Comparative Proteomic Studies in Rhodospirillum rubrum Grown under Different Nitrogen Conditions Tiago T. Selao, Stefan Nordlund, and Agneta Nore´n* Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Sweden Received November 19, 2007
Forty-four differentially expressed proteins have been identified in the photosynthetic diazotroph Rhodospirillum rubrum grown anaerobic and photoheterotrophically, with different nitrogen sources, using 2D-PAGE and MALDI-TOF, from gels containing an average of 679 ( 52 (in N+) and 619 ( 37 (in N-) protein spots for each gel. A higher level of expression was found under nitrogen-rich growth, for proteins involved in carbon metabolism (reductive tricarboxylic acid cycle, CO2 fixation, and poly-βhydroxybutyrate metabolism) and amino acid metabolism. The key enzymes RuBisCO and R-ketoglutarate synthase were found to be present in higher amounts in nitrogen-rich conditions. Ntr and Nif regulated proteins, such as glutamine synthetase and nitrogenase, were, as expected, induced under nitrogen-fixing conditions and glutamate dehydrogenase was down regulated. A novel 2Fe-2S ferredoxin with unknown function was identified from nitrogen-fixing cultures. In addition to differential expression, two of the identified proteins revealed variable pI values in response to the nitrogen source used. Keywords: Rhodospirillum rubrum • nitrogen fixation • comparative proteome • 2D-PAGE
1. Introduction The diazotrophic R-proteobacterium Rhodospirillum rubrum has the capacity to grow under a wide range of metabolic conditions. This versatility includes photoautotrophic, chemoautotrophic, photoautolithotrophic, as well as heterotrophic growth,1 which creates a demand for regulation both at a transcriptional as well as at metabolic level for the cell to be able to adapt to different environmental conditions. Regulation of nitrogen fixation in photosynthetic bacteria involves complex regulatory signal transduction with changes in nitrogen and energy levels as primary signals for regulation. The differential proteome of a free living nitrogen-fixing organism does contribute to the understanding of the effects on metabolism that a drastic change in fixed nitrogen would have. In other nitrogen-fixing photosynthetic bacteria such as the cyanobacteria Nostoc sp. PCC 731022 and Nostoc sp. PCC 71203 as well as the free-living R-proteobacterium Rhodopseudomonas palustris,4 global protein profiles have been provided either as whole-cell proteomes or as differential proteomes and this work focusses on the differential proteome of the soluble fraction of R. rubrum to obtain comparative data from photoheterotrophic nitrogen-fixing versus non nitrogen-fixing conditions. Nitrogen Metabolism. The two most abundant nitrogen sources for prokaryotes are ammonium ions and nitrate. R. rubrum does not grow on nitrate but ammonium ions or amino acids sustain growth. Atmospheric dinitrogen (N2) is only * To whom correspondence should be addressed. Agneta Nore´n, PhD, Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Sweden. E-mail:
[email protected]. Phone: +46 8 16 2592. Fax: +46 8 15 3679. 10.1021/pr700771u CCC: $40.75
2008 American Chemical Society
available to diazotrophic bacteria, such as R. rubrum, through the reaction catalyzed by the nitrogenase complex. N2 is reduced to ammonia in an ATP-dependent manner, which requires at least 16 ATP and 8 electrons for each N2 molecule reduced, making this a very energy demanding process for the cell. The ammonium ions produced are assimilated via glutamine synthetase (GS) and glutamate synthase (GOGAT) to be further used in cellular anabolic processes, e.g., nucleotide and protein synthesis.5 Nitrogenase (a highly conserved enzyme) is an oxygen-sensitive protein complex comprised of the molybdenum iron protein/dinitrogenase (NifDK) and the iron protein/dinitrogenase reductase (NifH), synthesized during nitrogen-poor and anaerobic conditions.6 The synthesis is dependent of a number of nitrogen fixation (nif) gene-encoded proteins that are involved in the formation and maturation of the apoproteins. Nitrogenase, per se, is under tight regulation at transcriptional level in all diazotrophs studied,6 and in some of these, including R. rubrum, it is also regulated at a metabolic level.7 Transcription of the nif genes is in R. rubrum and R. palustris (both free living, non-symbiotic, photosynthetic diazotrophs) under the control of the NifA protein which, in R. rubrum (as in Azospirillum brasilense 8), has been shown to interact with a signal transduction PII protein, GlnB.9 Expression of the nif genes in R. rubrum is not under the control of the NtrBC system, as it is the case of Rhodobacter capsulatus or Klebsiella pneumoniae6 and, also unlike R. capsulatus, homologue genes for the global regulatory proteins RegA/ RegB10 have never been found in the R. rubrum genome thus indicating an alternative global regulatory pathway balancing the carbon to nitrogen (C/N) ratio. Journal of Proteome Research 2008, 7, 3267–3275 3267 Published on Web 06/21/2008
research articles The PII protein family is present through a wide range of organisms, from bacteria such as Escherichia coli, where they were first discovered, to plants.11 Altogether three PII paralogues are present in R. rubrum, GlnB, GlnJ, and GlnK,12 and their functions have been thoroughly studied in our laboratory as well as in others.13,14 All three PII proteins in R. rubrum have been shown to be modified, in vitro, by uridylylylation depending on the presence of R-ketoglutarate, ATP, as well as divalent cations,15 and the homologues in Escherichia coli have been shown to bind R-ketoglutarate and ATP.16 As such, PII proteins are in the center of a regulatory network, sensing changes in carbon (through the binding of R-ketoglutarate) as well as being part of the signaling pathway for nitrogen availability through uridylylation/deuridylylation by the bifunctional enzyme GlnD. It was also shown that in E. coli PII proteins can sense the ATP/ ADP ratio,17 thus enabling the integration of carbon, nitrogen and energy signals within the same pathway. This integration enables the coordinated expression of several other enzymes involved in nitrogen assimilation. The expression of GlnJ in R. rubrum has been established to be regulated by the Nitrogen regulation (Ntr) system,18 i.e., it is only expressed under low nitrogen conditions (Wang, H. et al., personal communication). This PII paralog has also been shown to have a role in the posttranslational regulation of nitrogenase13 and interact with an ammonia channel protein.19 In Nostoc sp. PCC 7120, an equally intricate pathway for the regulation of fixed nitrogen levels is dependent on the interaction of a PII protein with the regulatory protein NtcA where the key metabolite R-ketoglutarate plays a central role in the signaling of carbon and nitrogen levels.20,21 Under nitrogen-rich conditions, the ammonium ions are further assimilated by glutamate dehydrogenase (GDH), which has a higher Km for NH4+ than GS.5 The expression of GDH has in E. coli been shown to be repressed by a transcriptional regulator (NAC, nitrogen assimilation control protein) in response to low nitrogen concentrations. nac transcription is regulated by the Ntr system in response to the nitrogen level in E. coli, thereby coupling GDH regulation to the general nitrogen regulatory system.22 The nitrogen regulatory mechanism for GDH in R. rubrum is yet unknown. Carbon