Analysis of the fnrL gene and its function in Rhodobacter capsulatus.

The fnr gene encodes a regulatory protein involved in the response to oxygen in a variety of bacterial genera. For example, it was previously shown that the anoxygenic, photosynthetic bacterium Rhodobacter sphaeroides requires the fnrL gene for growth under anaerobic, photosynthetic conditions. Additionally, the FnrL protein in R. sphaeroides is required for anaerobic growth in the dark with an alternative electron acceptor, but it is not essential for aerobic growth. In this study, the fnrL locus from Rhodobacter capsulatus was cloned and sequenced. Surprisingly, an R. capsulatus strain with the fnrL gene deleted grows like the wild type under either photosynthetic or aerobic conditions but does not grow anaerobically with alternative electron acceptors such as dimethyl sulfoxide (DMSO) or trimethylamine oxide. It is demonstrated that the c-type cytochrome induced upon anaerobic growth on DMSO is not synthesized in the R. capsulatus fnrL mutant. In contrast to wild-type strains, R. sphaeroides and R. capsulatus fnrL mutants do not synthesize the anaerobically, DMSO-induced reductase. Mechanisms that explain the basis for FnrL function in both organisms are discussed.

Differential levels of specific cytochrome c biogenesis proteins in response to oxygen: analysis of the ccl operon in Rhodobacter capsulatus.

The photosynthetic bacterium Rhodobacter capsulatus synthesizes c-type cytochromes under a variety of growth conditions. For example, under aerobic growth, c-type cytochromes are synthesized as part of an electron transport pathway, using oxygen as the terminal electron acceptor. Anaerobically in the light, R. capsulatus requires cytochrome bc1 and other c-type cytochromes for the photosynthetic electron transport pathway. It is shown here that the ccl1 and ccl2 genes of R. capsulatus are required for the synthesis of all c-type cytochromes, including the cytochrome c' protein of unknown function but of structural similarity to cytochrome b562. Polar and nonpolar mutations constructed in each gene demonstrated that the ccl12 genes form an operon. Expression of the ccl12 genes was examined by using lacZ and phoA fusions as translational reporters. Primer extension analysis was used to determine transcriptional control and the start site of the ccl12 promoter. Finally, antiserum to the Ccl2 protein was used to quantitate levels of Ccl2 under six different growth conditions. The Ccl2 protein is present at 20-fold-higher levels under conditions where oxygen is present. In contrast, other cytochromes c biogenesis proteins, HelA and HelX, previously shown to be part of an helABCDX operon, are at relatively similar levels under these six growth conditions. This discovery is discussed in terms of the physiology and evolution of cytochromes c biogenesis, with particular attention to oxidative environments.

Positive selection systems for discovery of novel polyester biosynthesis genes based on fatty acid detoxification.

The photosynthetic bacterium Rhodobacter capsulatus can grow with short- to long-chain fatty acids as the sole carbon source (R. G. Kranz, K. K. Gabbert, T. A. Locke, and M. T. Madigan, Appl. Environ. Microbiol. 63:3003-3009, 1997). Concomitant with growth on fatty acids is the production to high levels of the polyester storage compounds called polyhydroxyalkanoates (PHAs). Here, we describe colony screening and selection systems to analyze the production of PHAs in R. capsulatus. A screen with Nile red dissolved in acetone distinguishes between PHA producers and nonproducers. Unlike the wild type, an R. capsulatus PhaC- strain with the gene encoding PHA synthase deleted is unable to grow on solid media containing high concentrations of certain fatty acids. It is proposed that this deficiency is due to the inability of the PhaC- strain to detoxify the surrounding medium by consumption of fatty acids and their incorporation into PHAs. This fatty acid toxicity phenotype is used in selection for the cloning and characterization of heterologous phaC genes.

Polyhydroxyalkanoate production in Rhodobacter capsulatus: genes, mutants, expression, and physiology.

Like many other prokaryotes, the photosynthetic bacterium Rhodobacter capsulatus produces high levels of polyhydroxyalkanoates (PHAs) when a suitable carbon source is available. The three genes that are traditionally considered to be necessary in the PHA biosynthetic pathway, phaA (beta-ketothiolase), phaB (acetoacetylcoenzyme A reductase), and phaC (PHA synthase), were cloned from Rhodobacter capsulatus. In R. capsulatus, the phaAB genes are not linked to the phaC gene. Translational beta-galactosidase fusions to phaA and phaC were constructed and recombined into the chromosome. Both phaC and phaA were constitutively expressed regardless of whether PHA production was induced, suggesting that control is posttranslational at the enzymatic level. Consistent with this conclusion, it was shown that the R. capsulatus transcriptional nitrogen-sensing circuits were not involved in PHA synthesis. The doubling times of R. capsulatus transcriptional nitrogen-sensing circuits were not involved in PHA synthesis. The doubling times of R. capsulatus grown on numerous carbon sources were determined, indicating that this bacterium grows on C2 to C12 fatty acids. Grown on acetone, caproate, or heptanoate, wild-type R. capsulatus produced high levels of PHAs. Although a phaC deletion strain was unable to synthesize PHAs on any carbon source, phaA and phaAB deletion strains were able to produce PHAs, indicating that alternative routes for the synthesis of substrates for the synthase are present. The nutritional versatility and bioenergetic versatility of R. capsulatus, coupled with its ability to produce large amounts of PHAs and its genetic tractability, make it an attractive model for the study of PHA production.

