Acetate and CO2 assimilation by Methanothrix concilii.

Biosynthetic pathways in Methanothrix concilii, a recently isolated aceticlastic methanogen, were studied by 13C-nuclear magnetic resonance spectroscopy. Labeling patterns of amino acids, lipids, and carbohydrates were determined. Similar to other methanogens, acetate was carboxylated to pyruvate, which was further converted to amino acids by various biosynthetic pathways. The origin of carbon atoms in glutamate, proline, and arginine clearly showed that an incomplete tricarboxylic acid cycle operating in the oxidative direction was used for their biosynthesis. Isoleucine was synthesized via citramalate, which is a typical route for methanogens. As with Methanosarcina barkeri, an extensive exchange of the label between the carboxyl group of acetate and CO2 was observed. Lipids predominantly contained diphytanyl chains, the labeling of which indicated that biosynthesis proceeded through mevalonic acid. Labeling of the C-1,6 of glucose from [2-13C]acetate is consistent with a glucogenic route for carbohydrate biosynthesis. Except for the different origins of the methyl group of methionine, the metabolic properties of Methanothrix concilii are closely related to those of Methanosarcina barkeri.     

Metabolic flux analysis of the mixotrophic metabolisms in the green sulfur bacterium Chlorobaculum tepidum

The photosynthetic green sulfur bacterium Chlorobaculum tepidum assimilates CO(2) and organic carbon sources (acetate or pyruvate) during mixotrophic growth conditions through a unique carbon and energy metabolism. Using a (13)C-labeling approach, this study examined biosynthetic pathways and flux distributions in the central metabolism of C. tepidum. The isotopomer patterns of proteinogenic amino acids revealed an alternate pathway for isoleucine synthesis (via citramalate synthase, CimA, CT0612). A (13)C-assisted flux analysis indicated that carbons in biomass were mostly derived from CO(2) fixation via three key routes: the reductive tricarboxylic acid (RTCA) cycle, the pyruvate synthesis pathway via pyruvate:ferredoxin oxidoreductase, and the CO(2)-anaplerotic pathway via phosphoenolpyruvate carboxylase. During mixotrophic growth with acetate or pyruvate as carbon sources, acetyl-CoA was mainly produced from acetate (via acetyl-CoA synthetase) or citrate (via ATP citrate lyase). Pyruvate:ferredoxin oxidoreductase converted acetyl-CoA and CO(2) to pyruvate, and this growth-rate control reaction is driven by reduced ferredoxin generated during phototrophic growth. Most reactions in the RTCA cycle were reversible. The relative fluxes through the RTCA cycle were 80～100 units for mixotrophic cultures grown on acetate and 200～230 units for cultures grown on pyruvate. Under the same light conditions, the flux results suggested a trade-off between energy-demanding CO(2) fixation and biomass growth rate; C. tepidum fixed more CO(2) and had a higher biomass yield (Y(X/S), mole carbon in biomass/mole substrate) in pyruvate culture (Y(X/S) = 9.2) than in acetate culture (Y(X/S) = 6.4), but the biomass growth rate was slower in pyruvate culture than in acetate culture.     

Solution (sup13)C Nuclear Magnetic Resonance Spectroscopic Analysis of the Amino Acids of Methanosphaera stadtmanae: Biosynthesis and Origin of One-Carbon Units from Acetate and Carbon Dioxide

We found that general pathways for amino acid synthesis of Methanosphaera stadtmanae, a methanogen that forms CH(inf4) from H(inf2) and methanol, resembled those of methanogens that form CH(inf4) from CO(inf2) or from the methyl group of acetate. We determined the incorporation of (sup14)C-labeled CO(inf2), formate, methanol, methionine, serine, and acetate into cell macromolecules. Labeling of amino acid carbons was determined by solution nuclear magnetic resonance spectroscopy after growth with (sup13)C-labeled acetate, CO(inf2), serine, and methanol. The (alpha) and (beta) carbons of serine and alanine were formed from carboxyl and methyl carbons of acetate, respectively, and the amino acid carboxyl groups were formed from CO(inf2). This indicates that pyruvate was formed by reductive carboxylation of acetate. Labeling of the methyl carbon of methionine indicated that the major route of synthesis was from the hydroxymethyl carbon of serine that arises from the methyl carbon of acetate. Methanol was a minor source of the methyl of methionine. Unambiguous assignment was made of the sources of all carbons of histidine. Labeling of the histidine 7 position ((epsilon) carbon) was consistent with formation from the C-2 of the purine ring of ATP and the origin of the C-2 from a formyl unit derived from the hydroxymethyl carbon of serine.     

