Protein Synthesis Patterns Reveal a Complex Regulatory Response to

Protein Synthesis Patterns Reveal a Complex Regulatory Response to Singlet Oxygen in Rhodobacter Jens Glaeser,*,†,‡ Monica Zobawa,‡,§ Friedrich Lottspeich,‡,§ and Gabriele Klug†,‡ Institut fu ¨ r Mikrobiologie und Molekularbiologie, Justus-Liebig-Universita¨t Giessen, Heinrich-Buff-Ring 26-32, D-35392 Gieβen, Germany, and Max Planck Institut fu ¨ r Biochemie, Am Klopferspitz 18, D-82152 Martinsried, Germany Received November 23, 2006

Singlet oxygen (1O2) is a stress factor and signal in the facultative phototrophic bacterium Rhodobacter sphaeroides. In vivo protein labeling with L-[35S]-methionine and analysis by two-dimensional gel electrophoresis revealed that the synthesis of 61 proteins was changed in response to 1O2. After 1O2 treatment, protein synthesis patterns were distinct from those after H2O2 treatment but similar to those after high light exposure. This indicates regulatory mechanisms selective for different reactive oxygen species (ROS) and a response to light partly mediated by 1O2. Analysis of mutant strains support that the response to 1O2 is regulated mainly by rpoE (σE), but also a modulation of the σE dependent response by other factors and the existence of σE independent responses. The involvement of the RNA chaperon Hfq in the 1O2 response implies a role of small regulatory RNAs. Keywords: Rhodobacter • singlet oxygen • stress • regulation • protein synthesis • rpoE • sRNA

Introduction Microorganisms are frequently exposed to harmful agents that damage cellular components. In particular, oxygen appears to be a universal problem because reactive oxygen species (ROS), which are generated by unspecific transfer of electrons to molecular oxygen during redox processes, generate oxidative stress.1 A major source for these toxic compounds is the cellular metabolism, e.g., autoxidation of respiratory dehydrogenases and yet not identified cytoplasmic mechanisms.2,3 In addition, ROS are generated by the exposure of microorganisms to divalent metal ions, redox-active chemicals, and radiation. In contrast to other ROS, photochemical generation of 1O2 involves the transfer of energy from an excited-state photosensitizer molecule to oxygen.4 Despite the fact that many enzymatic reactions have been described that generate 1O2, photochemical reactions are assumed to be the major source in biological systems.5 By the transfer of energy to oxygen, the spin restriction is removed, which makes 1O2 a molecule that largely exceeds superoxide and H2O2 in reactivity. Until recently, 1O2 was believed to elicit damage only in close proximity of its site of generation, because of its very short half-life in cells.6 However, recent findings demonstrate a rather long half-life * To whom correspondence should be addressed. Mailing address, Institut fu ¨ r Mikrobiologie und Molekularbiologie, Heinrich-Buff-Ring 26-32, 35392 Gieβen, Germany; Phone, (+49) 641 99 355 57; Fax, (+49) 641 99 355 49; E-mail, Jens. [email protected]. † Justus Liebig Universita ¨ t Giessen. ‡ Further addresses and phone numbers. Prof. G. Klug: Phone, (+49) 641 99 355 42; E-mail, [email protected]. M. Zobawa and Dr. F. Lottspeich: Phone, +49 (89) 8578 2465; Fax, +49 (89) 8578 2802; E-mail, [email protected], [email protected]. § Max Planck Institut fu ¨ r Biochemie.

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of up to 3 µs and, therefore, diffusion over appreciable distances, which may need to be considered for intracellular signaling.7 Although the investigation of regulatory responses to oxidative stress has started in the past decade, a detailed picture of the molecular factors involved in regulation and the role of many newly synthesized proteins is lacking.1 Several defense systems are required for the survival of oxidative stress including the synthesis of sunscreens,8 ROS scavengers, and enzymes detoxifying ROS.1 Furthermore, repair systems for cellular components as DNA, proteins, and membranes are necessary.1,9 In phototrophic organisms, conditions of photo-oxidative stress are caused by pigment generated 1O2 and defense is provided by carotenoids, which quench 1O2 directly4 or quench excited chlorophylls to prevent 1O2 formation.10,11 Because defense and repair systems are energetically expensive and may influence the cellular redox homeostasis, a balanced regulation of their components is pivotal. Regulatory systems controlling gene expression in response to elevated levels of H2O2, superoxide, and organic peroxides have been described throughout the last years for several microorganisms. However, information on regulatory mechanisms responding to 1O2 exposure is scarce.12 Only a few case studies have been performed, which provide information on the signaling role of 1O in plants and animals.13,14 In Escherichia coli, OxyR was 2 suggested to be involved in 1O2 defense,15 and the induction of carotenoid biosynthesis by light is potentially 1O2 mediated in myxobacteria.16 Only recently, transcriptional regulation of genes in response to 1O2 was suggested to depend on the ECF sigma factor E (σE) in R. sphaeroides.17 Dissociation of σE and its anti-sigma factor ChrR in response to 1O2 exposure has been suggested as a possible, but in detail yet unknown, mechanism 10.1021/pr060624p CCC: $37.00

 2007 American Chemical Society

Regulatory Responses to Singlet Oxygen in Rhodobacter

that liberates σE and allows binding to target promoter sequences.17,18 In Rhodobacter, effects of H2O2 and superoxide on growth, survival, and gene regulation were investigated recently,19-22 which makes this bacterium an ideal system to elucidate the differences in the regulatory response to 1O2 and other ROS. Although carotenoids have a crucial role in the defense against 1O2,23 an adaptive response was induced in R. sphaeroides by exposure to 1O2 without an increase of the cellular carotenoid content.24 To investigate factors initiating this adaptation, changes of protein synthesis patterns were analyzed using radioactive labeling of proteins with L-[35S]-methionine and two-dimensional (2D) gel electrophoresis. Because the defense systems induced by different ROS may overlap in R. sphaeroides, we compared the effects of 1O2 and H2O2. Furthermore, we investigated the response to high light conditions, which initiate pigment-mediated 1O2 generation in vivo. Changes in protein synthesis patterns of mutants lacking either σE and its anti-sigma factor ChrR (strain TF18) or only ChrR (strain ∆ChrR) indicated that important regulatory mechanisms responding to 1O2 have been missed. Further experiments performed with a mutant lacking the RNA chaperone protein Hfq provided evidence that a σE independent regulatory mechanism is active in R. sphaeroides.