Molecular and immunological analysis of an ABC transporter complex required for cytochrome c biogenesis.

The helABC genes are predicted to encode an ATP-binding cassette (ABC) transporter necessary for heme export for ligation in bacterial cytochrome c biogenesis. The recent discoveries of homologs of the helB and helC genes in plant mitochondrial genomes suggest this is a highly conserved transporter in prokaryotes and some eukaryotes with the HelB and HelC proteins comprising the transmembrane components. Molecular genetic analysis in the Gram-negative bacterium Rhodobacter capsulatus was used to show that the helABC and helDX genes are part of an operon linked to the secDF genes. To facilitate analysis of this transporter, strains with non-polar deletions in each gene, epitope and reporter-tagged HelABCD proteins, and antisera specific to the HelA and HelX proteins were generated. We directly demonstrate that this transporter is present in the cytoplasmic membrane as an HelABCD complex. The HelB and HelC but not HelD proteins are necessary for the binding and stability of the HelA protein, the cytoplasmic subunit containing the ATP-binding region. In addition we show that the HelA protein co-immunoprecipitates with either the HelC or HelD proteins. Thus, the HelABCD heme export complex is distinguished by the presence of four membrane-associated subunits and represents a unique subfamily of ABC transporters.

Use of heme reporters for studies of cytochrome biosynthesis and heme transport.

Strains of Escherichia coli containing mutations in the cydDC genes are defective for synthesis of the heme proteins cytochrome bd and c-type cytochromes. The cydDC genes encode a putative heterodimeric ATP-binding cassette transporter that has been proposed to act as an exporter of heme to the periplasm. To more fully understand the role of this transporter (and other factors) in heme protein biosynthesis, we developed plasmids that produce various heme proteins (e.g., cytochrome b5, cytochrome b562, and hemoglobin) in the periplasm of E. coli. By using these reporters, it was shown that the steady-state levels of polypeptides of heme proteins known to be stable without heme (e.g., cytochrome b5 and hemoglobin apoprotein) are significantly reduced in a cydC mutant. Exogenous addition of hemin to the cydC mutant still resulted in < 10% of wild-type steady-state levels of apohemoglobin in the periplasm. Since the results of heme reporter studies are not consistent with lower heme availability (i.e., heme export) in a cydC mutant, we analyzed other properties of the periplasm in cydC mutants and compared them with those of the periplasm in cydAB (encoding cytochrome bd) mutants and wild-type cells. Our results led us to favor a hypothesis whereby cydDC mutants are defective in the reduction environment within the periplasmic space. Such an imbalance could lead to defects in the synthesis of heme-liganded proteins. The heme reporters were also used to analyze strains of E. coli with a defect in genes encoding homologs of a different ABC transporter (helABC). The helABC genes have previously been shown to be required for the assembly of c-type cytochromes in Rhodobacter capsulatus (R. G. Kranz, J. Bacteriol. 171:456-464, 1989; D. L. Beckman, D. R. Trawick, and R. G. Kranz, Genes Dev. 6:268-283, 1992). This locus was shown to be essential in E. coli for endogenous cytochrome c biogenesis but not cytochrome b562 synthesis. Consistent with these and previous results, it is proposed that the HelABC transporter is specifically involved in heme export for ligation (hel). This class of periplasmic cytochromes is proposed to require heme liganding before undergoing correct folding.

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.

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).

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.

Multidose vial contamination in anesthesia.

The intent of this research was to address the following question: Will an alteration in the drug aspiration technique cause a significant difference in the incidence of multidose vial contamination? The control group consisted of multidose vials collected at the end of each day from staff anesthetists. The use of these vials reflected the practice technique of a single needle and syringe for each vial. The vial, as well as needle and syringe, were used on all cases managed for the day. The experimental group consisted of multidose vials collected at the end of each day from the four investigators. The vials and syringes were utilized in the same manner as the control group with the exception that a new needle was used each time a vial was reentered. Upon completion of the collection period, guaiac testing, using Hemoccult slides and developer, was performed on a 0.1 cc sample from each vial. A multidose vial was considered positive for blood contamination if traces of blue appeared on the Hemoccult slide in a 15-minute period. A chi-square statistic was applied to the cumulative data. The control group consisted of 492 multidose vials. Of the 492 multidose vials tested, 11 were guaiac positive, 2.24%. The experimental group consisted of 369 multidose vials. Of the 369 multidose vials, one tested guaiac positive, 0.27%. A chi-square test on the cumulative data demonstrated a significant (p less than .05) difference between the two groups. The research demonstrated that occult blood may be contained within the used multidose vials suggesting that contaminated drug may then be injected into another patient.(ABSTRACT TRUNCATED AT 250 WORDS)