Metabolism. R. rubrum can grow anaerobically in the light on CO2 and H2 as carbon and electron sources, respectively.23 The assimilation of CO2 involves the CalvinBenson-Bassham (CBB) cycle where RuBisCO is the key enzyme for CO2 fixation. Under photoheterotrophic growth the CBB cycle has been suggested to work as an electron sink to maintain the redox balance in the cell in contrast to diazotrophical conditions, where much of the reducing power is utilized by nitrogenase.10 In addition to the CBB cycle, a bifunctional carbon monoxide dehydrogenase is present in R. rubrum, which converts CO to CO2 for further assimilation, thus providing an additional carbon source to this bacterium.24 As reported by Joshi et al., a RuBisCO R. rubrum mutant strain shows increased levels of CO dehydrogenase, an effect that could be reversed by overexpressing RuBisCO, hence demonstrating that a functional CBB cycle affects the control of the CO dehydrogenase levels.25 Photoheterotrophic growth of R. rubrum can be supported by the addition of acetate, pyruvate, succinate or malate.1 Carbohydrates, such as fructose, are fermented or otherwise metabolized under aerobic conditions with oxygen as the terminal electron acceptor. Fermentation has been established in R. rubrum by adding fructose without any accessory oxidant, 3268
Journal of Proteome Research • Vol. 7, No. 8, 2008
Selao et al. yielding succinate, acetate, hydrogen, and carbon dioxide as final products.26 The TCA cycle enzymes have been the subject of many investigations over the years, in R. rubrum.27,28 The oxidative TCA cycle has been shown to operate under aerobic and also under microaerophilic conditions but it should be pointed out that all TCA cycle enzymes are present under either anaerobic (in the light) as well as aerobic (in the dark) conditions.29 In addition to this oxidative TCA cycle, a reductive TCA (RTCA) cycle has been shown to operate in R. rubrum under anaerobic conditions, using succinate as a substrate, where two ferredoxin-dependent enzymes, R-ketoglutarate synthase and pyruvate synthase, play important roles.30 The presence of all the enzymes in this RTCA cycle was established by measuring their activities in R. rubrum extracts. Acetyl-CoA generated through the RTCA cycle was shown to be used for further synthesis of lipid or C3-C6 carbon compounds.27,31 When carbon is in excess, intracellular carbon is stored as poly-β-hydroxybutyrate (PHB) in R. rubrum. When needed, PHB is mobilized by a PHB depolymerase system consisting of a soluble PHB depolymerase (PhaZ1/Z2), a heat-stable activator (phasin, ApdA) and a 3-hydroxybutyrate dimer hydrolase.32 The synthesis of polyhydroxyalkanoates (PHA) in R. rubrum involves a polymerization of short to medium chain length PHA, carried out by PHA synthase (PhaC) when the cells are grown on a diverse range of carbon sources, which demonstrates the broad substrate specificity for PHA synthetase.33 In the nitrogen-fixing bacterium Sinorhizobium meliloti, the PHB cycle has been shown to involve the action of a bidirectional acetoacetyl-CoA reductase and the acetylCoA generated from PHB degradation is suggested to be further assimilated by the TCA cycle.34 In R. rubrum, the pathway through which acetyl-CoA is metabolized is dependent on several factors, such as aerobicity/anaerobicity, carbon and nitrogen sources, and whether or not light is available.1 As mentioned above, other proteomic studies have already been performed in cyanobacteria and R. palustris. These have shown that in the cyanobacterium Nostoc sp PCC 7120 grown under nitrogen-fixing conditions, proteins involved in energy metabolism and photosynthesis were up regulated, reflecting a demand for increased carbon supply. To be noted is the up regulation of glucose-6-phosphate dehydrogenase (G6PD) and a glycogen phosphorylase essential for generating glucose-1phosphate, both important enzymes for the oxidative pentose phosphate (OPP) pathway and the generation of NAD(P)H.3 In R. rubrum, however, the presence of G6PD has not been verified, while this pathway is not likely to be involved in the generation of reductants.35 The R. palustris protein expression profile under nitrogenfixing conditions revealed an expected up regulation of nitrogenregulated proteins as well as in the expression of a number of proteins involved in electron transfer that could be essential for the supply of reductants to the energy demanding nitrogenase reaction.4 The use of proteomic techniques, where the level of differentially expressed proteins shows how a primary signal affects the protein profile is an important tool to increase the understanding of the complex interplay between fixed carbon/ energy and nitrogen metabolism. This work provides additional information regarding the molecular regulation of nitrogen versus carbon signaling in the cell, in nitrogen-fixing versus non fixing (nitrogen-rich) cultures of R. rubrum.
research articles
Rhodospirillum rubrum Grown under Nitrogen Conditions
2. Materials and Methods 2.1. Growth Conditions and Cell Fractionation. Rhodospirillum rubrum S1 was grown photoheterotrophically, at 30 °C in minimal media, as previously described,36 using 28 mM ammonium chloride (N+, nitrogen-rich) or sparging with 95 % N2-5 % CO2 (N-, nitrogen-fixing) as nitrogen sources. Cells (3 independent cultures for each condition) were anaerobically harvested at OD600 1.5-2 by centrifugation at 3000g for 15 min in a Beckman JLA 8.1000 rotor and washed twice in degassed and nitrogen-sparged buffer (buffer A) containing 100 mM Tris (pH 7.5) and 1 mM dithionite. Whole cells were frozen in liquid nitrogen until further use. Cell pellets (ca. 5 g wet weight/L culture) were resuspended at a ratio of 3 mL buffer/g wet weight cell pellet in degassed buffer A supplemented with DNAse (final concentration of 20 µg mL-1), 1 mM phenylmethylsulfonyl fluoride (PMSF, Boehringer-Manheim) and one tablet of Complete Mini EDTA-free Protease Inhibitor (Roche) for each 50 mL of extract. Lysis was performed by passing the cell suspension twice through a French Pressure Cell, at 18 000 psi. The cell lysate was centrifuged at 100 000g for 90 minutes, using a Beckman Ti70 rotor, and the supernatant was recentrifuged to ensure full separation from the membrane fraction. Purified soluble fractions were frozen in liquid nitrogen until use. 2.2. Sample Preparation. The protein concentration of the soluble fractions was determined by the Lowry method, as previously described.37 Two-hundred micrograms of soluble protein fraction was treated with DNAse I, in the presence of 1 mM PMSF and 5 mM MgCl2, for 30 minutes, in a sealed vial with anaerobic atmosphere. Proteins were precipitated with 10 % TCA in acetone (final volume of 500 µL), for 45 min at -20 °C, centrifuged for 5 min at 12 000g, resuspended in 25 µL of double-distilled water (ddH2O) and washed with 90% acetone at -20 °C. The pellets were air-dried and resuspended in 250 µL rehydration solution containing 7 M urea, 2 M thiourea, 4% CHAPS (Calbiochem), 1% Triton X-100 (GE Healthcare), 0.5% IPG Buffer 4-7 L (GE Healthcare), 0.002% bromphenol blue, and 65 mM dithiothreitol (DTT). Samples were incubated at room temperature overnight and stored at -80 °C until further use. 2.3. Two-Dimensional Electrophoresis. Two-hundred micrograms of the protein samples (prepared as described above) were loaded on 13 cm strips, pH gradient 4-7 (GE Healthcare) and rehydrated for a minimum of 16 hours, at 20 °C. IEF was performed in an IPGphor apparatus (GE Healthcare), at 20 °C, in four steps: 500 V for 1500 Vhr, gradient from 500 to 1000 V for 1000 VHr, gradient from 1000 to 8000 V for 7500 VHrs, and 8000 V for 15 000 VHr (total of 25 000 Vhr). The focused strips were stored at -80 °C until further analysis. Prior to running the second dimension, the strips were incubated in 20 mL of re-equilibration solution (50 mM Tris, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and 0.002% bromphenol blue) with 65 mM DTT, for 15 min, followed by an alkylation step in reequilibration solution with 135 mM iodoacetamide. The second dimension was run in 10% acrylamide gels (18 cm × 14 cm × 1 mm), using 25 mM Tris, 20 mM glycine, 0,1% SDS as running buffer, at 4°C. Gels were run at 5 mA/gel for one hour, followed by 20 mA/gel until the end of the run. 2.4. Staining and Analysis. Gels were stained with “Blue Silver” stain, at room temperature for a minimum of 20 h, as previously described,38 scanned using a GS-800 Calibrated Densitometer (BioRad) and analyzed using PDQuest 7.3.0
