The 2.7 kilobase transcript of the puf operon of the photosynthetic bacterium Rhodobacter capsulatus has five open reading frames. The gene products of four of these open reading frames (pufB, A, L,and M) are well characterized as structural polypeptides of the reaction center (pufL and M) and the B870 light-harvesting antenna complex (pufB and A), The role of the pufX gene product has been unknown. By deleting the pufX gene from a plasmid carrying the puf operon and using this plasmid to reconstitute a strain of R. capsulatus which had the puf operon deleted, it was possible to characterize the pufX gene product. It was found that the pufX⁻ mutant was unable to grow photosynthetically until a secondary suppressor mutation had occurred. It appeared that either more than one type of suppressor mutation could occur or that one suppressor mutation could be accompanied by further mutations. To determine the nature of the lesion caused by the deletion of pufX, the structure of the photosynthetic unit and the ability of the subunits of the photosynthetic unit to accomplish energy and electron transfer of the mutant were compared to a pseudo-wild type. Spectrophotometric techniques, including fluorescence detection, reduced minus oxidized spectra, flash-induced absorbance change spectra, and ground state absorption spectra were used for these comparisons as well as biochemical assays. The biochemical assays measured the ability of chromatophores to transfer electrons from a quinone analog to horse-heart cytochrome c and to pump protons in response to light irradiation. The results of these comparisons indicated that the individual components of the photosynthetic unit functioned normally but that electron transfer between these components, specifically between the reaction center and the cytochrome b⁄c₁ complex, was impaired. It thus seemed likely that there was some structural defect in the photosynthetic unit. The structure of the photosynthetic units of pseudo-wild type and mutant strains was probed using sodium dodecyl sulfate-polyacrylamide gels, absorption spectroscopy, and electron microscopy. It was determined that the mutant had chromatophore vesicles that were about 50% larger than those of the pseudo-wild type and contained higher levels of reaction center and B870 light-harvesting antenna polypeptides. The suppressor mutants also had altered levels of polypeptides and showed differences in the way the expression of their B800-850 polypeptides was regulated. It was concluded that the pufX gene product plays a role in the correct assembly of the photosynthetic unit as a structural component of the unit and/or as a regulator of its assembly. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The internal organisation of bacteria is far more complex than originally thought. Many components of the cell have specific localisation patterns. Proteins are localised to many different regions of the cell by numerous mechanisms, and often their function depends on correct localisation. Bacterial and plasmid DNA are also highly organised and actively positioned. These tightly regulated positioning patterns ensure stable maintenance of genetic material. Members of the ParA/MinD family of ATPases are responsible for the segregation of a large number of bacterial chromosomes and plasmids. Recently members of this family have been shown to position and segregate protein complexes. One such complex is the cytoplasmic chemosensory cluster of Rhodobacter sphaeroides. This large complexes are segregated from a single cluster positioned at the mid-cell to two clusters at 1/4, 3/4 positions by the ParA homologue PpfA using the nucleoid as a scaffold. This ensures that each daughter cell inherits a cluster. This study sought to investigate this cytoplasmic chemosensory cluster, and its positioning and segregation by PpfA through the cell cycle. The use of fluorescence recovery after photobleaching revealed that like membrane bound chemoreceptor arrays the cytoplasmic cluster of R. sphaeroides is a highly stable complex. The difference seen between the cytoplasmic cluster and the data reported for the membrane bound cluster of Escherichia coli is probably due to the lack of membrane helping hold the array together. Investigation of the role of PpfA in segregation of the cytoplasmic cluster, using fluorescence imaging and single molecule tracking with a range of mutants through the cell cycle, suggest that it uses a mechanism unlike any reported for ParA homologues. Single molecule tracking of PpfA molecules shows that the chemoreceptor TlpT stabilises PpfA molecules resulting in slower diffusion of PpfA molecules at the cluster. The use of a ΔppfA mutant shows that PpfA restrains the movement of the cluster, together these results suggest a model in which TlpT stabilises PpfA’s interaction with the nucleoid and PpfA positions the cluster.

Zugl.: Bielefeld, Univ., Diss., 2009

Bochum, Univ., Diss., 2003. / Computerdatei im Fernzugriff.

