Biomedical subjects

R G Kranz

Publications and source records attributed to R G Kranz.

At least 37 records · Page 2Linked to original sources

PubMed1996

The temperature-sensitive growth and survival phenotypes of Escherichia coli cydDC and cydAB strains are due to deficiencies in cytochrome bd and are corrected by exogenous catalase and reducing agents.

The cydDC operon of Escherichia coli encodes an ATP-dependent transporter of unknown function that is required for cytochrome bd synthesis. Strains containing defects in either the cydD or cydC gene also demonstrate hypersensitivity to growth at high temperatures and the inability to exit the stationary phase at 37 degrees C. We wished to determine what is responsible for these hypersensitive phenotypes and whether they are due to a lack of the CydDC proteins or a defect of the cytochrome bd encoded by the cydAB genes. Using both K-12- and B-type strains of E. coli, we have compared the phenotypes of isogenic cydAB mutants and cydC mutants. In both K-12- and B-type backgrounds, the hypersensitive phenotypes are due to defects of cytochrome bd activity and not defects of the cydDC genes. We also found that the temperature-sensitive growth phenotypes can be suppressed by exogenous reducing agents, such as glutathione and cysteine. Strikingly, even the enzymes catalase and superoxide dismutase, when added exogenously, can correct the temperature-sensitive and stationary phase arrest phenotypes. We propose that the temperature sensitive growth phenotypes are due to a buildup of diffusible oxygen radicals brought on by the absence of cytochrome bd.

ATP-Binding Cassette Transporters↗

PubMed1994

Structure and expression of the alternative sigma factor, RpoN, in Rhodobacter capsulatus; physiological relevance of an autoactivated nifU2-rpoN superoperon.

The alternative sigma factor, RpoN (sigma 54) is responsible for recruiting core RNA polymerase to the promoters of genes required for diverse physiological functions in a variety of eubacterial species. The RpoN protein in Rhodobacter capsulatus is a putative sigma factor specific for nitrogen fixation (nif) genes. Insertional mutagenesis was used to define regions important for the function of the R. capsulatus RpoN protein. Insertions of four amino acids in the predicted helixturn-helix or in the highly conserved C-terminal eight amino acid residues (previously termed the RpoN box), and an in-frame deletion of the glutamine-rich N-terminus completely inactivated the R. capsulatus RpoN protein. Two separate insertions in the second hydrophobic heptad repeat, a putative leucine zipper, resulted in a partially functional RpoN protein. Eight other linkers in the rpoN open reading frame (ORF) resulted in a completely or partially functional RpoN protein. The rpoN gene in R. capsulatus is downstream from the nifHDKU2 genes, in a nifU2-rpoN operon. Results of genetic experiments on the nifU2-rpoN locus show that the rpoN gene is organized in a nifU2-rpoN superoperon. A primary promoter directly upstream of the rpoN ORF is responsible for the initial expression of rpoN. Deletion analysis and insertional mutagenesis were used to define the primary promoter to 50 bp, between 37 and 87 nucleotides upstream of the predicted rpoN translational start site. This primary promoter is expressed constitutively with respect to nitrogen, and it is necessary and sufficient for growth under nitrogen-limiting conditions typically used in the laboratory. A secondary promoter upstream of nifU2 is autoactivated by RpoN and NifA to increase the expression of rpoN, which ultimately results in higher expression of RpoN-dependent genes. Moreover, rpoN expression from this secondary promoter is physiologically beneficial under certain stressful conditions, such as nitrogen-limiting environments that contain high salt (> 50 mM NaCl) or low iron (< 400 nM FeSO4).

Alleles↗

PubMed1994

The Rhodobacter capsulatus glnB gene is regulated by NtrC at tandem rpoN-independent promoters.

The protein encoded by glnB of Rhodobacter capsulatus is part of a nitrogen-sensing cascade which regulates the expression of nitrogen fixation genes (nif). The expression of glnB was studied by using lacZ fusions, primer extension analysis, and in vitro DNase I footprinting. Our results suggest that glnB is transcribed from two promoters, one of which requires the R. capsulatus ntrC gene but is rpoN independent. Another promoter upstream of glnB is repressed by NtrC; purified R. capsulatus NtrC binds to sites that overlap this distal promoter region.

Bacterial Proteins↗

PubMed1994

A new type of NtrC transcriptional activator.