(BioRad). For the analysis, the best gels for each culture (best out of three) were chosen, creating a matchset with 3 gels for nitrogen-fixing (N-) and 3 gels for nitrogen-rich conditions (N+). The gels were normalized according to the “Total density in gel image” method and spot detection and matching, using a Gaussian model, ensued. The gels were manually treated to remove wrongly assigned or duplicated spots and image artifacts, until correlation coefficients of at least 0.8 could be obtained. Quantitative and statistical analysis (using Student′s t-test with a 95% level of confidence) between the two conditions was performed using, in relation to the normalized protein quantity, and spot selection lists that were generated. 2.5. In-Gel Digestion and Mass Spectrometry. In-gel digestion of selected spots was carried out as described by Candiano et al.,38 with the following modifications: following an overnight trypsin in-gel digestion at 37 °C, condensed material under the tube lids was removed and the gel pieces were rehydrated in 5 µL 0.25% formic acid, at 4 °C for 15 min. An equal volume of 70% ACN was subsequently added and the gel plugs were incubated for 15 minutes, at 4 °C. 1 µL of the supernatant was spotted directly on the MALDI plate, using 0.5 µL of a saturated solution of R-cyano-hydroxycinnamic acid in 70% ACN/1% trifluoroacetic acid (TFA), as matrix. Peptide digests were analyzed in a Voyager DE-STR mass spectrometer (Applied Biosystems), in positive reflector mode, using 20 kV as accelerating voltage. External calibration was performed using Calibration Mixture 2 (Sequazyme Peptide Mass Standards kit, Applied Biosystems) and 200 shots for each spectrum (4 sets of 50 shots, in 4 different locations in the sample spot) were collected. Spectra were treated in DataExplorer (Applied Biosystems), by applying advanced baseline correction, noise filtering and peak deisotoping. Internal calibration using the trypsin autolysis peaks (at 842.51 and 2211.10 m/z) was performed whenever possible. Peaks over 5% of the maximum peak intensity were selected and a mass list generated that was submitted to the Mascot search engine (http://www.matrixscience.com/search_form_select.html), using the NCBInr database limited to “Other Proteobacteria” (database from 15th July 2007, total of 1 443 531 sequences available), 1 missed trypsin cleavage allowed, 100 ppm mass tolerance, cysteine carbamidomethylation as fixed modification and methionine oxidation as optional. Only the hits with a MOWSE score higher than 71 (p < 0.05) and belonging to the predicted R. rubrum proteome were considered significant. Protein migration for the different spots was compared to theoretical pI and molecular mass values, as supplied by NCBInr database, to eliminate false positive identifications All reagents were of the highest purity available from the manufacturer. TCA, acetone, ACN, and TFA were purchased from Merck, SDS and formic acid were acquired from Scharlau, glycine was from VWR, and glycerol was from J. T. Baker. All other reagents, unless otherwise specified, were from SigmaAldrich.
3. Results and Discussion 3.1. General Considerations. We have identified 44 differentially expressed proteins from gels containing an average of 679 ( 52 (in N+) and 619 ( 37 (in N-) protein spots per gel. Comparative analysis of the protein expression patterns for both conditions (Figure 1) and their expression ratios (Table 1) show noticeable differences in the expression of many metabolic enzymes in N- (nitrogen-fixing) conditions, compared to N+ (nitrogen-rich), reflected in the distribution of Journal of Proteome Research • Vol. 7, No. 8, 2008 3269
research articles
Selao et al.
Figure 1. Representative 2D-PAGE gel images of soluble fractions from R. rubrum grown under nitrogen-fixing (left) and nitrogen-rich (right) conditions. Proteins identified by MALDI-TOF mass spectrometry are indicated by black arrows and their respective SSP identification numbers (numbers refer to Table 1).
protein expression through the different functional categories (Figure 2). In N- conditions, 50% of the identified proteins with up regulated expression are involved in nitrogen fixation and metabolism. On the other hand, in N+ conditions the major fraction (35%) of the proteins with a higher expression level represents proteins involved in carbon metabolism. It is also noticeable that the change in nitrogen source induces a response in the expression of enzymes related to carbon metabolism, as will be discussed. 3.2. Nitrogen Metabolism. As expected, dinitrogenase reductase and the beta chain of dinitrogenase were expressed only in N- conditions. NifX, a protein required for FeMoCo maturation,39 was also found to be expressed under the same conditions. Regulation of nitrogen fixation involves intricate regulatory pathways, some of which are still not completely understood in detail. In Klebsiella pneumoniae nitrogenase expression is regulated by the Ntr system.5,40 The activity of the NtrB/NtrC two-component system is regulated in accordance to the cellular pool of fixed nitrogen, through the interaction of GlnB with NtrB. NtrC, once phosphorylated by NtrB, activates transcription of NifA which in turn activates the transcription of other nif genes including nifHDK the cluster encoding nitrogenase proteins.6 In R. rubrum by contrast it has been shown that the NtrB/NtrC system is not essential for nif gene expression41 and that NifA activation depends upon the action of GlnB, one of the PII proteins in R. rubrum.42 The complete regulatory mechanism however still remains unclear. In R. rubrum nitrogenase is also regulated post-translationally, in response to such diverse signals as darkness or the presence of ammonium ions (the “switch-off” effectors). Previous studies in R. rubrum have demonstrated that dinitrogenase reductase is ADP-ribosylated, in response to “switch-off” effectors, on a specific arginine residue (Arg101), resulting in a decrease in nitrogenase activity.43 The activity is restored when demodification occurs and the effector is metabolized or removed. In the present study, we were able to identify both forms of dinitrogenase reductase (modified, inactive - spot #1306; unmodified, active - spot #2311, see Figure 1). To verify the presence of the modified 3270
Journal of Proteome Research • Vol. 7, No. 8, 2008
