in guayule. However, the application of each of the glycolate pathway inhibitors showed no significant effect on photosynthetic CO₂ fixation in the leaves. The inhibitors individually also reduced the incorporation of labeled glycolate, glyoxylate, and glycine into rubber, while the incorporation of serine and pyruvate was not affected. The effective inhibition of incorporation of glycolate pathway intermediates in the presence of AOA was due to an inhibition of glycine decarboxylase and serine hydroxymethyltransferase. It is concluded that serine is a putative photorespiratory intermediate in the biosynthesis of rubber via pyruvate and acetyl coenzyme A.     

On the mechanism of glycolate synthesis by Chromatium and Chlorella

When cultures of the photosynthetic bacterium, Chromatium vinosum, capable of photosynthesizing glycolate at about 10 μmol/mg of bacteriochlorophyll/h were exposed to atmospheres enriched with ¹⁸O₂, one atom of oxygen-18 was incorporated into the car?yl group of glycolate. Allowing for the small (3–5%) loss of oxygen-18 during the manipulations leading up to the mass spectrometric determination of the oxygen-18 content of the glycolate, the isotopic enrichment of the¹⁸O-labeled glycolate synthesized by Chromatium was substantially (at least 94%) the same as the isotopic enrichment of the ¹⁸O₂. Similar results were obtained with the green alga, Chlorella fusca. The close agreement between the isotopic enrichments of the glycolate and the oxygen with which it was synthesized was independent of the oxygen concentration. The major pathway of glycolate synthesis by Chromatium therefore involves reaction(s) which bring about the incorporation of one atom of molecular oxygen into the car?yl group of glycolate. The in vitro rate of ribulose 1,5-bisphosphate oxygenase in extracts of Chromatium, previously thought to be too low to account for the rates of glycolate synthesis in vivo, was shown to be adequate for this purpose when precautions were taken to fully activate the enzyme. Similarly, the activity of phosphoglycolate phosphatase, when assayed under optimal conditions, was also adequate to sustain the rates of glycolate formation observed i vivo. It is concluded that the oxygenolytic cleavage of ribulose 1,5-bisphosphate represents the major pathway of glycolate synthesis by Chromatium.     

Some Enzymes and Properties of the Reductive Carboxylic Acid Cycle Are Present in the Green Alga Chlamydomonas reinhardtii F-60

The reductive carboxylic acid cycle, the autotrophic pathway of CO₂ assimilation in prokaryotes (photosynthetic and nonphotosynthetic autotrophic bacteria), was investigated in Chlamydomonas reinhardtii F-60, an algal mutant lacking a complete photosynthetic carbon reduction pathway (C₃) due to a deficiency in phosphoribulokinase. Evidence was obtained consistent with the presence of the reductive carboxylic acid cycle in F-60. This conclusion is based on the fact that: (a) acetate approximately doubled CO₂ fixation in whole cells (4 micromoles per milligram chlorophyll per hour) and in chloroplasts (32 nanomoles per milligram chlorophyll per hour); and (b) pyruvate synthase, α-ketoglutarate synthase, and ATP-citrate lyase, three indicators of the cycle, were found in cell-free extracts.     

Triacylglyceride Production and Autophagous Responses in Chlamydomonas reinhardtii Depend on Resource Allocation and Carbon Source

To improve the economic viability of microalgal biodiesel, it will be essential to optimize the productivity of fuel molecules such as triacylglyceride (TAG) within the microalgal cell. To understand some of the triggers required for the metabolic switch to TAG production, we studied the effect of the carbon supply (acetate or CO₂) in Chlamydomonas reinhardtii (wild type and the starchless sta6 mutant) grown under low N availability. As expected, initial rates of TAG production were much higher when acetate was present than under strictly photosynthetic conditions, particularly for the sta6 mutant, which cannot allocate resources to starch. However, in both strains, TAG production plateaued after a few days in mixotrophic cultures, whereas under autotrophic conditions, TAG levels continued to rise. Moreover, the reduced growth of the sta6 mutant meant that the greatest productivity (measured as mg TAG liter⁻¹ day⁻¹) was found in the wild type growing autotrophically. Wild-type cells responded to low N by autophagy, as shown by degradation of polar (membrane) lipids and loss of photosynthetic pigments, and this was less in cells supplied with acetate. In contrast, little or no autophagy was observed in sta6 mutant cells, regardless of the carbon supply. Instead, very high levels of free fatty acids were observed in the sta6 mutant, suggesting considerable alteration in metabolism. These measurements show the importance of carbon supply and strain selection for lipid productivity. Our findings will be of use for industrial cultivation, where it will be preferable to use fast-growing wild-type strains supplied with gaseous CO₂ under autotrophic conditions rather than require an exogenous supply of organic carbon.     