The enteric NtrC (NRI) protein has been the paradigm for a class of bacterial enhancer-binding proteins (EBPs) that activate transcription of RNA polymerase containing the sigma 54 factor. Activators in the NtrC class are characterized by essentially three properties: (i) they bind to sites distant from the promoters that they activate (> 100 bp upstream of the transcriptional start site), (ii) they contain a conserved nucleotide-binding fold and exhibit ATPase activity that is required for activation, and (iii) they activate the sigma 54 RNA polymerase. We have characterized the NtrC protein from a photosynthetic bacterium, Rhodobacter capsulatus, which represents a metabolically versatile group of bacteria found in aquatic environments. We have shown that the R. capsulatus NtrC protein (RcNtrC) binds to two tandem sites that are distant from promoters that it activates, nifA1 and nifA2. These tandem binding sites are shown to be important for RcNtrC-dependent nitrogen regulation in vivo. Moreover, the conserved nucleotide-binding fold of RcNtrC is required to activate nifA1 and nifA2 but is not required for DNA binding of RcNtrC to upstream activation sequences. However, nifA1 and nifA2 genes do not require the sigma 54 for activation and do not contain the highly conserved nucleotides that are present in all sigma 54-type, EBP-activated promoters. Thus, the NtrC from this photosynthetic bacterium represents a novel member of the class of bacterial EBPs. It is probable that this class of EBPs is more versatile in prokaryotes than previously envisioned.

Adenosine Triphosphate↗

PubMed1993

Cytochromes c biogenesis in a photosynthetic bacterium requires a periplasmic thioredoxin-like protein.

Rhodobacter capsulatus is a Gram-negative photosynthetic bacterium that requires c-type cytochromes for photosynthetic electron transport. Our studies demonstrate that the gene helX is required for the biogenesis of c-type cytochromes in R. capsulatus. A helX chromosomal deletion mutant cannot grow photosynthetically, due to a deficiency of all c-type cytochromes. The predicted amino acid sequence of the helX gene product (176 residues) is related to that of thioredoxin and shares active-site homology with protein disulfide isomerase. Cytochrome c2-alkaline phosphatase gene fusions are used to show that HelX is not required for the transcription, translation, or secretion of apocytochrome c2. HelX-alkaline phosphatase and HelX-beta-galactosidase gene fusions are used to demonstrate that HelX is a periplasmic protein, which is consistent with the presence of a typical signal sequence in HelX. Based on these results, we propose HelX functions as a periplasmic disulfide oxidoreductase that is essential for cytochromes c biogenesis. This role is in accordance with the observation that both heme and the cysteines of apocytochromes c (Cys-Xaa-Yaa-Cys-His) must be in the reduced state for covalent linkage between the two moieties to occur.

Alkaline Phosphatase↗

PubMed1993

Sequence, genetic, and lacZ fusion analyses of a nifR3-ntrB-ntrC operon in Rhodobacter capsulatus.

Transcription of Rhodobacter capsulatus genes encoding the nitrogenase polypeptides (nifHDK) is repressed by fixed nitrogen and oxygen. Regulatory genes required to sense and relay the nitrogen status of the cell are glnB, ntrB (nifR2), and ntrC (nifR1). R. capsulatus nifA1 and nifA2 require ntrC for activation when fixed nitrogen is limiting. The polypeptides encoded by nifA1 and nifA2 along with the alternate sigma factor RpoN activate nifHDK and the remaining nif genes in the absence of both fixed nitrogen and oxygen. In this study we report the sequence and genetic analysis of the previously identified nifR3-ntrB-ntrC regulatory locus. nifR3 is predicted to encode a 324-amino-acid protein with significant homology to an upstream open reading frame cotranscribed with the Escherichia coli regulatory gene, fis. Analysis of ntrC-lacZ fusions and complementation data indicate that nifR3 ntrBC constitute a single operon. nifR3-lacZ fusions are expressed only when lacZ is in the proper reading frame with the predicted nifR3 gene product. Tn5, a kanamycin-resistance cassette, and miniMu insertions in nifR3 are polar on ntrBC (required for nif transcription). This gene organization suggests that the nifR3 gene product may be involved in nitrogen regulation, although nifR3 is not stringently required for nitrogen fixation when ntrBC are present on a multicopy plasmid. In addition, a R. capsulatus strain with a 22-nucleotide insert in the chromosomal nifR3 gene was constructed. This nifR3 strain is able to fix nitrogen and activate nifA1 and nifA2 genes, again supporting the hypothesis that nifR3 is not stringently required for ntrC-dependent gene activation in R. capsulatus.

