Purple Non-sulfur Bacterium Rhodobacter Capsulatus Research Articles

Genetic analysis of a bacterial genetic exchange element: the gene transfer agent of Rhodobacter capsulatus.

An unusual system of genetic exchange exists in the purple nonsulfur bacterium Rhodobacter capsulatus. DNA transmission is mediated by a small bacteriophage-like particle called the gene transfer agent (GTA) that transfers random 4.5-kb segments of the producing cell's genome to recipient cells, where allelic replacement occurs. This paper presents the results of gene cloning, analysis, and mutagenesis experiments that show that GTA resembles a defective prophage related to bacteriophages from diverse genera of bacteria, which has been adopted by R. capsulatus for genetic exchange. A pair of cellular proteins, CckA and CtrA, appear to constitute part of a sensor kinase/response regulator signaling pathway that is required for expression of GTA structural genes. This signaling pathway controls growth-phase-dependent regulation of GTA gene messages, yielding maximal gene expression in the stationary phase. We suggest that GTA is an ancient prophage remnant that has evolved in concert with the bacterial genome, resulting in a genetic exchange process controlled by the bacterial cell.

Primary alcohols and di-alcohols as growth substrates for the purple nonsulfur bacterium <i>Rhodobacter capsulatus</i><sup>†</sup>

Growth experiments were performed with the purple nonsulfur bacterium Rhodobacter capsulatus to test its ability to use aliphatic, methyl-substituted, and unsaturated alcohols, as well as di-alcohols, as carbon sources for growth. Both phototrophic and chemotrophic growth was observed on a wide variety of such alcohols. By contrast, secondary or tertiary alcohols, or primary alcohols containing an ethyl or propyl substituent, did not support growth. In addition, preculture history and serial subculturing were found to be important factors for obtaining reliable growth of R. capsulatus on alcohols. Collectively, these results suggest that the carbon nutritional diversity of Rhodobacter capsulatus is even greater than previously suspected and that besides metabolizing organic acids and fatty acids in nature, this species may also be a major consumer of alcohols.

DNA Binding Characteristics of RegA : A CONSTITUTIVELY ACTIVE ANAEROBIC ACTIVATOR OF PHOTOSYNTHESIS GENE EXPRESSION IN RHODOBACTER CAPSULATUS

In the purple non-sulfur bacterium Rhodobacter capsulatus, RegA and RegB comprise a two-component regulatory system that is required for maximal anaerobic transcription of key photosynthesis genes. RegB is a sensor kinase that uses ATP to phosphorylate its cognate response regulator, RegA. The mechanism under which RegA approximately P influences transcription of target genes has been unclear given that past attempts to demonstrate DNA binding activity by isolated RegA have failed. This led to a model invoking a role for RegA approximately P as an intermediate in a more complex multicomponent phosphoryl transfer cascade. In the present study, we describe the isolation of a mutant version of RegA (RegA*) which promotes high level expression of photosynthesis genes independent of RegB. DNase I footprint analyses show that purified RegA* binds to the promoters of the puf and puc operons at locations that are consistent with RegA functioning as a transcriptional activator for these operons. We conclude that RegA functions, like most members of the response regulator family, as a DNA-binding protein that directly affects the expression of its target genes.

Sulfide-Quinone Reductase from Rhodobacter capsulatus: PURIFICATION, CLONING, AND EXPRESSION

A sulfide-quinone oxidoreductase (SQR, EC 1.8.5.'.) has been purified to homogeneity from chromatophores of the non-sulfur purple bacterium Rhodobacter capsulatus DSM 155. It is composed of a single polypeptide with an apparent molecular mass of about 55 kDa, exhibiting absorption and fluorescence spectra typical for a flavoprotein and similar to the SQR from the cyanobacterium Oscillatoria limnetica. From N-terminal and tryptic peptide sequences of the pure protein a genomic DNA clone was obtained by polymerase chain reaction amplification. Its sequence contains an open reading frame of 1275 base pairs (EMBL nucleotide sequence data base, accession no. X97478X97478) encoding the SQR of R. capsulatus. The deduced polypeptide consists of 425 amino acid residues with a molecular mass of 47 kDa and a net charge of +9. The high similarity (72%)/identity (48%) between the N termini of the cyanobacterial and the bacterial enzyme was confirmed and extended. Both enzymes exhibit the FAD/NAD(P) binding betaalphabeta-fold (Wierenga, R. K., Terpstra, P., and Hol, W. G. S. (1986) J. Mol. Biol. 187, 101-107). The complete sequence of the SQR from R. capsulatus shows further similarity to flavoproteins, in particular glutathione reductase and lipoamide dehydrogenase. The cloned sqr was expressed in Escherichia coli in a functional form.