pH Dependence and Kinetics of Glycolate Uptake by Intact Pea Chloroplasts

Experiments in which [1-¹⁴C]glycolate uptake is carried out in conjunction with measurements of stromal pH indicate that only glycolic acid and not the glycolate anion is crossing the pea (Pisum sativum var. Progress No. 9, Agway) chloroplast envelope. This mechanism of glycolate transport appears to be too slow to account for observed photorespiratory carbon fluxes in C₃ plants.     

Mechanism of glycolate transport in spinach leaf chloroplasts

The incorporation of ¹⁴CO₂ into glycolate by intact spinach leaf (Spinacia oleracea L. var. Kyoho) chloroplasts exposed to ¹⁴CO₂ (NaH¹⁴CO₃, 1 millimolar) in the light was determined as a function of O₂ concentrations in the reaction media. A hyperbolic saturation curve was obtained, apparent K_m (O₂) of 0.28 millimolar, indicating that glycolate is produced predominantly by ribulose-1,5-bisphosphate carboxylase/oxygenase. A concentration gradient of glycolate was invariably observed between chloroplast stroma and the outside media surrounding chloroplasts during photosynthetic ¹⁴CO₂ fixation under an O₂ atmosphere.     

Effects of Different Inorganic Nitrogen Sources on Photosynthetic Carbon Metabolism in Primary Leaves of Non-nodulated Phaseolus vulgaris L

Young bean plants (Phaseolus vulgaris L. var Saxa) were fed with three different types of inorganic nitrogen, after being grown on nitrogen-free nutrient solution for 8 days. The pattern of ¹⁴CO₂ fixation was investigated in photosynthesizing primary leaf discs of 11-day-old plants (3 days with nitrogen source) and in a pulse-chase experiment in 13-day-old plants (5 days with nitrogen source).

Ammonium caused, in contrast to nitrate nutrition, a higher level of ¹⁴C incorporation into sugar phosphates but a lower incorporation of label into malate, glycolate, glycerate, aspartate, and alanine. The labeling kinetics of glycine and serine were little changed by the nitrogen source. Ammonium feeding also produced an increase in the ratio of extractable activities of ribulose-1,5-bisphosphate carboxylase to phosphoenolpyruvate carboxylase and an increase in dark respiration and the CO₂ compensation concentration. Net photosynthesis was higher in plants assimilating nitrate.

The results point to stimulated turnover of the photosynthetic carbon reduction cycle metabolites, reduced phosphoenolpyruvate carboxylation, and altered turnover rates within the photosynthetic carbon oxidation cycle in ammonium-fed plants. Mechanisms of the regulation of primary carbon metabolism are proposed and discussed.

     

Carbonic Anhydrase-Deficient Mutant of Chlamydomonas reinhardii Requires Elevated Carbon Dioxide Concentration for Photoautotrophic Growth

A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated which is deficient in carbonic anhydrase (EC 4.2.1.1) activity. This mutant strain, designated ca-1-12-1C (gene locus ca-1), was selected on the basis of a high CO₂ requirement for photoautotrophic growth. Photosynthesis by the mutant at atmospheric CO₂ concentration was very much reduced compared to wild type and, unlike wild type, was strongly inhibited by O₂. In contrast to a CO₂ compensation concentration of near zero in wild type at all O₂ concentrations examined, the mutant exhibited a high, O₂-stimulated CO₂ compensation concentration. Evidence of photorespiratory activity in the mutant but not in wild type was obtained from the analysis of photosynthetic products in the presence of ¹⁴CO₂. At air levels of CO₂ and O₂, the mutant synthesized large amounts of glycolate, while little glycolate was synthesized by wild type under identical conditions. Both mutant and wild type strains formed only small amounts of glycolate at saturating CO₂ concentration. At ambient CO₂, wild type accumulated inorganic carbon to a concentration several-fold higher than that in the suspension medium. The mutant cells accumulated inorganic carbon internally to a concentration 6-fold greater than found in wild type, yet photosynthesis was CO₂ limited. The mutant phenotype was mimicked by wild type cells treated with ethoxyzolamide, an inhibitor of carbonic anhydrase activity. These observations indicate a requirement for carbonic anhydrase-catalyzed dehydration of bicarbonate in maintaining high internal CO₂ concentrations and high photosynthesis rates. Thus, in wild type cells, carbonic anhydrase rapidly converts the bicarbonate taken up to CO₂, creating a high internal CO₂ concentration which stimulates photosynthesis and suppresses photorespiration. In mutant cells, bicarbonate is taken up rapidly but, because of a carbonic anhydrase deficiency, is not dehydrated at a rate sufficiently rapid to maintain a high internal CO₂ concentration.     

