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Journal of Bacteriology, October 2007, p. 7464-7474, Vol. 189, No. 20
0021-9193/07/$08.00+0     doi:10.1128/JB.00946-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Xiaohua Zeng,¹ Jung Hyeob Roh,¹ Stephen J. Callister,² Christine L. Tavano,³ Timothy J. Donohue,³ Mary S. Lipton,² and Samuel Kaplan^1*

Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, Texas 77030,¹ Biological Separations and Mass Spectrometry, Pacific Northwest National Laboratory, Richland, Washington 99352,² Department of Bacteriology, University of Wisconsin—Madison, Madison, Wisconsin 53706³

Received 14 June 2007/ Accepted 8 August 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 bc₁ 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.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 Rhodobacter sphaeroides as a model system. Solar energy is captured by the R. sphaeroides intracytoplasmic membrane (ICM), a specialized domain of the cytoplasmic membrane (CM) that contains proteins required for converting light into chemical energy. While the subunits of several supramolecular complexes within the R. sphaeroides ICM have been extensively studied by biochemical, biophysical, and molecular strategies, a complete delineation of the ICM composition has not been entertained (1, 14, 22, 25). Here, we test the hypothesis that numerous proteins, in addition to those already extensively studied, exist within the ICM and this enumeration and study will lead to a fuller understanding of photosynthesis. Recently, multichromatographic methods coupled with Fourier transform ion cyclotron resonance mass spectrometry have been applied to the analysis of R. sphaeroides membrane proteins to test the hypothesis stated above (2, 3). Using this approach, we have delineated many of the proteins associated with the R. sphaeroides ICM, to more fully understand the functional and structural organization of a photosynthetic membrane. Thus, we extend our characterization of the ICM into advancing the ability to directly harvest solar energy for the creation of biofuels or other human activities. This approach has proven successful by virtue of the direct demonstration that "newly" discovered gene products localized to the ICM, when removed by mutation, reveal an altered phenotype during photosynthetic growth.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Strains and growth conditions. All strains and plasmids used in this study are shown in Table 1. R. sphaeroides 2.4.1 (NC_007493) was grown in Sistrom's minimal medium A containing succinate (6). Cells designated aerobic cells were grown by sparging with 30% O₂, 69% N₂, and 1% CO₂; semiaerobic cells were grown by sparging with 2% O₂, 97% N₂, and 1% CO₂; photosynthetic cells were grown anaerobically at a light intensity of 3 W/m² by sparging with 95% N₂ and 5% CO₂; and dark-dimethyl sulfoxide (dark-DMSO) cells were grown anaerobically in the dark in the presence of 1% yeast extract and 0.5% DMSO.

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TABLE 1. Bacterial strains and plasmids

Determination of protein concentration and SDS-PAGE. Protein concentrations were determined using a bicinchoninic acid assay (Pierce, Rockford, IL) with bovine serum albumin as the standard. Polypeptide profiles of ICM vesicles were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (17).

Construction of in-frame mutations and plasmid derivatives. In-frame deletion mutations RSP1760 and RSP1467 were constructed by deleting an internal 636-bp fragment of AffyChip designation RSP1760 and a 1,170-bp fragment of RSP1467, respectively, according to the previous protocol using vector pLO1 (31) (Table 1). The resulting constructions were confirmed by DNA sequencing and were transferred into Escherichia coli S17-1. The conjugation and selection of mutant strains were carried out by using methods described previously (31). Plasmids pR1467his and pR1760his were constructed by fusing 10 histidine codons to the 3' terminus of the RSP1467 and RSP1760 genes, respectively (Table 1). All mutants were further confirmed by PCR amplification and DNA sequencing from the chromosome.

Cell fractionation and purification of photosynthetic complexes. Aerobic and photosynthetic cells were harvested at an optical density at 600 nm (OD₆₀₀) of 0.14 and 0.5, respectively, and broken by French press. The cell debris and unbroken cells were removed by centrifugation at 12,000 x g for 30 min, and the resulting crude cell extracts were centrifuged at 260,000 x g for 1 h to obtain the membrane fractions. To purify the ICM vesicles, the membrane fractions were resuspended in 0.1 M sodium phosphate buffer (pH 7.6) containing 0.01 M EDTA, applied to Sepharose 2B chromatography (5 by 60 cm), and subjected to sucrose gradient centrifugation (0.5 to 1.0 M) at 6,3500 x g for 10 h to obtain the purified ICM vesicles (9). To partially purify the cytochrome (cyt) bc₁, light-harvesting complex 1 (LH1), and LH2, the membranes were solubilized in 1% n-dodecyl-ß-maltoside and analyzed by sucrose gradient ultracentrifugation (0.2 to 0.7 M) at 100,000 x g for 16 h. The proteins were collected and analyzed by spectroscopic c-type cyt staining (25, 27, 31). To isolate the cytoplasm, inner membrane (IM), periplasm, and outer membrane (OM) cellular fractions, cells were treated with lysozyme followed by differential ultracentrifugation (27).

