J. Oelze

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

DNA sequence analysis of a 12236 by 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. Therefore, we propose to call these genes rnfA, rnfB, rnfC, rnfD, rnfE and rnfF (for Rhodobacter nitrogen fixation). Secondary structure predictions suggested that the rnf genes encode four potential membrane proteins and two putative iron-sulphur proteins, which contain cysteine motifs (C-X2-C-X2-C-X3-C-P) typical for [4Fe-4S] proteins. Comparison of the in vivo and in vitro nitrogenase activities of fdxN and rnf mutants suggested that the products encoded by these genes are involved in electron transport to nitrogenase. In addition, these mutants were shown to contain significantly reduced amounts of nitrogenase. The hypothesis that this new class of nitrogen fixation genes encodes components of an electron transfer system to nitrogenase was corroborated by analysing the effect of metronidazole. Both the fdxN and rnf mutants had higher growth yields in the presence of metronidazole than the wild type, suggesting that these mutants contained lower amounts of reduced ferredoxins.

Organization and Differentiation of Membranes of Phototrophic Bacteria

Publisher Summary It is generally accepted that phototrophic bacteria are recent representatives of an evolutionary ancient group of living organisms. One of the major arguments in favor of this hypothesis is the fact that all members of the phototrophic bacteria perform anoxygenic photosynthesis. It may be speculated that the acquisition of tetrapyrrole synthesis enabled original forms of these organisms to adapt to an aerobic energy metabolism employing cytochromes in a respiratory chain with oxygen as the electron acceptor. The chapter demonstrates that the photosynthetic apparatus of several species is localized in intracytoplasmic membranes, whereas the respiratory chain is contained predominantly in the cytoplasmic membrane. This cytological compartmentation of electron-transport chains cannot be generalized. Although photosynthetic bacteria exhibit considerable metabolic versatilities, all of them are largely restricted to ecological niches providing anaerobic conditions. Overall, the data reported in this chapter demonstrate essential similarities as well as specific differences in the responses of various members of the phototrophic bacteria toward changes in the environmental conditions.

Whole cell respiration and nitrogenase activities in Azotobacter vinelandii growing in oxygen controlled continuous culture

Azotobacter vinelandii strain OP was grown in continuous culture at various dissolved oxygen concentrations of air (100% air saturation of the medium=225 ±14 μM O2). Sucrose was added as carbon source and either dinitrogen or ammonia as nitrogen sources. Irrespective of the nitrogen source steady state cultures showed the following general responses with dissolved oxygen concentrations increasing from about 1% to 30% air saturation: (i) cell protein levels, (ii) the amount of cell protein formed per sucrose consumed as well as (iii) nitrogenase activity decreased by at least a factor of two while (iv) cellular respiration increased. At higher oxygen concentrations the parameters changed only slightly, if at all. Increasing the sucrose concentration in the inflowing medium (sR) from 3 g/l to 15 g/l increased the total level of cellular respiration with nitrogen-fixing cultures but was more pronounced with ammonium-assimilating cultures. With nitrogen-fixing cultures cell protein levels increased five-fold while the ratio of protein formed per sucrose consumed as well as cellular nitrogenase activity remained unaffected. With ammonium-assimilating cultures the cell protein level was only doubled and the level of cell protein formed per sucrose consumed was decreased at the higher sR.Increasing the dilution rate at a constant oxygen concentration of 45% air saturation resulted in an almost parallel increase of both cellular respiratory and nitrogenase activity at low and moderate dilution rates. At high dilution rates nitrogenase activity increased steeply over the respiratory activity. Nitrogen-fixing cultures adapted to various oxygen concentrations were subjected to oxygen stress by increasing the oxygen concentration for 7 min. In all cases, this resulted in a complete inhibition (‘switch-off’) of nitrogenase activity. Upon restoration of the original oxygen concentration nitrogenase activity returned to a decreased level. The discussion arrives at the conclusion that some of the results are incompatible with the concept of respiratory protection of nitrogenase.