Amino Acid Sequence↗

PubMed1992

DNA gyrase activities from Rhodobacter capsulatus: analysis of target(s) of coumarins and cloning of the gyrB locus.

Bacterial DNA gyrase is composed of two subunits, gyrase A and B, and is responsible for negatively supercoiling DNA in an ATP-dependent manner. The coumarin antibiotics novobiocin and coumermycin are known inhibitors of bacterial DNA gyrase in vivo and in vitro. We have cloned, mapped, and partially sequenced Rhodobacter capsulatus gyrB which encodes the gyrase B subunit that is presumably involved in binding to coumarins. DNA gyrase activities from crude extracts of R. capsulatus were detected and it was shown that the R. capsulatus activity is (1) inhibited by novobiocin and coumermycin, (2) ATP-dependent and, (3) present in highly aerated and anaerobically grown cells. We previously observed that when R. capsulatus coumermycin-resistant strains are continuously recultured on media containing coumermycin they sometimes acquired mutations in hel genes (i.e., cytochromes c biogenesis mutations). We discuss the possibility that coumarins may inhibit cytochromes c biogenesis as a second target in R. capsulatus via hel (i.e., a putative ATP-dependent heme exporter).

Adenosine Triphosphate↗

PubMed1992

Bacterial cytochromes c biogenesis.

We report the primary sequence analyses of two loci, hel and ccl, whose gene products are required specifically for the biogenesis of c-type cytochromes in the Gram-negative photosynthetic bacterium Rhodobacter capsulatus. Genetic and molecular analyses show that the hel locus contains at least four genes, helA, helB, helC, and orf52, and the ccl locus contains two genes, ccl1 and ccl2, that are essential for cytochromes c biogenesis. HelA is homologous to a class of proteins called ABC transporters and helA, helB, and helC are proposed to encode an export complex. Cytochrome c2-alkaline phosphatase gene fusions were used to show that apocytochrome c2 synthesis and secretion are not affected by the hel and ccl defects. Ccl1 and Ccl2 possess typical signal sequences to direct them to the periplasm. The periplasmic orientation of Ccl1 was confirmed using a Ccl1-alkaline phosphatase gene fusion. The Ccl1-alkaline phosphatase gene fusion analysis also demonstrated that Ccl1 does not require hel genes for its synthesis and secretion. Ccl1 is homologous to proteins encoded by chloroplast and mitochondrial genes, suggesting analogous functions in these organelles. Taken together, these results support the hypothesis that the hel-encoded proteins are required for the export of heme to the periplasm where it is subsequently ligated to the c-type apocytochromes.

Alkaline Phosphatase↗

PubMed1992

Analysis of the promoters and upstream sequences of nifA1 and nifA2 in Rhodobacter capsulatus; activation requires ntrC but not rpoN.

Transcription of Rhodobacter capsulatus genes encoding the nitrogenase polypeptides (nifHDK) is repressed by fixed nitrogen and oxygen. R. capsulatus nifA1 and nifA2 encode identical NIFA proteins that activate transcription of nifHDK and other nif genes. In this study, we report that nifA1-lacZ and nifA2-lacZ fusions are repressed in the presence of NH3 and activated to similar levels under nitrogen-deficient conditions. This nitrogen-controlled activation was dependent on R. capsulatus ntrC (which encodes a transcriptional activator) but not rpoN (which encodes an RNA polymerase sigma factor). We have used primer extension analyses of nifA1, nifA2 and nifH and deletion analyses of nifA1 and nifA2 upstream regions to define likely promoters and cis upstream activation sequences required for nitrogen control of these genes. Primer extension mapping confirmed that ntrC but not rpoN is required for nifA1 and nifA2 activation, and that nifA1 and nifA2 do not possess typical RPON-activated promoters.

Base Sequence↗

PubMed1991

A bacterial homolog to HPRT.

The deduced 182 amino acid sequence of an open reading frame in the photosynthetic bacterium Rhodobacter capsulatus shows significant similarity to the hypoxanthine-guanine phosphoribosyltransferases of other organisms. This similarity includes conserved amino acid residues involved in Lesch-Nyhan syndrome.