versus unmodified forms of dinitrogenase reductase, extracts from nitrogen-starved cell cultures, using argon, were analyzed as described above (data not shown). Spot #1306 was much reduced in intensity, which is in consistency with earlier results showing that these cells cannot be “switchedoff”7 and therefore the lower form represents the unmodified dinitrogenase reductase. Glutamine synthetase (GS) was found to be up regulated in nitrogen-fixing conditions compared to N+ conditions, at the same time glutamate dehydrogenase (GDH) was down regulated. These two proteins both assimilate ammonium ions in R. rubrum although as mentioned before GDH has a much higher Km for ammonium ions (1 mM) than GS,5 making it less efficient in conditions of nitrogen deficiency. GS is extensively regulated, transcriptionally as well as at the post-translational level, the latter by adenylylation of a specific tyrosine residue.5 In R. rubrum, previous work in our laboratory has shown that the glnBA operon (encoding GlnB and GS) is regulated by the NtrB/NtrC system in response to nitrogen levels in the cell.44 The transcription levels of the glnBA operon were shown to increase in nitrogen-fixing conditions, which is consistent with our results. Glutamate dehydrogenase expression has been shown to be under the control of the NAC protein in E. coli and K. pneumoniae, where NAC represses expression of the gdhA gene.22,45 A BLAST search,46 using the sequence of E. coli K12 nac gene as a template, does not reveal the presence of any homologue to this gene in R. rubrum. The control mechanism for GDH expression in response to nitrogen has not yet been elucidated in this organism. Regardless of how this regulation is attained, GDH expression increases in response to high ammonium concentrations in R. rubrum, as seen in this study, at the same time GS expression is down regulated and the active enzyme is adenylylated. Conversely, in nitrogen deficient conditions, as mentioned above, the glnBA operon is actively transcribed and GS is unmodified (active), whereas GDH expression is repressed. Previous studies in our laboratory have shown that NifJ, a pyruvate oxidoreductase, is part of an electron pathway to nitrogenase, even though it is not essential for nitrogen fixation
research articles
Rhodospirillum rubrum Grown under Nitrogen Conditions Table 1. Proteins in Soluble Fractions of R. rubrum Identified by MALDI-TOF Mass Spectrometry functional grouping
Nitrogen metabolism
SSP accession no. Mr (kDa)a pIa no.
2105 2609 5905 1306 2311 4613
Carbon metabolism
6807 5316 5210 8309 4708 7807 7703 6716 6712 6809 2810 4302 5609 0406
Aminoacid metabolism
5712 7217 5405 8415 4704 7506 8505 7708 5805 6509 4405 6408
Electron transfer
ABC-type transporters
1115 0122 5311 5203 1201 0301 8212
Photosynthesis
1213 5107
Stress response
4406
MOWSE coverage identified normalized normalized ratio score (%) peptides quantity N-b quantity N+b (N-/N+)
identification
17,689 4.96 83593619 Dinitrogenase FeMoCo biosynthesis protein (NifX) 52,503 5.15 83593421 Glutamine synthetase type I (GlnA) 178,347 5.56 83594992 NAD-Glutamate dehydrogenase (Gdh) 31,964 4.91 83592346 Nitrogenase Fe Protein (Nif H) 31,964 4.91 83592346 Nitrogenase Fe Protein (Nif H) 56,398 5.38 83592348 Nitrogenase MoFe beta chain (NifK) 128,446 5.59 453436 Pyruvate oxidoreductase (NifJ) 29,842 5.75 83594054 2-oxoglutarate synthase, beta subunit 29,842 5.75 83594054 2-oxoglutarate synthase, beta subunit 40,376 6.20 83593009 4-diphosphocytidyl-2Cmethyl-D-erythritol synthase 72,150 5.43 83594904 Acetate-CoA ligase 83,075 5.85 83592645 Acetoacetyl-Coa reductase 64,241 5.80 83591807 Acetolactate synthase, large subunit 64,686 5.57 83592644 Acyl-CoA dehydrogenase 58,791 5.76 83592652 AMP-dependent synthetase and ligase 85,760 5.69 83592302 Carbon-monoxide dehydrogenase 76,157 5.18 83594629 Glycoside hydrolase, family 3-like 40,553 5.31 83593738 Inositol phosphatase / Fructose-1,6bisphosphatase 50,735 5.60 83593735 RuBisCO, form II 42,981 5.09 83592547 Succinyl-CoA synthetase, beta subunit 70,015 5.55 83591934 Transketolase 29978 6.00 83594808 2,3,4,5-tetrahydropyridine-2carboxylate N-succinyltransferase 44,404 5.52 83594334 Aminotransferase, class I and II 37,761 6.27 83592532 Aspartate-semialdehyde dehydrogenase, USG-1 related 67,568 5.37 83593407 Aspartyl-tRNA synthetase 45,495 6.03 83593162 Glycine/Serine hydroxymethyltransferase 45,495 6.03 83593162 Glycine/Serine hydroxymethyltransferase 77,998 5.85 83594287 Glycine-tRNA ligase 105,786 5.51 83594296 Isoleucyl-tRNA synthetase, class Ia 47,718 5.60 83592123 O-acetylhomoserine / O-acetylserine sulfhydrylase 43,680 5.34 83594544 O-succinylhomoserine sulfhydrylase 39,525 5.61 83594852 Phenylalanyl-tRNA synthetase, alpha subunit 20,689 5.04 83592754 Alkyl hydroperoxyde/Thiol specific antioxidant/Mal allergen 13,831 4.76 83593251 Ferredoxin (Fdx), [2Fe-2S] cluster 38,678 5.58 83591815 NADH:flavin oxidoreductase / NADH oxidase 59,240 6.01 83593691 Extracellular solute-binding protein, family 5 28,170 5.08 83593752 Lipoprotein YaeC 36,744 4.88 83592472 Periplasmic binding protein (TroA) 37,231 6.41 83593812 Substrate-binding region, ABCtype glycine betaine transporter 24,578 5.05 83594406 Cobalamin adenosyltransferase 28,557 5.60 83592602 Photosystem I assembly protein (BtpA) 46,465 5.49 83592886 ClpX (ATPase regulatory subunit) 68,805 5.03 83594884 Heat shock protein 70 30,508 5.54 83593944 Universal stress protein A 50,821 4.99 83592562 ATP synthase F1, beta subunit
1702 6302 Oxidative 1502 phosphorylation Proteolysis 5508 52,356 5.68 83591794 Leucyl aminopeptidase Hypothetical 5101 21,242 5.48 83591697 Hypothetical protein Rru_A0357 proteins 7806 128,573 5.78 83591905 Hypothetical protein Rru_A0566
NDc
83
38
5
222.00
90
34
10
2314.20
865.60
2.67
208
19
23
41.00
180.10
0.23
93 94 134
32 36 29
8 9 7
12,855.90 11,889.60 12,382.40
157 98
23 37
18 6
593.80 177.60
431.20 ND
76
24
4
ND
255.10
154
38
12
44.50
202.60
0.22
99 122 95
28 22 19
16 13 9
1173.90 102.20 57.50
3,585.30 231.70 280.00
0.33 0.44 0.21
158 127
39 32
18 13
2073.80 1592.60
4,865.90 3,456.90
0.43 0.46
77
9
6
105 132
22 25
14 7
621.70 ND
1,269.80 59.70
0.49
142 77
26 19
10 6
45.10 59.90
828.10 455.90
0.05 0.13
157 106
43 25
21 7
1838.00 ND
5,280.80 32.50
0.35 -
79 90
24 29
6 10
64.10 788.10
358.50 2,271.40
0.18 0.35
91 121
12 50
7 7
153.40 1779.60
625.80 255.80
0.25 6.96
185
38
12
858.80
3,459.20
0.25
251 113 208
42 14 50
22 11 14
60.10 79.10 ND
295.40 227.20 230.40
0.20 0.35
142 83
28 29
8 9
458.80 253.20
1,540.40 0.77
0.30 194.22
105
43
7
2720.10
5,599.30
0.49
77 106
37 40
6 8
199.60 129.30
ND 1,304.10
0.10
151
46
19
472.50
188.00
2.51
119 111
34 28
8 8
1130.90 116.90
2,774.60 968.90
0.41 0.12
218
57
13
82.40
534.20
0.15
91 71
43 25
6 5
ND ND
84.10 95.80
109
21
8
ND
1,200.10
118 86 147
24 28 56
10 7 17
56.70 371.50 542.20
228.00 873.50 1,222.60
0.25 0.43 0.44
215 97
45 47
13 6
103.80 ND
522.90 151.00
0.20
113
13
10
ND
751.30
ND
ND ND ND 1.38
45.10
a Theoretical values. b Gels images were normalized using the “Total density in gel image” method, in the PDQuest 7.3.0 program. Raw spot quantities are assessed by multiplying the Gaussian spot heights by the standard deviations of the spot’s Gaussian distributions, both in the x and y axis: Raw spot quantity ) Height * σx * σy. By using this normalization method, each (raw, non-normalized) spot quantity in a gel is divided by the total density value of the gel image. c ND - not detectable.