Quantitative relationship between bacteriochlorophyll content, cytoplasmic membrane structure and chlorosome size in Chloroflexus aurantiacus

Continuous cultures of Chloroflexus aurantiacus were cultivated in a chemostat in the light with varying bacteriochlorophyll (BChl) a/c ratios by changing the growth rate. Under these culture conditions all cells were homogeneously and reproducibly equipped with chlorosomes. In order to determine the number and size of chlorosomes in relation to different BChl contents morphometric measurements were performed on electron micrographs. The linear increase of BChl a contents coincided with an increasing number of chlorosomes per membrane area and per bacterium rather than with an enlargement of the average size of chlorosomes. The numbers of chlorosomes and therefore the percentage of chlorosome-covered cytoplasmic membrane increased linearly with increasing BChl a contents. The average size of the baseplates was largely constant in all cultures (mean 3,222±836 nm2). However, within individual cells the size of baseplates varied by a factor of 3.0, especially by the variation of the length. The exponential increase in BChl c contents coincided with an increasing number of chlorosomes (up to a factor of 2.3) and an enlargement of the average chlorosome volume (up to a factor of 1.9). The number of BChl a molecules per chlorosome was about 1,484±165, thus the number of reaction centers per chlorosome was 58±12. The data suggest, firstly, that BChl a is confined to areas (cytoplasmic membrane plus baseplate) as represented by the chlorosome attachment sites; secondly, that the degree of packing of BChl c molecules within chlorosomes increases with increasing BChl c contents.

Membranes and Chlorosomes of Green Bacteria: Structure, Composition and Development

Although belonging to evolutionary distantly related groups, cells of members of the Chlorobiaceae and the Chloroflexaceae exhibit structurally and functionally comparable substructures. While the photochemical reaction center complex plus a light-harvesting unit are housed in the peripheral cytoplasmic membrane (CM) system, an accessory light-harvesting unit is localized in specialized structures, the chlorosomes, underlying the CM. In this chapter, the present knowledge on the fine structure of chlorosomes and the CM is reviewed. After a description of methods commonly employed to isolate chlorosomes and CM, data of chemical analyses of both subcellular fractions are detailed. In spite of considerable similarities in the overall ultrastructural and functional properties, the chemical composition reveals significant differences between chlorosomes and CM, when isolated from Chlorobium and Chloroflexus, respectively. The same holds true with respect to the supramolecular organization, particularly of chlorosomes and elements involved in their connection to the CM. Since the photosynthetic apparatus of green bacteria is composed of two different moieties, i.e. the chlorosomes and the CM-bound unit, the important questions arise how these different units are synthesized, how the synthesis of individual constituents is controlled and how the syntheses of chlorosomes and the CM-bound units are coordinated. In the present contribution these problems are approached on the basis of the pigments characteristic of both units. In addition, the knowledge on polypeptide formation is presented. These data are combined with the respective changes in number and size of chlorosomes. In spite of a considerable amount of detailed information available as yet, it is finally concluded that considerable research efforts are still required in order to understand the development of the biologically unique type of photosynthetic apparatus characteristic of the green bacteria.

The organization of bacteriochlorophyll c in chlorosomes from Chloroflexus aurantiacus and the structural role of carotenoids and protein. An absorption, linear dichroism, circular dichroism and Stark spectroscopy study.

The organization of bacteriochlorophyll c (BChl c) molecules was studied in normal and carotenoid-deficient chlorosomes isolated from the green phototrophic bacterium Chloroflexus aurantiacus. Carotenoid-deficient chlorosomes were obtained from cells grown in the presence of 60 µg of 2-hydroxybiphenyl per ml. At this concentration, BChl c synthesis was not affected while the formation of the 5.7 kDa chlorosome polypeptide was inhibited by about 50% (M. Foidl et al., submitted). Absorption, linear dichroism and circular dichroism spectroscopy showed that the organization of BChl c molecules with respect to each other as well as to the long axis of the chlorosomes was similar for both types of chlorosomes. Therefore, it is concluded that the organization of BChl c molecules is largely independent on the presence of the bulk of carotenoids as well as of at least half of the normal amount of the 5.7 kDa polypeptide. The Stark spectra of the chlorosomes, as characterized by a large difference polarizability for the ground- and excited states of the interacting BChl c molecules, were much more intense than those of individual pigments. It is proposed that this is caused by the strong overlap of BChl c molecules in the chlorosomes. In contrast to individual chlorophylls, BChl c in chlorosomes did not give rise to a significant difference permanent dipole moment for the ground- and excited states. This observation favors models for the BChl c organization which invoke the anti-parallel stacking of linear BChl c aggregates above those models in which linear BChl c aggregates are stacked in a parallel fashion. The difference between the Stark spectrum of carotenoid-deficient and WT chlorosomes indicates that the carotenoids are in the vicinity of the BChls.