Determination of light-harvesting complexes and pigments. The amount of B800-850 was measured at OD₈₄₉ to OD₉₀₀ ( = 96 ± 4 mM^–1 cm^–1), assuming using 3 mol of bacteriochlorophyll (Bchl) a per mol of complex, and the amount of B875 light-harvesting complexes was measured at OD₈₇₅ to OD₈₂₀ ( = 73 ± 2.5 mM^–1 cm^–1), assuming 2 mol of Bchl a per mol of complex, as described elsewhere (30). Carotenoids were extracted and quantitated as previously described (32), and the resulting carotenoids from the cells of the 100-ml growing culture were dissolved into a 40-µl mixture of acetone/methanol (7:2, vol/vol) and applied to high-pressure liquid chromatography (HPLC) analysis. Absorption spectra were analyzed with an SLM DW2000 double-beam UV-visible infrared spectrophotometer (SLM Aminco Instruments, Urbana, IL).

HPLC analysis of pigments was performed with a Spherisorb ODS2 (Rainin Instruments Company, Emeryville, CA) reverse-phase column (25 by 0.46 cm) equipped with a Shimadzu SPD-M20A diode array detector (Shimadzu Instruments Company, Emeryville, CA) as described previously (30).

Peptide purification, protein identification, and proteomic data processing. The proteins were digested with trypsin and applied to multichromatography coupled with Fourier transform ion cyclotron resonance mass spectrometry to identify peptides (2, 3). The identified peptides were compared to the annotated R. sphaeroides 2.4.1 genome (NCBI website or http://www.jgi.doe.gov) (18). A total of 12 replicates from 3 different ICM samples were analyzed to produce this proteomic data set. The proteomic profile of each protein includes the numbers of accurate mass and time (AMT) tags (peptides) and unique peptides and the protein percentage coverage for a protein in all 12 replicates; AMT tag is defined as a peptide detected using high mass measurement accuracy mass spectrometry and elution time measurements. Protein percentage coverage is calculated based on the number of amino acid residues observed divided by the total number of amino acid residues for the protein (3).

Since ICM vesicles are physically connected to the CM, and because of the sensitivity of these methods, we used other complementary approaches to analyze the ICM protein profiles. The ICM vesicles (40- to 60-nm diameter) were purified by spectral features as well as size (9), such that ribosomes (21-nm diameter) as well as fragments derived from the CM or OM are likely sources of contamination. Therefore, OM proteins, ribosomal proteins, and polypeptides predicted by their annotation to be involved in metabolism, transcription, or translation were considered to be contaminants and excluded from the final analysis of the ICM proteins. In addition, previous studies predict that <10% of the ICM fraction is derived from other subcellular fractions (9). To help distinguish ICM-detected proteins that may derive from other subcellular fractions, we also determined the proteome profile of periplasmic, cytoplasmic, OM, and IM samples from R. sphaeroides 2.4.1 cells by the same approach (2, 3). Thus, the abundance of each peptide within the ICM was compared to that in all other subcellular fractions. Peptides with an ICM abundance of >10% of that detected in other subcellular fractions were considered as candidates for further analysis.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
ICM proteome. To define the number and types of protein components present within a photosynthetic membrane, we generated a proteomic map of purified ICM vesicles from R. sphaeroides 2.4.1 utilizing the high-throughput AMT tag proteomic approach (2, 3). In this study, more than 1,500 peptides were detected and over 600 proteins were identified in association with the ICM vesicles. Out of these 600 proteins, 153 proteins had at least one peptide that was identified in all 12 ICM samples from three independently grown cultures of photosynthetic cells. By filtering the contaminant proteins from other subcellular fractions and ribosomes using the two complementary approaches (see Materials and Methods), 98 proteins were identified as being present in the ICM samples. The number of AMT tags (peptides) for these proteins, the number of unique peptides, and the protein percentage coverage for each protein in all 12 samples are provided in Table S1 in the supplemental material, to indirectly reflect the levels of each protein in these ICM vesicles.

To determine the precise subcellular localizations of the 98 proteins associated with the ICM, the proteomic profiles of the ICM were compared with those of the OM, the IM, a fraction containing both ICM and CM, the periplasm, and the cytoplasm (2). Among the 98 identified ICM proteins, 70 proteins which possessed a larger number of AMT tags and a higher percentage of protein coverage in the IM relative to the cytoplasm, periplasm, and OM were detected, suggesting that these proteins or some subset of these proteins are ICM or/and CM associated, since the IM sample analyzed in this study contains a "mixture" of the ICM and CM and is thus uniquely designated IM (9, 27). To help distinguish if an individual protein/protein complex was localized to the ICM or the CM, the proteomic profiles of the 70 proteins in the ICM vesicles were compared with those of the IM of photosynthetic or the CM of aerobic cells, which lack ICM (for direct comparison, the protein numbering used in Fig. 1A to D and Table S1 in the supplemental material are the same). The other 28 ICM proteins have higher AMT tag levels and a higher percentage of protein coverage in the OM, periplasm, or cytoplasm than in the CM or ICM (Table 2). Among these 28 proteins, six are periplasmic subunits of ABC or TRAP transporters; one is cyt c₂, which serves as a periplasmic electron carrier in the photosynthetic and respiratory pathways (5, 21); 12 are cytoplasmic enzymes for amino acid, carbohydrate, or cofactor metabolism or modification; one, PrrA, is a transcription factor in the PrrBA regulatory system for photosynthesis gene expression; and six are predicted OM proteins. Except for one protein (RSP0841), all 12 of the OM and periplasmic proteins are predicted by using SignalP 3.0 server software to contain a signal peptide; this further suggests a nonspecific association or engulfment of these proteins within the sealed membrane vesicles that comprise the ICM. Only two proteins (RSP2589 and RSP2352) have similar AMT tag levels detected across most subcellular fractions of photosynthetic cells, so it is difficult to distinguish their specific localization.