in heterotrophic conditions.47 Transcription of the nifJ gene occurs most probably from a putative σ54-dependent promoter, in R. rubrum (Cheng, J. et al., unpublished results). Northern blot analysis of nifJ transcription shows that under nitrogenfixing conditions (in the light) the protein is expressed with a 2-fold increase comparing to nitrogen-rich conditions.48 Our
results confirm this increase in expression in N- grown cells, compared to N+. 3.3. Carbon Metabolism. As mentioned above, many metabolic enzymes are down regulated in nitrogen-fixing conditions, in comparison to N+. A number of these are related to carbon fixation pathways (e.g., RuBisCO, CO dehydrogenase, transkeJournal of Proteome Research • Vol. 7, No. 8, 2008 3271
research articles
Selao et al.
Figure 2. Distribution of identified proteins by functional categories and differential expression compared to N+ (N- up regulated, left; N- down regulated, right). In bracketssnumber of proteins identified within each of the categories.
Figure 3. Zoomed 2D-PAGE gel images of the R-ketoglutarate synthase-containing spots (indicated by arrows), in nitrogen-rich (left) and nitrogen-fixing (right) conditions.
tolase or acetate-CoA ligase) and it is noteworthy that a change in nitrogen availability induces a response in carbon fixation pathways, most likely to keep the carbon to nitrogen (C/N) ratio at steady levels. A putative [2Fe-2S] ferredoxin was found to be expressed only during nitrogen-fixing conditions. This ferredoxin is different from those previously reported as being part of the main electron pathway to nitrogenase49 and has not been investigated in earlier work. As such, it cannot be linked to any particular metabolic pathway. In R. palustris a ferredoxin-like protein with a [2Fe-2S] was also shown to be present only under nitrogen-fixing conditions4 and an alignment between these two proteins (using CLUSTALW50) revealed stretches of high similarity, indicating a possible relation between them. We have identified two different spots (spots #5316 and #5210), one in each condition, as R-ketoglutarate synthase, an enzyme involved in carbon fixation through the reductive TCA cycle (RTCA). The two spots have the same molecular mass, as shown in the second dimension gel although the pI is slightly different between the two conditions (Figure 3). The level of expression is also more elevated in nitrogen-rich conditions. The RTCA cycle was first described by Evans and coworkers in 1966 and R-ketoglutarate synthase is one of the key enzymes, incorporating one CO2 molecule into succinyl-CoA, producing R-ketoglutarate.51 The activities of R-ketoglutarate synthase as well as of another key enzyme in this process, pyruvate synthase, have been demonstrated in R. rubrum extracts.30 Furthermore, the β-subunit of succinyl-CoA synthetase was also found at a lower level in nitrogen-fixing conditions, as opposed to nitrogen-rich growth. The genetic map of a R. rubrum CO2 fixation gene cluster includes cbbM (RuBisCO), cbbR (transcriptional regulator), cbbE (epimerase), cbbF (fructose-1,6-bisphosphatase), cbbP (phos3272
Journal of Proteome Research • Vol. 7, No. 8, 2008
phoribulokinase), and cbbT (transketolase).52 Out of these 6 proteins, we observed an increase in the expression of RuBisCO, fructose-1,6-bisphosphatase and transketolase when cells were grown in nitrogen-rich medium, indicating an increased activity of the reductive pentose phosphate pathway in these conditions. Transcriptional control of the cbb genes has been shown to be under the control of the CbbR activator, a member of the LysR transcriptional activator family, in Rhodobacter capsulatus,53 Rhodobacter sphaeroides54 and R. rubrum.52 CbbR is, in turn, regulated by the two-component system RegA/RegB, in Rh. capsulatus.55 R. rubrum has however some unique features as it only possesses one type of RuBisCO, formed exclusively by large subunits.52 This difference in RuBisCO expression levels in response to changes in nitrogen concentration could not be observed in similar studies in Nostoc sp. PCC 71203 or R. palustris.4 However, Badger and Price have previously demonstrated an increase in expression of the rbcLS genes, encoding RuBisCO, in Nostoc sp. PCC 7120 ammonium grown cells,56 but it should be noted that RuBisCO is not present in the heterocysts and this may account for the discrepancy between these results. Unlike Rh. capsulatus, R. rubrum does not have any homologues to the RegA/RegB proteins and deletion mutants in the cbbM gene are still capable of growing photoheterotrophically, with malate or succinate as carbon sources, thus suggesting an alternative carbon fixation pathway involved, proposed by Falcone and Tabita to be the RTCA cycle.52 RuBisCO has also been suggested to play a role in the control of the redox balance, in both Rh. capsulatus57 and R. rubrum, where mutant strains with deletions in the cbb operon showed increased expression of nitrogenase, probably in order to maintain cellular redox balance.10 Previous work in our laboratory has shown that also in a mutant in the FixABCX electron pathway to nitrogenase the expression of RuBisCO in nitrogenfixing conditions is up regulated.58 Nitrogenase is as previously mentioned an important electron sink providing an alternative to RuBisCO as an electron acceptor under nitrogen-fixing conditions. The transcriptional regulation of these two enzymes is therefore considered to be coordinated although the details for this regulatory mechanism still remain to be elucidated. The carbon storage compound poly-β-hydroxybutyrate (PHB) is synthesized by PHB synthetase and degraded by the PHB depolymerase system in R. rubrum.32,33 In our experiments, higher nitrogen availability (i.e., growth in N+) results in an
research articles
Rhodospirillum rubrum Grown under Nitrogen Conditions increase in the expression of 3 proteins linked to the butanoate metabolism, acetoacetyl-CoA reductase, acyl-CoA dehydrogenase and acetolactate synthase. Acetoacetyl-CoA reductase and acyl-CoA dehydrogenase are found in the same gene cluster in the R. rubrum genome, which along with our current results suggests a probable up regulation of this pathway under nitrogen-rich photoheterotrophic growth. The increase in acetyl-CoA formed via the RTCA or reductive pentose pathway would then be further metabolized via PHB formation under nitrogen-rich conditions, as a means to balance the redox state in the cell. 3.4. Amino Acid Metabolism. A number of proteins related to amino acid synthesis and conversion, as well as protein expression, are down regulated in nitrogen-fixing conditions. These findings are in accordance with the general trend, i.e., a decrease in metabolic functions when R. rubrum is grown in nitrogen limiting conditions. A number of proteins, such as enzymes involved in methionine biosynthesis and the aspartate biosynthetic pathway, are among those seen at a lower level of expression in N- conditions. We were also able to identify two different spots with the