J. Barry Jackson

The Role of Auxiliary Oxidants in Maintaining Redox Balance During Phototrophic Growth of Rhodobacter-Capsulatus On Propionate or Butyrate

Phototrophic growth of Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata) under anaerobic conditions with either butyrate or propionate as carbonsource was dependent on the presence of either CO2 or an auxiliary oxidant. NO-3, N2O, trimethylamine-N-oxide (TMAO) or dimethylsulphoxide (DMSO) were effective provided the appropriate anaerobic respiratory pathway was present. NO-3was reduced extensively to NO-3, TMAO to trimethylamine and DMSO to dimethylsulphide under these conditions. Analysis of culture fluids by nuclear magnetic resonance showed that two moles of TMAO or DMSO were reduced per mole of butyrate utilized and one mole of either oxidant was reduced per mole of propionate consumed. The growth rate of Rb. capsulatus on succinate or malate as carbon source was enhanced by TMAO in cultures at low light intensity but not at high light intensities. A new function for anaerobic respiration during photosynthesis is proposed: it permits reducing equivalents from reduced substrates to pass to auxiliary oxidants present in the medium. The use of CO2 or auxiliary oxidants under phototrophic conditions may be influence by the availability of energy from light. It is suggested that the nuclear magnetic resonance methodology developed could have further applications in studies of bacterial physiology.

Rationalization of properties of nitrate reductases in Rhodopseudomonas capsulata

Abstract1.The properties of nitrate reductase activities have been compared in several strains of Rhodopseudomonas capsulata grown phototrophically in the presence of nitrate as sole nitrogen source.2.Strains AD2 and BK5 resemble the spontaneous mutant N22DNAR+ (described by McEwan et al. 1982 FEBS Lett. 150, 277\2-280) in that reduction of nitrate was inhibited by either illumination or oxygen but not by NH4+, and that electron flow to nitrate under dark anaerobic conditions generated a cytoplasmic membrane potential (as judged by an electrochromic shift in the absorbance spectrum of endogenous carotenoid pigments). In contrast disappearance of nitrate from suspensions of strains N22 and St. Louis was dependent upon illumination and was inhibited by NH4+. Membrane potentials were not generated by addition of nitrate in the dark to N22, St. Louis or strain Kbl.3.Nitrate reductase was shown to be located in the periplasmic space of both strain AD2 and mutant N22DNAR+. The nitrate reductase activity in cells of AD2 and N22DNAR+ was relatively insensitive to azide, with 0.5mM azide required for 50% inhibition. The nitrate reductase of strain BK5 was more strongly associated with the cytoplasmic membrane and no conclusion could be reached about whether it was located on the periplasmic or cytoplasmic surface. In BK5 cells nitrate reductase activity was sensitive to low concentrations of azide (50% inhibition with 2 \gmM azide). It is proposed that functionally the nitrate reductase activity in strains AD2, BK5 and N22DNAR+ has identical roles. These roles are suggested to include:(i)The first step in the assimilation of nitrate.(ii).Provision of an alternative electron acceptor to oxygen for generating a membrane potential.(iii).A mechanism for disposing of excess reducing equivalents in the maintenance of balanced growth.This type of nitrate reductase, especially in AD2 and N22DNAR+, appears to resemble that described in a denitrifying strain of Rps. sphaeroides, but to differ markedly from its membrane-bound counterpart in other bacteria including the denitrifying Paracoccus denitrificans and Escherichia coli.4.In other strains of Rps. capsulata including St. Louis, N22 and Kbl, only an assimilatory nitrate reductase, whose activity in intact cells is relatively sensitive to azide, is present in anaerobic, phototrophic cultures grown with nitrate as nitrogen source. As this reductase cannot be detected after breakage of cells, no conclusion can be made as to its location in the cell.

