Application of the accurate mass and time tag approach to the proteome analysis of sub-cellular fractions obtained from Rhodobacter sphaeroides 2.4.1. Aerobic and photosynthetic cell cultures

The high-throughput accurate mass and time (AMT) tag proteomic approach was utilized to characterize the proteomes for cytoplasm, cytoplasmic membrane, periplasm, and outer membrane fractions from aerobic and photosynthetic cultures of the gram-nagtive bacterium Rhodobacter sphaeroides 2.4.1. In addition, we analyzed the proteins within purified chromatophore fractions that house the photosynthetic apparatus from photosynthetically grown cells. In total, 8,300 peptides were identified with high confidence from at least one subcellular fraction from either cell culture. These peptides were derived from 1,514 genes or 35% percent of proteins predicted to be encoded by the genome. A significant number of these proteins were detected within a single subcellular fraction and their localization was compared to in silico predictions. However, the majority of proteins were observed in multiple subcellular fractions, and the most likely subcellular localization for these proteins was investigated using a Z-score analysis of estimated protein abundance along with clustering techniques. Good (81%) agreement was observed between the experimental results and in silico predictions. The AMT tag approach provides localization evidence for those proteins that have no predicted localization information, those annotated as putative proteins, and/or for those proteins annotated as hypothetical and conserved hypothetical.     

The significance of type II and PrxQ peroxiredoxins for antioxidative stress response in the purple bacterium Rhodobacter sphaeroides

Two peroxiredoxins, classified as Type II and PrxQ, were characterized in the purple non-sulfur photosynthetic bacterium Rhodobacter sphaeroides. Both recombinant proteins showed remarkable thioredoxin-dependent peroxidase activity with broad substrate specificity in vitro. Nevertheless, PrxQ of R. sphaeroides, unlike typical PrxQs studied to date, does not contain one of the two conserved catalytic Cys residues. We found that R. sphaeroides PrxQ and other PrxQ-like proteins from several organisms conserve a different second Cys residue, indicating that these proteins should be categorized into a novel PrxQ subfamily. Disruption of either the Type II or PrxQ gene in R. sphaeroides had a dramatic effect on cell viability when the cells were grown under aerobic light or oxidative stress conditions created by exogenous addition of reactive oxygen species to the medium. Growth rates of the mutants were significantly decreased compared with that of wild type under aerobic but not anaerobic conditions. These results indicate that the peroxiredoxins are crucial for antioxidative stress response in this bacterium. The gene disruptants also demonstrated reduced levels of photopigment synthesis, suggesting that the peroxiredoxins are directly or indirectly involved in regulated synthesis of the photosynthetic apparatus.     

Effect of selenite on growth and protein synthesis in the phototrophic bacterium Rhodobacter sphaeroides

The effect of selenite on the growth rate and protein synthesis has been investigated in Rhodobacter sphaeroides. This photosynthetic bacterium efficiently reduces selenite with intracellular accumulation under both dark aerobic and anaerobic photosynthetic conditions. Addition of 1 mM selenite under these two growth conditions does not affect the final cell density, although a marked slowdown in growth rate is observed under aerobic growth. The proteome analysis of selenite response by two-dimensional gel electrophoresis shows an enhanced synthesis of some chaperones, an elongation factor, and enzymes associated to oxidative stress. The induction of these antioxidant proteins confirms that the major toxic effect of selenite is the formation of reactive oxygen species during its metabolism. In addition, we show that one mutant unable to precipitate selenite, selected from a transposon library, is affected in the smoK gene. This encodes a constituent of a putative ABC transporter implicated in the uptake of polyols. This mutant is less sensitive to selenite and does not express stress proteins identified in the wild type in response to selenite. This suggests that the entry of selenite into the cytoplasm is mediated by a polyol transporter in R. sphaeroides.     