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FIG. 1. Proteomic profiles of 70 ICM- and IM-associated proteins identified in ICM vesicles of R. sphaeroides grown anaerobically in the light at 3 W/m2. (A and B) Comparison of the numbers of AMT tags for the 70 ICM- and IM-associated proteins identified in the ICM with those identified in the IM of the photosynthetic cells and with those identified in the IM of aerobic cells, respectively. (C and D) Comparison of protein coverage percentages for 70 ICM- and IM-associated proteins identified in the ICM with those identified in the IM of photosynthetic cells and with those identified in the IM of aerobic cells, respectively. (E) Relative RNA abundance (microarray data) from genes corresponding to 70 ICM- and IM-associated proteins in the photosynthetic cells (http://www.rhodobacter.org) (24). The same number is used for each gene or its protein. 1 to 10, the ICM-unique proteins; 11 to 28, the ICM-localized proteins; and 29 to 70, the CM-enriched proteins. AE, aerobic cells; 3W, photosynthetic cells.

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TABLE 2. Subcellular distribution of AMT tags for 28 ICM-identified proteins which are possibly localized in the periplasm, cytoplasm, or OM of R. sphaeroides cellsa

ICM-unique proteins. Theoretically, ICM-unique proteins should be detected only in the ICM vesicles, and their abundance in the ICM should be greater than that detected in the IM of photosynthetic cells, a fraction of the cellular membrane in which CM and ICM are physically contiguous (27). However, no ICM-unique protein should be present in the CM of aerobic cells, since aerobic cells lack the ICM and its pigment-protein complexes (9). Therefore, we established several criteria which must be met to define a protein as an ICM-unique protein, namely, (i) the number of AMT tags for this protein in the ICM should be >5-fold greater than that in the CM of aerobic cells, (ii) the number of AMT tags or percentage of protein coverage for the protein in the ICM should be similar to or at least more than 90% of that found in the IM of photosynthetic cells, and (iii) many or a majority of the subunits of a given protein complex should be found in the ICM. Using these criteria, we can identify 10 ICM-unique proteins (Fig. 1A to D and Table 3, protein no. 1 to 11). Among the 10 ICM-unique proteins, 7 are known subunits of pigment-protein complexes, including the ß and polypeptides of LH2 (no. 3 and 6) and LH1 (no. 9), the M, L, and H subunits (no. 1, 4, and 7) of the photochemical reaction center (RC), and the assembly factor, PufX (no. 8) for the LH1-RC-PufX core complex. RSP0258 and RSP0314 include all the peptides identified for both and ß subunits of LH1 and LH2, respectively, in this study. Each subunit of these known photosynthetic complexes has a similar percentage of protein coverage in the ICM relative to that in the IM of photosynthetic cells (Fig. 1C) and much lower or no detectable protein coverage in the CM of aerobic cells (Fig. 1D). The agreement of these results with previous reports that LH2, LH1, and the RC complex are unique ICM proteins (1, 10, 25, 29) validates the methods used to define this group of proteins and allows us to query this data set for other polypeptides with similar distributions. Thus, these internal controls are distributed as expected. Had we not found these as described, then our approach would not have been valid.

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TABLE 3. Classification of 70 ICM- and IM-associated proteins identified in ICM vesicles of R. sphaeroides cellsa

Using the same criteria, the other three ICM-unique proteins contained two proteins of unknown function (RSP1760 and RSP6124; no. 2 and 5, respectively) and a putative D-alanyl-D-alanine carboxypeptidase (no. 10, RSP3246). However, RSP3246 and RSP6124 have similar AMT tag levels and percent protein coverage in the cytoplasm of photosynthetic cells relative to that in the ICM or IM of photosynthetic cells (data not shown), and topological predictions (TMHMM server, v2.0) indicate that they lack a transmembrane domain. Thus, it appears that RSP3246 and RSP6124 are primarily cytoplasmic, but some fraction of these may be associated with the ICM, possibly because they play an unknown role in either formation or function of this membrane. Thus, these two ICM-unique proteins should be subjected to further study.

