Biosynthetic mechanism of glycolate in Chromatium I. Glycolate pathway

The pattern of radioactivity distribution in several amino acidsof Chromatium cells exposed to ¹⁴CO₂ was determined. By transferringthe bacterial cells from an atmosphere of nitrogen to oxygenthere occurred a transient decrease of ¹⁴CO₂ incorporation intoaspartate and glutamate, whereas that into glycine showed aprominent increase. The labeling of both serine and alaninedid not show a marked change under such conditions. The, activitiesof glycolate oxidase and glycolate dehydrogenase in crude extractsof the bacterial cells were very low. The formation of glycolic acid only occurred during the oxidativemetabolism of Chromatium cells grown on bicarbonate as a C source,being negligibly small in bacteria under nitrogen or after growthon malate or acetate. The activities of both ribulose- 1,5-bisphosphateoxygenase and phosphoglycolate phosphatase in the extract preparedfrom the bicarbonate-grown bacterial cells were very low andapparently could not account for the glycolic acid formationthrough these enzymic reactions. Metabolic patterns of glycolicacid in Chromatium are discussed in relation to the photorespiratoryphenomenon. (Received February 24, 1975; )     

Biosynthetic mechanism of glycolate in Chromatium II. Enzymic mechanism of glycolate formation by a transketolase system

The mechanism for the biosynthesis of glycolate in the photosyntheticbacterium Chromatium was studied. The enzymic nature of glycolatesynthesis from fructose-6-P and ribose-5-P by a cell-free bacterialextract, strictly dependent on the presence of ferricyanidein the assay mixture, was established. Removal of thiamine pyrophosphatefrom the assay mixture did not alter the magnitude of glycolateformation. By examining the reaction products from ¹⁴C-fructose-6-Pand ¹⁴C-ribose-5-P, the operating mechanism involved in glycolateformation was judged to be the oxidative breakdown of the glycolaldehyde-transketolaseaddition product by ferricyanide. The role of transketolasein the oxidative formation of glycolate in the photosynthesizingcells of Chromatium is discussed. ¹ This is paper XXXI in the series "Structure and Function ofChloroplast Proteins". Paper XXX is reference (5). The researchwas supported in part by grants from the Ministry of Educationof Japan (986009), the Toray Science Foundation (Tokyo), andthe Naito Science Foundation(Tokyo). (Received May 22, 1975; )     

Biosynthetic mechanism of glycolate in Chromatium 6. Glycolate formation and metabolism under low O21

Under low O₂ (0.05 mM O₂), there was no measurable excretionof glycolate or glycine by Chromatium cells, unlike the caseof their incubation under high (0.7 mM) O₂ However, upon additionof non-radioactive glycolate and glycine to the suspension medium,there occurred a measurable incorporation of ¹⁴CO₂ into thesecompounds, which were then excreted extracellularly; the totalradioactivities measured were approximately 15% of the totalCO₂ fixed photosynthetically. This phenomenon could be as cribedto the dilution of the intracellular pools by the compoundsadded. The results indicate that under low O₂ the glycolatemolecules produced are metabolically further transformed inthe bacterial cells. The incorporation of ¹⁴CO₂ into the extracellularglycolate fraction was maximal at 0.3 mM glycolate in both highand low O₂. Presumably, glycolate formed in the bacterial cellsunder both the high and low O₂ is metabolized in a similar manner,although the excess glycolate and glycine molecules are rapidlyexcreted. During glycolate metabolism CO₂ was evolved from anisonicotinylhydrazide-sensitive reaction, suggesting that thepathway <glycolate glycine . CO₂ was similar in green plants.The results thus indicate that studies on glycolate and glycinemetabolism in the anaerobic bacterium, Chromatium, provide auseful model system for elucidating the mechanism of photorespirationin green plants. (Received May 19, 1978; )     

Biosynthetic mechanism of glycolate in Chromatium III. Effects of {alpha}-hydroxy-2-pyridinemethanesulfonate, glycidate and cyanide on glycolate excretion

Effects of -hydroxy-2-pyridinemethanesulfonate (-HPMS), 2,3-epoxypropionate(glycidate), and cyanide on the photosynthetic activities ofChromatiumwere studied. -HPMS stimulated photosynthetic CO₂fixation in the bacterial cells in both N₂ and O₂ environments.The formation and subsequent excretion of both glycolate andglycine in the O₂ atmosphere were markedly enhanced by -HPMS.In contrast to a recent report by Zelitch [Arch. Biochem. Biophys.163: 367–377 (1974) ] that glycidate specifically inhibitsglycolate formation in tobacco leaf disks, we found that ithad no influence on CO₂ fixation by Chromatium in either N₂or O₂ atmosphere, and that the synthesis and extracellular excretionof glycolate were markedly stimulated by glycidate treatment.Cyanide (0.01–1 mM) exerted a marked inhibitory effecton photosynthetic CO₂ fixation in N². In O₂ atmosphere, photosynthesiswas stimulated by 0.01 mM cyanide, and inhibited by it abovethis level. Both the incorporation of ¹⁴CO₂ into glycolate andthe total synthesis of glycolate in the light were also enhancedby 0.01 mM cyanide, and strongly inhibited above that concentration. ¹This is paper XXXVI in the series "Structure and Function ofChloroplast Proteins," and the research supported in part bygrants from the Ministry of Education of Japan (No. 111912),the Toray Science Foundation (Tokyo) and the Naito Science Foundation(Tokyo). (Received May 31, 1976; )     

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.     