13C-NMR study of autotrophic CO2 fixation in Thermoproteus neutrophilus

The pathway of autotrophic CO2 fixation has been investigated in the extremely thermophilic sulfur-respiring anaerobic archaebacterium Thermoproteus neutrophilus. [1,4-13C2]Succinate was used as a tracer since this compound was incorporated in small amounts virtually into all cell compounds without affecting the organism's ability to synthesize all cell constituents from CO2. Three representative amino acids, glutamate, aspartate and alanine were isolated from cells after growth for several generations in the presence of [1,4-13C2]succinate and their labelling patterns were determined by 13C NMR spectroscopy. The data is consistent with CO2 fixation by a reductive citric acid cycle, as proposed earlier for the green sulfur bacterium Chlorobium limicola, the sulfate-reducing Desulfobacter hydrogenophilus and the microaerophilic Knallgasbacterium Hydrogenobacter thermophilus. The presence of a reductive citric acid cycle in archaebacteria indicates that this CO2 fixation mechanism which is an alternative to the Calvin cycle is present in many anaerobic or facultative anaerobic microorganisms.     

Evolution of the biosynthesis of the branched-chain amino acids

The origin of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threonine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from -ketoisovaleric acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use of the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.     

Beta-aminoglutaric acid is a major soluble component of Methanococcus thermolithotrophicus

13C- and 15N-NMR spectroscopy have been used to identify beta-aminoglutaric acid (beta-glutamic) as a major soluble component of the thermophilic, autotrophic marine methanogen Methanococcus thermolithotrophicus. This rare, non-protein amino acid has been recognized as a major dissolved free amino acid in marine sediments, but the microorganism responsible for its production has not previously been identified. The concentration of beta-aminoglutarate (beta-glutamate) is about one half that of free alpha-glutamate and increases (relative to the alpha-isomer) as cells enter the stationary phase. Analysis of the 13C label distribution in a 13CO2-pulse/12CO2-chase experiment shows that label enters the beta-aminoglutarate pool after it has decayed from other small soluble molecules. This implies that beta-aminoglutarate is a catabolic product of the cells. Preliminary biosynthesis studies with labeled precursors indicate that only a single acetate moiety is incorporated in this unusual compound. This information is used to suggest possible biosynthetic pathways.     

Carbon isotope-labelling experiments indicate that ladderane lipids of anammox bacteria are synthesized by a previously undescribed, novel pathway

Ladderane lipids are unusual membrane lipids of bacteria that anaerobically oxidize ammonium to dinitrogen gas (anammox). Ladderane lipids contain linearly concatenated cyclobutane rings for which the pathway of biosynthesis is currently unknown. To investigate the possible biosynthetic routes of these lipids, 2-¹³C-labelled acetate was added to a culture of the anammox bacterium Candidatus Brocadia fulgida. Labelling patterns obtained by high-field ¹³C nuclear magnetic resonance spectroscopy of isolated lipids indicated that C . Brocadia fulgida synthesizes C_16:0 and iso C_16:0 fatty acids according to the known pathway of type II fatty acid biosynthesis. The ¹³C-labelling pattern of the C₈ alkyl chain of the C₂₀ [3] ladderane monoether also indicated the use of this route. However, carbon atoms in the cyclobutane rings and the cyclohexane ring were nonspecifically labelled and did not correspond to known patterns of fatty acid synthesis. Taken together, our results indicate that it is unlikely that ladderane lipids are formed from the cyclization of polyunsaturated fatty acids as hypothesized previously and suggest an alternative, although as yet unknown, pathway of biosynthesis.     

Mevalonic acid is partially synthesized from amino acids in Halobacterium cutirubrum: a 13C nuclear magnetic resonance study.