Materials and Methods Bacterial Strains and Growth Conditions. Rhodobacter strains were cultivated at 32 °C in minimal salt medium containing malate as a carbon source.25 Semiaerobic cultures were grown in Erlenmeyer flasks with continuous shaking at 140 rpm resulting in a concentration of dissolved oxygen of approximately 25 µM. For aerobic growth, cultures were gassed with air resulting in a concentration of dissolved oxygen of approximately 180 µM. When necessary, trimethoprim (50 µg mL-1) was added to liquid and solid growth media, which contained 1.6% agar. R. sphaeroides strains 2.4.1,26 TF18,27 and ∆ChrR28 were used throughout this work. E. coli strains were grown at 37 °C in LB medium under continuous shaking at 180 rpm or on solid growth media. Construction of a R. sphaeroides hfq- Mutant. R. sphaeroides strain 2.4.1hfq::ΩSp was generated by transferring the suicide plasmid pPHU2.4.1hfq::ΩSp in R. sphaeroides 2.4.1 and screening for insertion of the omega (Ω)-spectinomycin cassette into the chromosome by homologous recombination. In brief, parts of the hfq gene of R. sphaeroides 2.4.1, together with upstream and downstream sequences, were amplified by PCR using oligonucleotides 2.4.1hfqupEcoRI (5′-CTTCGAATTCTCGATTGCCG-3′), 2.4.1hfq+50BamHI (5′-TAACGGGATCCTTGGCCTTC-3′), 2.4.1hfq+150BamHI (5′-CACGGGATCCCGACCATCATG-3′), and 2.4.1hfqdownSphI (5′-CGAGCATGCGCCCGGTCCGG-3′). The obtained PCR fragments were cloned into pPHU28129 using the appropriate restriction endonucleases. Then the Ω-spectinomycin cassette obtained from plasmid pPH45Ω30 was inserted into the BamHI restriction site to obtain the plasmid pPHU2.4.1∆hfq::ΩSp. The plasmid pPHU2.4.1∆hfq:: ΩSp was transferred into E. coli strain S17-1,31 and mobilized from E. coli strain SM1032 containing the plasmid pPHU2.4.1∆hfq::ΩSp into R. sphaeroides 2.4.1 wild-type strain by triparental conjugation. Conjugants were selected on malate minimal medium agar plates containing 10 µg spectinomycin mL-1. Southern blot analysis of chromosomal DNA was carried out to confirm the double crossover event of the Ω-spectinomycin cassette into the R. sphaeroides chromosome. By inser-

research articles tion of the Ω-spectinomycin cassette, 165 bp of the 310 bp R. sphaeroides hfq gene were deleted. Complementation of the R. sphaeroides hfq Deletion Mutant. For complementation of the R. sphaeroides hfq deletion mutant, a 1.1 kb PCR fragment containing the entire hfq gene along with 400 bp of the upstream sequence and 400 bp of the downstream sequence was amplified using the oligonucleotides 2.4.1hfqupEcoRI and 2.4.1hfqdownSphI. The obtained PCR fragment was cloned into the pDrive vector (Qiagen, Hilden, Germany). Digestion of the pDrive vector containing the insert with KpnI and XbaI followed by cloning with the same restriction sites into plasmid pRK415, obtained plasmid p2.4.1hfq. This plasmid was subsequently transformed in E. coli S17-1 and conjugated with strain 2.4.1hfq::ΩSp to obtain the complemented strain 2.4.1hfq. High Light and Oxidative Stress Conditions. To obtain pigmented cells, cultures were grown semiaerobically overnight, diluted to an optical density of 0.2 (λ ) 660 nm) and allowed to double once under aerobic growth conditions in darkened flat glass bottles.24 For illumination with high light intensities (800 W m-2), cultures were placed in a water bath at 32 °C in front of a white light halogen bulb (500 W, Osram, Germany). Light intensity was monitored with a LI-250 light meter connected to a LI-200 pyranometer sensor with a sensitivity range of 400-1100 nm (Li Cor, Lincoln, Neb., USA). Throughout experiments, light intensities in front of (I1) and behind (I2) the cultures were measured and mean values of light intensity within the bacterial cultures (I) were calculated as:33 I ) (I1 - I2)/ln (I1/I2) Light intensity was adjusted throughout the experiments to compensate changes in turbidity due to bacterial growth. Photo-oxidative stress was generated as described earlier24 by adding 1O2 producing methylene blue (Sigma-Aldrich, Seelze, Germany) to liquid cultures at a final concentration of 0.2 µM prior to high light exposure. Oxidative stress was generated by adding H2O2 (Roth, Karlsruhe, Germany) at a final concentration of 1 mM to aerobically growing cultures kept in the dark. Radioactive Labeling of Proteins. Samples of 7 mL were retrieved from shift experiments with Rhodobacter cultures, 10 µCi mL-1 L-[35S]-methionine (Amersham Biosciences, Freiburg, Germany) was added, and samples were incubated for 10 min under the experimental condition. The samples were cooled on ice after incubation, cells were harvested by centrifugation at 10 000g for 10 min at 4 °C and stored at -20 °C until further processing. Extraction of Soluble Proteins and Quantification of Radioactive Label. Harvested cells were washed once with 50 mM Tris-Cl buffer pH 7.5 and resuspended in the same buffer containing 1 mM Phenylmethylsulfonylfluorid (PMSF, Roth) and 1 mM EDTA (Sigma-Aldrich). Disruption of cells was performed on ice in a volume of 600 µL with an ultrasonic disintegrator (Sonopuls GM70, Bandelin, Berlin, Germany). Three cycles of 30 s were used for disruption with a setting of 70 for both amplitude and interval. Remaining intact cells and cell debris were removed by centrifugation at 10 000g for 20 min at 4 °C, and 450 µL of the supernatant was used for ultracentrifugation at 100 000g for 60 min at 4 °C. The colorless supernatant containing the soluble protein fraction was collected and stored at -20 °C until further processing. For quantification of radioactive labeling, 10 µL aliquots of the Journal of Proteome Research • Vol. 6, No. 7, 2007 2461