Die morphogenese des photosyntheseapparates von Rhodospirillum rubrum. I. Die isolierung und charakterisierung von zwei membransystemen

Abstract 1. 1. Two pigmented bands from cell-free extracts of light-growth Rhodospirillum rubrum could be isolated in a linear gradient of Ficoll by zone centrifugation. 2. 2. As shown by several methods, the lower band of the gradient contained chromatophores (thylakoids) in a highly purified form. 3. 3. In the upper band of the gradient, particles with an average diameter of 360 A could be identified by electron microscopy. 4. 4. Electrophoresis of the contents of the upper band in agar gel (0.5%) made it possible to separate two zones. Whereas only protein could be determined in the faster migrating zone, protein, pigments, lipids and activity of succinate dehydrogenase could be determined in the slower migrating zone. 5. 5. The chromatophores (thylakoids) of the lower band were split by treatment with phenol-formic acid-water (2:1:1, v/v/v) and then separated by gel electrophoresis in polyacrylamide into 5 main subunits. 6. 6. When the upper band of the gradient was treated with phenol-formic acid-water and separated by gel electrophoresis in polacrylamide, only one of the 5 main subunits was obtained. This was characteristic of cytoplasmic membrane. 7. 7. The upper band of the gradient could be isolated from cultures grown under anaerobic light conditions, from cultures grown aerobically in the dark and from the carotenoid-free mutant strain of R. rubrum M 46, which forms only few chromatophores. 8. 8. The results indicate that the upper band of the Ficoll gradient consists mainly of mechanically disrupted cytoplasmic membrane. 9. 9. Morphogenetic relationships between cytoplasmic membrane and chromatophores are discussed.

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. When grown with glutamate as an N source and either d,l-malate or l-lactate as carbon sources, the efficiency of H2 production by the HupL mutant was higher than 90%, whereas wild-type cultures exhibited efficiencies of 54% (with d,l-malate) and 64% (with l-lactate), respectively. With NHinf4sup+as the N source, efficiencies of H2 production were 70% (mutant) and 52% (wild type).

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. Analyses were performed after polyacrylamide gel electrophoresis of chromatophore proteins solubilized with sodium dodecyl sulfate. Reversible light induced proton uptake by partially digested chromatophores was used as a criterion for the integrity of the permeability barrier and thus, as evidence for proteolysis only of proteins outside of this barrier. Trypsin or alpha-chymotrypsin completely cleaved four proteins which were identified as the heavy subunit of succinate dehydrogenase (Mr = 64 000), the alpha- and beta-subunits of coupling factor ATPase (Mr = 55 000 and 51 000), and the heavy (H) subunit of photochemical reaction centers (Mr = 31 000). alpha-Chymotrypsin, in addition, attacked the protein (Mr = 9000) of light harvesting bacteriochlorophyll preparations. By enzymatic iodination, the same proteins were labeled as were digested with trypsin or alpha-chymotrypsin except for the protein of Mr = 9000. In addition, significant label was incorporated into three more proteins, one of which (Mr = 41 000) could be identified as a major protein of the cell wall. The complete cleavage with trypsin of four proteins exposed at the surface indicated that isolated chromatophores were homogeneously oriented regardless of the method employed for cell breakage, i.e., passage through a French pressure cell at different forces or osmotic shock of sphaeroplasts.

Control of nitrogen fixation by oxygen in purple nonsulfur bacteria

Abstract 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. Current data show that oxygen controls nitrogen fixation at least at the levels of (1) transcription of nif genes, (2) the accumulation of the three different nitrogenase polypeptides, (3) the cellular activity of nitrogen fixation. In Rba. capsulatus, activation of the nifH promoter is the least oxygen-sensitive step, and nitrogen fixation is the most oxygen-sensitive step. ADP-Ribosylation of nitrogenase, occurring under conditions of ammonium-dependent inactivation of the enzyme, is not observed when Rba. capsulatus is exposed either suddenly or at a steady state to increased oxygen concentrations. Future research is required to understand the mechanisms of protection of nitrogenase against oxygen damage, and also the mechanisms by which oxygen controls the formation and activity of nitrogenase; this will add significantly to the biologically important question of how cells deal with the presence of toxic oxygen.