13C nuclear magnetic resonance revealed an unusual pathway for the biosynthesis of lipids in Halobacterium cutirubrum and H. halobium. Mevalonic acid was not synthesized from three acetyl-coenzyme A molecules, as has been suggested previously, and the branch-methyl and methine carbons in phytanyl chains were derived from neither acetate nor glycerol. Instead, they were supplied by the degradation of amino acids, in particular of lysine. Presumably, two different types of two-carbon fragments were used simultaneously by halobacteria for the biosynthesis of mevalonate. The labeling pattern of squalene supported the above conclusions. Based on these data, a general scheme is proposed to account for the contribution of lysine-to-lipid biosynthesis.     

Metabolic Pathways in Methanococcus jannaschii and Other Methanogenic Bacteria

Eleven strains of methanogenic bacteria were divided into two groups on the basis of the directionality (oxidative or reductive) of their citric acid pathways. These pathways were readily identified for most methanogens from the patterns of carbon atom labeling in glutamate, following growth in the presence of [2-¹³C]acetate. All used noncyclic pathways, but members of the family Methanosarcinaceae were the only methanogens found to use the oxidative direction. Methanococcus jannaschii failed to incorporate carbon from acetate despite transmembrane equilibration comparable to other weak acids. This organism was devoid of detectable activities of the acetate-incorporating enzymes acetyl coenzyme A synthetase, acetate kinase, and phosphotransacetylase. However, incorporation of [1-¹³C]-, [2-¹³C]-, or [3-¹³C]pyruvate during the growth of M. jannaschii was possible and resulted in labeling patterns indicative of a noncyclic citric acid pathway operating in the reductive direction to synthesize amino acids. Carbohydrates were labeled consistent with glucogenesis from pyruvate. Leucine, isoleucine, phenylalanine, lysine, formate, glycerol, and mevalonate were incorporated when supplied to the growth medium. Lysine was preferentially incorporated into the lipid fraction, suggesting a role as a phytanyl chain precursor.     

Unusual enzymes involved in five pathways of glutamate fermentation

Anaerobic bacteria from the orders Clostridiales and Fusobacteriales are able to ferment glutamate by at least five different pathways, most of which contain enzymes with radicals in their catalytic pathways. The first two pathways proceed to ammonia, acetate and pyruvate via the coenzyme B12-dependent glutamate mutase, which catalyses the re-arrangement of the linear carbon skeleton to that of the branched-chain amino acid (2S,3S)-3-methylaspartate. Pyruvate then disproportionates either to CO2 and butyrate or to CO2, acetate and propionate. In the third pathway, glutamate again is converted to ammonia, CO2, acetate and butyrate. The key intermediate is (R)-2-hydroxyglutaryl-CoA, which is dehydrated to glutaconyl-CoA, followed by decarboxylation to crotonyl-CoA. The unusual dehydratase, containing an iron-sulfur cluster, is activated by an ATP-dependent one-electron reduction. The remaining two pathways require more then one organism for the complete catabolism of glutamate to short chain fatty acids. Decarboxylation of glutamate leads to 4-aminobutyrate, which is fermented by a second organism via the fourth pathway to acetate and butyrate, again mediated by an unusual dehydratase which catalyses the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The fifth pathway is the only one without decarboxylation, since the gamma-carboxylate of glutamate is reduced to the amino group of delta-aminovalerate, which then is fermented to acetate, propionate and valerate. The pathway involves the oxidative dehydration of 5-hydroxyvaleryl-CoA to 2,4-pentadienoyl-CoA followed by reduction to 3-pentenoyl-CoA and isomerisation to 2-pentenoyl-CoA.     

13C-NMR study of autotrophic CO2 fixation pathways in the sulfur-reducing Archaebacterium Thermoproteus neutrophilus and in the phototrophic Eubacterium Chloroflexus aurantiacus.