research articles soluble protein extract were added to 1 mL rotiszint scintillation cocktail (Roth), and radioactivity was measured in a Beckmann LS-6500 scintillation counter (Beckmann Coulter, Fullerton, CA). Protein concentrations of non-labeled samples were determined according to the method of Bradford.34 Two-Dimensional Gel Electrophoresis. Samples of 300 µg of non-labeled protein or 1.5 × 106 cpm of radioactively labeled protein solution were treated with 30 µg of RNase A and 5 units of RQ1 DNase I (Promega, Madison, WI) to remove nucleic acids. Proteins were precipitated with one volume of 10% trichloroacetic acid solution, and precipitates were washed twice with the same solution and once with chilled acetone. Protein pellets were dried and then solubilized in 320 µL of sample buffer containing 7 M urea, 2 M thiourea, 50 mM DTT, 2% Triton X-100 (v/v), 0.2% carrier ampholites pH 3-10, and traces of bromphenol blue. All chemicals were purchased from Roth except the carrier ampholites were obtained from Amersham Biosciences. Samples were applied to 17 cm immobilized pH gradient (IPG) strips with a linear separation range of pH 4-7 (Readystrip, Biorad, Hercules, CA). After overnight rehydration, isoelectric focusing was carried out at 20 °C in a onestep method for 60 000 V-h in a Protean IEF Cell according to the manufactures description (Biorad). First dimension IPG strips were stored at -20 °C after IEF or soaked for 10 min in a solution containing 50 mM Tris-HCl, pH 8.8, 6 M urea, 20% (w/v) glycerol, 2% (w/v) SDS, and 2% DTT (w/v) and afterward for 10 min in the same buffer containing 2.5% (w/v) iodoacetamide instead of DTT. IPG strips were transferred to 12% SDS-polyacrylamide gels and sealed with 1% (w/v) low-melting agarose. Electrophoresis was performed for 1 h at 5 mA and then over night at 7 mA per gel using 2-fold concentrated Trisglycine running buffer.35 After fixation with 25% (v/v) isopropanol and 10% (v/v) acetic acid, gels containing non-labeled proteins were stained with 100 mg L-1 coomassie brilliant blue G-250 (Serva, Heidelberg Germany). Gels containing 35S-labeled proteins were dehydrated with 50% (v/v) ethanol, dried and exposed to phosphoimaging screens for 48-72 h. Phosphoimages were read with a Molecular Imager FX (Biorad) set to a resolution of 100 µm. Protein Spot Quantification and Identification. Protein spots on digital phosphoimages were compared using the Delta2D software version 3.3 as described by the manufactures description (Decodon, Greifswald, Germany). Instead of using a cutoff value, protein spots were included in the analysis when a significant change of protein synthesis rate was observed as indicated by the t-test statistics available in the software. For MALDI analysis, a Proteomics analyzer 4700 (MALDITOF/TOF, Applied Biosystems, Darmstadt, Germany) was used. The matrix applied was alpha-matrix (alpha-Cyano-4-hydroxycinnamic acid) in overlay technique at 5 mg mL-1 in 50% Acetonitrile, 0.1% TFA, 0.45 µL sample, and 0.45 µL matrix solution. The MALDI-MS and MS/MS measurements were performed with a 355 nm Nb-YAG-laser in positive reflector mode with 20 kV acceleration voltage. Sometimes the spectra were accumulated to get better peaklists for the MASCOTSearch in the NCBI database. RNA Extraction and Quantitative Real Time RT-PCR. Cell samples from growth experiments were rapidly cooled on ice and harvested by centrifugation at 10 000g in a cooled centrifuge. Total RNA was isolated by the hot phenol method and quantified by photometric analysis at a wavelength of 260 nm. Samples were treated with RQ1 RNase-free DNase I (Promega), to remove contaminating DNA. Contamination of DNA was 2462

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checked by PCR amplification of RNA samples. A final concentration of 4 ng µl-1 of total RNA was used for real time RTPCR amplifying the following genes with primers indicated by sequence: rpoE: rpoE-A 5′-GTCTGGCAGAAGGCTCAT-3′ and rpoE-B 5′-GTTCTCCTGCTGCATCTC-3′, ggt: ggt-A 5′- GACCTCGCATATCTCCAT-3′ and ggt-B 5′-AGTCGGTGACTTCGTTGT-3′, RSP0799: RSP0799-A 5′-GAACAATTACGCCTTCTC-3′ and RSP0799-B 5′-CATCAGCTGGTAGCTCTC-3′, RSP0847: RSP0847-A 5′-GTGCTTCTCCACGAGGTT-3′ and RSP0847-B 5′ACTCCGTGACGACAAGAC-3′. For normalization of mRNA levels, the rpoZ gene was used, which encodes the ω-subunit of RNA-polymerase of R. sphaeroides.36 Primers applied for analyzing rpoZ mRNA levels were 2.4.1rpoZ-A 5′-ATCGCGGAAGAGACCCAGAG-3′ and 2.4.1rpoZ-B 5′-GAGCAGCGCCATCTGATCCT-3′.19 A one-step RT-PCR kit (Qiagen) was used for reverse transcription followed by PCR as described in the manufacturers manual. SYBR Green I (Sigma-Aldrich) was added in a final dilution of 1:50 000 to the master mix. For individual reactions, master mix and RNA solution were mixed in a final volume of 10 µL and relative quantification of mRNA transcripts was performed in a Rotor-Gene 3000 real time PCR cycler (Corbett Research, Sydney, Australia). For data analysis, slope correction and dynamic tube normalization options were applied in the rotor-gene software version 6.0 (Corbett Research). Crossing point (Cp) values representing the number of cycles where fluorescence signals started to increase in real time RT-PCR were determined for all genes with a fluorescence threshold of 0.002. Relative expression of rpoE, RSP0799, and ggt mRNA was calculated relative to the expression of untreated samples and relative to rpoZ.37 Real time PCR efficiencies were determined by applying serial dilutions of mRNA between a final concentration of 8 and 0.2 ng µL-1. PCR efficiencies were 2.02 for rpoZ, 1.98 for ggt, 2.31 for RSP0799, and 2.04 for RSP0847. In Silico Promoter Analysis. A genome wide search for σE binding sites was performed with PRODORIC, a software tool suitable for regulon studies available at http://prodoric.tubs.de,38,39 using the previously published σE promoter target sequences.17,40 Statistical Analysis. Statistical analysis for comparison of Cpvalues obtained for individual genes under different stress conditions was performed with student’s t-test using Microsoft Excel (Microsoft, Redmond, California). In all cases, significance was assumed if p > 0.95. Statistically significant differences in protein synthesis were tested with the student’s t-test option available in the Delta2D software. Proteins affected by 1O2 were included in the analysis if changes in protein synthesis were significant (p > 0.9).

Results and Discussion Effect of 1O2 on Protein Synthesis Patterns. Changes in protein synthesis patterns were observed when exponentially growing R. sphaeroides cultures were exposed to 1O2 generated by methylene blue in the light (Figure 1A). For 61 proteins representing ∼12% of all protein spots, changes in protein synthesis rates were significant (Figure 1A, Supplemental Table 1 (see Supporting Information)). Of proteins responding to 1O2, a number of 41 were increased and 19 decreased in synthesis. Synthesis rates of many proteins changed within 15 min upon exposure of R. sphaeroides cultures to 1O2 (Figure 2). The majority of proteins reached the largest synthesis rate within 15-60 min and, in rare cases, after 120 min of exposure (data not shown). Individual proteins showed different kinetics in


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Figure 1. Protein synthesis pattern changes by exposure to 1O2 and H2O2. Protein extracts were prepared from cells growing exponentially in the presence of methylene blue and high light (0.2 µM, 800 W m-2) for 60 or 15 min after the addition of H2O2 (1 mM). For radioactive labeling, 10 µCi L-[35S]-methionine was added to 7 mL samples of the cultures and incubated for 10 min. Superimposed images were generated by combining the digitalized autoradiograms in the Delta2D software. Blue colored spots (control conditions) indicate proteins decreased in synthesis, and orange colored spots (treatments) indicate newly synthesized proteins. Spots synthesized with and without treatment are colored in black and indicate no or only small changes in protein synthesis. Proteins depicted with RSP numbers have hypothetical function, which is indicated in Supplemental Table 2 (see Supporting Information). *: Protein encoding genes with increased mRNA levels in response to elevated σE levels.17 Journal of Proteome Research • Vol. 6, No. 7, 2007 2463

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Figure 2. Rapid induction of protein synthesis by 1O2 exposure. The fold change in protein synthesis of individual proteins normalized to dark controls are depicted in A and B. Relative changes in protein synthesis rates represent the mean of three independent experiments. All values showed significant difference to the dark control as tested by the Delta2D software version 3.3 (t-test, p > 0.9). A value of 1 indicates no change in protein synthesis.