same relative molecular mass as the enzyme glycine hydroxymethyltransferase (Figure 1, spots #7506 and #8505). It appears that in nitrogen-fixing conditions this protein exists mainly in a more acidic form, whereas in nitrogen-rich medium a different form with a more basic pI is more abundant. Glycine hydroxymethyltransferase (or serine hydroxymethyltransferase, SHMT) is responsible for the interconversion of glycine to serine, with the concomitant transfer of a methyl group from tetrahydrofolate intermediates.59 In many organisms SHMT, besides converting these two amino acids, is also responsible for regulating the pool of folate intermediates.59 In addition to this, as shown for methanogen species (e.g. Clostridium sp. grown under anaerobic conditions60), the carbon fixation involving one-carbon compounds is linked to tetrahydrofolate and thereby SMHT via CO dehydrogenase/acetyl-CoA synthase, through the Wood-Ljungdahl pathway, producing acetyl-CoA. 2 molecules of CO2 are converted to 2 molecules of CO by CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) and 1 of the CO molecules is utilized to produce acetyl-CoA, with a methylated corrinoid-containing protein as methyl donor. This reaction is catalyzed by the CODH/ACS protein, which is as mentioned earlier up regulated in N+ grown cells, compared to N-. Methylation of the corrinoid-containing protein has been shown in Clostridium thermoaceticum to be dependent the tetrahydrofolate pool, replenished by a methyltransferase.61 SHMT has also been reported to be essential for the establishment of effective symbiosis between the nitrogen-fixing bacterium Bradyrhizobium japonicum and its host and it has been hypothesized that either an insufficient supply of glycine or a disturbed C1 metabolism was responsible for this effect.62 Nonetheless, the relation between SMHT, CODH, and the Wood-Ljungdahl pathway still needs additional experimental data to be clearly established. 3.5. Transport. Following the general trend, the expression of several periplasmic components of ABC-type transporters was down regulated in N-, in comparison to N+. Only one of these proteins was found to be up regulated under nitrogenfixing conditions. It is annotated in the NCBI database as belonging to family 5 of the extracellular solute-binding protein branch, containing a cleavable signal peptide sequence as predicted by SignalP.63 A similarity search, using the BlastP algorithm,46 identified several extracellular peptide-binding
proteins as the closest homologues. A conserved domain search, using CD-Search,64 showed the similarities between the conserved domains for proteins of the OppA, DppA,65,66 and NikA67 families and the R. rubrum equivalent. These proteins are involved in the transport of oligopeptides, dipeptides, or nickel ions, respectively.
4. Conclusions We have studied the response in protein expression when R. rubrum is subjected to different nitrogen sources. This is the first time a differential study of the proteome of this organism has been performed using 2D-PAGE and MALDI-TOF mass spectrometry. We have identified 44 differentially expressed proteins in gels containing a total average of 679 ( 52 (in N+) and 619 ( 37 (in N-) protein spots for each gel. Our results show that there is an overall decrease in protein expression when R. rubrum is grown in nitrogen-fixing conditions compared to cells grown in nitrogen-rich medium. Experiencing a higher level of fixed nitrogen results in a higher level of proteins involved in carbon metabolism, which could be interpreted as a cellular response to balance the redox state when grown in nitrogen-rich medium supplied with malate. As the anaerobic photosynthesis provides enough energy to maintain growth, the surplus of reductant liberated is funneled to the carbon metabolism in nitrogen-rich conditions, whereas during nitrogen fixation, the reducing equivalents are instead used for the reduction of N2 to NH4+ by nitrogenase. We have observed a higher level of expression of proteins involved in the reductive TCA cycle, CO2 fixation, and PHB metabolism and, subsequently, of proteins involved in amino acid metabolism, in ammonium rich cultures as compared to nitrogenfixing conditions. The proteins induced in nitrogen-fixing conditions are mainly found in the nitrogen fixation and assimilation pathways of R. rubrum, which is coherent with the well-known Ntr and Nif regulated expression of these proteins, as in the case of glutamine synthetase and nitrogenase. In all three organisms where differential nitrogen dependent expression has been studied (Nostoc sp. PCC 7120, R. palustris and R. rubrum), the presence of nif gene products identified is restricted to only a fraction of the total number expected from the genome sequences. The abundant nitrogenase proteins (NifH, NifD, and NifK) are reported in all three studies, and in addition to those, NifX in R. palustris and R. rubrum and NifS, NifT, and NifA in R. palustris have also been identified.3,4 The analyses were performed either by 2D-PAGE, followed by MALDI-TOF, or by LC-ESI-MS/MS, with or without iTRAQ labeled samples, thus highlighting the difficulty in detecting low-abundance proteins. A transport protein and an electron transfer protein were found to be expressed only in nitrogen-fixing conditions. This demonstrates the existence of other classes of proteins whose expression is regulated in response to nitrogen status, although the mechanism responsible for this regulatory network is yet unknown. This study allows for a broader view of the regulatory networks responding to nitrogen availability in R. rubrum. We have shown the “cross talk” between nitrogen and carbon signaling for protein expression, because a major effect of the different nitrogen sources is a response in the expression pattern of many enzymes related to carbon metabolism. The results from nitrogen-dependent differential proteomes from Nostoc sp. PCC 7120,3 Nostoc sp. PCC 73102,2 and R. rubrum Journal of Proteome Research • Vol. 7, No. 8, 2008 3273
research articles certainly reveal an effect of the fixed nitrogen availability on the expression profile of proteins related to carbon metabolism and a study in R. palustris shows a higher expression of electron transfer proteins under nitrogen-fixing conditions.4 Altogether, these results demonstrate the importance for the cell to keep the C/N ratio as well as the redox balance. Due to the characteristics of the techniques employed in this study, only a fraction (roughly 18%) of the total putative ORFs in the R. rubrum genome can be detected. The membrane proteins, as well as proteins of low-abundance, have not yet been identified. The analysis of these particular proteomes, using different methodological approaches, should provide important insights into the complex metabolic and regulatory networks in this bacterium. Abbreviations: 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; FeMoCo, iron-molybdenum cofactor; RuBisCO, ribulose-bisphosphate carboxylase/oxidase.
