Phototaxis and membrane potential in the photosynthetic bacterium Rhodospirillum rubrum.

Cells of the photosynthetic bacterium Rhodospirillum rubrum cultivated anaerobically in light show phototaxis. The behavior of individual cells in response to the phenomenon is reversal(s) of the swimming direction when the intensity of the light available to them abruptly decreases. The tactic response was inhibited by antimycin, an inhibitor of the photosynthetic electron transfer system. The inhibitory effect of antimycin was overcome by phenazine methosulfate. Motility of the cells was not impaired by antimycin under aerobic conditions. Valinomycin plus potassium also inhibited their phototactic response; however, valinomycin or potassium alone had no effect. A change in membrane potential of the cells was measured as an absorbance change of carotenoid. Changes in the membrane potential caused by "on-off" light were prevented by antimycin and by valinomycin plus potassium, but not by antimycin plus phenazine methosulfate nor valinomycin or potassium alone. The results indicated that the phototactic response of R. rubrum is mediated by a sudden change in electron flow in the photosynthetic electron transfer system, and that the membrane potential plays an important role in manifestation of the response.     

The fixABCX genes in Rhodospirillum rubrum encode a putative membrane complex participating in electron transfer to nitrogenase

In our efforts to identify the components participating in electron transport to nitrogenase in Rhodospirillum rubrum, we used mini-Tn5 mutagenesis followed by metronidazole selection. One of the mutants isolated, SNT-1, exhibited a decreased growth rate and about 25% of the in vivo nitrogenase activity compared to the wild-type values. The in vitro nitrogenase activity was essentially wild type, indicating that the mutation affects electron transport to nitrogenase. Sequencing showed that the Tn5 insertion is located in a region with a high level of similarity to fixC, and extended sequencing revealed additional putative fix genes, in the order fixABCX. Complementation of SNT-1 with the whole fix gene cluster in trans restored wild-type nitrogenase activity and growth. Using Western blotting, we demonstrated that expression of fixA and fixB occurs only under conditions under which nitrogenase also is expressed. SNT-1 was further shown to produce larger amounts of both ribulose 1,5-bisphosphate carboxylase/oxygenase and polyhydroxy alkanoates than the wild type, indicating that the redox status is affected in this mutant. Using Western blotting, we found that FixA and FixB are soluble proteins, whereas FixC most likely is a transmembrane protein. We propose that the fixABCX genes encode a membrane protein complex that plays a central role in electron transfer to nitrogenase in R. rubrum. Furthermore, we suggest that FixC is the link between nitrogen fixation and the proton motive force generated in the photosynthetic reactions.     

The inhibition of photosynthetic electron transfer in Rhodospirillum rubrum by N ,N' -dicyclohexylcarbodiimide

N ,N' -Dicyclohexylcarbodiimide (DCCD) at concentrations above 0.1 mM inhibits light-induced generation of a membrane potential in the course of cyclic and non-cyclic electron transfer, as well as light-induced oxygen uptake due to interaction of photoreduced secondary (loosely bound) ubiquinone with O2 in Rhodospirillum rubrum chromatophores. Similarly to o-phenanthroline, DCCD blocks the electron transfer in the chromatophores between the primary (tightly bound) and secondary ubiquinones.     

Studies on the light-dependent synthesis of inorganic pyrophosphate by Rhodospirillum rubrum chromatophores

Characteristics of inorganic pyrophosphate synthesis from inorganic orthophosphate were examined in chromatophores of Rhodospirillum rubrum. The application of an ADP-glucose pyrophosphorylase-trapping system has shown in an unequivocal fashion that pyrophosphate is a product of a light-dependent reaction utilizing P(i) as the substrate. Only very limited pyrophosphate synthesis takes place in the dark. The rates of synthesis of both ATP and pyrophosphate were studied under conditions in which the membrane-bound adenosine triphosphatase and pyrophosphatase activities would normally make these substances unstable. The maximum rate of pyrophosphate synthesis was 25% of that for ATP synthesis, with maximum activation of pyrophosphate synthesis occurring at a lower light-intensity than that required for ATP synthesis. As a result, at low light-intensity the rate of pyrophosphate formation approached that of ATP. Maximal rates of synthesis of both pyrophosphate and ATP were attained only on the addition of an exogenous reducing agent. Conditions for optimum pyrophosphate synthesis required about one-half of the concentration of the reductant required for maximum ATP synthesis. Consistent with previous reports, oligomycin inhibited ATP synthesis, but had little influence on the rate of pyrophosphate synthesis. In membrane particles that retained pyrophosphatase activity but were treated to remove adenosine triphosphatase activity and the ability to photophosphorylate ADP, oligomycin stimulated light-dependent pyrophosphate synthesis by nearly 250%. The influence of Mg(2+) concentration, pH and various inhibitors and uncouplers on pyrophosphate synthesis was studied. The results are discussed with respect to the mechanism and function of electron-transport-coupled energy conservation in R. rubrum chromatophores.     