response to 1O2 (Figure 2). For example, the synthesis rates of proteins RSP0799 (GloB), RSP2802 (AcrA) and RSP0847 (OmpR) increased very rapidly and decreased after a maximum at 30 or 60 min. In contrast, synthesis rates for RSP0392 (GloA), RSP3272 (Ggt), and RSP3342 (Bfr) increased more slowly and stayed at a high level or decreased slightly after a maximum was reached. However, based on differences in synthesis, kinetics categories of proteins responding differently to 1O2 could not be defined. Elevated levels of σE mRNA were observed by exposure of R. sphaeroides to 1O2,17 and therefore, it has been suggested that σE dependent genes should mediate the adaptation to 1O2 exposure. Subsequent transcriptome analysis of the anti-σE factor mutant ∆ChrR,28 which exhibits elevated σE expression, revealed that genes of 60 operons increased in mRNA levels.17 Of the 61 genes encoding proteins affected in synthesis rate by 1O2 exposure in this study (Figure 1), only 8 showed elevated mRNA levels in strain ∆ChrR.17 Discrepancies between transcriptome and proteome approach were observed earlier for stress responses in other organisms41 and may be explained by general differences of the respective methods. Gel-based proteome approaches usually represent a subset of proteins encoded by genes detected by increased mRNA levels in simultaneous transcriptome approaches.41 2464


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It is remarkable that, despite the known limitations of gel based proteome studies, 53 proteins responded to 1O2 that were encoded by genes not responding to elevated σE levels in R. sphaeroides. This result indicates that either the σE-mediated response has not been fully elucidated or further factors exist that induce the synthesis of proteins upon 1O2 exposure. In addition, the transcriptome study may be biased, because a single microarray experiment was used to characterize the σE regulon.17 Regulatory mechanisms acting at the post-transcriptional level cannot be observed using microarray data and may cause the unusual result. Especially small RNAs (sRNAs) are known to influence gene expression by affecting mRNA translation in several bacteria. As an example, in E. coli, the oxyS sRNA is strongly induced by H2O242 and basepairs to the fhlA and other mRNAs, thereby repressing the translation of the FhlA regulator.43 The transcriptome analysis of strain ∆ChrR indicated that genes induced by 1O2 depend on σE.17 Because ∆ChrR cultures investigated by transcriptome analysis were not exposed to 1O2,17 σE independent gene regulation was missed. Our studies provide strong evidence that regulatory factors other than σE are involved in the response to 1O2. Comparison of the Response to 1O2 and H2O2. In this study, we addressed the question, if the regulatory response triggered on the proteome level by 1O2 is similar to the response upon H2O2 addition. Different effects of both ROS became evident by comparing protein synthesis patterns depicting the response to 1O2 and H2O2 superimposed to patterns derived from control cultures (Figure 1A, B). Exposure of R. sphaeroides to 1O2 affected mostly different cytoplasmic proteins than H2O2. To investigate those differences in greater detail, relative changes of protein synthesis rates were plotted pair wise for individual proteins (Figure 3A). The cloud-like appearance of the pairwise plotted values support the view that the majority of proteins affected by 1O2 were only marginally or not affected by H2O2. However, proteins as, e.g., RSP1591 (Gst), RSP1096 (PpqL), RSP0799 (GloB), and RSP3342 (Bfr), were increased in synthesis rate by both ROS (Supplemental Table 1, Supporting Information). In total, 14 proteins were clearly affected in a similar manner by both ROS (Figure 3A). Therefore, a restricted correlation of the response to 1O2 and H2O2 was observed and will be discussed in detail below. High Light Induced Proteome Responses are Mostly 1O2 Mediated. Generation of 1O2 was observed in pigmented cultures of R. sphaeroides when exposed to high light intensities under oxic conditions,24 and pigment-mediated 1O2 generation was suggested to trigger σE expression.17 These observations imply that the response to high light conditions is triggered by 1O in R. sphaeroides. The majority of all proteins responding 2 to 1O2 exposure were indeed triggered by high light conditions, although the change in synthesis rates was smaller under high light conditions (Figure 3B). This may be explained by rather low amounts of 1O2 generated by photosynthetic pigments when carotenoids are present,24 which is supported by the observations that the σE dependent response is predominantly mounted when carotenoid levels are low in R. sphaeroides.17 Evidently, the regulatory response triggered by 1O2 can be induced by exposure of pigmented R. sphaeroides cultures to high light, which indicates the relevance of the observed response under natural conditions. Genome-Wide Search for σE Target Sequences. It was unclear if genes encoding proteins affected in synthesis rates by 1O2 detected in this study contain a σE target promoter. Therefore, the genome of R. sphaeroides strain 2.4.1 was

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Figure 3. Pair-wise correlation analysis of changes in protein synthesis rates. The correlation plots show relative changes in synthesis of proteins significantly affected by treatment with 1O2, high light, and H2O2. All values were normalized to values obtained with untreated cultures of wild-type strain 2.4.1 (WT). Relative changes in protein synthesis obtained for the treatments of strains 2.4.1, TF18, and ∆ChrR are plotted pair-wise for each protein against values obtained upon 1O2 exposure in strain 2.4.1. (A) Correlation of 1O2 and H2O2 in strain 2.4.1. (B) Correlation of 1O2 and high light in strain 2.4.1. (C) Correlation of 1O2 treatment in strain 2.4.1 and strain ∆ChrR. (D) Correlation of 1O2 treatment in strain 2.4.1 and strain TF18. Only proteins significantly increased by exposure to 1O2 (t-test, p > 0.9) are depicted in A-D. Table 1. Comparison of σE Target Sequencesa gene

locus tag

rpoHII -

RSP_0601b

rpoE rpoE phrA cycA (paaJ) (dsbG)

sequence

ATG-distance

RSP_1091b

TGATCC(N15)CCGTA TGATCC(N15)CCGTA

41 29

RSP_1092b RSP_1091b RSP_1092b RSP_2143b RSP_0296b RSP_2685 RSP_1852b RSP_6222 RSP_3184 RSP_1465

TGATCC(N15)CCGTA TGATCC(N15)CCGTA TGATCC(N15)CCGTA TGATCC(N15)GCGTA TGATCC(N15)CAGTA TGATCC(N14)AGGTA TGATCT(N15)CCGTA TGATCT(N15)CCGTA TGATCT(N14)GCGTA TGATCT(N14)CGGTA

161 85 105 24 63 344 78 47 162 217

function

σ38 putative cyclopropane/ cyclopropene fatty acid synthesis protein σE σE photolyase cytochrome c2 hypothetical UV endonuclease hypothetical putative thiolase hypothetical

a To find σE target sequences, previously published promoter sequences for σE were used in a genome-wide search using PRODORIC.38 Target sequences are arranged according to the rank obtained in PRODORIC; most conserved sequences are shown in the Table first and least conserved sequences last. Bold face letters indicate newly identified promoter sequences. b Previously identified in refs 17 and 40.