Enzymic formation of glycolate in Chromatium. Role of superoxide radical in a transketolase-type mechanism.

Chromatophores prepared from Chromatium exhibit a light-dependent O2 uptake in the presence of reduced 2,6-dichlorophenolindophenol, the maximum rate observed being 10.8 micronmol (mg of Bchl)-1 h-1 (air-saturated condition). As it was found that the uptake of O2 was markedly inhibited by superoxide dismutase, it is suggested that molecular oxygen is subject to light-dependent monovalent reduction, resulting in the formation of the superoxide anion radical (O2-). By coupling baker's yeast transketolase with illuminated chromatophore preparations, it was demonstrated that [U-14C]-fructose 6-phosphate (6-P) is oxidatively split to produce glycolate, and that the reaction was markedly inhibited by superoxide dismutase and less strongly by catalase. A coupled system containing yeast transketolase and xanthine plus xanthine oxidase showed a similar oxidative formation of glycolate from [U-14C] fructose 6-P. It is thus suggested that photogenerated O2- serves as an oxidant in the transketolase-catalyzed formation of glycolate from the alpha, beta-dihydroxyethyl (C2) thiamine pyrophosphate complex, whereas H2O2 is not an efficient oxidant. The rate of glycolate formation in vitro utilizing O2- does not account for the in vivo rate of glycolate photosynthesis in Chromatium cells exposed to an O2 atmosphere (10 micronmol (mg of Bchl)-1 h-1). However, the enhancement of glycolate formation by the autoxidizable electron acceptor methyl viologen in Chromatium cells in O2, as well as the strong suppression by 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron), an O2- scavenger, suggest that O2- is involved in the light-dependent formation of glycolate in vivo.     

Biosynthetic mechanism of ribulose-1,5-bisphosphate carboxylase in the purple photosynthetic bacterium, Chromatium vinosum. III. Absence of extrachromosomal DNA

Inducible formation of ribulose-1,5-bisphosphate (RuBP) carboxylase in the cells of Chromatium vinosum under autotrophic conditions was not affected by six different inhibitors of DNA synthesis. Photosynthetic CO2 fixation and RuBP carboxylase activities were not influenced by seven reagents known to eliminate plasmids. Plasmids were not detectable by agarose gel electrophoresis employing either the cleared lysate or alkaline sodium dodecyl sulfate method, nor were they detected by ethidium bromide-CsCl density gradient centrifugation. Overall experimental results tend to indicate that plasmids are absent in the Chromatium cells and that the induction of RuBP carboxylase is presumably not regulated in the DNA replication process.     

Biosynthetic mechanism of ribulose-1,5-bisphosphate carboxylase in the purple photosynthetic bacterium,Chromatium vinosum: I. Inducible formation

The nature of the inducible formation of enzymes engaged in the photosynthetic CO₂ fixation was examined in Chromatium vinosum during its autotropic development. Although the activity of RuBP carboxylase was the lowest among several enzyme activities examined, it was enhanced 2.5 times during a 5-hr incubation, while other enzyme activities were little altered. The enhancement of the RuBP carboxylase activity was dependent on the presence of reduced sulfur compounds in the incubation medium and illumination (>100 lx). The increase in enzyme activity, however, was repressed by CO₂ or pyruvate. Furthermore, O₂ markedly reduced the enzyme activity. In order to prove whether or not the enhancement of RuBP carboxylase activity was attributable to the biosynthesis of the enzyme, the incorporation of [³⁵S]methionine into RuBP carboxylase was followed by immunoprecipitation analysis. The incorporation was dependent on the reduced sulfur compounds, and was repressed by elevating the CO₂ level.     

Biosynthetic mechanism of ribulose-1,5-bisphosphate carboxylase in the purple photosynthetic bacterium,Chromatium vinosum: II. Biosynthesis of constituent subunits

[³⁵S]Methionine-labeled free subunits A and B of RuBP carboxylase were present in barely detectable amounts; the radioactivity in the free subunit B was approximately 1/150th of that in the subunit B contained in the holoenzyme of RuBP carboxylase. The turnover rates of subunits A and B in the holoenzyme were equal at each time during the incubation period. The ratio of subunit A to subunit B was constant throughout the incubation time both in quantity and in the level of [³H]leucine and [³⁵S]methionine incorporated. CO₂ contained in the incubation medium suppressed [³⁵S]methionine incorporation into both subunits A and B equally. These results suggest that the biosynthesis of subunits A and B is completely synchronized and may be regulated by identical mechanisms.     