The unresolved autotrophic CO2 fixation pathways in the sulfur-reducing Archaebacterium Thermoproteus neutrophilus and in the phototrophic Eubacterium Chloroflexus aurantiacus have been investigated. Autotrophically growing cultures were labelled with [1,4-13C1]succinate, and the 13C pattern in cell constituents was determined by 1H- and 13C-NMR spectroscopy of purified amino acids and other cell constituents. In both organisms succinate contributed to less than 10% of cell carbon, the major part of carbon originated from CO2. All cell constituents became 13C-labelled, but different patterns were observed in the two organisms. This proves that two different cyclic CO2 fixation pathways are operating in autotrophic carbon assimilation in both of which succinate is an intermediate. The 13C-labelling pattern in T. neutrophilus is consistent with the operation of a reductive citric acid cycle and rules out any other known autotrophic CO2 fixation pathway. Surprisingly, the proffered [1,4-13C1]succinate was partially converted to double-labelled [3,4-13C2]glutamate, but not to double-labelled aspartate. These findings suggest that the conversion of citrate to 2-oxoglutarate is readily reversible under the growth conditions used, and a reversible citrate cleavage reaction is proposed. The 13C-labelling pattern in C. aurantiacus disagrees with any of the established CO2 fixation pathways; it therefore demands a novel autotrophic CO2 fixation cycle in which 3-hydroxypropionate and succinate are likely intermediates. The bacterium excreted substantial amounts of 3-hydroxypropionate (5 mM) and succinate (0.5 mM) at the end of autotrophic growth. Autotrophically grown Chloroflexus cells contained acetyl-CoA carboxylase and propionyl-CoA carboxylase activity. These enzymes are proposed to be the main CO2-fixing enzymes resulting in malonyl-CoA and methylmalonyl-CoA formation; from these carboxylation products 3-hydroxypropionate and succinate, respectively, can be formed.     

Amino Acid Biosynthesis in the Halophilic Archaeon Haloarcula hispanica

Biosynthesis of proteinogenic amino acids in the extremely halophilic archaeon Haloarcula hispanica was explored by using biosynthetically directed fractional 13C labeling with a mixture of 90% unlabeled and 10% uniformly 13C-labeled glycerol. The resulting 13C-labeling patterns in the amino acids were analyzed by two-dimensional 13C,1H correlation spectroscopy. The experimental data provided evidence for a split pathway for isoleucine biosynthesis, with 56% of the total Ile originating from threonine and pyruvate via the threonine pathway and 44% originating from pyruvate and acetyl coenzyme A via the pyruvate pathway. In addition, the diaminopimelate pathway involving diaminopimelate dehydrogenase was shown to lead to lysine biosynthesis and an analysis of the 13C-labeling pattern in tyrosine indicated novel biosynthetic pathways that have so far not been further characterized. For the 17 other proteinogenic amino acids, the data were consistent with data for commonly found biosynthetic pathways. A comparison of our data with the amino acid metabolisms of eucarya and bacteria supports the theory that pathways for synthesis of proteinogenic amino acids were established before ancient cells diverged into archaea, bacteria, and eucarya.     

Flux analysis of central metabolic pathways in Geobacter metallireducens during reduction of soluble Fe(III)-nitrilotriacetic acid

We analyzed the carbon fluxes in the central metabolism of Geobacter metallireducens strain GS-15 using 13C isotopomer modeling. Acetate labeled in the first or second position was the sole carbon source, and Fe-nitrilotriacetic acid was the sole terminal electron acceptor. The measured labeled acetate uptake rate was 21 mmol/g (dry weight)/h in the exponential growth phase. The resulting isotope labeling pattern of amino acids allowed an accurate determination of the in vivo global metabolic reaction rates (fluxes) through the central metabolic pathways using a computational isotopomer model. The tracer experiments showed that G. metallireducens contained complete biosynthesis pathways for essential metabolism, and this strain might also have an unusual isoleucine biosynthesis route (using acetyl coenzyme A and pyruvate as the precursors). The model indicated that over 90% of the acetate was completely oxidized to CO2 via a complete tricarboxylic acid cycle while reducing iron. Pyruvate carboxylase and phosphoenolpyruvate (PEP) carboxykinase were present under these conditions, but enzymes in the glyoxylate shunt and malic enzyme were absent. Gluconeogenesis and the pentose phosphate pathway were mainly employed for biosynthesis and accounted for less than 3% of total carbon consumption. The model also indicated surprisingly high reversibility in the reaction between oxoglutarate and succinate. This step operates close to the thermodynamic equilibrium, possibly because succinate is synthesized via a transferase reaction, and the conversion of oxoglutarate to succinate is a rate-limiting step for carbon metabolism. These findings enable a better understanding of the relationship between genome annotation and extant metabolic pathways in G. metallireducens.     