searched for σE target promoters with a consensus matrix based on previously published σE target sequences.17,40 By applying the regulon analysis tool of the PRODORIC software,38,39 previously detected σE target sequences were supported. Only two additional genes, RSP1464 (DsbG) and RSP1852, detected in our study contained a σE target promoter (Table 1), and three more genes, RSP2685, RSP3184 (paaJ), and RSP6222, contained a less conserved σE target promoter but were not detected by our proteome or a previous transcriptome study17 (Table 1). In conclusion, our results support earlier data that few genes

triggered by 1O2 exhibit binding sites recognized by σE, which indicates that further regulatory molecules are necessary for the response to 1O2. Exposure to 1O2-Induced σE Dependent and Independent Responses. The impact of σE on changes in synthesis of individual proteins is not clear and therefore needs to be elucidated in further detail. For this purpose, the response to 1 O2 was investigated in the anti-σE deficient strain ∆ChrR with and without treatment of 1O2. Protein synthesis patterns observed without 1O2 treatment of strain ∆ChrR were partially Journal of Proteome Research • Vol. 6, No. 7, 2007 2465

research articles similar to pattern with 1O2 treatment of wild-type strain 2.4.1. This is shown by the similar distribution of pair-wise plotted changes in protein synthesis rates (Figure 3C, Supplemental Table 1 (see Supporting Information)). A highly similar response to 1O2 treated wild-type strain 2.4.1 was observed after treatment of strain ∆ChrR with 1O2. Several proteins (e.g., RSP0558, RSP2275, and RSP2802 (AcrA)) that were not affected by elevated levels of σE in strain ∆ChrR were synthesized upon 1 O2 treatment (Figure 3C). In addition, the change of protein synthesis rates observed in ∆ChrR was lower for many proteins in absence of 1O2. These results indicate a σE independent 1O2 response in R. sphaeroides and that regulatory molecules may exist that affect the σE dependent induction of protein synthesis. Forty-two proteins were affected by 1O2 exposure in strain 2.4.1 and elevated σE levels in strain ∆ChrR (Supplemental Table 1, Supporting Information), but few of the genes encoding those proteins responded to elevated σE levels in an earlier study.17 This implies that important features of the σE-mediated response to 1O2 were missed. Also it was previously not recognized that synthesis of several proteins was repressed in strain 2.4.1 by exposure to 1O2 and by elevated σE levels in strain ∆ChrR (Figure 3B, C). Repression of protein synthesis was potentially mediated by regulatory factors such as RSP0601 (σ38),40 RSP2030 (putative histidine sensor kinase), RSP1409 (TspO),44 RSP0847 (OmpR),45 and RSP1825 (TldD),46 which were increased in protein synthesis by 1O2 or showed elevated levels of mRNA in strain ∆ChrR17 (Supplemental Table 1, Supporting Information). To verify these results, changes in protein synthesis rates induced by 1O2 were investigated in strain TF18, which lacks the genes encoding σE and ChrR. Only a few proteins affected by 1O2 exposure in wild-type strain 2.4.1 were affected in strain TF18 when 1O2 treatment was omitted, e.g., RSP0558, RSP0799 (GloB), and RSP1387 (Figure 3D). The cloud like distribution of the pair-wise plotted values indicates that most proteins were not or marginally affected when σE is lacking. More important, the majority of all proteins affected by 1O2 exposure in wildtype strain 2.4.1 changed in synthesis rate in strain TF18 when exposed to 1O2. However relative changes were smaller compared to strain ∆ChrR or strain 2.4.1 in most cases (Supplemental Table 2, Supporting Information). These results strongly support that a σE independent response to 1O2 indeed exists in R. sphaeroides. Definition of Regulatory Categories in Response to 1O2. It will assist further investigations to define functional categories to discriminate between σE dependent and independent response and to take into account changes in the σE dependent responses that are conditional. Consequently, three categories are defined for the regulatory response to 1O2, which are termed “σE dependent”, “σE conditional dependent”, and “σE independent” (Figure 4, 5 and Supplemental Table 1 (see Supporting Information)). Category 1 (σE dependent response): In this category, proteins were typically affected in synthesis rate by elevated levels of σE (strain ∆ChrR) regardless if cells were exposed to 1 O2. In contrast, protein synthesis rates were not affected when σE was absent (strain TF18). Hence, the response of genes encoding category 1 proteins fully depends on the presence of σE. This category is represented by RSP3272, which encodes a γ-glutamyl-transpeptidase (Ggt). Characteristic changes in protein synthesis rates are depicted by differences in spot intensities on 2D gel sections (Figure 4) and subsequent quantification of spots on several gels (Figure 5). Proteins 2466


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Figure 4. Definition of regulatory categories and comparison of the response to 1O2, high light and H2O2. Sections of single 2Dgel images containing characteristic proteins for each category are shown. Left panels indicate difference in Ggt synthesis rates, which are σE dependent. Middle panels indicate difference in RSP0799 (GloB) protein synthesis rates, which are σE conditional dependent. Right panels indicate differences in OmpR synthesis rates, which indicate a σE independent response. Protein spots indicated by arrows illustrate the response to 1O2, high light conditions, and H2O2. For comparison, all images belonging to one category were normalized to a protein spot not changed in intensity throughout all experiments.

belonging to this category were usually strongly affected in synthesis rate by 1O2 and high light and some were increased in synthesis by H2O2. Category 2 (σE conditional dependent response): Proteins of this category were affected in synthesis by elevated σE levels (strain ∆ChrR) without 1O2 treatment. However, some proteins were also affected by 1O2 treatment in strain ∆ChrR. Synthesis of category 2 proteins was also affected in absence of σE (strain TF18) upon exposure to 1O2, which clearly distinguishes them from category 1 proteins. This category is represented by RSP0799, encoding a putative zinc-dependent hydrolase with glyoxalase function (GloB) (Figures 4 and 5). In comparison to category 1, a smaller number of proteins conditionally dependent on σE are affected in synthesis rate by high light but more by H2O2 (Supplemental Table 1, Supporting Information). Category 3 (σE independent response): Proteins were considered to be σE independently regulated when 1O2 affected their rate of synthesis regardless if σE was present (strain ∆ChrR)

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Figure 5. Changes of protein synthesis in regulatory categories. Fold changes in synthesis of proteins characterizing regulatory categories (Figure 4) upon 1O2, high light, and H2O2 exposure and for experiments with mutant strains TF18 and ∆ChrR are depicted as average of three independent experiments. Relative changes in protein synthesis were determined by normalization of protein spot intensities in treatments and mutant strains to values obtained in untreated wild-type strain 2.4.1. Changes larger than 1 indicate an increase of protein synthesis, and a value equal to 1 indicate no change in protein synthesis. Asterisks indicate changes that were significant (t-test, p > 0.9) compared to untreated wild-type cultures. For clarity, values for RSP0847 (OmpR) were multiplied by a factor of 2. Numbers indicating fold changes for all proteins are shown in Supplemental Table 1 (see Supporting Information).

