DNA sequence analysis of the photosynthesis region of Rhodobacter sphaeroides 2.4.1

This paper describes the DNA sequence of the photosynthesis region of Rhodobacter sphaeroides 2.4.1^T. The photosynthesis gene cluster is located within a ~73 kb AseI genomic DNA fragment containing the puf, puhA, cycA and puc operons. A total of 65 open reading frames (ORFs) have been identified, of which 61 showed significant similarity to genes/proteins of other organisms while only four did not reveal any significant sequence similarity to any gene/protein sequences in the database. The data were compared with the corresponding genes/ORFs from a different strain of R.sphaeroides and Rhodobacter capsulatus, a close relative of R.sphaeroides. A detailed analysis of the gene organization in the photosynthesis region revealed a similar gene order in both species with some notable differences located to the pucBAC=cycA region. In addition, photosynthesis gene regulatory protein (PpsR, FNR, IHF) binding motifs in upstream sequences of a number of photosynthesis genes have been identified and shown to differ between these two species. The difference in gene organization relative to pucBAC and cycA suggests that this region originated independently of the photosynthesis gene cluster of R.sphaeroides.     

Revised Sequence and Annotation of the Rhodobacter sphaeroides 2.4.1 Genome

The DNA sequences of chromosomes I and II of Rhodobacter sphaeroides strain 2.4.1 have been revised, and the annotation of the entire genomic sequence, including both chromosomes and the five plasmids, has been updated. Errors in the originally published sequence have been corrected, and ∼11% of the coding regions in the original sequence have been affected by the revised annotation.     

Analysis of hemF gene function and expression in Rhodobacter sphaeroides 2.4.1

The hemF gene of Rhodobacter sphaeroides 2.4.1 is predicted to code for an oxygen-dependent coproporphyrinogen III oxidase. We found that a HemF- mutant strain is unable to grow under aerobic conditions. We also determined that hemF expression is controlled by oxygen, which is mediated, at least in part, by the response regulatory protein PrrA.     

DNA sequence duplication in Rhodobacter sphaeroides 2.4.1: evidence of an ancient partnership between chromosomes I and II

The complex genome of Rhodobacter sphaeroides 2.4.1, composed of chromosomes I (CI) and II (CII), has been sequenced and assembled. We present data demonstrating that the R. sphaeroides genome possesses an extensive amount of exact DNA sequence duplication, 111 kb or approximately 2.7% of the total chromosomal DNA. The chromosomal DNA sequence duplications were aligned to each other by using MUMmer. Frequency and size distribution analyses of the exact DNA duplications revealed that the interchromosomal duplications occurred prior to the intrachromosomal duplications. Most of the DNA sequence duplications in the R. sphaeroides genome occurred early in species history, whereas more recent sequence duplications are rarely found. To uncover the history of gene duplications in the R. sphaeroides genome, 44 gene duplications were sampled and then analyzed for DNA sequence similarity against orthologous DNA sequences. Phylogenetic analysis revealed that approximately 80% of the total gene duplications examined displayed type A phylogenetic relationships; i.e., one copy of each member of a duplicate pair was more similar to its orthologue, found in a species closely related to R. sphaeroides, than to its duplicate, counterpart allele. The data reported here demonstrate that a massive level of gene duplications occurred prior to the origin of the R. sphaeroides 2.4.1 lineage. These findings lead to the conclusion that there is an ancient partnership between CI and CII of R. sphaeroides 2.4.1.     

Proteomic characterization of the Rhodobacter sphaeroides 2.4.1 photosynthetic membrane: identification of new proteins

The Rhodobacter sphaeroides intracytoplasmic membrane (ICM) is an inducible membrane that is dedicated to the major events of bacterial photosynthesis, including harvesting light energy, separating primary charges, and transporting electrons. In this study, multichromatographic methods coupled with Fourier transform ion cyclotron resonance mass spectrometry, combined with subcellular fractionation, was used to test the hypothesis that the photosynthetic membrane of R. sphaeroides 2.4.1 contains a significant number of heretofore unidentified proteins in addition to the integral membrane pigment-protein complexes, including light-harvesting complexes 1 and 2, the photochemical reaction center, and the cytochrome bc(1) complex described previously. Purified ICM vesicles are shown to be enriched in several abundant, newly identified membrane proteins, including a protein of unknown function (AffyChip designation RSP1760) and a possible alkane hydroxylase (RSP1467). When the genes encoding these proteins are mutated, specific photosynthetic phenotypes are noted, illustrating the potential new insights into solar energy utilization to be gained by this proteomic blueprint of the ICM. In addition, proteins necessary for other cellular functions, such as ATP synthesis, respiration, solute transport, protein translocation, and other physiological processes, were also identified to be in association with the ICM. This study is the first to provide a more global view of the protein composition of a photosynthetic membrane from any source. This protein blueprint also provides insights into potential mechanisms for the assembly of the pigment-protein complexes of the photosynthetic apparatus, the formation of the lipid bilayer that houses these integral membrane proteins, and the possible functional interactions of ICM proteins with activities that reside in domains outside this specialized bioenergetic membrane.     