The distribution of NADH oxidase in the membrane system of Rhodospirillum rubrum

Summary The activity of NADH oxidase in cell fractions of R. rubrum was measured. In cells grown photosynthetically (anaerobic in the light) and semiaerobically in the dark, the highest activity was found to be in the 19,000xg pellet. This consisted preponderantly of cytoplasmic membrane and cell wall material. Bchl was more enriched in the purified thylakoids than the NADH oxidase activity.In extracts of cells grown aerobically the oxidase activity was higher than in cells grown anaerobically or semiaerobically. The highest activity was recovered in the 220,000xg pellet. The data suggest that NADH oxidase as well as bacteri ochlorophyll can be localized in the total intracellular membrane system. However, the distribution is inhomogeneous. Most of the NADH oxidase activity is localized in the c. m. region and most of bchl in the thylakoids.Abbreviations c. m. cytoplasmic membrane - th. Thylakoid - bchl bacteriochlorophyll - Tris Tris (hydroxymethyl) amino-methane     

The effect of transfer from low to high light intensity on electron transport in Rhodospirillum rubrum membranes

The effects of transfer from low to high ligh intensity on membrane bound electrontransport reactions of Rhodospirillum rubrum were investigated. The experiments were performed with cultures which did not form bacteriochlorophyll (Bchl) for about two cell mass doublings during the initial phase of adaptation to high light intensity. Lack of Bchl synthesis causes a decrease of Bchl contents of cells and membranes. Also, the cellular amounts of photosynthetically active intracytoplasmic membranes decrease.In crude membrane fractions containing both cytoplasmic and intracytoplasmic membranes the initial activities of NADH oxidizing reactions increase only slightly (about 1.2 times) per protein, but the initial activities of succinate oxidizing reactions decrease (multiplied by a factor of 0.7). On a Bchl basis activities of NADH oxidizing reactions increase 3.4 times while activities of succinate dependent reactions increase 1.9 times. With isolated intracytoplasmic membranes activities of NADH as well as succinate dependent reactions increase to a comparable extent on a Bchl basis (about 1.8 times) and stay nearly constant on a protein basis. Cytochrome c oxidase responds like succinate dependent reactions. The data indicate that in cells growing under the conditions applied NADH oxidizing electron transport systems are incorporated into both, cytoplasmic and intracytoplasmic membranes, while incorporation of succinate oxidizing systems is confined to intracytoplasmic membranes only.Activities of photophosphorylation and succinate dependent NAD⁺ reduction in the light increase per Bchl about 1.8 times. On a Bchl basis increases of the fast light induced on reactions at 422 nm and increases of soluble cytochrome c ₂ levels are comparable to increases of photophosphorylations and succinate dependent activities. But increases of slow light off reactions at 428 nm and of b-type cytochrome levels become three times greater then increases of cytochrome c ₂ reactions and levels. These results infer that although electrontransport reactions of intracytoplasmic membranes change correlated to each other, Bchl, cytochrome c ₂ and b-type cytochromes cellular levels are independent of each other. Furthermore, the data indicate that cytochrome c ₂ rather than b-type cytochrome is involved with steps rate limiting for photophosphorylation.Abbreviations Bchl bacteriochlorophyll - DCIP 2,6-dichlorophenolindophenol     

Bioconversion of synthesis gas to hydrogen using a light-dependent photosynthetic bacterium,Rhodospirillum rubrum

Biological hydrogen production from synthesis gas was carried out in batch culture. The phototrophic anaerobic bacterium, Rhodospirillum rubrum was used to oxidize CO and water to CO₂ and hydrogen. The bacteria were grown under anaerobic conditions in liquid medium; also acetate was used as carbon source in presence of synthesis gas. Biological hydrogen production was catalysed by R. rubrum via the water–gas shift reaction. A light-dependent cell growth modelled with a desired rate of hydrogen production and CO uptake was determined. The effect of light intensity on microbial cell growth was also studied at 500, 1,000 and 1,500 m.cd. A complete conversion of CO to hydrogen and maximum light efficiency were obtained with an acetate concentration of 1 g/l and light intensity of 500 m.cd. Utilization of the carbon monoxide from the gas phase was often considered as a mass transfer limited process, which needed to diffuse through the gas–liquid interface and then further diffuse into liquid medium prior to reaction. The results from this study showed that maximum cell propagation and hydrogen production were achieved with a limited light intensity of 1,000 m.cd. It was also found that high-light intensity may interfere with cell metabolism. In low-light intensity and substrate concentration, no inhibition was observed, however at extreme conditions, non-competitive inhibition was identified. The adverse effect of high-light intensity was shown at 5,000 m.cd, where the CO conversion drastically dropped to as low as 21%. Maximum CO conversion of 98% and maximum yield of 86% with an acetate concentration of 1.5 g/l and a light intensity of 1,000 m.cd were achieved.     

The Structure of Rhodospirillum rubrum

Cells from serial cultures of R. rubrum, grown anaerobically in the light, were harvested at intervals from ½ to 15 days and sectioned for electron microscopy by conventional methods. Cells of this species possess a multilayered outer envelope, and the external cell surface is differentiated into ridges extending parallel or obliquely to the long axis of the cell. Cells from very young cultures resemble non-photosynthetic bacteria and contain only a granular cytoplasm, scattered high-density particles, and low-density areas corresponding to the chromatin areas observed by light microscopy. They contain neither the chromatophores nor the lamellar systems assumed by previous investigators to be characteristic of this species when grown anaerobically in the light. Chromatophores appear in cells from cultures older than about 12 hours, while systems of paired lamellae appear along with the chromatophores in cells from cultures older than about 8 days. Divergent opinions concerning the occurrence of chromatophores or lamellae in this species can be resolved on the basis of the age of cultures used in previous studies. Other changes occurring in cells from cultures of increasing age include the appearance of granular and reticulate cytoplasmic bodies and vacuoles, extension of the chromatin areas, and the appearance of a single membrane enclosing several chromatophores.