Figure 6. Expression kinetics of selected genes affected by 1O2. Changes in mRNA levels upon exposure to 1O2 were followed by using real time RT-PCR for genes encoding proteins shown in Figures 4 and 5 and for rpoE, which encodes σE. Measurements were carried out for three independent experiments with very similar results. For clarity, only one graph is shown for each gene analyzed.

or absent (strain TF18). However, elevated levels of σE alone (strain ∆ChrR) did not affect protein synthesis rates. As an example, changes in protein synthesis are indicated by differences in spot intensity for RSP0847 (OmpR, Figures 4 and 5), which putatively regulates the expression of outer membrane proteins. Only a few proteins in category 3 were significantly increased in synthesis rate by high light and some were affected by H2O2 addition (Supplemental Table 1, Supporting Information). Unclassified proteins: A number of 8 proteins were affected in synthesis by 1O2 exposure in wild-type strain 2.4.1 but not in strains ∆ChrR or TF18. Therefore, they do not belong to categories 1-3 (Supplemental Table 1, Supporting Information). The response to 1O2 of those proteins was clearly σE independent; however, the lack of response in both mutants remains obscure and needs further investigation. 1O Dependent Changes of mRNA Levels in Regulatory 2 Categories. Relative changes in mRNA levels were investigated for genes encoding proteins used for definition of regulatory categories (Figures 4 and 5) to verify the obtained results (Figure 6). In addition, relative changes for rpoE mRNA levels were analyzed. Clearly, rpoE mRNA levels increased within a few minutes after exposure to 1O2, indicating the instant response of the σE/ChrR system with respect to transcription. The changes in mRNA levels after induction by 1O2 for RSP3272 (ggt), RSP0799 (gloB), and RSP0847 (ompR) were very similar (Figure 6). However, protein synthesis kinetics were only similar for category 1 and 2 (RSP3272 and RSP0799) but different for category 3 (RSP0847). Regulatory Categories Respond Differently to 1O2, High Light, and H2O2. For the understanding of how 1O2 induced

Table 2. Correlation Analysis of Changes in Protein Synthesis Ratesa conditions

linear regression coefficient

WT, 1O2 vs

all proteins

category 1 σE dependent

category 2 σE conditional dependent

category 3 σE independent

WT, H2O2 WT, high light ∆ChrR ∆ChrR, 1O2 TF18 TF18, 1O2 nc

0.10 0.76 0.71 0.81 0.02 0.05 61

0.26 0.92 0.77 0.86 0.21b 0.10b 14

0.03 0.18 0.66 0.85 0.23 0.32 19

0.05† 0.16 0.08 0.65 0.00 0.60 18

a Correlation coefficients were obtained from a linear regression performed either for all proteins responding to 1O2 or proteins in the respective regulatory categories. b Negative slope. c n, number of proteins analyzed.

changes in protein synthesis correlates with high light and H2O2 responses, a linear regression analysis was performed for the defined regulatory categories. Responses to H2O2: A correlation for the response to 1O2 and H2O2 was not observed when all proteins affected by 1O2 were used for linear regression (Figure 1B, Table 2). The same picture holds true for categories 2 and 3, but a weak increase in correlation was indicated by a value of 0.28 for category 1 (σE dependent proteins). This finding reflects the fact that some proteins showed the same response when exposed to 1O2 or H2O2 (Figure 3A). Because it has been suggested that σE is specifically triggered by 1O2 but not by other ROS,17 this finding may be contradictory to previous results and questions the specificity of the σE-mediated response. A lacZ reporter gene fusion was used to prove the inability of H2O2, paraquat, and diamide to activate σE expression.17 Recent results obtained in our laboratory show that in vivo expression studies using β-galactosidase as a reporter are strongly affected by oxidative stress conditions.47 The β-galactosidase activity may be lost or is strongly reduced by exposure to ROS, and therefore, β-galactosidase reporter fusions are not useful to study oxidative stress effects in Rhodobacter. Earlier transcriptome analysis Journal of Proteome Research • Vol. 6, No. 7, 2007 2467

research articles support that the addition of H2O2 increased transcription of σE and genes containing a σE target promoter.40,48 Interestingly, mRNA levels of rpoHII (σ38) and several genes containing a putative σ38 target promoter also increased after H2O2 addition,40,48 which could explain the observed response if σ38 is triggered by H2O2 independently. A sigma factor cascade involving σ38 induction by σE may exist for two reasons. First, σ38 expression was increased by elevated σE levels and second, σ38 contains a σE target promoter.17 Obviously, the regulation of genes affected by both 1O2 and H2O2 is more complex, and the question, how genes are regulated that are induced by both ROS, needs to be elucidated. Response to high light: Pair-wise plotted changes in protein synthesis indicated that the response to high light is potentially mediated by 1O2 (Figure 3A). This is supported by a high correlation coefficient of 0.76 as obtained when all proteins affected by 1O2 were used for linear regression analysis (Table 1). However, the synthesis of two proteins, RSP1410 and RSP2802 (AcrA), was not induced by high light but only by the exposure to 1O2. In addition, three proteins, RSP2241 (HisH), RSP3317 (TetR), and RSP3571 (ZnuA), were induced by high light conditions only and were repressed by 1O2 (Supplemental Table 1, Supporting Information). Values for TetR and ZnuA were not included in the correlation analysis, because synthesis was virtually absent in the presence of 1O2 and extremely high under high light conditions. Proteins of category 1 (σE dependent proteins) exhibited the highest correlation value (0.92), and categories 2 and 3 showed drastically decreased correlation coefficients, indicating no or only a weak response to high light in both categories (Table 1). These results imply that only a part of the response to methylene blue generated 1O2 can be triggered by 1O2 generated in the photosystem. Because methylene blue is soluble in water, it may elicit stress response throughout the cytoplasm in contrast to the membrane bound photosystem. High light induced synthesis of proteins might tolerate moderate 1O2, however, higher levels of 1O2 as generated by methylene blue evidently repressed their synthesis, supporting the presence of a well-balanced regulation. Characteristics of σE independent responses to 1O2: Changes in protein synthesis patterns of strains ∆ChrR and TF18 indicated a σE independent regulatory mechanism responding to 1O2 and a mechanism affecting the σE dependent response (Figure 3B, C). The changes of protein synthesis rates observed without 1O2 treatment in strain ∆ChrR strongly correlated with the response to 1O2 treatment in wild-type strain 2.4.1 in categories 1 and 2 but not in category 3 (Table 2). Upon 1O2 treatment of strain ∆ChrR, the correlation coefficient slightly increased for categories 1 and 2 and 8-fold for category 3. Therefore, induction of category 1 and 2 proteins strongly depends on σE, and synthesis of category 3 proteins was clearly σE independent. As expected, a correlation for changes in protein synthesis patterns was lacking between 1O2 exposed wild-type strain 2.4.1 and untreated strain TF18 among all categories. When strain TF18 was treated with 1O2, induction of protein synthesis was not observed for category 1 proteins. This finding independently shows that protein synthesis in this category strictly depends on the presence of σE. Further increase in protein synthesis by 1O2 exposure of strain ∆ChrR may therefore be mediated by a σE independent mechanism. In contrast, the correlation of changes in protein synthesis was increased for strain TF18 in category 2 and strongly increased in category 3 upon 1O2 treatment (Table 2). This finding provides further evidence for a σE independent response to 1O2 2468


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Figure 7. Synthesis of proteins upon 1O2 and H2O2 exposure is affected in a mutant lacking the RNA chaperon Hfq. Relative changes in synthesis of protein RSP2275 in wild-type strain 2.4.1 and strain 2.4.1 hfq are shown on 2D-gel image sections. On all images, protein spots corresponding to RSP2275 are circled, and images were normalized for spot intensities using protein spots not changed in intensities by treatment with 1O2 and H2O2 or in the hfqmutant.