Modifying the endogenous electron fluxes of Rhodobacter sphaeroides 2.4.1 for improved electricity generation

The purple bacteria Rhodobacter sphaeroides serve as a promising biocatalyst in the photo-microbial fuel cell system (photo-MFC). This gram-negative species performs highly efficient anoxygenic photosynthesis that ensures an anaerobic environment in the anode compartment. Previous studies incorporating R. sphaeroides into photo-MFC were conducted using platinum as the anode electrode. In this study, we detected a steady current generation of R. sphaeroides in a bioelectrochemical system where glassy carbon was the working electrode and a typical growth medium was the electrolyte. The bioelectricity generation synchronized with the supplementation of reduced carbon source and showed immediate response to illumination, which strongly indicated the correlation between the observed current and the cytoplasmic quinone activity. Modifications of the endogenous electron flows mediated by quinone pool are shown to have significantly enhanced the bioelectricity generation. We anticipate that the findings in this study would advance future optimization of R. sphaeroides as an anode strain, as well as facilitate the study of bioenergetics in photosynthetic bacteria.     

Reaction centers from three herbicide-resistant mutants of Rhodobacter sphaeroides 2.4.1: sequence analysis and preliminary characterization

Many herbicides that inhibit photosynthesis in plants also inhibit photosynthesis in bacteria. We have isolated three mutants of the photosynthetic bacterium Rhodobacter sphaeroides that were selected for increased resistance to the herbicide terbutryne. All three mutants also showed increased resistance to the known electron transfer inhibitor o-phenanthroline. The primary structures of the mutants were determined by recombinant DNA techniques. All mutations were located on the gene coding for the L-subunit resulting in these changes Ile²²⁹ Met, Ser²²³ Pro and Tyr²²² Gly. The mutations of Ser²²³ is analogous to the mutation of Ser²⁶⁴ in the D1 subunit of photosystem II in green plants, strengthening the functional analogy between D1 and the bacterial L-subunit. The changed amino acids of the mutant strains form part of the binding pocket for the secondary quinone, Q _b . This is consistent with the idea that the herbicides are competitive inhibitors for the Q _b binding site. The reaction centers of the mutants were characterized with respect to electron transfer rates, inhibition constants of terbutryne and o-phenanthroline, and binding constants of the quinone UQ₀ and the inhibitors. By correlating these results with the three-dimensional structure obtained from x-ray analysis by Allen et al. (1987a, 1987b), the likely positions of o-phenanthroline and terbutryne were deduced. These correspond to the positions deduced by Michel et al. (1986a) for Rhodopseudomonas viridis.Abbreviations ATP adenosine 5-triphosphate - Bchl bacteriochlorophyll - Bphe bacteriopheophytin - bp basepair - cyt c²⁺ reduced form of cytochrome c - DEAE diethylami-noethyl - EDTA ethylenediamine tetraacetic acid - Fe²⁺ non-heme iron atom - LDAO lauryl dimethylamine oxide - Pipes piperazine-N,N-bis-2-ethane-sulfonic acid - PSII photosystem II - RC reaction center - SDS sodium dodecylsulfate - Tris tris(hydroxy-methyl)aminomethane - UQ₀ 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50     

From redox flow to gene regulation: role of the PrrC protein of Rhodobacter sphaeroides 2.4.1

Activation of photosynthesis (PS) gene expression by the PrrBA two-component activation system in Rhodobacter sphaeroides 2.4.1 results from the interruption of an inhibitory signal originating from the cbb(3) cytochrome c oxidase via its interaction with oxygen, in conjunction with the Rdx redox proteins. The CcoQ protein, encoded by the ccoNOQP operon, which encodes the cbb(3) cytochrome c oxidase, was shown to act as a "transponder" that conveys the signal derived from reductant flow through cbb(3) to oxygen, to the Prr system. To further define the elements comprising this signal transduction pathway we considered the prrC gene product, which to date possessed no definable role in this signal transduction pathway despite its being part of the prrBCA gene cluster. Similar to mutations in cbb(3) and rdx, suitably constructed prrC deletion mutations lead to PS gene expression in the presence of high oxygen. Unlike mutations that remove cbb(3) terminal oxidase activity or Rdx function, the PrrC deletion mutant shows no effect upon cbb(3) activity, nor does it affect the ratio of the carotenoid (Crt) spheroidene (SE) to spheroidenone (SO). Thus, the PrrC deletion mutant behaves identically to the CcoQ deletion mutant. Taking these and previous results together, we suggest that PrrC is located upstream of the two-component PrrBA activation system in the signal transduction pathway but downstream of the cbb(3) cytochrome c oxidase and its "transponder" CcoQ. The PrrC deletion mutant was also shown to lead to an increase in the DorA protein under aerobic conditions as was shown earlier for the cbb(3) mutant. Finally, PrrC is a member of a highly conserved family of proteins found in both prokaryotes and eukaryotes, and this appears to be the first instance in which a direct regulatory role has been ascribed to a member of this protein family.     

Topological analysis of the membrane-localized redox-responsive sensor kinase PrrB from Rhodobacter sphaeroides 2.4.1.

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.