It is interesting to identify RSP1760, a membrane protein with unknown function with regard to the ICM. The RSP1760 gene (on C1, coding sequence [CDS] 342569.0.343225) is located 1.6 Mb counterclockwise from the photosynthesis gene cluster. No clue could be obtained as to its function by analyzing the nearby DNA sequences in chromosome 1. To further characterize its relation to the ICM, we made an in-frame deletion mutation of the RSP1760 gene (RSP1760). The mutation results in the failure of cell growth under photosynthetic conditions, but the cells of RSP1760 can grow aerobically as well as anaerobically in the dark with DMSO (data not shown). As shown in Table 4, little light-harvesting complex is detected under dark-DMSO conditions in which ICM is produced in abundance in wild-type cells. We also detected some carotenoids from mutant strain RSP1760. There are two major carotenoids (spheroidene and spheroidenone) in R. sphaeroides, anaerobic or dark-DMSO grown cells containing mainly spheroidene and semiaerobic cells containing predominantly spheroidenone (30). As shown in Fig. 2 the amount of carotenoids was found to be less than 10% of that observed for the wild type (Fig. 2) under semiaerobic or dark-DMSO conditions. To directly localize protein RSP1760 to the ICM, polyhistidines were tagged to the C terminus of RSP1760, and the construction (pR1760his) expressing the His-tagged RSP1760 was introduced into mutant strain RSP1760 to produce strain RSP1760 (pR1760his), which has a wild-type phenotype. These data directly show that the original in-frame mutation is not polar and that the absence of photosynthetic growth is the direct effect of the in-frame deletion of the RSP1760 gene. The mutant was grown aerobically and anaerobically in the presence of light at 3 W/m², and anti-His antibody was used to detect the protein RSP1760his. As shown in Fig. 3, no RSP1760 was observed in aerobic cells (lane 2); however, a strong band was observed in anaerobic cells (lane 3), which is coincident with the microarray and proteomic data (see below). Furthermore, protein RSP1760 is present in the ICM (lane 7) at a much higher level than that observed for the IM (lane 6). These results verify that the protein encoded by the RSP1760 gene is required for the formation of the ICM, and it is localized to the ICM, although its role in ICM formation, structure, and function remains to be determined.

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TABLE 4. Spectral complex formation of R. sphaeroides 2.4.1 and mutant strain RSP1467 and RSP1760a

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FIG. 2. HPLC analysis of carotenoids extracted from 100-ml cultures of cells of R. sphaeroides 2.4.1 and mutant strains RSP1760 and RSP1467 according to the method described in Materials and Methods. The peak indicates an elution profile monitored at 486 nm, and the elution times for spheroidenone (SO) and spheroidene (SE) are 14.5 min and 15.5 min, respectively. Semi, semiaerobic.

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FIG. 3. Western blot analysis of His-tagged alkane hydroxylase (46.0 kDa; RSP1467) and an unknown protein (25 kDa; RSP1760) in IM and ICM of the cells of mutant strains RSP1760(pR1760his) and RSP1467(pR1467his) grown aerobically or anaerobically in the presence of light intensity at 3 W/m2. Lane 1, protein standards; lane 2, RSP1760-His in aerobic cells; lane 3, RSP1760-His in anaerobic cells; lane 4, RSP1467-His in aerobic cells; lane 5, RSP1467-His in anaerobic cells; lane 6, RSP1760-His in the IM of anaerobic cells; lane 7, RSP1760-His in ICM of anaerobic cells; lane 8, RSP1467-His in the IM of anaerobic cells; lane 9, RSP1467-His in ICM of anaerobic cells.

Potential ICM-localized proteins. The ICM- and CM-associated proteins not designated as unique to the ICM were divided into potential ICM- and CM-localized proteins. Since microscopic studies indicated that the total area of the ICM is estimated to be similar to or less than that of the CM of intact cells (9) and proteins in the IM are comprised of proteins found in both CM and ICM, a protein with a number of AMT tags in the ICM that is >50% of that found in the IM of photosynthetic cells is considered to be ICM localized (Fig. 1A and D and Table 2). By these criteria, the 18 ICM-localized proteins (Fig. 1 and Table S1 in the supplemental material, protein no. 11 to 28) include four subunits of the cyt bc₁ complex (cyt b [no. 20], c₁ [no. 16], subunit IV [no. 15], and the Rieske iron-sulfur subunit [no. 14]), three proteins for solute or lipid transport and translocation (no. 21, 26, and 27), a putative alkane hydroxylase (no. 13), spheroidene monooxygenase (no. 11), a generic methyltransferase (no. 12), four proteins for amino acid, carbohydrate, lipid, and protein metabolism or modification (no. 17, 19, 22, and 23, respectively), a homolog of the YlmG cell wall/division protein (no. 28), and three proteins of unknown function (no. 18 [RSP2908], 24 [RSP3637], and 25 [RSP6001]). Among these 18 proteins, the cyt bc₁ complex (no. 14 to 16 and 20), spheroidene monooxygenase (no. 11), and two proteins with general functions (an alkane hydroxylase [no. 13] and a generic methyltransferase [no. 12]) were found to have significantly higher levels of AMT tags and percent protein coverage in the ICM than the CM of aerobic cells (Fig. 1B and D), but they each have either a lower or a similar level of AMT tags and percentage of protein coverage in the ICM and the IM of photosynthetic cells (Fig. 1A and C). These results indicate that the latter seven proteins are likely to be abundant in both the ICM and the IM of photosynthetic cells and may play an important role in ICM structure-function.