Acknowledgment. This work was supported by grants from the Carl Tryggers Foundation to A.N., from the Swedish Research Council to S.N., and from Fundac¸a˜o para a Cieˆncia e a Tecnologia-Portugal, through the PhD fellowship SFRH/BD/23183/2005 to T.T.S. We thank Leopold Ilag and Gianluca Maddalo, from the Department of Analytical Chemistry at Stockholm University, for technical support and helpful advice as well as Pedro Teixeira, Anders Jonsson, He Wang, and Tomas Edgren for helpful comments and discussion.
Selao et al.
(13)
(14)
(15)
(16) (17) (18)
(19) (20)
(21)
References (1) Gest, H. Metabolic patterns in photosynthetic bacteria. Bacteriol. Rev. 1951, 15, 183–210. (2) Ran, L.; Huang, F.; Ekman, M.; Klint, J.; Bergman, B. Proteomic analyses of the photoauto- and diazotrophically grown cyanobacterium Nostoc sp. PCC 73102. Microbiology 2007, 153, 608–618. (3) Stensjo, K.; Ow, S. Y.; Barrios-Llerena, M. E.; Lindblad, P.; Wright, P. C. An iTRAQ-based quantitative analysis to elaborate the proteomic response of Nostoc sp. PCC 7120 under nitrogen fixing conditions. J. Proteome Res. 2007, 6, 621–635. (4) VerBerkmoes, N. C.; Shah, M. B.; Lankford, P. K.; Pelletier, D. A.; Strader, M. B.; Tabb, D. L.; McDonald, W. H.; Barton, J. W.; Hurst, G. B.; Hauser, L.; Davison, B. H.; Beatty, J. T.; Harwood, C. S.; Tabita, F. R.; Hettich, R. L.; Larimer, F. W. Determination and comparison of the baseline proteomes of the versatile microbe Rhodopseudomonas palustris under its major metabolic states. J. Proteome Res. 2006, 5, 287–298. (5) Merrick, M. J.; Edwards, R. A. Nitrogen control in bacteria. Microbiol. Rev. 1995, 59, 604–622. (6) Dixon, R.; Kahn, D. Genetic regulation of biological nitrogen fixation. Nat. Rev. Microbiol. 2004, 2, 621–631. (7) Nordlund, S.; Ludden, P. W. Post-translational regulation of nitrogenase in photosynthetic bacteria. In Genetics and regulation of nitrogen fixation in free-living bacteria, Klipp, W.; Masephol, B.; Gallon, J. R.; Newton, W. E., Eds.; Kluwer Academic Publisher: The Netherlands, 2004; pp 175-196. (8) Arau ´ jo, L. M.; Monteiro, R. A.; Souza, E. M.; Steffens, M. B.; Rigo, L. U.; Pedrosa, F. O.; Chubatsu, L. S. GlnB is specifically required for Azospirillum brasilense NifA activity in Escherichia coli. Res. Microbiol. 2004, 155, 491–495. (9) Zhang, Y.; Pohlmann, E. L.; Roberts, G. P. Identification of critical residues in GlnB for its activation of NifA activity in the photosynthetic bacterium Rhodospirillum rubrum. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 2782–2787. (10) Joshi, H. M.; Tabita, F. R. A global two component signal transduction system that integrates the control of photosynthesis, carbon dioxide assimilation and nitrogen fixation. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14515–14520. (11) Arconde´guy, T.; Jack, R.; Merrick, M. J. P(II) signal transduction proteins, pivotal players in microbial nitrogen control. Microbiol. Mol. Biol. Rev. 2001, 65, 80–105. (12) Zhang, Y.; Pohlmann, E. L.; Ludden, P. W.; Roberts, G. P. Functional characterization of three GlnB homologs in the photosynthetic
3274
Journal of Proteome Research • Vol. 7, No. 8, 2008
(22) (23)
(24) (25)
(26) (27) (28) (29)
(30) (31) (32)
(33)
bacterium Rhodospirillum rubrum: roles in sensing ammonium and energy status. J. Bacteriol. 2001, 183, 6159–6168. Zhang, Y.; Pohlmann, E. L.; Halbleib, C. M.; Ludden, P. W.; Roberts, G. P. Effect of P(II) and its homolog GlnK on reversible ADPribosylation of dinitrogenase reductase by heterologous expression of the Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyl transferase-dinitrogenase reductase-activating glycohydrolase regulatory system in Klebsiella pneumoniae. J. Bacteriol. 2001, 183, 1610–1620. Jonsson, A.; Teixeira, P. F.; Nordlund, S. The activity of adenylyltransferase in Rhodospirillum rubrum is only affected by alphaketoglutarate and unmodified PII proteins, but not by glutamine, in vitro. FEBS J. 2007, 274, 2449–2460. Jonsson, A.; Nordlund, S. In vitro studies of the uridylylation of the three PII protein paralogs from Rhodospirillum rubrum: the transferase activity of R. rubrum GlnD is regulated by alphaketoglutarate and divalent cations but not by glutamine. J. Bacteriol. 2007, 189, 3471–3478. Kamberov, E. S.; Atkinson, M. R.; Ninfa, A. J. The Escherichia coli PII signal transduction protein is activated upon binding 2-ketoglutarate and ATP. J. Biol. Chem. 1995, 270, 17797–17807. Jiang, P.; Ninfa, A. J. Escherichia coli PII signal transduction protein controlling nitrogen assimilation acts as a sensor of adenylate energy charge in vitro. Biochemistry 2007, 46, 12979–12996. Zhang, Y.; Pohlmann, E. L.; Roberts, G. P. GlnD is essential for NifA activation, NtrB/NtrC-regulated gene expression, and posttranslational regulation of nitrogenase activity in the photosynthetic, nitrogen-fixing bacterium Rhodospirillum rubrum. J Bacteriol 2005, 187, 1254–1265. Wolfe, D. M.; Zhang, Y.; Roberts, G. P. Specificity and regulation of interaction between the PII and AmtB1 proteins in Rhodospirillum rubrum. J Bacteriol 2007, 189, 6861–6869. Jiang, F.; Mannervik, B.; Bergman, B. Evidence for redox regulation of the transcription factor NtcA, acting both as an activator and a repressor, in the cyanobacterium Anabaena PCC 7120. Biochem J 1997, 327, 513–517. Zhang, Y.; Pu, H.; Wang, Q.; Cheng, S.; Zhao, W.; Zhang, Y.; Zhao, J. PII is important in regulation of nitrogen metabolism but not required for heterocyst formation in the cyanobacterium Anabaena sp. PCC 7120. J Biol Chem 2007, 282, 33641–33648. Camarena, L.; Poggio, S.; Garcia, N.; Osorio, A. Transcriptional repression of gdhA in Escherichia coli is mediated by the Nac protein. FEMS Microbiol. Lett. 1998, 167, 51–56. Gest, H.; Kamen, M. D.; Bregoff, H. M. Studies on the metabolism of photosynthetic bacteria. V. Photoproduction of hydrogen and nitrogen fixation by Rhodospirillum rubrum. J. Biol. Chem. 1949, 182, 153–170. Bonam, D.; Murrell, S. A.; Ludden, P. W. Carbon monoxide dehydrogenase from Rhodospirillum rubrum. J. Bacteriol. 