Characterisation of a Rrhodobacter sphaeroides gene that encodes a product resembling Eescherichia coli cytochrome b(561) and R. sphaeroides cytochrome b(562)

Analysis of the photoactive yellow protein (pyp) gene region of Rhodobacter sphaeroides has revealed the presence of an additional open reading frame, orfD, that had not previously been identified. Here we report the location of this new gene and the predicted amino acid sequence of the encoded protein. The translation product resembles a group of small cytochrome b-like proteins, including Escherichia coli cytochrome b(561), R. sphaeroides cytochrome b(562), and two new cytochrome b(561)-like proteins identified using the E. coli genome sequence, for which functions have not yet been established. To determine OrfD function in R. sphaeroides, an orfD mutant was constructed. The OrfD mutant exhibited growth rates and yields very similar to those of the wild-type strain when grown under a variety of growth conditions. Respiration rates, reduced-minus-oxidised spectra and levels of photosynthetic complexes were also very similar in the two strains. Although the role of OrfD was therefore not determined here, we demonstrate that the orfD gene is expressed in R. sphaeroides under aerobic, semi-aerobic and photosynthetic growth conditions.     

Induction of the photosynthetic membranes of Rhodopseudomonas sphaeroides: biochemical and morphological studies

Cells of Rhodopseudomonas sphaeroides grown in a 25% O2 atmosphere were rapidly subjected to total anaerobiosis in the presence of light to study the progression of events associated with the de novo synthesis of the inducible intracytoplasmic membrane (ICM). This abrupt change in physiological conditions resulted in the immediate cessation of cell growth and whole cell protein, DNA, and phospholipid accumulation. Detectable cell growth and whole cell protein accumulation resumed ca. 12 h later. Bulk phospholipid accumulation paralleled cell growth, but the synthesis of individual phospholipid species during the adaptation period suggested the existence of a specific regulatory site in phospholipid synthesis at the level of the phosphatidylethanolamine methyltransferase system. Freeze-fracture electron microscopy showed that aerobic cells contain small indentations within the cell membrane that appear to be converted into discrete ICM invaginations within 1 h after the imposition of anaerobiosis. Microscopic examination also revealed a series of morphological changes in ICM structure and organization during the lag period before the initiation of photosynthetic growth. Bacteriochlorophyll synthesis and the formation of the two light-harvesting bacteriochlorophyll-protein complexes of R. sphaeroides (B800-850 and B875) occurred coordinately within 2 h after the shift to anaerobic conditions. Using antibodies prepared against various ICM-specific polypeptides, the synthesis of reaction center proteins and the polypeptides associated with the B800-850 complex was monitored. The reaction center H polypeptide was immunochemically detected at low levels in the cell membrane of aerobic cells, which contained no detectable ICM or bacteriochlorophyll. The results are discussed in terms of the oxygen-dependent regulation of gene expression in R. sphaeroides and the possible role of the reaction center H polypeptide and the cell membrane indentations in the site-specific assembly of ICM pigment-protein complexes during the de novo synthesis of the ICM.     

Sequential assembly of photosynthetic units in Rhodobacter sphaeroides as revealed by fast repetition rate analysis of variable bacteriochlorophyll a fluorescence

The development of functional photosynthetic units in Rhodobacter sphaeroides was followed by near infra-red fast repetition rate (IRFRR) fluorescence measurements that were correlated to absorption spectroscopy, electron microscopy and pigment analyses. To induce the formation of intracytoplasmic membranes (ICM) (greening), cells grown aerobically both in batch culture and in a carbon-limited chemostat were transferred to semiaerobic conditions. In both aerobic cultures, a low level of photosynthetic complexes was observed, which were composed of the reaction center and the LH1 core antenna. Interestingly, in the batch cultures the reaction centers were essentially inactive in forward electron transfer and exhibited low photochemical yields F(V)/F(M), whereas the chemostat culture displayed functional reaction centers with a rather rapid (1-2 ms) electron transfer turnover, as well as a high F(V)/F(M) of approximately 0.8. In both cases, the transfer to semiaerobiosis resulted in rapid induction of bacteriochlorophyll a synthesis that was reflected by both an increase in the number of LH1-reaction center and peripheral LH2 antenna complexes. These studies establish that photosynthetic units are assembled in a sequential manner, where the appearance of the LH1-reaction center cores is followed by the activation of functional electron transfer, and finally by the accumulation of the LH2 complexes.     