In an attempt to test the roles of these potential ICM-localized proteins, an alkane hydroxylase (RSP1467, no. 13) was selected for further study as to its effect on ICM formation. The RSP1467 gene (on C1, CDS 52800.0.54071) is located 1.9 Mb counterclockwise from the photosynthesis gene cluster, and an in-frame deletion of the RSP1467 gene encoding the alkane hydroxylase was constructed (RSP1467). The mutant was found to be capable of forming an ICM and had levels of light-harvesting complexes (Table 4) that were similar to or slightly lower than to those of wild-type cells grown anaerobically in the presence of a light intensity of 3 W/m² or in dark-DMSO conditions. Similar amounts of carotenoids (Fig. 2) were observed for mutant RSP1467 relative to those of wild-type R. sphaeroides 2.4.1 under semiaerobic or Dark-DMSO conditions. However, the doubling time for the RSP1467 mutant growth is 30% of that of the wild type under anaerobic conditions in the presence of saturating light intensities (100 W/m²) (Fig. 4). Its growth rate is similar to that of the wild type under aerobic or anaerobic dark-DMSO conditions, suggesting that the loss of RSP1467 leads to a specific defect in photosynthetic growth. To directly localize protein RSP1467, His-tagged RSP1467 was introduced into the mutant strain RSP1467. As shown in Fig. 4, the growth rate of the strain RSP1467(pR1467his) is similar to that of the wild type, again demonstrating that this mutation is not polar. The Western blot analysis in Fig. 3 indicates that no RSP1467 was observed for aerobic cells (lane 4); however, a strong band was observed for anaerobic cells (lane 5), which verifies the proteomic analysis. Further, protein RSP1467 is observed to be present in both the ICM (lane 9) and the IM (lane 8). These results verify the presence of RSP1467 in the ICM, and it appears that the absence of the protein encoded by the RSP1467 gene results in changes exclusive to photosynthetic growth, perhaps by affecting the formation/function of the ICM.

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FIG. 4. Growth curves for R. sphaeroides 2.4.1 and mutant strains RSP1467 and RSP1467(pR1467his). Cells were grown anaerobically in the presence of light at 100 W/m2.

Another protein of interest is spheroidene monooxygenase, a known enzyme in Crt biosynthesis, a pigment within the light-harvesting and RC complexes of the ICM (16). Since it appears that spheroidene monooxygenase was the only pigment biosynthesis enzyme found at high levels in the ICM, we compared the proteomic profiles for other enzymes of Crt and tetrapyrrole (Bchl) biosynthesis in the CM, cytoplasm, and ICM in an effort to predict where these pigments might be synthesized. This analysis predicts that enzymes for Crt, tetrapyrrole, or Bchl biosynthesis, such as spheroidene monooxygenase (no. 11, RSP0272), hydroxyneurosporene methyltransferase (no. 59, RSP0264), magnesium-protoporphyrin IX monomethylase (no. 55, RSP0281), a HemY-like membrane protein (no. 38, RSP1508), uroporphyrinogen decarboxylase (no. 83, RSP0680), and 5-aminolevulinate synthase (no. 89, RSP2984), were detected in the ICM (Table 5). However, many of these proteins have higher levels of AMT tags and protein coverage in the cytoplasm or the CM of photosynthetic cells. In contrast, spheroidene monooxygenase (CrtA) appears to be enriched in both the CM and ICM relative to the cytoplasm of photosynthetic cells (Table 5), suggesting that this protein is present at high levels in the ICM. These other pigment biosynthesis enzymes are present in relatively small amounts in the ICM or CM, and it appears that their overall abundance in photosynthetic cells is low (Fig. 1A and C and Table 5).

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TABLE 5. Subcellular location of known enzymes in tetrapyrrole, Bchl, and Crt biosynthesis in R. sphaeroides cellsa

This analysis suggests that ICM vesicles contain high levels of photosynthetic complexes and the cyt bc₁ complex, but relatively lower levels of enzymes for Crt and Bchl biosynthesis or other physiological functions, like solute transport across the membrane. This is not an unexpected result from previous studies (1, 4).