The Effect of pH on the Products of Photosynthesis in CO(2) by Chloroplast Preparations from Acetabularia mediterranea

The effect of pH on the pathways of carbon in photosynthesis was examined in chloroplast preparations from Acetabularia mediterranea. The flow of carbon into a number of photosynthetic intermediates, particularly sucrose, glycine, serine, glycolate, and the insoluble fraction, was strongly influenced by pH. At higher pH a much larger portion of the ¹⁴C entered intermediates of the glycolate pathway. Although maximal apparent photosynthesis occurred at pH 7.6 to 7.7, cytoplasmic pH was found to be 8.0 to 8.4, using indicators. The pattern of distribution of ¹⁴C in intermediates of whole cells was closest to that in chloroplasts at the higher pH range.     

Changes in Chlorophyll a/b Ratio and Products of CO(2) Fixation by Algae Grown in Blue or Red Light

Chlamydomonas and Chlorella were grown for 10 days in white light. 955 μw/cm² blue light (400-500 mμ) or 685 μw/cm² red light (above 600 mμ). Rates of growth in blue or red light were initially slow, but increased over a period of 5 days until normal growth rates were reestablished. During this adaptation period in blue light, total chlorophyll per volume of algae increased 20% while the chlorophyll a/b ratio decreased. In red light no change was observed in the total amount of chlorophyll or in the chlorophyll a/b ratio. After adaptation to growth in blue light and upon exposure to ¹⁴CO₂ with either blue or white light for 3 to 10 minutes, 30 to 36% of the total soluble fixed ¹⁴C accumulated in glycolate-¹⁴C which was the major product. However, with 1 minute experiments, it was shown that phosphate esters of the photosynthetic carbon cycle were labeled before the glycolate. Glycolate accumulation by algae grown in blue light occurred even at low light intensity. After growth of the algae in red light, ¹⁴C accumulated in malate, aspartate, glutamate and alanine, whereas glycolate contained less than 3% of the soluble ¹⁴C fraction.     

Fixation of O(2) during Photorespiration: Kinetic and Steady-State Studies of the Photorespiratory Carbon Oxidation Cycle with Intact Leaves and Isolated Chloroplasts of C(3) Plants

Mass spectrometric techniques were used to trace the incorporation of [¹⁸O]oxygen into metabolites of the photorespiratory pathway. Glycolate, glycine, and serine extracted from leaves of the C₃ plants, Spinacia oleracea L., Atriplex hastata, and Helianthus annuus which had been exposed to [¹⁸O]oxygen at the CO₂ compensation point were heavily labeled with ¹⁸O. In each case one, and only one of the carboxyl oxygens was labeled. The abundance of ¹⁸O in this oxygen of glycolate reached 50 to 70% of that of the oxygen provided after only 5 to 10 seconds exposure to [¹⁸O]oxygen. Glycine and serine attained the same final enrichment after 40 and 180 seconds, respectively. This confirms that glycine and serine are synthesized from glycolate.

The labeling of photorespiratory intermediates in intact leaves reached a mean of 59% of that of the oxygen provided in the feedings. This indicates that at least 59% of the glycolate photorespired is synthesized with the fixation of molecular oxygen. This estimate is certainly conservative owing to the dilution of labeled oxygen at the site of glycolate synthesis by photosynthetic oxygen. We examined the yield of ¹⁸O in glycolate synthesized in vitro by isolated intact spinach chloroplasts in a system which permitted direct sampling of the isotopic composition of the oxygen at the site of synthesis. The isotopic enrichment of glycolate from such experiments was 90 to 95% of that of the oxygen present during the incubation.

The carboxyl oxygens of 3-phosphoglycerate also became labeled with ¹⁸O in 20- and 40-minute feedings with [¹⁸O]oxygen to intact leaves at the CO₂ compensation point. Control experiments indicated that this label was probably due to direct synthesis of 3-phosphoglycerate from glycolate during photorespiration. The mean enrichment of 3-phosphoglycerate was 14 ± 4% of that of glycine or serine, its precursors of the photorespiratory pathway, in 10 separate feeding experiments. It is argued that this constant dilution of label indicates a constant stoichiometric balance between photorespiratory and photosynthetic sources of 3-phosphoglycerate at the CO₂ compensation point.