13C-NMR study of CO2-fixation during the heterotrophic growth in Chlorobium thiosulfatophilum

The functioning of the biosynthetic pathways of the amino acids alanine, glycine, aspartic acid, glutamic acid and tyrosine, and of nucleosides in the photosynthetic bacterium Chlorobium thiosulfatophilum during heterotrophic growth on ¹³CO₂ and unlabelled acetate was investigated using ¹³C-NMR as the method for determination of the labelling patterns of the separated substances. On the basis of the analysis of the multiplet structure of the spectra of the tightly-coupled systems, the conclusion was drawn that the Calvin cycle does not function in the experimental conditions used. The labelling pattern of the glutamic acid indicated that about 30% of the amino acid molecules were synthesized through the reactions of the reductive carboxylic acid cycle, the remaining 70% being derived from oxaloacetate and exogenous acetate through the reactions of the Krebs cycle. Labelling patterns of the nucleosides were in agreement with their known biosynthetic pathways.     

Alternative pathways for biosynthesis of leucine and other amino acids in Bacteroides ruminicola and Bacteroides fragilis

Bacteroides ruminicola is one of several species of anaerobes that are able to reductively carboxylate isovalerate (or isovaleryl-coenzyme A) to synthesize alpha-ketoisocaproate and thus leucine. When isovalerate was not supplied to growing B. ruminicola cultures, carbon from [U-14C]glucose was used for the synthesis of leucine and other cellular amino acids. When unlabeled isovalerate was available, however, utilization of [U-14C]glucose or [2-14C]acetate for leucine synthesis was markedly and specifically reduced. Enzyme assays indicated that the key enzyme of the common isopropylmalate (IPM) pathway for leucine biosynthesis, IPM synthase, was present in B. ruminicola cell extracts. The specific activity of IPM synthase was reduced when leucine was added to the growth medium but was increased by the addition of isoleucine plus valine, whereas the addition of isovalerate had little or no effect. The activity of B. ruminicola IPM synthase was strongly inhibited by leucine, the end product of the pathway. It seems unlikely that the moderate inhibition of the enzyme by isovalerate adequately explains the regulation of carbon flow by isovalerate in growing cultures. Bacteroides fragilis apparently also uses either the isovalerate carboxylation or the IPM pathway for leucine biosynthesis. Furthermore, both of these organisms synthesize isoleucine and phenylalanine, using carbon from 2-methylbutyrate and phenylacetate, respectively, in preference to synthesis of these amino acids de novo from glucose. Thus, it appears that these organisms have the ability to regulate alternative pathways for the biosynthesis of certain amino acids and that pathways involving reductive carboxylations are likely to be favored in their natural habitats.     

Alternative pathways for biosynthesis of leucine and other amino acids in Bacteroides ruminicola and Bacteroides fragilis.

Bacteroides ruminicola is one of several species of anaerobes that are able to reductively carboxylate isovalerate (or isovaleryl-coenzyme A) to synthesize alpha-ketoisocaproate and thus leucine. When isovalerate was not supplied to growing B. ruminicola cultures, carbon from [U-14C]glucose was used for the synthesis of leucine and other cellular amino acids. When unlabeled isovalerate was available, however, utilization of [U-14C]glucose or [2-14C]acetate for leucine synthesis was markedly and specifically reduced. Enzyme assays indicated that the key enzyme of the common isopropylmalate (IPM) pathway for leucine biosynthesis, IPM synthase, was present in B. ruminicola cell extracts. The specific activity of IPM synthase was reduced when leucine was added to the growth medium but was increased by the addition of isoleucine plus valine, whereas the addition of isovalerate had little or no effect. The activity of B. ruminicola IPM synthase was strongly inhibited by leucine, the end product of the pathway. It seems unlikely that the moderate inhibition of the enzyme by isovalerate adequately explains the regulation of carbon flow by isovalerate in growing cultures. Bacteroides fragilis apparently also uses either the isovalerate carboxylation or the IPM pathway for leucine biosynthesis. Furthermore, both of these organisms synthesize isoleucine and phenylalanine, using carbon from 2-methylbutyrate and phenylacetate, respectively, in preference to synthesis of these amino acids de novo from glucose. Thus, it appears that these organisms have the ability to regulate alternative pathways for the biosynthesis of certain amino acids and that pathways involving reductive carboxylations are likely to be favored in their natural habitats.     