Bochum, Universiẗat, Diss., 2003.

Universiẗat, Diss., 2001--Stuttgart. / Gedr. Ausg. im Inst. für Technische Biochemie der Univ. Stuttgart.

Rhodobacter capsulatus photosynthesis gene was isolated by creating in-frame fusions in a lacZ transcriptional/translational vector, and selecting for those that directed oxygen-regulated levels of β-galactosidase in R. capsulatus. One lacZ fusion isolate was used to identify an open reading frame (ORF) of unknown function and flanking sequences that promoted initiation of transcription. Interposon mutagenesis experiments identified the ORF as the bchC gene, which encodes an enzyme that catalyses the penultimate step in the biosynthesis of bacteriochlorophyll a, and also showed that the bchC gene formed an operon with the bchA gene. The nucleotide sequence of this bchC gene and its 5' regulatory region were determined. The deduced amino acid sequence showed that the bchC gene encodes a 33 kDA protein that has hydrophobic segments that could interact with a lipid membrane, and that this putative BchC protein contains a potential bacteriochlorophyll a binding site. Deletion analysis, S1-nuclease protection, and primer extension experiments showed that promoter activity was associated with sequences to which a 5' end mapped, and that these sequences had significant similarity to the proposed promoter regions of several other R. capsulatus photosynthesis genes. RNA blotting and S1-nuclease protection end-mapping experiments using bipartite probes provided direct evidence that the mRNA transcripts of the bchCA operon overlap those of the two flanking operons, the crtEF and the puf operons, such that the crtEF, bchCA, and puf operons may be cotranscribable, and that RNA polymerase may initiate transcription at one of several promoters. The significance of these overlapping mRNAs was evaluated using two interposon mutant strains, one that prevented crtEF transcripts from overlapping those the bchCA and puf operons, and the other that prevented both crtEF and bchCA transcripts from overlapping those of the puf operon. The results suggested that transcriptional readthrough stimulates promoter activity. Moreover, a pufB::lac'Z fusion could be expressed from the bchCA promoter equally as well as from the puf promoter, suggesting that these overlapping transcripts are functionally significant in the chromosomal context. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The primary amino acid sequences of the L, M, and H photosynthetic reaction center peptide subunits from a number of purple non-sulfur bacteria, including Rhodopseudomonas viridis, Rhodobacter sphaeroides, and Rhodobacter capsulatus have been previously shown to be highly homologous, and detailed X-ray crystallographic analyses of reaction centers from two species of purple non-sulfur bacteria, Rps. viridis and R. sphaeroides have shown that all recognized structural and functional features are conserved. Experiments were undertaken to determine whether genes encoding reaction center and light harvesting peptide subunits from one species could be functionally expressed in other species. Plasmid-borne copies of R sphaeroides and Rps. viridis pigment binding-peptide genes were independently introduced into a photosynthetically incompetent R. capsulatus mutant host strain, deficient in all known pigment-binding peptide genes. The R. sphaeroides puf operon, which encodes the L and M subunits of the reaction center as well as both peptide subunits of light harvesting complex I, was shown to be capable of complementing the mutant R. capsulatus host. Hybrid reaction centers, comprised of R. sphaeroides-encoded L and M subunits and an R. capsulatus-encoded H subunit, were formed in addition to the R. sphaeroides-encoded LHI complexes. These hybrid cells were capable of photosynthetic growth, but their slower growth rates under low light conditions and their higher fluorescence emission levels relative to cells containing native complexes, indicated an impairment in energy transduction. The Rps. viridis puf operon was found to be incapable of functional expression in the R. capsulatus mutant host. Introduction of a plasmid-borne copy of the Rps. viridis puhA gene, which encodes the H subunit of the reaction center, into host cells already containing the Rps. viridis puf operon, such that all structural peptides of the Rps. viridis reaction center were present, still did not permit stable assembly of Rps. viridis photosynthetic complexes. RNA blot analysis demonstrated that the barrier to functional expression was not at the level of transcription. Differences between Rps. viridis and R. sphaeroides that may account for their differing abilities to complement the R. capsulatus mutant host strain are discussed. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

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