Electron flow to dimethylsulphoxide or trimethylamine-N-oxide generates a membrane potential in Rhodopseudomonas capsulata

Under dark and essentially anaerobic conditions electron flow to either dimethylsulphoxide or trimethylamine-N-oxide in cells of Rhodopseudomonas capsulata has been shown to generate a membrane potential. This conclusion is based on the observation of a red shift in the carotenoid absorption band which is a well characterised indicator of membrane potential in this bacterium. The magnitude of the dimethylsulphoxide- or trimethylamine-N-oxide-dependent membrane potential was reduced either by a protonophore uncoupler of oxidative phosphorylation or synergistically by a combination of a protonophore plus rotenone, an inhibitor of electron flow from NADH dehydrogenase. These findings, together with the observation that venturicidin, an inhibitor of the proton translocating ATPase, did not reduce the membrane potential, show that electron flow to dimethylsulphoxide or trimethylamine-N-oxide is coupled to proton translocation. Thus contrary to some previous proposals dark and anaerobic growth of Rps. capsulata in the presence of dimethylsulphoxide or trimethylamine-N-oxide cannot be regarded as purely fermentative.

The effect of venturicidin on light and oxygen-dependent electron transport, proton translocation, membrane potential development and ATP synthesis in intact cells of Rhodopseudomonas capsulata

Venturicidin behaves as an orthodox energy transfer inhibitor in intact cells of Rhodopseudomonas capsulata as judged by the following criteria. 1. It led to inhibition of respiration. Inhibition was relieved by low concentrations of uncoupling agent. 2. It enhanced light-induced and oxygen dependent H+ efflux. 3. It stimulated light-induced and oxygen dependent carotenoid band shifts. The rate of decay of the band shifts after short flash excitation was decreased in the presence of venturicidin. 4. It stimulated light-induced and oxygen dependent butyltriphenylphosphonium uptake. 5. It inhibited the rise in cellular ATP concentration accompanying either photosynthesis or respiration.

Kinetics and stoichiometry of proton binding in Rhodopseudomonas sphaeroides chromatophores

A currently popular preoccupation in bioenergetics is the stoichiometry of proton (H ÷) binding or release during electron transfer in mitochondria [1,2], chloroplasts [3-5] and bacterial membranes (see [6] for references). Chromatophores from the photosynthetic bacterium Rhodopseudomonas sphaeroides possess a unique combination of advantages [7]. They permit the determination of the time-resolved binding of protons following flash-activation as well as the number of H ÷ bound for an electron moving through their ubiquinone-cytochromes b/c2 (Q-b/c~) oxidoreductase. Because the system is cyclic and there is no input of external oxidising or reducing equivalents, the redox state of the Q-b/c~ system can be adjusted prior to activation and the flash-induced reactions can be referred to this starting state. Microsecond H + binding accompanying electron transport was first revealed in photosynthetic bacteria by Chance et al. [8]. Chemiosmotic models [9] predicted the existence of a second, slower, antimycin sensitive phase of proton binding. This was later detected by Cogdell et al. [10] but strangely, only in the presence of valinomycin and K ÷ ions. In a previous paper [11] we further characterized the rapid (tl/2 ~ 120/Is, pH 7.0), antimycin insensitive proton binding ( designated H~) and established that

A nitrate reductase activity in Rhodopseudomonas capsulata linked to electron transfer and generation of a membrane potential

We have isolated from a laboratory strain of Rhodopseudomonas capsulata a spontaneous mutant possessing a dissimilatory NO− 3 reductase activity. Reduction of NO− 3 under dark and anaerobic conditions generated a membrane potential, and was inhibited by rotenone, oxygen and illumination.

Identification of cytochromes involved in electron transport to trimethylamine N-oxide/dimethylsulphoxide reductase in Rhodobacter capsulatus

The role of cytochromes in the electron-transport pathway to trimethylamine N -oxide (TMAO)/dimethylsulphoxide (DMSO) reductase in the photosynthetic bacterium Rhodobacter capsulatus was investigated. Reduced-minus-oxidized difference spectra in intact cells with TMAO or DMSO as oxidant revealed cytochrome absorbance changes with a maximum at 559 nm and a shoulder between 548 nm and 556 nm. The former change indicates a role for a 6-type cytochrome and the latter for a c-type cytochrome, both of which are distinct from the cytochrome bc 1 complex. Cytochrome c -556 was identified in a bacterial periplasmic fraction as a redox component which couldbe oxidised by TMAO or DMSO. Cytochrome c -556 was the only cytochrome species which co-fractionated with TMAO/DMSO reductase following gel filtration of a post-chromatophore supernatant produced after French presstreatment of intact cells. The mid-point redox potential (pH 7.6) of cytochrome c -556 was + 105 mV ( n = 1). It is suggested that TMAO/DMSO reductase and cytochrome c -556 form a structural and functional association in the periplasm of Rhodobacter capsulatus .

