Respiratory protection of nitrogenase in Azotobacter species: is a widely held hypothesis unequivocally supported by experimental evidence?

The hypothesis of respiratory protection, originally formulated on the basis of results obtained with Azotobacter species, postulates that consumption of O(2) at the surface of diazotrophic prokaryotes protects nitrogenase from inactivation by O(2). Accordingly, it is assumed that, at increased ambient O(2) concentrations, nitrogenase activity depends on increased activities of a largely uncoupled respiratory electron transport system. The present review compiles evidence indicating that cellular O(2) consumption as well as both the activity and the formation of the respiratory system of Azotobacter vinelandii are controlled by the C/N ratio, that is to say the ratio at which the organism consumes the substrate (i.

Nitrogenase activity and regeneration of the cellular ATP pool in Azotobacter vinelandii adapted to different oxygen concentrations.

The in vivo activity of nitrogenase under aerobiosis was studied with diazotrophic chemostat cultures of Azotobacter vinelandii grown under glucose- or phosphate-limited conditions at different dilution rates (Ds, representing the growth rate mu) and different dissolved oxygen concentrations. Under steady-state conditions, the concentration as well as the cellular level of ATP increased in glucose-limited cultures when D was increased. Irrespective of the type of growth limitation or the dissolved oxygen concentration, the steady-state concentrations of ATP and of dinitrogen fixed by nitrogenase increased in direct proportion to each other.

Control of nitrogen fixation by oxygen in purple nonsulfur bacteria.

Some members of the facultatively phototrophic bacteria are able to grow diazotrophically in the presence of oxygen. As in other diazotrophs, the nitrogenase of the phototrophic bacteria is highly sensitive to oxygen; therefore, both the function and the expression of nitrogenase are strictly controlled by oxygen. This review focuses on the different levels of oxygen control in the two most extensively studied facultatively phototrophic bacteria, Rhodospirillum rubrum and Rhodobacter capsulatus.

Hydrogenase does not confer significant benefits to Azotobacter vinelandii growing diazotrophically under conditions of glucose limitation.

The presumed beneficial effect of hydrogenase on growth of diazotrophic bacteria was reinvestigated with carbon-limited chemostat cultures of the hydrogenase-deficient mutant hoxKG of Azotobacter vinelandii and its parent. The results revealed that hydrogen recycling was too low to benefit the cellular energy metabolism or activities of nitrogenase and respiration.

Cellular ATP levels and nitrogenase switchoff upon oxygen stress in chemostat cultures of Azotobacter vinelandii.

When Azotobacter vinelandii, growing diazotrophically in chemostat culture, was subjected to sudden increases in the ambient oxygen concentration (oxygen stress), nitrogenase activity was switched off and cellular ATP pools decreased at rates depending on the stress level. Following a fast decrease, the ATP pool approached a lower level. When the stress was released, these effects were reversed.

Bacteriochlorophyllc formation and chlorosome development inChloroflexus aurantiacus.

The dependence of chlorosome development on bacteriochlorophyll (BChl)c synthesis was studied with the phototrophic green bacteriumChloroflexus aurantiacus. By selecting defined culture conditions, three possibilities could be identified. Upon addition of 5-aminolevulinic acid, cells of resting cultures increased their specific BChlc contents as well as the volumes of already existing chlorosomes.

Optimizing photoheterotrophic H2 production by Rhodobacter capsulatus upon interposon mutagenesis in the hupL gene.

In Rhodobacter capsulatus, the hupL gene encoding the large subunit of the uptake-hydrogenase (Hup) enzyme complex was mutated by insertion of an interposon. The mutant neither synthesized an active hydrogenase nor grew photoautotrophically. Under conditions of nitrogen (N) limitation, photoheterotrophic cultures of the wild type and the mutant evolved H2 by activity of the nitrogenase enzyme complex.

Identification of a new class of nitrogen fixation genes in Rhodobacter capsulatus: a putative membrane complex involved in electron transport to nitrogenase.

DNA sequence analysis of a 12236 bp fragment, which is located upstream of nifE in Rhodobacter capsulatus nif region A, revealed the presence of ten open reading frames. With the exception of fdxC and fdxN, which encode a plant-type and a bacterial-type ferredoxin, the deduced products of these coding regions exhibited no significant homology to known proteins. Analysis of defined insertion and deletion mutants demonstrated that six of these genes were required for nitrogen fixation.

Light and oxygen regulation of the synthesis of bacteriochlorophylls a and c in Chloroflexus aurantiacus.

Control of the synthesis of bacteriochlorophylls (Bchls) a and c by light and oxygen was studied in Chloroflexus aurantiacus grown in batch or chemostat culture with serine as the growth-limiting substrate. For comparison, inhibition by gabaculine of the formation of selected tetrapyrroles was studied. The inhibitory effect of gabaculine decreased in the following order of tetrapyrrole formation: coproporphyrin greater than Bchl c greater than Bchl a.

Control of diauxic growth of Azotobacter vinelandii on acetate and glucose.

Batch cultures of Azotobacter vinelandii were inoculated with cells pregrown on either acetate or glucose. When they were subsequently grown on a mixture of acetate and glucose, typical diauxic growth was observed, with preferential uptake of acetate in the first and glucose in the second phase of growth. Extracts from acetate-pregrown cells exhibited high acetate kinase activity in the first phase of growth.

Dependence of nitrogenase switch-off upon oxygen stress on the nitrogenase activity in Azotobacter vinelandii.