Site-Directed Mutations Near the L-Subunit D-Helix of the Purple Bacterial Reaction Center: A Partial Model for the Primary Donor of Photosystem II

We have engineered a photosynthetically competent mutant of the purple non-sulfur bacterium Rhodobacter capsulatus which seeks to mimic the behavior of the primary electron donor (P) of the plant photosystem II (PS II) reaction center (RC). To construct this mutant (denoted D1-ILMH), four residues in the bacterial L subunit were mutagenized, such that an 11-residue segment was made identical to the analogous segment from the D1 subunit of PS II. The electronic properties of the bacteriochlorophyll (Bchl) dimer which constitutes the primary donor are substantially altered by these modifications, to the degree that the dimer becomes functionally much more "monomeric". The changes include (1) an increase in the values of the zero-field splitting (ZFS) parameters, as measured by electron paramagnetic resonance (EPR), for the spin-polarized triplet state, 3P, from /D/ = 185 x 10(-4) cm(-1) and /E/ = 31 x 10(-4) cm(-1) in wild-type (WT) chromatophore membranes to /D/ = 200 x 10(-4) cm(-1) and /E/ = 44 x 10(-4) cm(-1) in the mutant and (2) an increase in the EPR line width of the oxidized state, P+, from 0.97 mT in WT to 1.09 mT in D1-ILMH RCs. However, unlike the PS II primary donor (P680), the orientation of 3P in the D1-ILMH mutant is the same as in WT bacteria and does not display the unusual orientation found for PS II. And whereas the redox couple P/P+ has a very high midpoint potential in PS II, P/P+ in the D1-ILMH mutant has a lower midpoint (90 mV more negative) than in WT Rb. capsulatus. In addition, Raman measurements indicate that the hydrogen bond between HisL168 and the C2 acetyl carbonyl oxygen of the Bchl on the active electron transfer pathway (P(A)) is absent in the mutant, due to the fact that HisL168 in the WT sequence has been replaced by a leucine in D1-ILMH. However, the Raman data also reveal the presence of a new hydrogen bond in the D1-ILMH RCs, between the C9 keto carbonyl oxygen of P(A) and an unknown hydrogen-bond donor. Thus, although the protein environment around one of the Bchls of the special pair is significantly changed in D1-ILMH, the chimeric RC does not, as a result of these changes, have a primary donor that is oriented like the one in PS II.

Organization and regulation of genes encoding the molybdenum nitrogenase and the alternative nitrogenase in Rhodobacter capsulatus

The phototrophic non-sulfur purple bacterium Rhodobacter capsulatus is able to fix atmospheric dinitrogen either via a conventional molybdenum nitrogenase or via an alternative iron-only nitrogenase. At least 53 genes are involved in the synthesis and regulation of these two nitrogenase systems, most of which are clustered in four regions widely spread in the genome. Expression of both nitrogenase systems is regulated at the transcriptional level by NifR1 and NifR2, homologues of NtrC and NtrB, respectively. However, this ntr system is only involved in the regulation of the two nitrogenase systems and the high-affinity molybdenum transport system and is not required for utilization of other N sources such as proline and arginine. In contrast to enteric bacteria, the R. capsulatus NtrC homologue does not act in concert with the alternative sigma factor RpoN (σ54). Nitrogen fixation in R. capsulatus is regulated at the transcriptional level and also at the post-translational level. The draTG gene products are responsible for covalent modification of the dinitrogenase reductases of both nitrogenase systems. In addition, mutations in hvrA, a gene previously described as being responsible for low-light activation of the photosynthetic apparatus, also affect regulation of nitrogen fixation.

Localization of nitrate reductase genes in a 115-kb plasmid ofRhodobacter capsulatusand restoration of NIT+character in nitrate reductase negative mutant or wild-type strains by conjugative transfer of the endogenous plasmid

The capacity to reduce nitrate (NIT+ character) of wild-type and mutant strains of the purple nonsulfur bacterium Rhodobacter capsulatus was analysed by the methods of plasmid genetics and by DNA-DNA hybridization techniques. By conjugative introduction of the endogenous 115-kb plasmid of strain AD2 into a plasmid-free NIT− mutant of the same strain the missing assimilatory nitrate reductase activity was restored. By analogous experimental techniques, the capacity to reduce nitrate was also temporarily established in the Rb. capsulatus NIT− wild-type strain B10. DNA-DNA hybridization experiments with the narGHIJ operon of Escherichia coli and napA of Alcaligenes eutrophus yielded positive signals with an 11-kb EcoRI fragment of the AD2 plasmid.