Amino Acid Sequence↗

PubMed1991

A bacterial homolog to the mitochondrial enoyl-CoA hydratase.

A 257-amino acid (aa) open reading frame in the photosynthetic bacterium, Rhodobacter capsulatus, shows significant homology to the mitochondrial enoyl-CoA hydratase (290 aa). This similarity in size and sequence suggests that R. capsulatus oxidizes fatty acids using specific components, more like the mitochondrial system than the multifunctional component system of Escherichia coli.

Amino Acid Sequence↗

PubMed1990

Transcriptional regulatory cascade of nitrogen-fixation genes in anoxygenic photosynthetic bacteria: oxygen- and nitrogen-responsive factors.

Many photosynthetic bacteria from aquatic and terrestrial habitats reduce atmospheric dinitrogen to ammonia. The synthesis of proteins required for nitrogen fixation in these microorganisms is repressed by fixed nitrogen or oxygen. Studies on the purple non-sulphur phototroph Rhodobacter capsulatus have helped to clarify this transcriptional control and to define the factors involved in this regulation. The molecular mechanisms by which the nitrogen and oxygen status of the cell are relayed into nif gene expression or repression involve many trans- and cis-acting factors. The roles of these factors in the nif regulatory cascade of R. capsulatus are summarized. Two levels of control are present. The first level of control involves the nitrogen sensing circuitry in which at least four proteins act in a cascade. Upon nitrogen deficiency, genes involved in the second level of control are transcriptionally activated. These genes encode regulatory proteins that subsequently activate transcription of all other nif genes under anaerobic conditions. The R. capsulatus cascade is compared to the nif regulatory cascade in Klebsiella pneumoniae, highlighting both common and unique aspects.

Bacteria↗

PubMed1990

Inactivation, sequence, and lacZ fusion analysis of a regulatory locus required for repression of nitrogen fixation genes in Rhodobacter capsulatus.

Transcription of the genes that code for proteins involved in nitrogen fixation in free-living diazotrophs is typically repressed by high internal oxygen concentrations or exogenous fixed nitrogen. The DNA sequence of a regulatory locus required for repression of Rhodobacter capsulatus nitrogen fixation genes was determined. It was shown that this locus, defined by Tn5 insertions and by ethyl methanesulfonate-derived mutations, is homologous to the glnB gene of other organisms. The R. capsulatus glnB gene was upstream of glnA, the gene for glutamine synthetase, in a glnBA operon. beta-Galactosidase expression from an R. capsulatus glnBA-lacZ translational fusion was increased twofold in cells induced by nitrogen limitation relative to that in cells under nitrogen-sufficient conditions. R. capsulatus nifR1, a gene that was previously shown to be homologous to ntrC and that is required for transcription of nitrogen fixation genes, was responsible for approximately 50% of the transcriptional activation of this glnBA fusion in cells induced under nitrogen-limiting conditions. R. capsulatus GLNB, NIFR1, and NIFR2 (a protein homologous to NTRB) were proposed to transduce the nitrogen status in the cell into repression or activation of other R. capsulatus nif genes. Repression of nif genes in response to oxygen was still present in R. capsulatus glnB mutants and must have occurred at a different level of control in the regulatory circuit.

Base Sequence↗

PubMed1989

Isolation of mutants and genes involved in cytochromes c biosynthesis in Rhodobacter capsulatus.

Mutants of the photosynthetic bacterium Rhodobacter capsulatus that have combined deficiencies in the cytochrome b/c1 complex and other c-type cytochromes have been isolated. These mutants were unable to grow anaerobically in the light or dark but could grow aerobically. Cosmids with R. capsulatus wild-type DNA that complement the mutants have been used to construct genetic and physical maps of the affected genes. Complementation profiles with Tn5 and mini-Mu insertions in these cosmids and subcloned fragments from them indicated that at least three genes (called helA, helB, and helC) are involved in the defects in cytochromes c biosynthesis. The genes are clustered, and helC is transcribed away from helA and helB. Stable insertion mutants in each gene were constructed. It is postulated that helA, helB, and helC are involved in posttranslational processing during cytochromes c synthesis.

Blotting, Southern↗

PubMed1988

Ammonia-constitutive nitrogen fixation mutants of Rhodobacter capsulatus.