CM-enriched proteins. Among the 42 ICM- and CM-associated proteins (Fig. 1 and Table S1 in the supplemental material, protein no. 29 to 70) are nine subunits for solute transport or protein translocation (no. 39, 46, 48, 50, 54, 58, 60, 62, and 70), seven subunits for the F₀F₁-ATPase (no. 30, 31, 34, 41, 45, 47, and 66), subunits associated with four respiratory electron carriers (no. 33, 43, 44, and 67), three enzymes in Bchl or Crt biosynthesis (no. 38, 55, and 59), 16 proteins for amino acid, carbohydrate, lipid, or protein metabolism (no. 29, 32, 35 to 37, 40, 42, 51 to 53, 57, 61, 64, 65, 68, and 69), and three proteins of unknown function (no. 49 [RSP2697], 56 [RSP1762], and 63 [RSP3985]) (Table 3 and Table S1 in the supplemental material). One of the major differences between the predicted protein profiles of the ICM and the CM is the presence of these CM-enriched proteins (Fig. 1A), which are predicted to be at low levels in the ICM. However, 14 of the CM-enriched proteins, including six subunits of the ATP synthase (no. 30, 31, 34, 41, 45, and 47), three subunits of respiratory enzymes (no. 33, 43, and 44), two subunits of the succinate dehydrogenase, two solute transporters (no. 39 and 50), and an unknown protein (no. 49) were also found (>4 peptides covering >25% of their amino acid sequence) in the ICM (Fig. 1C), suggesting that some of the CM-enriched proteins are also present in the ICM. This observation, i.e., that a protein may have more than one membrane location, is reasonable. These results agree with a previous report that the ATP synthase and some respiratory electron carriers are present in the ICM due to their role in either the bioenergetic light reactions of photosynthesis or the control of expression of the photosynthetic apparatus (4, 5, 20). The identification of the cyt cbb₃-type cyt oxidase, a terminal oxidase with a high affinity for oxygen, in both the IM and ICM, is also in accordance with its bioenergetic and regulatory role(s) at low oxygen tensions (23). This finding is an important result, since there are considerable data (23) revealing the importance of the cbb₃ complex to ICM structure and function.

Transcriptional profiles of genes encoding ICM-identified proteins. From global gene expression analysis previously published (24) (http://www.rhodobacter.org), the RNA abundance of the 98 ICM-identified proteins and the 70 ICM- or CM-associated proteins (Fig. 1E) are known and listed in Table S1 in the supplemental material to make a comparison between the microarray and the proteomic data. The gene expression data indicate that RNA abundance of 25 of the 70 ICM-associated gene products increased at low oxygen tensions (mRNA level, >4,000) that are known to promote assembly of the photosynthetic apparatus (24). Among the proteins encoded by the genes with high RNA levels in photosynthetic cells are subunits of the ICM-unique proteins LH2, LH1, and RC, the cyt bc₁ complex (no. 1, 5, 11, 14, 21, 23, 25, and 29), spheroidene monooxygenase (no. 11), a general function generic methyltransferase (no. 12), alkane hydroxylase (no. 13), and one unknown protein (no. 2, RSP1760). This provides additional support for the hypothesis that these 15 proteins are ICM-abundant proteins. Conversely, another 10 proteins, including a 5-aminovulinate synthase (no. 86), five subunits of ABC transporters or protein translocases (no. 39, 60, 62, 71, 72, and 76), cyt c₂ (no. 77), one protein for cysteine/methionine metabolism (no. 78), and one protein of unknown function (no. 56), are unlikely to be ICM-abundant proteins, since higher AMT tag levels and percent protein coverage were found for these proteins in the CM, cytoplasm, or periplasm of photosynthetic cells relative to ICM vesicles. We know that cyt c₂ (5, 21), a periplasmic protein, is entrapped within the lumen of the sealed ICM vesicles; thus, the conclusions of our analysis are consistent with this prior knowledge and further suggest the distribution of the proteins reported here.

Protein profiles of the ICM vesicles by SDS-PAGE electrophoresis. To further validate the proteomic predictions by comparing their abundances in the ICM with those in the IM, we confirmed the identity of the subunits of the LH2, LH1, and RC by partial purification and the cyt bc₁ complex by c-type cyt staining and apparent protein size (1, 7, 29). From this analysis, we can assign proteins to the known RC subunits (31, 34, and 28 kDa), cyt bc₁ complex polypeptides cyt b, cyt c₁, Rieske FeS protein, and subunit IV (50, 31, 20, and 14 kDa, respectively), LH2 and LH1 and ß subunits, and PufX protein (6 to 10 kDa) (14) (Fig. 2). Our analysis also predicts that the abundant proteins of 46, 35, and 25 kDa are likely to be the ICM-unique protein (RSP1760, 25 kDa), a possible alkane hydroxylase (46 kDa), spheroidene monooxygenase (36 kDa), and a generic methyltransferase (35 kDa), all of unknown function in activity or assembly of the photosynthetic membrane. Apparently, more proteins are found in the IM than in the ICM; however, it is impossible to assign proteins to other polypeptides of the IM or ICM at this time. According to Fig. 5, all well-known ICM-unique proteins, including ß subunits for LH1 and LH2, assembly factor PufX, L, H, and M subunits for RC, as well as an unknown protein, RSP1760 (Fig. 3, lanes 6 and 7), were found to be present in the ICM with a greater abundance than those found in the IM; on the other hand, the potential ICM-localized proteins, including subunits of the cyt bc₁ complex, a possible alkane hydroxylase (46 kDa) (Fig. 3, lanes 8 and 9), spheroidene monooxygenase (36 kDa), and a generic methyltransferase (35 kDa), are present in both the ICM and the IM with similar abundances. This result suggests that the proteomic prediction is indicative of their actual levels in the ICM and IM as detected by SDS-PAGE and Western blot analysis. In Fig. 5, which represents the more standard approach to viewing the ICM protein profile, we are now able to describe the likely presence of numerous additional proteins not previously identified.

