Reidun Sirevåg

Mechanisms of CO2 fixation in bacterial photosynthesis studied by the carbon isotope fractionation technique.

Abstract1. The carbon isotope discrimination properties of a representative of each of the three types of photosynthetic bacteria Chlorobium thiosulfatophilum, Rhodospirillum rubrum and Chromatium and of the C3-alga Chlamydomonas reinhardii were determined by measuring the ratio of 13CO2 to 12CO2 incorporated during photoautotrophic growth. 2. Chromatium and R. rubrum had isotope selection properties similar to those of C3-plants, whereas Chlorobium was significantly different. 3. The results suggest that Chromatium and R. rubrum assimilate CO2 mainly via ribulose 1,5-diphosphate carboxylase and the associated reactions of the reductive pentose phosphate cycle, whereas Chlorobium utilizes other mechanisms. Such mechanisms would include the ferredoxin-linked carboxylation enzymes and associated reactions of the reductive carboxylic acid cycle.

Structural Basis for Thermophilic Protein Stability: Structures of Thermophilic and Mesophilic Malate Dehydrogenases

The three-dimensional structure of four malate dehydrogenases (MDH) from thermophilic and mesophilic phototropic bacteria have been determined by X-ray crystallography and the corresponding structures compared. In contrast to the dimeric quaternary structure of most MDHs, these MDHs are tetramers and are structurally related to tetrameric malate dehydrogenases from Archaea and to lactate dehydrogenases. The tetramers are dimers of dimers, where the structures of each subunit and the dimers are similar to the dimeric malate dehydrogenases. The difference in optimal growth temperature of the corresponding organisms is relatively small, ranging from 32 to 55 degrees C. Nevertheless, on the basis of the four crystal structures, a number of factors that are likely to contribute to the relative thermostability in the present series have been identified. It appears from the results obtained, that the difference in thermostability between MDH from the mesophilic Chlorobium vibrioforme on one hand and from the moderate thermophile Chlorobium tepidum on the other hand is mainly due to the presence of polar residues that form additional hydrogen bonds within each subunit. Furthermore, for the even more thermostable Chloroflexus aurantiacus MDH, the use of charged residues to form additional ionic interactions across the dimer-dimer interface is favored. This enzyme has a favorable intercalation of His-Trp as well as additional aromatic contacts at the monomer-monomer interface in each dimer. A structural alignment of tetrameric and dimeric prokaryotic MDHs reveal that structural elements that differ among dimeric and tetrameric MDHs are located in a few loop regions.

Quantitative and structural characteristics of lipids in Chlorobium and Chloroflexus

The lipid compositions of Chlorobium limicola (4 strains) and Chloroflexus aurantiacus (2 strains) have been compared. Both species contained straight-chain, saturated and monosaturated fatty acids as their main fatty acid constituents but the patterns were distinctly different. Chlorobium contained acids of chain-length essentally in the range C12−C18 with n-tetradecanoate, hexadecenoate and n-hexadecanoate predominating. Chloroflexus was characterized by the presence of significant amounts of C17 and C18−C20 fatty acids not detected in Chlorobium. The latter, on the other hand, contained hydroxylated and cyclopropane-substituted acids not detected in Chloroflexus. Simple wax esters (C28−C38) were found solely in Chloroflexus and accounted for 2.5–3.0% of the cell dry weight. Their fatty acid constituents ranged from C12−C19 (both saturated and monounsaturated isomers) whereas the alcohols were generally saturated and of chain-length C16−C19. Waxes in the range C34−C36 accounted for more than 60% of the total.The polar lipid patterns of the two genera also showed marked differences. All strains contained phosphatidyl-glycerol, monogalactosyl diglyceride and sulfoquinovosyldiglyceride. Chlorobium contained in addition cardiolipin, phosphatidylethanolamine, the unidentified “glycolipid II” and several other unidentified glycolipids, whereas phosphatidyl inositol and a diglycosyl diglyceride were specific for Chloroflexus. The latter lipid contained equimolar amounts of glucose and galactose.Phenol-water extraction yielded material comprising 14% of the dry cell weight for Chlorobium but only 2.5% for Chloroflexus. The Chlorobium material contained two 3-hydroxy fatty acids and several uncommon sugars (not identified). The analytical results were inconclusive regarding occurrence of 2-keto-3-deoxyoctonate. No typical lipopoly-saccharide constituents were found in Chloroflexus.

Further studies on carbon dioxide fixation in Chlorobium

The pathway of carbon in photosynthesis of Chlorobium thiosulfatophilum strain 8346 was examined. After exposure of the cells to 14CO2, chromatography and radioautography indicated that malic, α-oxoglutaric, pyruvic, oxaloacetic, glutamic, and aspartic acids were the first stable products of photosynthesis. After 5 sec of photosynthesis, no radioactivity was detected in the area corresponding to phosphate esters on the chromatograms. 2. After 30 sec of exposure of the cells to 14CO2, all except one of the intermediates of the reductive carboxylic acid cycle were detectable on x-ray films of radioautograms. 3. An experiment was performed which showed that the specific activities of the individual intermediates of the reductive carboxylic acid cycle were within the same range, thus suggesting that the intermediates of the cycle were in isotopic equilibrium with each other. 4. Succinic acid showed a high specific activity after exposure of the cells to 14CO2, indicating that this compound might be formed by a direct carboxylation reaction as well as by the reactions of the reductive tricarboxylic acid cycle. 5. Cells of Chlorobium were shown to contain isocitrate and citrate in amounts comparable to those found in aerobic bacteria. 6. The two enzymes specific for the reductive pentose phosphate cycle, RuDP-carboxylase and phosphoribulokinase, were not detected in cell-free extracts of the organism. 7. The presence of aconitate hydratase and isocitric dehydrogenase in amounts consistent with the operation of the reductive carboxylic acid cycle was confirmed. The level of these enzymes was not repressed when acetate was present in the medium, nor was their activity inhibited by acetate or acetyl-CoA when these substances were added to cell-free extracts. 8. The data presented here are discussed in relation to the reductive pentose phosphate cycle and the reductive tricarboxylic acid cycle, or other possible mechanisms of CO2-fixation in Chlorobium.