in R. sphaeroides. Also, the correlation analysis supports the finding that proteins in category 2 were indeed conditional dependent on σE and can therefore also be induced by a different, yet unknown, regulatory molecule. The increased correlation coefficients observed in strain TF18 upon 1O2 exposure show that the σE independent response (category 3) and the modulation of the σE dependent response (category 2) is common, because 61% of all proteins affected by 1O2 in strain 2.4.1 were found in those categories. In conclusion, the obtained results imply that regulatory molecules are present in R. sphaeroides that affect the σE mediated response to 1O2 but do not independently induce protein synthesis. In addition, regulatory molecules inducing protein synthesis upon 1O2 treatment may not only rescue the σE dependent response, but may act in concert or independently of σE. Interestingly, protein RSP2275 showed a decreased synthesis without 1O2 treatment and induction of protein synthesis upon 1O2 treatment in both ∆ChrR and TF18 and RSP2802 only in strain TF18. This finding underlines the σE independent response (RSP2275) and implies an overlap of different regulatory pathways responding to 1O2 (RSP2802). In conclusion, the observations suggest that putatively several regulatory molecules mediate the 1O2 response independent or in concert with σE, which needs to be investigated in greater detail in the future. Hfq is Involved in Regulation of 1O2 Induced Genes. Hfq is an RNA chaperone that is widely distributed in bacteria and binds small RNAs (sRNAs) that convey regulatory function by binding to mRNAs expressed, e.g., under stress conditions.49,50 To test the role of Hfq for the induction of protein synthesis in response to 1O2, an hfq deletion mutant was exposed to 1O2 and H2O2. The deletion of hfq in R. sphaeroides affected the sensitivity to 1O2 but not toward H2O2. In detail, treatment of the R. sphaeroides hfqmutant with 1O2 generated by methylene blue in the light increased inhibition in zone diffusion assays by 20% compared to wild-type cells, but the complemented strain 2.4.1hfq showed the same response as the wildtype strain 2.4.1 (data not shown). Further analysis of the hfqmutant showed that protein synthesis for several proteins has drastically changed. Obviously, Hfq mediates part of the σE independent response in R. sphaeroides, as shown for the synthesis of RSP2275 (Figure 7). Without oxidative stress treatment, the synthesis of RSP2275 is strongly (126-fold) increased in the hfqmutant when compared with the synthesis in cultures of wild-type strain 2.4.1. This clearly shows that the presence of Hfq repressed the synthesis of RSP2275 in

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R. sphaeroides under no-stress conditions. Hfq acts as an RNA chaperone and mediates the binding of sRNAs to their target mRNAs. This result therefore indicates an important function of sRNAs in the regulation of 1O2 triggered genes. Addition of H2O2 did not increase protein synthesis for RSP2275 in cultures of wild-type strain 2.4.1. In the hfqmutant, synthesis of RSP2275 was increased when compared to wild-type 2.4.1 cultures. However, when compared to the untreated control, synthesis of RSP2275 was obviously decreased. Exposure to 1O2 induced the synthesis of the RSP2275 protein in cultures of wild-type 2.4.1, but synthesis could not be observed in the hfqmutant (Figure 7). In conclusion, Hfq certainly plays an important role in oxidative stress response in R. sphaeroides. The contrasting effects on 1O2 and H2O2 exposure as well as the discrepancies between transcriptome and proteome response to both ROS may be caused by post-transcriptional regulation mediated by binding of small regulatory RNAs. However, the presence of sRNAs and their role in response to oxidative and photo-oxidative stress needs to be further investigated in R. sphaeroides. Putative Function of Proteins Responding to 1O2. Important cellular components as lipids, proteins, DNA, and RNA are targets for oxidative damage by 1O2. Proteins are the primary target of 1O2 damage since they are the most abundant cellular components and some amino acid residues react more rapidly with 1O2 than other biomolecules.5 The regulatory response to 1 O2 observed in R. sphaeroides supports this expectation because increased synthesis rates were observed for proteins involved in folding, replacement, repair, or degradation of proteins (Supplemental Table 2, Supporting Information). Two molecular chaperones, RSP1207 (HslO) and RSP1219 (GrpE), may be involved in the response to 1O2. Synthesis of HslO is also induced by H2O2, but GrpE is specifically responding to 1 O2 in a σE independent manner (Supplemental Table 1, Supporting Information). However, a higher turnover of those chaperones may also be induced because they are damaged by ROS or as a secondary effect of damages generated by oxidative stress. The reaction of 1O2 with amino acid residues occurs rapidly with Cys, His, Met, Tyr, and Trp at physiological pH values and generates toxic intermediates as protein peroxides, sulfoxide and cysteic acid residues in vivo.5 Putatively, increased synthesis of alkylhydroperoxide reductase RSP2973 (AhpC), which may be involved in degradation of organic peroxides,51,52 is a response to the generation of organic peroxides. This response is supported by induction of membrane-bound glutathione-peroxidase Gpx (RSP2389) upon 1O2 exposure.17,24 Gpx was also increased in mRNA levels by 1O2 exposure in other organisms,53 and bovine Gpx was shown to efficiently degrade peptide peroxides in vitro.54 Peroxidation of amino acid residues may lead to a loss of function of proteins. Proteins damaged by peroxidation need to be cleaved into peptides, which are more efficiently detoxified by, e.g., Gpx.54 In R. sphaeroides, this could be accomplished by proteases RSP0197 (ClpP)55,56 and RSP0355 and the peptidase RSP1096-1097 (PpqL). The synthesis of proteases, peptidases, and peroxidases (Supplemental Table 2, Supporting Information) shows that the proposed mode of protein peroxide detoxification elucidated in vitro5,54,57 potentially takes place in R. sphaeroides. An increased synthesis of ABC-transporter subunits as RSP2275 and RSP2802 (AcrA) may indicate that small toxic waste products of biomolecules may potentially be excreted. Both proteins belong to a branched amino acid