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FIG. 5. SDS-PAGE analysis of ICM vesicles and IM (100 µg protein). Membranes were solubilized and separated on a 15% acrylamide gel before being stained with Coomassie blue. The arrows on the right of the gel lane represent the protein sizes predicted from molecular weight, c-type cyt staining, or partial purification of membrane complexes, and the band positions for Possible alkane hydroxylase (46.0 kDa; RSP1467) and an unknown protein (25 kDa; RSP1760) were detected by anti-histidine antibodies. The relative intensity of each band in ICM vesicles or IM is also indicated; the numbers on the left indicate the sizes of the protein standards.

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This study tests the hypothesis that the ICM of R. sphaeroides contains numerous proteins in addition to those which have been most extensively studied. Our approach, since possible functions and potential candidate proteins were unknown, was to obtain a proteomic profile of purified ICM. We determined the protein composition of the R. sphaeroides ICM, because it is easy to obtain large quantities of highly purified membrane vesicles and because it is one of the best-studied photosynthetic membranes from a structural, functional, and genetic standpoint (23, 25). The accurate AMT tag proteomics system identified many integral membrane proteins of different sizes or abundances within the photosynthetic membrane.

We detected each of the pigment-binding proteins involved in light energy capture (LH2, LH1) and its conversion into chemical energy as major components of the ICM. This finding agrees with past biochemical and spectroscopic studies, as well as the model for the topology of the photosynthetic membranes as derived from atomic force microscopy of the ICM (1, 5, 11, 13, 14, 25, 29). These findings also serve as a strong positive control for other conclusions concerning the proteomic blueprint of the ICM. Other abundant ICM proteins include subunits of the cyt bc₁ complex, the F₀F₁ ATPase, and other membrane-bound respiratory enzymes. The presence of these enzymes in the ICM is consistent with the ability of these sealed membrane vesicles to catalyze light-dependent formation of ATP, a proton gradient (8), or a passage for reducing power from organic substrates to oxygen (11, 19). The fact that past biochemical or functional analyses of the ICM agree with the proteomic blueprint provides confidence for assigning additional proteins to these vesicles and for formulating new hypotheses about photosynthetic membrane assembly or function.

Newly identified ICM proteins. Other abundant ICM proteins include a possible alkane hydroxylase, spheroidene monooxygenase, and a generic methyltransferase. The role of the predicted alkane hydroxylase and the generic methyltransferase in photosynthesis, function, or formation of ICM components remains to be determined. However, the presence of spheroidene monooxygenase, the last enzyme in Crt biosynthesis, within the ICM is important in the conversion of spheroidene to spheroidenone, the two major carotenoids that have functional and regulatory roles in R. sphaeroides photosynthesis. While a role for spheroidene monooxygenases in Crt synthesis has been established by biochemical and genetic analyses (12), there was no a priori reason to expect this enzyme, predicted to be soluble based on its amino acid sequence, to be associated with the photosynthetic membranes. However, we know that spheroidene monooxygenase activity is sensitive to the "redox" state of the cell, so its possible association with the ICM positions its activity to respond to the reducing power produced from photosynthetic activity (23).

Additional ICM-associated proteins included homologs of cell division proteins (RSP1360, RSP0356). Previous analysis shows that the R. sphaeroides photosynthetic membrane is derived as invaginations from the CM (9). Thus, these and other ICM proteins of unknown function could aid in the formation of photosynthetic membrane invaginations; control of protein assembly by oxygen tension, light intensity, or cell cycle; and partitioning to daughter cells (6, 16, 24). Studies of these possibilities are under way.

Other proteins needed for photosynthetic membrane function. Previous analyses of photosynthesis in R. sphaeroides identified a 70-kb region of the genome, the so-called photosynthesis gene cluster, that encodes many gene products which are required for photosynthetic growth (6, 16, 23). Seven of the pigment-binding proteins encoded by this 70-kb region are within the ICM. However, 54 identified proteins in the ICM are encoded by previously uncharacterized genes that lie outside the photosynthesis gene cluster (6, 16, 23). The numerous, previously uncharacterized ICM proteins that are encoded outside this gene cluster illustrate the importance of determining the function of these polypeptides in photosynthetic growth, ICM activity, or ICM structure.

In addition, 21 of the genes within the photosynthesis gene cluster encode pigment biosynthesis enzymes that are enriched in the CM of photosynthetic cells. There was also no a priori reason to predict that the Bchl and Crt biosynthetic enzymes would not be present in the ICM, since the products of these pathways are critical to photosynthetic membrane function. The R. sphaeroides ICM is physically contiguous with the CM of photosynthetic cells (9), so this localization of pigment biosynthesis enzymes to the CM also predicts that pigments are synthesized in a membrane domain other than the one in which they function.