Localization of nitrate reductase genes in a 115-kb plasmid of Rhodobacter capsulatus and restoration of NIT+ character in nitrate reductase negative mutant or wild-type strains by conjugative transfer of the endogenous plasmid

The capacity to reduce nitrate (NIT+ character) of wild-type and mutant strains of the purple nonsulfur bacterium Rhodobacter capsulatus was analysed by the methods of plasmid genetics and by DNA-DNA hybridization techniques. By conjugative introduction of the endogenous 115-kb plasmid of strain AD2 into a plasmid-free NIT− mutant of the same strain the missing assimilatory nitrate reductase activity was restored. By analogous experimental techniques, the capacity to reduce nitrate was also temporarily established in the Rb. capsulatus NIT− wild-type strain B10. DNA-DNA hybridization experiments with the narGHIJ operon of Escherichia coli and napA of Alcaligenes eutrophus yielded positive signals with an 11-kb EcoRI fragment of the AD2 plasmid.

The ubiquinol:cytochrome [formula omitted] oxidoreductase of Chromatium vinosum

Chromatophore membranes isolated from the photosynthetic purple sulfur bacterium Chromatium vinosum exhibit a quinol:cytochrome c 2( c) oxidoreductase activity that is sensitive to two specific inhibitors of cytochrome bc 1 complexes, antimycin A and myxothiazol. Digests of C. vinosum DNA hybridize to probes constructed from portions of the pet(fbc) operons that code for the cytochrome bc 1 complexes of the photosynthetic purple non-sulfur bacteria Rhodobacter capsulatus, Rhodobacter sphaeroides and Rhodospirillum rubrum. Despite the fact that it has not yet proven possible to isolate a detergent-solubilized, purified cytochrome bc 1 complex from C. vinosum, these new results, when combined with spectroscopic and kinetic data available from the literature, indicate that this purple sulfur bacterium contains a cytochrome bc 1 complex similar in structure to the well-characterized complexes found in purple non-sulfur bacteria. Equine cytochrome c and cytochromes c 2 isolated from the purple non-sulfur bacteria Rhodospirillum rubrum and Rhodopseudomonas viridis all function as effective electron acceptors from the C. vinosum cytochrome bc 1 complex. In contrast, HiPIP (high-potential iron protein) isolated from two purple sulfur bacteria, C. vinosum and Chromatium tepidum, are reduced only at low rates by quinol in the presence of C. vinosum membranes and their reduction is not inhibited by either antimycin A or myxothiazol. These observations support the hypothesis that a protein structurally related to cytochrome c 2, rather than HiPIP, is the physiological electron acceptor for the C. vinosum cytochrome bc 1 complex. Although it has not yet proven possible to purify the putative C. vinosum cytochrome c 2 to homogeneity, we report here that digests of C. vinosum DNA hybridize with probes constructed from portions of the cycA genes coding for cytochromes c 2 from Rb. capsulatus, Rps. viridis and Rb. sphaeroides. These DNA hybridization results provide further support for the hypothesis that a cytochrome c 2-like protein is present in C. vinosum.

Regulation of nitrate reductase inRhodobacter capsulatus E1F1

The photosynthetic purple non-sulfur nitrate-assimilating bacteriumRhodobacter capsulatus E1F1 has an adaptive nitrate reductase activity inducible by either nitrate or nitrite and molybdenum traces. Nitrate reductase induction by nitrate did not occur in media with nitrate and ammonium, which showed no effect if nitrite was the inductor instead of nitrate or in the presence ofl-methionine-dl-sulfoximine (MSX) plus nitrate. In vivo, tungstate inhibited nitrate reductase activity, and this was not recovered upon addition of molybdenum unless de novo protein synthesis took place. Nitrate reductase was also repressed in nitrogen-starved cells or after the addition of azaserine to cells growing phototrophically with nitrate. Moreover, higher rates of nitrate reductase induction and nitrite excretion were found in illuminated cells grown with nitrate under air than in those grown under argon.