Direct observation with an electrode of uncoupler-sensitive assimilatory nitrate uptake by Rhodopseudomonas capsulata

A major deficiency in our understanding of bacterial transport is the question of how nitrate enters bacteria, to act either as an electron acceptor or as a source of nitrogen. One of the reasons for the dearth of information about nitrate transport is probably an experimental one; transport is generally studied using radioisotopes, but the radioisotope of nitrogen, 13N, has a half-life of only 10 min. Despite this unhelpful property 13N has been successfully used in order to study nitrate uptake into Pseudomonasjluorescens [I]. The bacteria were grown aerobically with nitrate as the source of nitrogen and therefore, as there was evidently repression of any transport systems associated with nitrate respiration in this denitrifying organism, the first measurements of assimilatory nitrate uptake in bacteria were made [ 11. Here, we report that the assimilatory uptake of nitrate by Rhodopseudomonas capsulata, which does not denitrify, can be readily and continuously followed using a nitrate-specific electrode. Use of this method has shown that nitrate uptake is rapidly inhibited upon collapsing the proton electrochemical gradient across the plasma membrane either by addition of a protonophore uncoupler, or by transferring the cells from an illuminated to a darkened state. These data indicate that nitrate uptake is directly dependent on the proton electrochemical gradient rather than on the availability of intra-cellular AlT. It is also shown that nitrate uptake is rapidly but reversibly inhibited by ammonium ions, but that the ammonium analogue

The activities of two pathways of nitrate reduction in Rhodopseudomonas capsulata

Abstract(1)The disappearance of nitrate from suspensions of intact, washed cells of Rhodopseudomonas capsulata strain N22DNAR+ was measured with an ion selective electrode. In samples taken from phototrophic cultures grown to late exponential phase, nitrate disappearance was partially inhibited by light but was not affected by the presence of ammonium. Nitrate disappearance from samples from low density cultures in the early exponential phase of growth was first inhibited and later stimulated by light. In these cells ammonium ions inhibited the light-dependent but not the dark disappearance of nitrate. It is concluded that cells in the early exponential phase of growth possess both an ammonium-sensitive, assimilatory pathway for nitrate reduction (NRI) and an ammonium-insensitive pathway for nitrate reduction (NRII) which is linked to respiratory electron flow and energy conservation. In cells harvested in late exponential phase only the respiratory pathway for pitrate reduction is detectable.(2)Nitrate reduction, as judged by the oxidation of reduced methyl viologen by anaerobic cell suspensions, was measured at high rates in those strains of R. capsulata (AD2, BK5, N22DNAR+) which are believed to possess NRII activity but not in those strains (Kbl, R3, N22) which only manifest the ammonium-sensitive NRI pathway. On this basis we have used nitrate-dependent oxidation of reduced methyl viologen as a diagnostic test for the nitrate reductase of NRII in cells harvested from cultures of R. capsulata strain AD2. The activity was readily detectable in cells from cultures grown aerobically in the dark with ammonium nitrate as source of nitrogen. When the oxygen supply to the culture was withdrawn, the level of methyl viologen-dependent nitrate reductase increased considerably and nitrite accumulated in the culture medium. Upon reconnecting the oxygen supply, methyl viologen-dependent nitrate reductase activity decreased and the reduction of nitrate to nitrite in the culture was inhibited. It is concluded that the respiratory nitrate reductase activity is regulated by the availability of electron transport pathways that are linked to the generation of a proton electrochemical gradient.