Oxygen uptake sufficient to account for about half of the rate of ¹⁸O fixation into glycine in the intact leaves was observed with intact spinach chloroplasts. Oxygen uptake and production by intact leaves at the CO₂ compensation point indicate about 1.9 oxygen exchanged per glycolate photorespired. The fixation of molecular oxygen into glycolate plus the peroxisomal oxidation of glycolate to glyoxylate and the mitochondrial conversion of glycine to serine can account for up to 1.75 oxygen taken up per glycolate.

These studies provide new evidence which supports the current formulation of the pathway of photorespiration and its relation to photosynthetic metabolism. The experiments described also suggest new approaches using stable isotope techniques to study the rate of photorespiration and the balance between photorespiration and photosynthesis in vivo.

     

Photosynthetic products of division synchronized cultures of euglena

Rates and products of photosynthetic ¹⁴CO₂ fixation by division synchronized cultures of Euglena gracilis strain Z were determined over the cycle. Rate of ¹⁴CO₂ fixation doubled in a continuous manner throughout the light phase followed by a slight reduction of photosynthetic capacity in the dark phase. Greater ¹⁴C incorporation into the nucleic acid-polysaccharide fraction occurred with mature cells. Products of ¹⁴CO₂ fixation varied markedly over the cycle: although with mature cells ¹⁴C-labeled sucrose was not detected, with dividing cells this was the main sugar labeled; in young cells ¹⁴C maltose was formed. Cells removed at end of dark phase accumulated ¹⁴C in glycolate, whereas at other stages over the cycle less ¹⁴C was present in glycolate, and this was accompanied by a rapid incorporation of ¹⁴C into glycine and serine. Glycerate was an early and major product of photosynthesis with cells at the mature stage of the cycle.     

A Mutant of Nicotiana sylvestris Lacking Serine:Glyoxylate Aminotransferase: Substrate Specificity of the Enzyme and Fate of [2-C]Glycolate in Plants with Genetically Altered Enzyme Levels

The photorespiratory mutant of Nicotiana sylvestris, NS 349, lacking serine:glyoxylate aminotransferase (SGAT) grows in 1% CO₂ but not in normal air (NA McHale, EA Havir, I Zelitch 1988 Theor Appl Genet. In press). Alanine:hydroxypyruvate and asparagine:hydroxypyruvate aminotransferase activities were also lacking in the mutant, and plants heterozygous with respect to SGAT which grow in normal air had 50% of the activities present in homozygous plants. Therefore, all these activities are associated with the same enzyme. On feeding [2-¹⁴C]glycolate to leaf discs in the light, NS 349 showed reduced incorporation of radioactivity into the neutral and organic acid fractions and increased incorporation into the amino acid fraction, principally into serine. The effect of reducing SGAT by 50% in heterozygous plants produced little change in the metabolism of [2-¹⁴C]glycolate, showing there is a large excess of this enzyme in wild-type plants.     

Identification of genes required for recycling reducing power during photosynthetic growth

Photosynthetic organisms have the unique ability to transform light energy into reducing power. We study the requirements for photosynthesis in the alpha-proteobacterium Rhodobacter sphaeroides. Global gene expression analysis found that approximately 50 uncharacterized genes were regulated by changes in light intensity and O\2 tension, similar to the expression of genes known to be required for photosynthetic growth of this bacterium. These uncharacterized genes included RSP4157 to -4159, which appeared to be cotranscribed and map to plasmid P004. A mutant containing a polar insertion in RSP4157, CT01, was able to grow via photosynthesis under autotrophic conditions using H2 as an electron donor and CO2 as a carbon source. However, CT01 was unable to grow photoheterotrophically in a succinate-based medium unless compounds that could be used to recycle reducing power (the external electron acceptor dimethyl sulfoxide (DMSO) or CO2 were provided. This suggests that the insertion in RSP4157 caused a defect in recycling reducing power during photosynthetic growth when a fixed carbon source was present. CT01 had decreased levels of RNA for genes encoding putative glycolate degradation functions. We found that exogenous glycolate also rescued photoheterotrophic growth of CT01, leading us to propose that CO2 produced from glycolate metabolism can be used by the Calvin cycle to recycle reducing power generated in the photosynthetic apparatus. The ability of glycolate, CO2, or DMSO to support photoheterotrophic growth of CT01 suggests that one or more products of RSP4157 to -4159 serve a previously unknown role in recycling reducing power under photosynthetic conditions.