昆虫神经肽PBAN的细胞内信号转导

昆虫性信息素多数为长链的不饱和醇、醋酸酯、醛或酮类,链长一般为10-20碳,主要在性信息素腺体内由乙酰辅酶A经过脂肪酸合成、碳链缩短、去饱和以及碳酰基的还原修饰等步骤合成的;而性信息素合成激活肽(pheromone biosynthesis activating neuropeptide,PBAN)是由昆虫食管下神经节中的部分神经细胞合成和分泌的神经肽,通常由33个氨基酸组成,在C-末端有一个相同的五肽序列,主要调控性信息素的生物合成。有关PBAN的细胞内信号转导是近几年的研究热点,研究显示 PBAN首先与性信息素腺体细胞表面的G蛋白偶联受体结合,随后依据昆虫种类的不同,其细胞内信号转导方式主要有三种:(1)以cAMP信号传导途径进行信号转导;(2)以cAMP和磷脂酰肌醇信号传导途径共同进行信号转导;(3)主要以Ca2 为第二信使进行信号传导。     

NMR study of 13CO2 incorporation into short-chain fatty acids by pig large-intestinal flora

The nuclear magnetic resonance technique was used to study carbon dioxide reduction by the pig large-intestinal flora. Washed bacterial cell suspensions were incubated for 6 and 15 h under 13CO2 and H2 as the gas phase and with a buffer containing NaH13CO3 and cellobiose and amino acids (casein hydrolysate) as substrates. Methane was produced in all incubation media. Significant amounts of single- as well as multiple-labelled acetate and butyrate were formed, demonstrating synthesis of acetate from H2 + CO2. Propionate was labelled mainly on the carboxyl group, which was attributed to an enzymatic exchange of the carboxyl group of propionate with 13CO2. These results indicate that the reduction of CO2 to acetate may be an important pathway for microbial production of acetate in the pig large intestine even in the presence of methanogenesis.     

13C-NMR study of acetate assimilation in Thermoproteus neutrophilus

Acetate assimilation into amino acids and the functioning of central biosynthetic pathways in the extremely thermophilic anaerobic archaebacterium Thermoproteus neutrophilus was investigated using 13C NMR as the method for determination of the labelling patterns. Acetate was assimilated via reductive carboxylation of acetyl-CoA to pyruvate and pyruvate conversion to phosphoenolpyruvate which was further carboxylated to oxaloacetate. 2-Oxoglutarate was mainly formed via citrate. However, the labelling patterns of glutamic acid and alanine were in agreement with the concurrent synthesis of about 15% 2-oxoglutarate and 5% pyruvate through the reductive citric acid cycle. A scrambling phenomenon occurring in aspartate and all amino acids derived through oxaloacetate was observed. The labelling patterns of amino acids were in agreement with their standard biosynthetic pathways, with two remarkable exceptions: isoleucine was synthesized via the citramalate pathway and lysine was synthesized via the 2-aminoadipate pathway which has previously been reported only in eukaryotic microorganisms.     

Photosynthesis in Rhodospirillum rubrum. II. Photoheterotrophic Carbon Dioxide Fixation

The contribution of the reductive pentose phosphate cycle to the photometabolism of carbon dioxide and to carbon metabolism in Rhodospirillum rubrum grown photoheterotrophically with l-malate as the carbon source is nil, unlike autotrophically grown R. rubrum. Glycolic acid appears to be the first stable product of CO(2) fixation in R. rubrum cultured photoheterotrophically on l-malate. The results obtained in (14)CO(2) fixation experiments suggest that the photometabolism of CO(2) through glycolate into malate is a major pathway of CO(2) fixation in such cells. However, l-malate was a much more efficient precursor of phosphate esters, and of glutamic acid, than was carbon dioxide; l-malate is therefore, in this case, a far more important source of cell carbon than is carbon dioxide.The products of the light-dependent incorporation of CO(2) and of acetate were investigated in R. rubrum grown photoheterotrophically on acetate. Carboxylation reactions and the reductive pentose phosphate cycle are apparently of greater significance in the photometabolism of acetate heterotrophs than in malate heterotrophs; the photometabolism of the acetate photoheterotrophs seems to be intermediate between the photoheterotrophy of malate heterotrophs and strict autotrophy.