Analysis of the fnrL gene and its function in Rhodobacter capsulatus.

The fnr gene encodes a regulatory protein involved in the response to oxygen in a variety of bacterial genera. For example, it was previously shown that the anoxygenic, photosynthetic bacterium Rhodobacter sphaeroides requires the fnrL gene for growth under anaerobic, photosynthetic conditions. Additionally, the FnrL protein in R. sphaeroides is required for anaerobic growth in the dark with an alternative electron acceptor, but it is not essential for aerobic growth. In this study, the fnrL locus from Rhodobacter capsulatus was cloned and sequenced. Surprisingly, an R. capsulatus strain with the fnrL gene deleted grows like the wild type under either photosynthetic or aerobic conditions but does not grow anaerobically with alternative electron acceptors such as dimethyl sulfoxide (DMSO) or trimethylamine oxide. It is demonstrated that the c-type cytochrome induced upon anaerobic growth on DMSO is not synthesized in the R. capsulatus fnrL mutant. In contrast to wild-type strains, R. sphaeroides and R. capsulatus fnrL mutants do not synthesize the anaerobically, DMSO-induced reductase. Mechanisms that explain the basis for FnrL function in both organisms are discussed.     

Light-dependent regulation of the synthesis of soluble and intracytoplasmic membrane proteins of Rhodopseudomonas sphaeroides.

Cells of Rhodopseudomonas sphaeroides grown under saturating light conditions (30 W/m2) and then shifted to low light intensity (3 W/m2) required 2.5 h to adapt to the new lower light conditions. After the shift, cell growth, whole cell protein accumulation, and bacteriochlorophyll accumulation ceased immediately. Approximately midway into the adaptation period, bacteriochlorophyll synthesis commenced at a new, higher rate, which continued through the beginning of the low-light growth period until new steady-state levels were reached. Immediately after the downshift, the rate of cellular protein synthesis declined to 22% of its preshift rate. Pulse-labeling of protein throughout the adaptation period and comparison with a steady-state prelabel culture revealed that synthesis of two of the three light-harvesting proteins, as well as two additional high-molecular-weight photosynthetic membrane proteins, was derepressed three- to fivefold compared with bulk cellular protein. Finally, the synthesis of at least three soluble proteins showed light-dependent regulation after the light downshift. These results are discussed in terms of the light-dependent regulation of synthesis of the photosynthetic membrane macromolecular components and the division of protein synthesis between the photosynthetic membranes and the soluble cell phase.     

Bundle sheath proteins are more sensitive to oxidative damage than those of the mesophyll in maize leaves exposed to paraquat or low temperatures

In maize leaves growth at low temperatures causes decreases in maximum catalytic activities of photosynthetic enzymes and reduced amounts of proteins, rather than effects on regulation or co-ordination of the photosynthetic processes. To test the hypothesis that differential localization of antioxidants between the different types of photosynthetic cell in maize leaves is a major determinant of the extreme sensitivity of maize leaves to chilling damage, oxidative damage to proteins, induced by incubation of maize leaves with paraquat, has been measured and compared with the effects incurred by growth at low temperatures. While the increase in protein carbonyl groups caused by paraquat treatment was much greater than that caused by low temperature growth conditions, most carbonyl groups were detected on bundle sheath proteins in both stress conditions. With one or two exceptions proteins located in the mesophyll tissues were free of protein carbonyl groups in both situations. Paraquat treatment caused a complete loss of the psaA gene products, modified the photosystem II reaction centre polypeptide, D1, and increased the number of peptides arising from breakdown of ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco). In contrast, growth at 15 degrees C increased the abundance (but not number) of Rubisco breakdown products and decreased that of the psaB gene product while the psaA gene product and PEP carboxylase were largely unaffected. Since bundle sheath proteins are more susceptible to oxidative damage than those located in the mesophyll cells, strategies for achieving a more balanced system of antioxidant defence may be effective in improving chilling tolerance in maize.     