transporter family and to a multidrug efflux pump, respectively, which may suit this function.58,59 Reduced glutathione plays very likely a pivotal role in the defense against 1O2 in R. sphaeroides. This is supported by the increase in synthesis of several proteins that carry out glutathione-dependent reactions. Proteins involved in the glutathione-dependent degradation of methylglyoxal were strongly increased in synthesis by 1O2 exposure, as RSP0392 (Glyoxalase I, Glo A), RSP0799 (putative Glyoxalase II, GloB), and RSP 2294 (Glyoxalase II, GloB). Methylglyoxal is generated when catabolic and anabolic reactions are imbalanced,60 which occurs under oxidative stress conditions. The photosynthetic apparatus is constantly generating 1O2.4 Therefore, membrane-associated and periplasmic proteins should be affected because they are in close proximity to the place of 1O2 generation. Membrane proteins were not analyzed in this study, but two oxidative stress related periplasmic proteins were increased in synthesis by 1O2. The protein RSP3272 (γ-glutamyl-transferase, Ggt) is involved in regeneration of GSH in the periplasm and, therefore, regenerating reducing power and ensuring redox homeostasis.61 Also RSP1464 (DsbG) a member of the DsbA family was increased in synthesis by 1O2 exposure. In E. coli, dsbG encodes a periplasmic disulfide isomerase and exhibits chaperone activity62 but appears not to be essential.63 Therefore, it is likely that increased synthesis of DsbG is triggered by oxidative stress conditions to prevent misfolding and aggregation of proteins in the periplasm, which is facilitated by 1O2 exposure.5 Induction of katE and Scavengers of Divalent Metal Ions: Indirect Effects of 1O2. Interestingly, RSP2779 (Katalase, KatE) was increased in synthesis by 1O2 exposure. Because katE is also induced by the addition of H2O2 and is expressed in exponential phase,19,48 it is conceivable that this effect is indirect. KatE is potentially synthesized at a higher rate, because exposure to 1O2 specifically modifies the structure and therefore the function of catalases.64 Consequently, catalase synthesis rates are increased, to maintain cellular levels of H2O2 at low concentrations to prevent damages of iron sulfur clusters and subsequent generation of hydroxyl radicals by the Fenton reaction.65 An increased formation of H2O2 as a potential side effect of 1O2 exposure is also indicated by increased synthesis of proteins that scavenge or excrete Fe(II) as RSP3342 (Bacterioferritin, Bfr), RSP0904 (SitA), and RSP2913 (AfuA). In contrast, uptake mechanisms for Fe(II) and Zn(II) were decreased, as indicated by the decreased RSP0132 (MetQ) and RSP3571 (ZnuA) synthesis (Supplemental Table 2, Supporting Information).

Conclusions Our data support the existence of a well-balanced regulatory network responding to 1O2 in R. sphaeroides. The σE/∆ChrR system triggers the expression of the many genes (grouped to category 1) encoding proteins with changed synthesis rates under photo-oxidative stress. It is, however, evident that other regulatory molecules (i) affect the σE dependent response of some genes (category 2) and (ii) regulate gene expression independently from σE (category 3). Only category 1 genes showed a limited overlap of H2O2 and 1O2 responses, suggesting that σE is involved in transmission of both stimuli. A strong correlation between 1O2 and high light response in category 1 supports the view that high light elicits a σE dependent response by generating 1O2. As one of the additional factors that are part of the σE independent response to 1O2 in R. Journal of Proteome Research • Vol. 6, No. 7, 2007 2469

research articles sphaeroides, we identified the RNA chaperone Hfq. This implies a role of small regulatory RNAs that affect the expression of target genes at a post-transcriptional level, which needs to be elucidated in more detail.

Acknowledgment. We thank Prof. Timothy Donohoue for providing Rhodobacter strains TF18 and ∆ChrR, Dr. Richard Mu ¨ nch for help with PRODORIC, and Prof. K.-H. Mu ¨ hling for providing access to the Delta2D software. The hfq- strain was constructed by Dr. Tanja Zeller, Kerstin Haberzettl, and Anne Vomberg. Supporting Information Available: Supporting material includes two tables with the proteins identified in this study. Supplemental Table 1 includes all information on the changes in synthesis for individual proteins by 1O2, high light, and H2O2 in wild-type strain 2.4.1 and for changes in mutant strains TF18 and ∆ChrR with and without treatment of 1O2. Proteins have been listed according to the regulatory categories defined in the manuscript. In Supplemental Table 2, all proteins identified are listed according to functional categories. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Imlay, J. A. Pathways of Oxidative Damage. Annu. Rev. Microbiol. 2003, 57, 395-418. (2) Messner, K. R.; Imlay, J. A. Mechanism of superoxide and hydrogen peroxide formation by fumarate reductase, succinate dehydrogenase, and aspartate oxidase. J. Biol. Chem. 2002, 277(45), 42563-42571. (3) Seaver, L. C.; Imlay, J. A. Are respiratory enzymes the primary sources of intracellular hydrogen peroxide? J. Biol. Chem. 2004, 279(47), 48742-48750. (4) Krieger-Liszkay, A. Singlet oxygen production in photosynthesis. J. Exp. Bot. 2005, 56(411), 337-346. (5) Davies, M. J. Singlet oxygen-mediated damage to proteins and its consequences. Biochem. Biophys. Res. Commun. 2003, 305(3), 761-770. (6) Gorman, A. A.; Rodgers, M. A. Current perspectives of singlet oxygen detection in biological environments. J. Photochem. Photobiol. B: Biol. 1992, 14(3), 159-176. (7) Skovsen, E.; Snyder, J. W.; Lambert, J. D. C.; Ogilby, P. R. Lifetime and diffusion of singlet oxygen in a cell. J. Phys. Chem. B 2005, 109(18), 8570-8573. (8) Shick, J. M.; Dunlap, W. C. Mycosporine-like amino acids and related gadusols: Biosynthesis, accumulation, and UV-protective functions in aquatic organisms. Annu. Rev. Physiol. 2002, 64, 223-262. (9) Storz, G.; Zheng, M. Oxidative stress. In Bacterial Stress Responses; Storz, G., Hengge-Aronis, R., Eds.; American Society for Microbiology: Washington, 2000; pp 47-59. (10) Cogdell, R. J.; Frank, H. A. How carotenoids function in photosynthetic bacteria. Biochim. Biophys. Acta 1987, 895(2), 63-79. (11) Cogdell, R. J.; Howard, T. D.; Bittl, R.; Schlodder, E.; Geisenheimer, I.; Lubitz, W. How carotenoids protect bacterial photosynthesis. Philos. Trans. R. Soc. London, Ser. B 2000, 355(1402), 1345-1349. (12) Kochevar, I. E. Singlet oxygen signaling: from intimate to global. Sci. STKE 2004, 221, e7. (13) Laloi, C.; Apel, K.; Danon, A. Reactive oxygen signalling: The latest news. Curr. Opin. Plant Biol. 2004, 7(3), 323-328. (14) Klotz, L. O.; Kro¨ncke, K. D.; Sies, H. Singlet oxygen-induced signaling effects in mammalian cells. Photochem. Photobiol. Sci. 2003, 2(2), 88-94. (15) Sun, Y. K.; Eun, J. K.; Park, J. W. Control of singlet oxygen-induced oxidative damage in Escherichia coli. J. Biochem. Mol. Biol. 2002, 35(4), 353-357. (16) Whitworth, D. E.; Hodgson, D. A. Light-induced carotenogenesis in Myxococcus xanthus: Evidence that CarS acts as an antirepressor of CarA. Mol. Microbiol. 2001, 42(3), 809-819. (17) Anthony, J. R.; Warczak, K. L.; Donohue, T. J. A transcriptional response to singlet oxygen, a toxic byproduct of photosynthesis. Proc. Natl. Acad. Sci. U.S.A. 2005, 102(18), 6502-6507. (18) Anthony, J. R.; Newman, J. D.; Donohue, T. J. Interactions between the Rhodobacter sphaeroides ECF sigma factor, σE, and its anti-sigma factor, ChrR. J. Mol. Biol. 2004, 341(2), 345-360.

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Protein Synthesis Patterns Reveal a Complex Regulatory Response to

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