Proteomic Characterization of the Rhodobacter sphaeroides 2.4.1 Photosynthetic Membrane: Identification of New Proteins

Abstract

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.An important aspect of renewable energy production is the capability of microorganisms to directly convert solar energy into usable biofuels. The ability to convert solar energy into chemical energy is the defining property of photosynthetic systems. This critical biological function occurs in either a specialized organelle, such as the chloroplast, or a unique membrane system in prokaryotes. Each of these photosynthetic structures employ a series of (bacterio)chlorophyll-and carotenoid-containing membrane protein complexes, which convert solar energy into chemical energy. These bioenergetic systems also contain electron carriers or enzymes to generate a transmembrane electrochemical gradient and synthesize ATP (20). Despite the global importance of solar energy utilization, there are significant gaps in our understanding of the full complement of entities needed for the function and assembly of a photosynthetic membrane.In an effort to bridge this understanding gap, we have applied advanced proteomic technologies to the characterization of the protein components of a photosynthetic membrane from the bacterium Rhodob...

Role of the Global Transcriptional Regulator PrrA inRhodobacter sphaeroides2.4.1: Combined Transcriptome and Proteome Analysis

“…Relative quantitative comparisons between proteomes associated with each cell culture were based upon two "label-free" proteomic approaches: (i) the total number of peptides detected for a protein ("mass tag") (77), and (ii) the sum of abundances of peptides (determined by integrating the areas under each peak of the LC-FTICR-MS spectra for a detected peptide) for a protein ("abundance") (8) in four replicates emanating from WT and mutant PrrA2 cultures and expressed as either a "mass tag" or an "abundance" ratio between WT and PrrA2 cells (1). By comparing these abundance metrics for each specific protein only to itself in WT cells, as opposed to PrrA2 cells, we eliminated the bias inherent to the proportionality of protein mass to its abundance in terms of the number of specific peptides detected.…”

“…For some proteins, discrepancies between both quantitative approaches were observed, which was not surprising because of the variability associated with "label-free" quantitative proteomic approaches in general (6). In light of this variability, both approaches were used for comparison with transcriptome measurements, as has been previously described (77) . FIG.…”

The Use of Chromatin Immunoprecipitation to Define PpsR Binding Activity in Rhodobacter sphaeroides 2.4.1

“…Extracts containing equal amounts of protein (as determined with a bicinchoninic acid assay kit from Pierce used as recommended by the manufacturer) were used to determine the spectral data with a UV2450 spectrophotometer (Shimadzu Corp., Columbia, MD). The B800-850 and B875 complex levels were determined using the data collected, as reported previously (52).…”

“…The R. sphaeroides strains and plasmid used in this study are shown in Table 1. R. sphaeroides strains were grown aerobically in Sistrom's minimal medium A containing succinate with a gas mixture containing 30% O 2 , 69% N 2 , and 1% CO 2 and anaerobically in the dark in Sistrom's minimal medium A supplemented with yeast extract (0.1%, wt/vol) and DMSO (0.5%, vol/vol) with a gas mixture containing 95% N 2 and 5% CO 2 (45,52). Aerobic and anaerobic cells were harvested at optical densities at 600 nm of 0.2 Ϯ 0.05 and 0.45 Ϯ 0.05, respectively.…”

Regulation of Gene Expression by PrrA in Rhodobacter sphaeroides 2.4.1: Role of Polyamines and DNA Topology

“…Rhodobacter sphaeroides 2.4.1 is a purple nonsulfur bacterium which belongs to the Alpha-3-proteobacteria, whose members exhibit diversity in both their morphological and metabolic characteristics. With the availability of its genomic DNA sequence (http://www.rhodobacter.org) and the R. sphaeroides GeneChip (67), transcriptome and proteome analyses have been performed to investigate global cellular expression profiles in response to changes in growth conditions, as well as to the presence of specific mutations (1,5,6,8,9,29,61,72,89).…”