Growth of Rhodopseudomonas capsulata on l- and d-malic acid

d-malate replaced l-malate in supporting both photosynthetic (anaerobic, light) and heterotrophic (aerobic, dark) growth of Rhodopseudomonas capsulata. Growth rates and cell yields were nearly equivalent with both enantiomorphs. Addition of glucose to malate culture media increased the growth rate and doubled the cell yield of heterotrophic cultures, but had little effect on photosynthetic cultures. Aerobically-grown cells showed a higher level of substrate-dependent oxygen uptake with l-malate than with d-malate. This preference for l-malate occured even in cells grown on d-malate. No malic racemase activity was detected in extracts of heterotrophically- or photosynthetically-grown cells.     

Growth of Rhodopseudomonas capsulata on l- and d-malic acid

d-malate replaced l-malate in supporting both photosynthetic (anaerobic, light) and heterotrophic (aerobic, dark) growth of Rhodopseudomonas capsulata. Growth rates and cell yields were nearly equivalent with both enantiomorphs. Addition of glucose to malate culture media increased the growth rate and doubled the cell yield of heterotrophic cultures, but had little effect on photosynthetic cultures. Aerobically-grown cells showed a higher level of substrate-dependent oxygen uptake with l-malate than with d-malate. This preference for l-malate occured even in cells grown on d-malate. No malic racemase activity was detected in extracts of heterotrophically- or photosynthetically-grown cells.     

Polar localization of CheA2 in Rhodobacter sphaeroides requires specific Che homologs

Rhodobacter sphaeroides is a motile bacterium that has multiple chemotaxis genes organized predominantly in three major operons (cheOp(1), cheOp(2), and cheOp(3)). The chemoreceptor proteins are clustered at two distinct locations, the cell poles and in one or more cytoplasmic clusters. One intriguing possibility is that the physically distinct chemoreceptor clusters are each composed of a defined subset of specific chemotaxis proteins, including the chemoreceptors themselves plus specific CheW and CheA proteins. Here we report the subcellular localization of one such protein, CheA(2), under aerobic and photoheterotrophic growth conditions. CheA(2) is predominantly clustered and localized at the cell poles under both growth conditions. Furthermore, its localization is dependent upon one or more genes in cheOp(2) but not those of cheOp(1) or cheOp(3). In E. coli, the polar localization of CheA depends upon CheW. The R. sphaeroides cheOp(2) contains two cheW genes. Interestingly, CheW(2) is required under both aerobic and photoheterotrophic conditions, whereas CheW(3) is not required under aerobic conditions but appears to play a modest role under photoheterotrophic conditions. This suggests that R. sphaeroides contains at least two distinct chemotaxis complexes, possibly composed of proteins dedicated for each subcellular location. Furthermore, the composition of these spatially distinct complexes may change under different growth conditions.     

Molecular genetic analysis suggesting interactions between AppA and PpsR in regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1.

The AppA protein plays an essential regulatory role in development of the photosynthetic apparatus in the anoxygenic phototrophic bacterium Rhodobacter sphaeroides 2.4.1 (M. Gomelsky and S. Kaplan, J. Bacteriol. 177:4609-4618, 1995). To gain additional insight into both the role and site of action of AppA in the regulatory network governing photosynthesis gene expression, we investigated the relationships between AppA and other known regulators of photosynthesis gene expression. We determined that AppA is dispensable for development of the photosynthetic apparatus in a ppsR null background, where PpsR is an aerobic repressor of genes involved in photopigment biosynthesis and puc operon expression. Moreover, all suppressors of an appA null mutation thus far isolated, showing improved photosynthetic growth, were found to contain mutations in the ppsR gene. Because ppsR gene expression in R. sphaeroides 2.4.1 appears to be largely independent of growth conditions, we suggest that regulation of repressor activity occurs predominately at the protein level. We have also found that PpsR functions as a repressor not only under aerobic but under anaerobic photosynthetic conditions and thereby is involved in regulating the abundance of the light harvesting complex II, depending on light intensity. It seems likely therefore, that PpsR responds to an integral signal (e.g., changes in redox potential) produced either by changes in oxygen tension or light intensity. The profile of the isolated suppressor mutations in PpsR is in accord with this proposition. We propose that AppA may be involved in a redox-dependent modulation of PpsR repressor activity.     