The R. sphaeroides ICM is composed of a typical lipid bilayer. The phospholipid biosynthesis enzymes, which are known or predicted to be integral membrane proteins, are not present in the ICM but rather are found in the CM of photosynthetic cells (27). This finding confirms the previous hypothesis, based solely on enzyme specific activity measurements, that the CM of photosynthetic cells is the site of phospholipid synthesis (27, 28). It also supports the hypothesis that lipids must be transferred from their site of synthesis (CM of photosynthetic cells) into the ICM. In this regard, we note that RSP0246, which is predicted to encode a homolog of a lipid transporter, is present in the ICM. Thus, it is possible that results of future experiments will show that this and other uncharacterized proteins aid the mobilization of lipids from their site of synthesis into the ICM (27, 28).

Proteins shared by the ICM and the CM of photosynthetic cells. Our data also indicate that several integral membrane proteins (cyt bc₁ complex, F₀F₁ ATPase, and other respiratory enzymes [dehydrogenases, oxidases] or additional activities [chaperones, transporters, subunits of preprotein translocases, etc.]) are found in both the ICM and the CM of photosynthetic cells. This finding illustrates the fact that many critical membrane activities are common to the CM and ICM of photosynthetic cells. It also reinforces the importance of determining how individual integral membrane protein complexes are targeted selectively to the ICM (pigment protein complexes involved in light energy capture) or to the physically contiguous IM of photosynthetic cells (lipid biosynthesis, etc.) or are shared between the two domains of this bilayer.

Importantly, as a proof of principle, two of the newly identified genes (the RSP1760 and RSP1467 genes) selected to yield protein products involved in ICM structure/function were mutated and shown to produce detectable defects in photosynthetic growth, indicating that the approach taken here has and will continue to advance our knowledge of what is needed for solar energy capture by R. sphaeroides 2.4.1. The RSP1760 and RSP1467 genes are located at 1.6 Mb and 1.9 Mb counterclockwise from the photosynthesis gene cluster on chromosome 1, respectively. A search of the database on the NCBI website showed that the RSP1760 gene is highly conserved in Alphaproteobacteria but that no function has been revealed. Although it is not found in several other photosynthetic bacteria, it is found in Paracoccus spp. and members of the Roseobacter as well as Silicibacter spp. Importantly, R. sphaeroides shows more identities and similarities to these three groups of organisms than to other photosynthetic bacteria and other members of the Alphaproteobacteria. The function of RSP1760 remains to be determined, but the fact that its absence leads to a photosynthesis minus phenotype may assist us in this endeavor.

In summary, this analysis of ICM protein composition has provided important new insights into the activities and function of this photosynthetic membrane. It also sets the stage for future analysis of newly identified ICM proteins and their role in photosynthesis or ICM structure/function. In addition, proteomic and functional analysis of the ICM from wild-type and mutant strains can now be used to further our understanding of the supramolecular organization of photosynthetic membrane proteins, the assembly of these complexes, and their functional or structural relationship to the process of solar energy utilization, as well as the preempting of these membranes for biofuel syntheses. Finally, we should note that this is the first report of the protein profiles of a subcellular fraction of known high purity. Thus, we predict that experimental and computational methods used to define this ICM protein blueprint will be of use in the analysis of other fractions from prokaryotic and eukaryotic cells. Many important questions are raised by this analysis, and their answers are likely to shed light on the structural organization of photosynthetic tissue.

 
These studies were initiated with support from the Department of Energy (DOE) (ER63232-1018220-0007203 to Timothy J. Donohue and Samuel Kaplan and DE-AC05-76RL01830 to Mary Lipton). Sample processing and data analyses were completed with support from the DOE (DE-FG02-05ER15653) or the National Institutes of Health (GM075273 to Timothy J. Donohue, GM43488 to Mary Lipton, and GM15590 to Samuel Kaplan) and were performed in the Environmental Molecular Sciences Laboratory (a national scientific user facility sponsored by the U.S. DOE Office of Biological and Environmental Research) located at Pacific Northwest National Laboratory, operated by Battelle for the DOE.

Jesus Eraso gave numerous useful discussions regarding the experiments and data analysis.

 
* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston TX, 77030. Phone: (713) 500-5502. Fax: (713) 500-5499. E-mail: Samuel.Kaplan{at}uth.tmc.edu

Published ahead of print on 17 August 2007.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, October 2007, p. 7464-7474, Vol. 189, No. 20
0021-9193/07/$08.00+0     doi:10.1128/JB.00946-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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• Bruscella, P., Eraso, J. M., Roh, J. H., Kaplan, S. (2008). The Use of Chromatin Immunoprecipitation to Define PpsR Binding Activity in Rhodobacter sphaeroides 2.4.1. J. Bacteriol. 190: 6817-6828 [Abstract] [Full Text]  
• Eraso, J. M., Roh, J. H., Zeng, X., Callister, S. J., Lipton, M. S., Kaplan, S. (2008). Role of the Global Transcriptional Regulator PrrA in Rhodobacter sphaeroides 2.4.1: Combined Transcriptome and Proteome Analysis. J. Bacteriol. 190: 4831-4848 [Abstract] [Full Text]  

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