Azotobacter vinelandii was grown diazotrophically in chemostat cultures limited by sucrose, citrate, or acetate. Specific activities of cellular oxygen consumption (qO2) and nitrogenase (acetylene reduction) were measured in situ at different dilution rates (D, representing the specific growth rate mu at steady state). Sucrose-limited cultures exhibited linear relationships between qO2 and D, each of which, however, depended on the dissolved oxygen concentration in the range of 12 to 192 microM O2.

Regulation of tetrapyrrole synthesis by light in chemostat cultures of Rhodobacter sphaeroides.

Control of bacteriochlorophyll (Bchl), magnesium protoporphyrin monomethyl ester (MgPME), cytochromes, and coproporphyrin by light was studied with chemostat cultures of Rhodobacter sphaeroides growing at a constant dilution rate. By increasing the growth-limiting light energy flux from 10 to 55 W/m2, specific Bchl contents decreased from 19.3 to 7.

Levels and activities of nitrogenase proteins in Azotobacter vinelandii grown at different dissolved oxygen concentrations.

Azotobacter vinelandii was grown diazotrophically at different dissolved oxygen concentrations (in the range of 3 to 216 microM) in sucrose-limited continuous culture. The specific nitrogenase activity, measured on the basis of acetylene reduction in situ, was dependent solely on the growth rate and was largely independent of oxygen and sucrose concentration. FeMo (Av1) and Fe (Av2) nitrogenase proteins were quantified after Western blotting (immunoblotting).

Reversible and irreversible inactivation of cellular nitrogenase upon oxygen stress in Azotobacter vinelandii growing in oxygen controlled continuous culture.

Azotobacter vinelandii growing in oxygen controlled chemostat culture was subjected to sudden increases of ambient oxygen concentrations (oxygen stress) after adaptation to different oxygen concentrations adjustable with air (100% air saturation corresponds to 225 +/- 14 microM O2). Inactivations of cellular nitrogenase during stress (switch off) as well as after release of stress (switch on) were evaluated in vivo as depending on stress duration and stress height (delta pO2). Switch off was at its final extent within 1 min of stress.

Control of the formation of bacteriochlorophyll, and B875- and B850-bacteriochlorophyll complexes in Rhodopseudomonas sphaeroides mutant strain H5.

Rhodopseudomonas sphaeroides mutant H5 lacking 5-aminolevulinic acid synthase was employed to study the control of the formation of total bacteriochlorophyll as well as of the B875- and B850-bacteriochlorophyll protein complexes. The organisms were grown phototrophically in a chemostat where cell protein formation was limited by iron ions and bacteriochlorophyll by 5-aminolevulinic acid. 0.

Localization and activities of nitrogenase, glutamine synthetase and glutamate synthase in Azotobacter vinelandii grown in oxygen-controlled continuous culture.

Azotobacter vinelandii was grown in oxygen-controlled continuous cultures under conditions of dinitrogen fixation. Different oxygen concentrations were adjusted with air. Cell-free extracts were employed to study the oxygen dependency of the intracellular distribution and activity of the following enzymes: nitrogenase, glutamine synthetase and glutamate synthase.

Temperature dependence of growth and membrane-bound activities of Chloroflexus aurantiacus energy metabolism.

The temperature dependence of various activities related to the energy metabolism of isolated membranes and whole cells of the thermophilic bacterium Chloroflexus aurantiacus was determined after phototrophic growth at either 40, 50, or 60 degrees C. The data obtained were expressed by use of Arrhenius plots. Maximum activities were determined at about 65 degrees C for succinate 2,4-dichlorophenol-indophenol reductase as well as NADH oxidase and at about 70 degrees C for Mg-ATPase and for light-induced proton extrusion by cells.

Differences in the architecture of cytoplasmic and intracytoplasmic membranes of three chemotrophically and phototrophically grown species of the Rhodospirillaceae.

Freeze-fracture faces of membranes of either chemotrophically or phototrophically grown Rhodospirillum rubrum, Rhodopseudomonas sphaeroides, and Rhodospirillum tenue were analyzed. All three species differed from each other with respect to size as well as numerical density (number per square micrometer) of intramembrane particles. In R.

The dependency of proton extrusion in the light on the developmental stage of the photosynthetic apparatus in Rhodospirillum rubrum.

The rate of proton extrusion by whole cells of Rhodospirillum rubrum is constant on a bacteriochlorophyll basis only above cellular bacteriochlorophyll concentrations of about 10 nmol bacteriochlorophyll per mg cell protein. At specific bacteriochlorophyll cellular levels below this value, the rate of proton extrusion per bacteriochlorophyll increases. Correspondingly, membrane preparations isolated from these cells exhibit increases in the rate of proton uptake on a pigment basis.

Formation of intracytoplasmic membranes in chemotrophic mecillinam sphaeroplasts of Rhodospirillum rubrum.

Chemotrophically growing cells of Rhodospirillum rubrum were transformed into sphaeroplasts of irregular shape under the influence of the penicillin antibiotic mecillinam. On a cell protein basis, respiratory activities of whole sphaeroplasts were slightly decreased as compared with the untreated control. But, on a membrane protein basis various activities of the respiratory chain were largely identical in membrane preparations from untreated cells and sphaeroplasts, respectively.

Proteins exposed at the surface of chromatophores of Rhodospirillum rubrum: the orientation of isolated chromatophores.

The exposure of proteins at the surface of isolated chromatophores (i.e., the cytoplasmic face of intracytoplasmic membranes) of Rhodospirillum rubrum was studied by proteolysis as well as by enzymatic iodination with 125I.