Mutants of R. capsulatus that express nif genes constitutively with respect to ammonia were studied in order to define better the circuit that regulates nif gene transcription. One mutant class could be complemented in trans by a cosmid clone containing a wild-type gene (nifR5) defined by Tn5 inserts as being no longer than 1.6 kb. The nifR5 gene is unlinked to previously described nif genes. A second mutant class could not be complemented by the wild-type cosmid library. For one mutant in this class, a nifH::lac fusion was used to select further mutants that were Lac-. Only two of these could be complemented in trans to Lac+; the complementing gene was nifR4, which is analogous to the ntrA gene of enterobacteria. Both complemented strains were Nifc. Therefore these mutations do not bypass the need for the nifR4 gene product. A third class of constitutive mutant was found by selecting Nif+ revertants of a Nif- strain deleted for the nifR1 and nifR2 genes. The nifR1 and nifR2 genes are homologues of enterobacterial ntrC and ntrB genes, respectively. Not all of the Nif+ revertants were constitutive; some were regulated normally by ammonia. We suspect that the latter revertants use alternate Ntr systems to activate nif gene transcription, a suggestion consistent with the observation that numerous bands in Southern blots of total DNA of R. capsulatus are identified by Escherichia coli ntr gene probes.

Ammonia↗

PubMed1986

Characterization of monoclonal antibodies directed against pyruvate oxidase from Escherichia coli: modulation of antibody-induced inhibition by enzyme conformation.

Monoclonal antibodies have been prepared against pyruvate oxidase, a flavoprotein dehydrogenase isolated from Escherichia coli. Six monoclonals were obtained, but only one was found to bind to the native form of the enzyme. This monoclonal, 1I1, was a potent inhibitor. Although this antibody inhibited the unactivated and lipid-activated forms of the enzyme, it had much less of an inhibitory effect on the protease-activated form of the enzyme, although the antibody still bound to this form. Hence, the coupling between antibody binding and the conformation at the active site can itself be modulated by the conformation of the protein.

Antibodies, Monoclonal↗

PubMed1986

Anaerobic regulation of nitrogen-fixation genes in Rhodopseudomonas capsulata.

A Rhodopseudomonas capsulata nifH::lacZ gene fusion was used to isolate constitutive mutants of R. capsulata, unable to repress nif gene transcription anaerobically with every fixed-nitrogen source tested. When these nifc strains were grown aerobically, nif gene transcription was repressed. These results indicate that the regulation of nif gene transcription by fixed nitrogen is different from the regulation by oxygen. Under anaerobic conditions, nif gene transcription in both R. capsulata and Klebsiella pneumoniae is specifically prevented by inhibitors of DNA gyrase [DNA topoisomerase type II (ATP-hydrolyzing), EC 5.99.1.3]. A recent study has shown that anaerobically grown Salmonella typhimurium have high DNA gyrase activity, whereas aerobically grown cells have high DNA topoisomerase type I (EC 5.99.1.2) activity and DNA that is more relaxed [Yamamoto, N. & Droffner, M. L. (1985) Proc. Natl. Acad. Sci. USA 82, 2077-2081]. In view of these results, we suggest that the control of nif gene transcription in response to oxygen is determined by the action of DNA gyrase and DNA topoisomerase I. Thus, although nitrogen control of nif gene expression requires the products of regulatory genes for which constitutive mutations can be isolated, oxygen appears instead to prevent the adoption of a DNA conformation necessary, directly or indirectly, for nif gene transcription.

Anaerobiosis↗

PubMed1985

Isolation and organization of genes for nitrogen fixation in Rhodopseudomonas capsulata.

A library of Rhodopseudomonas capsulata chromosomal DNA was constructed in the broad host range cosmid vector pLAFR1. The library was used to isolate nitrogen fixation genes by complementation of R. capsulata Nif- mutants. Four complementing regions were localized on different cloned DNA fragments by Tn5 and mini-Mu mutagenesis. Additional nif genes were identified by recombination of transposons from the nif cosmids into the R. capsulata chromosome resulting in the creation of new Nif- mutations. Most of the newly cloned DNA fragments containing nif genes were found to be unlinked to any other by Southern hybridization of the cloned DNA to chromosomal DNA blots. One of the new fragments was linked to the nifHDK genes. Another cluster spanning 10-12 kilobase pairs contained a number of nif genes, possibly as many as eight.

Chromosome Mapping↗