ABSTRACT
Photosynthesis gene expression in Rhodobacter sphaeroides is controlled in part by the two-component (Prr) regulatory system composed of a membrane-bound sensor kinase (PrrB) and a response regulator (PrrA). Hydropathy profile-based computer analysis predicted that the PrrB polypeptide could contain six membrane-spanning domains at its amino terminus and a hydrophilic, cytoplasmic carboxyl terminus. Both the localization and the topology of the PrrB sensor kinase have been studied by generating a series of gene fusions with the Escherichia coli periplasmically localized alkaline phosphatase and the cytoplasmic beta-galactosidase. Eighteen prrB-phoA and five prrB-lacZ fusions were constructed and expressed in both E. coli and R. sphaeroides. Enzymatic activity assays and immunoblot analyses were performed to identify and to localize the different segments of PrrB in the membrane. The data obtained in E. coli generally correlated with the data obtained in R. sphaeroides and support the computer predictions. On the basis of the theoretical model and the results provided by these studies, a topological model for the membrane localization of the PrrB polypeptide is proposed.
Subject(s)
Cell Membrane/enzymology , Membrane Proteins/chemistry , Protein Kinases/chemistry , Rhodobacter sphaeroides/enzymology , Alkaline Phosphatase/genetics , Amino Acid Sequence , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Histidine Kinase , Membrane Proteins/genetics , Models, Molecular , Molecular Sequence Data , Oxidation-Reduction , Protein Conformation , Protein Kinases/genetics , Recombinant Fusion Proteins , Rhodobacter sphaeroides/genetics , Signal Transduction , beta-Galactosidase/geneticsABSTRACT
Carotenoids are essential to protection against photooxidative damage in photosynthetic and non-photosynthetic organisms. In a previous study, we reported the disruption of crtD and crtC carotenoid genes in the purple bacterium Rubrivivax gelatinosus, resulting in mutants that synthesized carotenoid intermediates. Here, carotenoid-less mutants have been constructed by disruption of the crtB gene. To study the biological role of carotenoids in photoprotection, the wild-type and the three carotenoid mutants were grown under different conditions. When exposed to photooxidative stress, only the carotenoid-less strains (crtB-) gave rise with a high frequency to four classes of mutants. In the first class, carotenoid biosynthesis was partially restored. The second class corresponded to photosynthetic-deficient mutants. The third class corresponded to mutants in which the LHI antenna level was decreased. In the fourth class, synthesis of the photosynthetic apparatus was inhibited only in aerobiosis. Molecular analyses indicated that the oxidative stress induced mutations and illegitimate recombination. Illegitimate recombination events produced either functional or non-functional chimeric genes. The R. gelatinosus crtB- strain could be very useful for studies of the SOS response and of illegitimate recombination induced by oxidants in bacteria.
Subject(s)
Alkyl and Aryl Transferases , Carotenoids/genetics , Recombination, Genetic , Rhodospirillaceae/genetics , Rhodospirillaceae/metabolism , Aerobiosis , Amino Acid Sequence , Bacteriochlorophylls/genetics , Carotenoids/biosynthesis , Genes, Bacterial , Geranylgeranyl-Diphosphate Geranylgeranyltransferase , Molecular Sequence Data , Mutation , Oxidative Stress , Phenotype , Photochemistry , Photosynthesis/genetics , Rhodospirillaceae/radiation effects , Sequence Homology, Amino Acid , Transferases/genetics , Transferases/metabolismABSTRACT
Rubrivivax gelatinosus mutants affected in the carotenoid biosynthesis pathways were created by interposon mutagenesis within the puf operon. Genetic and biochemical analysis of several constructed mutants suggest that at least crtC is localized downstream of the puf operon and that it is cotranscribed with this operon. Sequence analysis confirmed the genetic data and showed the presence of crtD and crtC genes downstream of the puf operon, a localization different from that known for other purple bacteria. Inactivation of the crtD gene indicated that the two crt genes are cotranscribed and that they are involved not only in the hydroxyspheroidene biosynthesis pathway as in Rhodobacter sphaeroides and R. capsulatus, but also in the spirilloxanthin biosynthesis pathway. Carotenoid genes implicated in the spirilloxanthin biosynthesis pathway were thus identified for the first time. Furthermore, analysis of carotenoid synthesis in the mutants gave genetic evidence that crtD and crtC genes are cotranscribed with the puf operon using the oxygen-regulated puf promoter.
Subject(s)
Alkyl and Aryl Transferases , Genes, Bacterial , Operon , Oxidoreductases/genetics , Rhodospirillaceae/genetics , Amino Acid Sequence , Carotenoids/metabolism , Cloning, Molecular , DNA Transposable Elements , Geranylgeranyl-Diphosphate Geranylgeranyltransferase , Molecular Sequence Data , Mutagenesis , Rhodospirillaceae/metabolism , Sequence Homology, Amino AcidABSTRACT
Gene transfer systems were developed in Rubrivivax (Rx.) gelatinosus S1. First, a system for conjugative transfer of mobilizable plasmids from Escherichia coli to Rx. gelatinosus S1 was established. Secondly, optimal conditions for the transformation of Rx. gelatinosus S1 by electroporation were determined. A delta puf strain was constructed. Complementation with the puf operon from a wild-type strain cloned in a replicative plasmid restored photosynthetic growth. Two insertion strains were also selected. All the strains constructed were green, due to a change in carotenoid content. Characterization of these strains provides genetic evidence for a "superoperon" organization in this bacterium.
Subject(s)
Bacterial Proteins , Gene Deletion , Gene Transfer Techniques , Light-Harvesting Protein Complexes , Photosynthetic Reaction Center Complex Proteins/genetics , Rhodospirillaceae/genetics , DNA Transposable Elements , Electroporation , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Genetic Complementation Test , Phenotype , Plasmids/genetics , Transformation, BacterialABSTRACT
Rubrivivax gelatinosus is a facultative phototrophic non-sulfur bacterium belonging to the beta subclass of the purple bacteria. A terbutryn-resistant mutant of R. gelatinosus has been isolated and characterized. Increased resistance levels to terbutryn (300-fold), atrazine (6-fold) and o-phenanthroline (3-fold) were observed for the mutant compared with wild type. Sequence analysis of the mutant revealed a new mutation in the pufL gene coding for the L subunit of the reaction centre (RC) at codon 192 leading to an amino-acid substitution from Gly in the wild type to Asp in the mutant. This substitution is located in the D helix of the L subunit, suggesting an interaction between terbutryn and this part of the polypeptide in the RC of R. gelatinosus. This is the first report of a mutation leading to herbicide resistance and affecting the D helix in purple bacteria. Furthermore R. gelatinosus wild type is highly sensitive to o-phenanthroline compared with other purple bacteria (Rhodobacter capsulatus and Rhodobacter sphaeroides). Sequence comparison of the L subunit from six purple bacteria in which o-phenanthroline sensitivity was measured suggests that SerL226 might be responsible for this phenotype.