Acetate metabolism in purple non-sulfur bacteria

The photosynthetic non-sulfur purple bacterium Rhodobacter capsulatus E1F1 can grow on acetate or dl-malate photoheterotrophically under anerobic conditions or chemoheterotrophically in the dark in the presence of dioxygen. Bacterial cells grown under both anaerobic and aerobic conditions exhibited high amounts of the tricarboxylic acid cycle enzymes especially in dark-aerobic cultures. A high activity of isocitrate lyase was found in cells of R. capsulatus E1F1 and, to a lesser extent, in those of R. capsulatus IP2, Rhodobacter sphaeroides and Rhodospirillum rubrum grown photoheterotrophically on acetate under anaerobic conditions. The second enzyme of the glyoxylate shunt, malate synthase, appears to be constitutive. Itaconate, a powerful inhibitor of isocitrate lyase, severely inhibited growth of R. capsulatus, R. rubrum and R. sphaeroides on acetate, thus corroborating a physiological role of the enzyme in acetate metabolism by Rhodospirillaceae. * Abbreviations: Abbreviations ISL : isocitrate lyase MS : malate synthase DTE : dithioerythrytol pHMB : p -hydroxymercuribenzoate DTNB : 5,5′-dithiobis(2-nitrobenzoic acid)

Properties and synthesis regulation of NAD(P)H dehydrogenases from Rhodobacter capsulatus

Three NAD(P)H dehydrogenases were found and purified from a soluble fraction of cells of the purple non-sulfur bacterium Rhodobacter capsulatus, strain B10. Molecular mass of NAD(P)H, NADPH and NADH dehydrogenases are 67 000 (4 · 18 000), 35 000 and 39 000, and the isoelectric points are 4.6, 4.3 and 4.5, respectively. NAD(P)H dehydrogenase is characterized by a higher sensitivity to quinacrine, NADPH dehydrogenase by its sensitivity to p-chloromercuribenzoate and NADH dehydrogenase by its sensitivity to sodium arsenite. In contrast to the other two enzymes, NAD(P)H dehydrogenase is capable of oxidizing NADPH as well as NADH, but the ratio of their oxidation rates depends on the pH. All NAD(P)H dehydrogenases reacted with ferricyanide, 2,6-dichlorophenolindophenol, benzoquinone and naphthoquinone, but did not exhibit transhydrogenase, reductase or oxidase activity. Moreover, NADH dehydrogenase was also capable of reducing FAD and FMN. NAD(P)H and NADH dehydrogenases possessed cytochrome- c reductase activity, which was stimulated by menadione and ubiquinone Q 1. The activity of NAD(P)H and NADH dehydrogenases depended on culture-growth conditions. The activity of NAD(P)H dehydrogenase from cells grown under chemoheterotrophic aerobic conditions was the lowest and it increased notably under photoheterotrophic anaerobic conditions upon lactate or malate growth limitation. The activity of NADH dehydrogenase was higher from the cells grown under photoheterotrophic anaerobic conditions upon nitrate growth limitation and under chemoheterotrophic aerobic conditions. NADPH dehydrogenase synthesis dependence on R. capsulatus growth conditions was insignificant.

Redox properties and active center of phototrophic bacteria hydrogenases

It is shown that the activity of phototrophic bacteria hydrogenases depends on the redox potential (Eh) of the medium. Hydrogenase from the purple sulfur bacterium Thiocapsa roseopersicina strain BBS reversibly activates H2 at Eh less than -290 mV (pH 7.0). When Eh is increased from -290 to -170 mV, the enzyme is converted into an inactive form which is accompanied by one-electron oxidation of its Fe-S cluster. In contrast, the hydrogenases of the purple nonsulfur bacterium Rhodobacter capsulatus B10 and the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum exhibit maximum activity at Eh greater than -300 mV, favourable only for H2 uptake. When Eh decreases the activities of these enzymes drop dramatically; this accounts for their unidirectional effect directed mainly towards H2 uptake. Such dependence on Eh of activity of hydrogenases from these bacteria correlates with their physiological function in the metabolism of phototrophic bacteria, i.e. with the catalysis of the H2 uptake reaction. Hydrogenases from purple bacteria contain nickel and a single Fe-S cluster. Metal chelators do not affect the activity of these enzymes, which indicates that iron and nickel are tightly bound to the apoprotein. Sulfhydryl compounds irreversibly inactivate T. roseopersicina hydrogenase by 30-40% in the presence of sulfide. Acetylene and carbon monoxide are reversible inhibitors of the enzyme. EPR and inhibitory analysis indicate a direct interaction of H2 with the nickel ion in the active center of the T. roseopersicina hydrogenase.