Analysis of the Salmonella typhimurium proteome through environmental response toward infectious conditions

Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium) is a facultative intracellular pathogen that causes approximately 8,000 reported cases of acute gastroenteritis and diarrhea each year in the United States. Although many successful physiological, biochemical, and genetic approaches have been taken to determine the key virulence determinants encoded by this organism, the sheer number of uncharacterized reading frames observed within the S. enterica genome suggests that many more virulence factors remain to be discovered. We used a liquid chromatography-mass spectrometry-based "bottom-up" proteomic approach to generate a more complete picture of the gene products that S. typhimurium synthesizes under typical laboratory conditions as well as in culture media that are known to induce expression of virulence genes. When grown to logarithmic phase in rich medium, S. typhimurium is known to express many genes that are required for invasion of epithelial cells. Conversely stationary phase cultures of S. typhimurium express genes that are needed for both systemic infection and growth within infected macrophages. Lastly bacteria grown in an acidic, magnesium-depleted minimal medium (MgM) designed to mimic the phagocytic vacuole have been shown to up-regulate virulence gene expression. Initial comparisons of protein abundances from bacteria grown under each of these conditions indicated that the majority of proteins do not change significantly. However, we observed subsets of proteins whose expression was largely restricted to one of the three culture conditions. For example, cells grown in MgM had a higher abundance of Mg(2+) transport proteins than found in other growth conditions. A second more virulent S. typhimurium strain (14028) was also cultured under these same growth conditions, and the results were directly compared with those obtained for strain LT2. This comparison offered a unique opportunity to contrast protein populations in these closely related bacteria. Among a number of proteins displaying a higher abundance in strain 14028 were the products of the pdu operon, which encodes enzymes required for propanediol utilization. These pdu operon proteins were validated in culture and during macrophage infection. Our work provides further support for earlier observations that suggest pdu gene expression contributes to S. typhimurium pathogenesis.     

Correlation between inhibition of photosynthesis and growth ofChlorella treated with methyl parathion

When methyl parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate), an organophosphorous insecticide, was added to an exponentially growing culture ofChlorella protothecoides and the effects were followed for 12 days, the following observations were made: a) In autotrophic culture the cell number and the chlorophyll content decreased as compared to the control. These changes paralleled the inhibition of the rate of net photosynthesis, suggesting that the photosynthetic apparatus was the primary target of the insecticide action. b) The inhibition of cell growth (on cell number basis) also occurred in the case of heterotrophic culture at 100μM insecticide concentration but the inhibition was less as compared to that of an autotrophic culture. c) The cell diameter in treated culture increased by 10–20% in both autotrophic and heterotrophic cultures. The observations, (b) and (c) suggest that apart from the photosynthetic apparatus, the insecticide has other sites of action, but the sensitivity of these sites to the insecticide is less than that of the photosynthetic apparatus     

In silico inference of inclusion membrane protein family in obligate intracellular parasites chlamydiae.

Chlamydiae are obligate intracellular pathogens that proliferate only inside a vacuole, called an inclusion. Chlamydial Inc proteins are known to be a major component of the inclusion membrane, but little is known about the gene number and function. The Inc proteins share very low sequence similarity but a similar hydropathy profile among them. Using the hydropathy profile, we computationally searched the open reading frames (ORFs) having a similar profile and predicted 90 and 36 ORFs (Inc-like ORFs) as candidates for Inc proteins in Chlamydia pneumoniae J138 and Chlamydia trachomatis serovar D, respectively. On the other hand, only a few Inc-like ORFs were found in organisms other than chlamydiae, suggesting that the Inc-like ORFs are specific to chlamydiae. Comparative genome analysis also revealed that the Inc-like ORFs have multiplied and diverged as paralogues and orthologues in the chlamydial genomes, and that some Inc-like ORFs lacked the N-terminal portion or encoded the split form. The data suggest that these gene products constitute a large protein family and may play an important role in chlamydial infection, growth and survival in the host cell.