Aspects of carbon metabolism in Chloroflexus

When fluoroacetate was added to aerobic, washed cells of Chloroflexus, O2 uptake was strongly inhibited and citrate accumulated. Under anaerobic conditions in the light, fluoroacetate inhibited CO2 uptake and caused citrate accumulation. The results are taken as evidence for the operation of a tricarboxylic acid cycle in Chloroflexus both under aerobic conditions in the dark and anaerobically in the light. 2. Organic compounds are assimilated into the storage materials polyglucose and poly-β-hydroxybutyric acid by washed cells of Chloroflexus. The type of storage product formed from acetate depends upon the availability of reducing power. 3. Low activities of the key enzymes of the reductive pentose phosphate cycle, ribulose-1,5-bisphosphate carboxylase and phosphoribulokinase were detected in cell free extracts of photoheterotrophically grown Chloroflexus.

Carbon Dioxide-Fixation in Photosynthetic Green Sulfur Bacteria

The main products of carbon dioxide-fixation in washed suspensions of Chlorobium thiosulfatophilum are a polyglucose, α-ketoglutarate, and α-keto-β-methylvalerate. All of these can be formed by a mechanism involving the reductive carboxylic acid cycle. The reductive pentose phosphate cycle appears to play a quantitatively minor role in carbon dioxide-fixation under these conditions.

Synthesis, storage and degradation of polyglucose in Chlorobium thiosulfatophilum.

Cultures of Chlorobium thiosulfatophilum form polyglucose during growth. The polyglucose is laid down within the cells as rosette-like granules, which are made up from smaller grains. The size of each granule appears to be limited to less than 30 nm, since an increase in polyglucose content leads to more granules being formed rather than an increase in granule size.The polyglucose in washed cells is fermented in the dark to acetate, propionate, caproate and succinate, of which acetate by far comprises the largest fraction (68%). During incubation of washed cells without hydrogen donor, the level of polyglucose decreases regardless of whether the cells are incubated in the dark or in the light. Since the products formed from polyglucose under the two different conditions are not the same, it is suggested that polyglucose in the dark serves as an energy source, whereas when in the light the role of polyglucose is mainly to provide the cell with reducing power.

Ribulose 1,5-diphosphate carboxylase and Chlorobium thiosulfatophilum

Abstract1. Cell-free extracts of the photosynthetic bacterium Chlorobium thiosulfatophilum, strains 8327 and Tassajara, were assayed for ribulose 1,5-diphosphate (RuDP) carboxylase and phosphoribulokinase-the two enzymes peculiar to the reductive pentose phosphate cycle. 2. RuDP carboxylase was consistently absent in strain 8327. The Tassajara strain showed a low RuDP-dependent CO2 fixation activity that was somewhat higher in cells following transatlantic air shipment than in freshly grown cells. The stability and behaviour of this activity in sucrose density gradients were similar to those described by other workers. 3. The radioactive carboxylation products formed in the presence of RuDP by enzyme preparations from the Tassajara strain did not include 3-phosphoglycerate-the known product of the RuDP carboxylase reaction, but instead consisted of the unrelated acids glutamate, aspartate and malate. 4. Phosphoribulokinase was absent in all preparations of the two Chlorobium strains tested. By contrast, phosphoribulokinase as well as RuDP carboxylase were readily demonstrated in preparations from pea chloroplasts and the photosynthetic bacterium Rhodospirillum rubrum. 5. It is concluded that C. thiosulfatophilum appears to lack RuDP carboxylase, phosphoribulokinase, and hence, the reductive pentose phosphate cycle.

Carbon Metabolism in Green Bacteria

The only common feature of the two families of green bacteria, Chlorobiaceae (green sulfur bacteria) and Chloroflexaceae (green gliding bacteria), is their type of light harvesting chlorophyll and the organization of these pigments into chlorosomes. In most other respects, including metabolism, photosynthetic apparatus and phylogeny, they are very different and far apart. Each of the two most studied genera possesses a unique pathway for autotrophic fixation of CO2: the reductive tricarboxylic acid cycle used by Chlorobium and the newly discovered 3-hydroxypropionate cycle used by Chloroflexus.

Field Evaluation of a Semiautomated Method for Rapid and Simple Analysis of Recreational Water Microbiological Quality

ABSTRACT An early warning system using a rapid enzymatic semiautomated method suitable for fecal coliform detection in recreational waters within 8 h was developed further and evaluated in this study. This rapid method was compared to the standard method followed in the United Kingdom. We used 1,011 samples originating from 206 different locations in Wales. When we assessed the presence or absence of fecal coliforms, targeting very low levels of contamination, we obtained 83.9% agreement between the rapid method and the lauryl sulfate broth-membrane filtration technique, whereas direct confirmation of the samples processed by the rapid method showed 89.3% agreement. Environmental enzymatic background activity was found to be the main limiting factor for this method. Owing to a specific and integrated handling of the results by the software of the instrument, the percentage of false-positive results (a consequence of enzymatic background) was successfully limited to 2.9% by the direct confirmation evaluation. However, 7.8% false-negative results due to “late-growers” had to be accepted in order to produce results within a working day. At present, the method can be used in a more conservative way to assess the environmental threshold of 100 CFU of fecal coliforms per 100 ml in recreational waters. The implications of our findings with regard to the applicability of rapid enzymatic methods are discussed.