Glycine-serine transformation in the photosynthetic bacterium Chromatium vinosum

The metabolic transformation of glycine into serine in the photosyntheticbacterium Chromatium vinosum was accompanied by the evolutionof CO₂ due to decarboxylation of glycine. Isonicotinylhydrazideinhibited both ¹⁴CO₂ evolution and the formation of ¹⁴C-serinefrom ¹⁴C-glycine. The results indicate that a glycine-serinetransformation reaction takes place which is analogous to thatoccurring in green leaf tissues. Glycine may be metabolisedthrough serine by this reaction. The light stimulation of ¹⁴CO₂evolution and ¹⁴C-serine formation from 14C-glycine by the Chromatiumcells are judged to be results of the light-induced enhancementof ¹⁴C-glycine uptake by the bacterial cells. ¹This is paper 53 in the series "Structure and Function of ChloroplastProteins" and paper 7 of the series "Biosynthetic Mechanismof Glycolate in Chromatium". Paper 6 of the latter series isRef. 3 by Asami and Akazawa (1978). ²This study was aided by research grants from the Ministry ofEducation, Science and Culture of Japan and the Nissan ScienceFoundation (Tokyo). ³Postdoctoral Fellow (1980) of the Japan Society for the Promotionof Science. (Received May 20, 1980; )     

Chromatium glycolicum sp. nov., a moderately halophilic purple sulfur bacterium that uses glycolate as substrate

From the microbial mats that develop in Solar Lake, a new purple sulfur bacterium was isolated. This strain (Chromatium strain SL 3201) was morphologically similar to Chromatium gracile and Chromatium minutissimum. Chromatium SL 3201 was found to be a moderate halophile with a growth range between 2 and 20% NaCl (optimum 4–5% NaCl) and was able to grow photo-organotrophically using glycolate and glycerol. It is the first described phototrophic sulfur bacterium able to use glycolate. According to NaCl requirements and utilization of organic compounds, the strain is not related to any known species of the genus Chromatium. On the basis of its 16S rRNA gene sequence, it clusters with other Chromatium species and is most similar to Chromatium salexigens and Chr. gracile, but it is sufficiently separated to be considered as a new species of the genus. It is, therefore, described as Chromatium glycolicum sp. nov. Received: 17 June 1996 / Accepted: 4 November 1996     

Photorespiration in diatoms. IV. Two pathways of glycolate metabolism in synchronized cultures of Cylindrotheca fusiformis.

Cylindrotheca fusiformis is shown to be able to convert glycolate to glycerate via tartronic semialdehyde as well as by the better known route involving transamination to glycine. Enzymes related to photorespiration were compared in light-dark synchronized cultures of C. fusiformis kept in continuous light in a complete synthetic seawater medium or starved for nitrogen or silicon. Glycolate oxidation remained constant throughout the cell cycle and was unaffected by starvation. Transamination of glyoxylate was stimulated by light, inhibited during nitrogen starvation, and dramatically stimulated by reintroduction of nitrate to the medium. Glyoxylate carboligase was also stimulated by light and inhibited during nitrogen-starvation but only partially recovered activity after reintroduction of nitrate.     

Magnetic studies of Chromatium flavocytochrome C552. A mechanism for heme-flavin interaction.

Electron paramagnetic resonance and magnetic susceptibility studies of Chromatium flavocytochrome C552 and its diheme flavin-free subunit at temperatures below 45 degrees K are reported. The results show that in the intact protein and the subunit the two low-spin (S = 1/2) heme irons are distinguishable, giving rise to separate EPR signals. In the intact protein only, one of the heme irons exists in two different low spin environments in the pH range 5.5 to 10.5, while the other remains in a constant environment. Factors influencing the variable heme iron environment also influence flavin reactivity, indicating the existence of a mechanism for heme-flavin interaction.     

Interspecies transformation in Bacillus: mechanism of heterologous intergenote transformation.

Bacillus subtilis-Bacillus globigii hybrids were made by integration of the B. globigii aromatic region (aroB to aroE) as an intergenote in the B. subtillis chromosome. Transformation of the heterologous intergenote by B. subtillis DNA (or vice versa) occurred at about 10% of the frequency of homologous transformation by hybrid donors into the same region. Heterologous intergenote crosses were unusually sensitive to shear fragmentations of donor DNA to sizes less than 30 X 10(6) to 40 X 10(6) daltons. In all cases, the entire intergenote was transferred en bloc. Homologous transformation of intergenote markers by B. globigii DNA was not unusually shear sensitive, and linkage was normal for markers in the intergenote. A model is proposed in which efficient heterologous intergenote transformation occurs by recognition and base pairing of homologous DNA sequences of both flanks of the intergenote.