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.     

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.     

In Vivo Analysis of Cobinamide Salvaging in Rhodobacter sphaeroides Strain 2.4.1

The genome of Rhodobacter sphaeroides encodes the components of two distinct pathways for salvaging cobinamide (Cbi), a precursor of adenosylcobalamin (AdoCbl, coenzyme B₁₂). One pathway, conserved among bacteria, depends on a bifunctional kinase/guanylyltransferase (CobP) enzyme to convert adenosylcobinamide (AdoCbi) to AdoCbi-phosphate (AdoCbi-P), an intermediate in de novo AdoCbl biosynthesis. The other pathway, of archaeal origin, depends on an AdoCbi amidohydrolase (CbiZ) enzyme to generate adenosylcobyric acid (AdoCby), which is converted to AdoCbi-P by the AdoCbi-P synthetase (CobD) enzyme. Here we report that R. sphaeroides strain 2.4.1 synthesizes AdoCbl de novo and that it salvages Cbi using both of the predicted Cbi salvaging pathways. AdoCbl produced by R. sphaeroides was identified and quantified by high-performance liquid chromatography and bioassay. The deletion of cobB (encoding an essential enzyme of the de novo corrin ring biosynthetic pathway) resulted in a strain of R. sphaeroides that would not grow on acetate in the absence of exogenous corrinoids. The results from a nutritional analysis showed that the presence of either CbiZ or CobP was necessary and sufficient for Cbi salvaging, that CbiZ-dependent Cbi salvaging depended on the presence of CobD, and that CobP-dependent Cbi salvaging occurred in a cbiZ⁺ strain. Possible reasons why R. sphaeroides maintains two distinct pathways for Cbi salvaging are discussed.Cobamides, such as adenosylcobalamin (AdoCbl, coenzyme B₁₂), are a group of complex cobalt-containing cyclic tetrapyrrole cofactors whose biosynthesis by bacteria and archaea requires substantial genetic information (>25 genes) (reviewed in references 25, 47, and 56). Two pathways for the de novo synthesis of the corrin ring have been described on the basis of the timing of cobalt insertion into the ring. The late cobalt insertion or aerobic pathway has been well studied in Pseudomonas denitrificans (9), while the early cobalt insertion or anaerobic pathway has been best studied in Salmonella enterica serovar Typhimurium LT2 (25). Many organisms, including those that synthesize AdoCbl de novo, salvage incomplete corrinoids (e.g., cobinamide [Cbi]) from their environments and use them as precursors for the synthesis of complete cobamide cofactors. Cbi is not an intermediate of the de novo AdoCbl biosynthesis pathway but can be converted into one by a process known as Cbi salvaging (Fig. ​(Fig.1)1) (24).Open in a separate windowFIG. 1.Abbreviated view of cobinamide salvaging pathways. Corrin ring-containing intermediates are in bold text. The letter A indicates the de novo corrin ring biosynthesis pathway. Abbreviations: Ado-, adenosyl-; AP, 1-amino-2-propanol; AP-P, 1-amino-2-propanol-phosphate; CobB, hydrogenobyrinic acid a,c-diamide synthase; CobD, adenosylcobinamide-phosphate synthetase; CobP, NTP:adenosylcobinamide kinase, GTP:adenosylcobinamide-phosphate guanylyltransferase; CobY, GTP:adenosylcobinamide-phosphate guanylyltransferase; CbiZ, adenosylcobinamide amidohydrolase. Functional groups are indicated as follows: Me, methyl; Ac, acetamide; and Pr, propionamide.The first step of Cbi salvaging is adenosylation of the molecule to adenosylcobinamide (AdoCbi) (24). The adenosyltransferases which catalyze this reaction are broadly distributed throughout the three domains of life (13, 14, 20, 32, 38). Two distinct pathways for converting AdoCbi into an intermediate of the de novo AdoCbl biosynthesis pathway have been described for prokaryotes. One, which is to date found only in bacteria, relies on a bifunctional nucleoside triphosphate (NTP):AdoCbi kinase (EC 2.7.7.62), GTP:AdoCbi-phosphate (AdoCbi-P) guanylyltransferase (EC 2.7.1.156) enzyme (called CobP in P. denitrificans and CobU in S. Typhimurium), which phosphorylates AdoCbi to AdoCbi-P and converts AdoCbi-P to AdoCbi-GDP (10, 41, 55).Previous work from our laboratory has shown that archaea lack the bifunctional NTP:AdoCbi kinase, GTP:AdoCbi-P guanylyltransferase enzyme and rely on a second pathway for Cbi salvaging (54, 62). In this pathway, AdoCbi is converted to adenosylcobyric acid (AdoCby) by an AdoCbi amidohydrolase (EC 3.5.1.90) known as CbiZ (58, 59, 62). The conversion of AdoCbi-P to AdoCbi-GDP for de novo AdoCbl biosynthesis in archaea is catalyzed by a monofunctional GTP:AdoCbi-P guanylyltransferase (EC 2.7.7.62) called CobY (54, 60), which has not been found in any bacterium.We recently showed that a small percentage of bacterial genomes encode orthologs of both CobP-type and CbiZ-type Cbi salvaging enzymes, raising the question of why these organisms might contain two redundant Cbi salvaging systems (29). A phylogenetic analysis showed that CbiZ has its roots in the archaea and that the cbiZ gene was acquired by several bacterial lineages via horizontal gene transfer.We previously showed that the CbiZ and CobP enzymes from the photosynthetic alphaproteobacterium Rhodobacter sphaeroides are functional in vitro and in vivo in a heterologous complementation system (29). However, the question of how the two Cbi salvaging systems might function in R. sphaeroides remained unresolved.In this paper, we show that R. sphaeroides 2.4.1 synthesizes substantial amounts of cobalamin (Cbl) and that it salvages incomplete corrinoids from its environment. We present in vivo genetic evidence that both the bacterial-type CobP-dependent and archaeal-type CbiZ-dependent Cbi salvaging pathways are functional in this organism. This work represents the first in vivo genetic analysis of coenzyme B₁₂ synthesis and salvaging in R. sphaeroides.     

The photosynthetic apparatus of Rhodobacter sphaeroides.

Functional and ultrastructural studies have indicated that the components of the photosynthetic apparatus of Rhodobacter sphaeroides are highly organized. This organization favors rapid electron transfer that is unimpeded by reactant diffusion. The light-harvesting complexes only partially surround the photochemical reaction center, which ensures an efficient shuttling of quinones between the photochemical reaction center and the bc1 complex.     

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.     

The flagellar muramidase from the photosynthetic bacterium Rhodobacter sphaeroides

We have characterized open reading frame RSP0072, which is located within the flgG operon in Rhodobacter sphaeroides. The amino acid sequence analysis of this gene product showed the presence of a soluble lytic transglycosylase domain. The deletion of the N-terminal region (90 amino acids) of the product of RSP0072 yields a leaky nonmotile phenotype, as determined by swarm assays in soft agar. Electron micrographs revealed the lack of flagella in mutant cells. The purified wild-type protein showed lytic activity on extracts of Micrococcus luteus. In contrast, no lytic activity was observed when the residues E57 or E83 were replaced by alanine. Affinity blotting suggests that the protein encoded by RSP0072 interacts with the flagellar rod-scaffolding protein FlgJ, which lacks the muramidase domain present in FlgJ of many bacteria. We propose that the product of RSP0072 is a flagellar muramidase that is exported to the periplasm via the Sec pathway, where it interacts with FlgJ to open a gap in the peptidoglycan layer for the subsequent penetration of the nascent flagellar structure.     

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     

Supramolecular organization of the photosynthetic apparatus of Rhodobacter sphaeroides.

Native tubular membranes were purified from the purple non-sulfur bacterium Rhodobacter sphaeroides. These tubular structures contain all the membrane components of the photosynthetic apparatus, in the relative ratio of one cytochrome bc1 complex to two reaction centers, and approximately 24 bacteriochlorophyll molecules per reaction center. Electron micrographs of negative-stained membranes diffract up to 25 A and allow the calculation of a projection map at 20 A. The unit cell (a = 198 A, b = 120 A and gamma = 103 degrees) contains an elongated S-shaped supercomplex presenting a pseudo-2-fold symmetry. Comparison with density maps of isolated reaction center and light-harvesting complexes allowed interpretation of the projection map. Each supercomplex is composed of light-harvesting 1 complexes that take the form of two C-shaped structures of approximately 112 A in external diameter, facing each other on the open side and enclosing the two reaction centers. The remaining positive density is tentatively attributed to one cytochrome bc1 complex. These features shed new light on the association of the reaction center and the light-harvesting complexes. In particular, the organization of the light-harvesting complexes in C-shaped structures ensures an efficient exchange of ubihydroquinone/ubiquinone between the reaction center and the cytochrome bc1 complex.     

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.     

The assembly and organisation of photosynthetic membranes in Rhodobacter sphaeroides.

Recent AFM data demonstrate that mature photosynthetic membranes of R. sphaeroides are composed of rows of dimeric RC-LH1-PufX complexes with some LH2 complexes 'sandwiched' between these rows of core complexes, and others in discrete LH2-only domains which might form the light-responsive complement of the LH2 antenna. The present work applies membrane fractionation, radiolabelling and LDS-PAGE techniques to investigate the response of R. sphaeroides to lowered light intensity. The kinetics underlying this adaptation to low light conditions were revealed by radiolabelling with the bacteriochlorophyll (bchl) biosynthetic precursor, delta-aminolevulinate, which allowed us to measure only the bchls synthesised after the light intensity shift. We show that (1) the increase in LH2 antenna size is mainly restricted to the mature ICM membrane fraction, and the antenna composition of the precursor upper pigmented band (UPB) membrane remains constant, (2) the precursor UPB membrane is enriched in bchl synthase, the terminal enzyme of the bchl biosynthetic pathway, and (3) the LH2 and the complexes of intermediate migration in LDS-PAGE exhibit completely different labelling kinetics. Thus, new photosynthetic complexes, mainly LH2, are synthesised and assembled at the membrane initiation UPB sites, where the LH2 rings pack between the rows of dimeric cores fostering new LH2-LH1 interactions. Mature membranes also assemble new LH2 rings, but in this case the 'sandwich' regions between the rows of core dimers are already fully occupied and the bulk antenna pool is the favoured location for these new LH2 complexes.