1984, 159, 693–699. Joshi, H. M.; Tabita, F. R. Induction of carbon monoxide dehydrogenase to facilitate redox balancing in a ribulose bisphosphate carboxylase/oxygenase-deficient mutant strain of Rhodospirillum rubrum. Arch. Microbiol. 2000, 173, 193–199. Schultz, J. E.; Weaver, P. F. Fermentation and anaerobic respiration by Rhodospirillum rubrum and Rhodopseudomonas capsulata. J. Bacteriol. 1982, 149, 181–190. Anderson, L.; Fuller, R. C. Photosynthesis in Rhodospirillum rubrum. 3. Metabolic control of reductive pentose phosphate and tricarboxylic acid cycle enzymes. Plant Physiol. 1967, 42, 497–509. Tayeh, M. A.; Madigan, M. T. Malate dehydrogenase in phototrophic purple bacteria: purification, molecular weight, and quaternary structure. J. Bacteriol. 1987, 169, 4196–4202. Grammel, H.; Gilles, E. D.; Ghosh, R. Microaerophilic cooperation of reductive and oxidative pathways allows maximal photosynthetic membrane biosynthesis in Rhodospirillum rubrum. Appl. Environ. Microbiol. 2003, 69, 6577–6586. Buchanan, B. B.; Evans, M. C.; Arnon, D. I. Ferredoxin-dependent carbon assimilation in Rhodospirillum rubrum. Arch Mikrobiol 1967, 59, 32–40. Buchanan, B. B.; Arnon, D. I. A reverse KREBS cycle in photosynthesis: consensus at last. Photosynth Res 1990, 24, 47–53. Handrick, R.; Reinhardt, S.; Kimmig, P.; Jendrossek, D. The ”intracellular” poly(3-hydroxybutyrate) (PHB) depolymerase of Rhodospirillum rubrum is a periplasm-located protein with specificity for native PHB and with structural similarity to extracellular PHB depolymerases. J. Bacteriol. 2004, 186, 7243–7253. Clemente, T.; Shah, D.; Tran, M.; Stark, D.; Padgette, S.; Dennis, D.; Bruckener, K.; Steinbuchel, A.; Mitsky, T. Sequence of PHA synthase gene from two strains of Rhodospirillum rubrum and in vivo substrate specificity of four PHA synthases across two
research articles
Rhodospirillum rubrum Grown under Nitrogen Conditions
(34) (35)
(36)
(37) (38)
(39)
(40)
(41)
(42)
(43)
(44)
(45)
(46) (47)
(48)
(49)
heterologous expression systems. Appl. Microbiol. Biotechnol. 2000, 53, 420–429. Trainer, M. A.; Charles, T. C. The role of PHB metabolism in the symbiosis of rhizobia with legumes. Appl. Microbiol. Biotechnol. 2006, 71, 377–386. Grammel, H.; Gilles, E. D.; Ghosh, R. Microaerophilic cooperation of reductive and oxidative pathways allows maximal photosynthetic membrane biosynthesis in Rhodospirillum rubrum. Appl. Environ. Microbiol. 2003, 11, 6577–6586. Ormerod, J. G.; Ormerod, K. S.; Gest, H. Light-dependent utilization of organic compounds and photoproduction of molecular hydrogen by photosynthetic bacteria; relationships with nitrogen metabolism. Arch. Biochem. Biophys. 1961, 94, 449–463. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193, 265–275. Candiano, G.; Bruschi, M.; Musante, L.; Santucci, L.; Ghiggeri, G. M.; Carnemolla, B.; Orecchia, P.; Zardi, L.; Righetti, P. G. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 2004, 25, 1327–1333. Hernandez, J. A.; Igarashi, R. Y.; Soboh, B.; Curatti, L.; Dean, D. R.; Ludden, P. W.; M, R. L. NifX and NifEN exchange NifB cofactor and the VK-cluster, a newly isolated intermediate of the ironmolybdenum cofactor biosynthetic pathway. Mol. Microbiol. 2007, 63, 177–192. Ninfa, A. J.; Atkinson, M. R.; Kamberov, E. S.; Feng, J.; Ninfa, E. G. Control of nitrogen assimilation by the NRI-NRII two-component system of enteric bacteria; American Society for Microbiology: Washington, D.C., 1995; pp 67-88. Zhang, Y.; Cummings, A. D.; Burris, R. H.; Ludden, P. W.; Roberts, G. P. Effect of an ntrBC mutation on the posttranslational regulation of nitrogenase activity in Rhodospirillum rubrum. J. Bacteriol. 1995, 177, 5322–5326. Zhang, Y.; Pohlmann, E. L.; Ludden, P. W.; Roberts, G. P. Mutagenesis and functional characterization of the glnB, glnA, and nifA genes from the photosynthetic bacterium Rhodospirillum rubrum. J. Bacteriol. 2000, 182, 983–992. Ludden, P. W.; Burris, R. H. Purification and properties of nitrogenase from Rhodospirillum rubrum, and evidence for phosphate, ribose and an adenine-like unit covalently bound to the iron protein. Biochem. J. 1978, 175, 251–259. Cheng, J.; Johansson, M.; Nordlund, S. Expression of P(II) and glutamine synthetase is regulated by P(II), the ntrBC products, and processing of the glnBA mRNA in Rhodospirillum rubrum. J. Bacteriol. 1999, 181, 6530–6534. Rosario, C. J.; Bender, R. A. Importance of tetramer formation by the nitrogen assimilation control protein for strong repression of glutamate dehydrogenase formation in Klebsiella pneumoniae. J. Bacteriol. 2005, 187, 8291–8299. Altschul, S. F.; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403–410. Edgren, T.; Nordlund, S. Two pathways of electron transport to nitrogenase in Rhodospirillum rubrum: the major pathway is dependent on the fix gene products. FEMS Microbiol. Lett. 2006, 260, 30–35. Brostedt, E. Electron transport to nitrogenase in the photosynthetic bacterium Rhodospirillum rubrum. PhD thesis, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden, 1994. Edgren, T.; Nordlund, S. Electron transport to nitrogenase in Rhodospirillum rubrum: identification of a new fdxN gene encoding the primary electron donor to nitrogenase. FEMS Microbiol. Lett. 2005, 245, 345–351.
(50) Higgins, D.; Thompson, J.; Gibson, T.; Thompson, J. D.; Higgins, D. G.; Gibson, T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22, 4673–4680. (51) Evans, M. C.; Buchanan, B. B.; Arnon, D. I. A new ferredoxindependent carbon reduction cycle in a photosynthetic bacterium. Proc. Natl. Acad. Sci. U.S.A. 1966, 55, 928–934. (52) Falcone, D. L.; Tabita, F. R. Complementation analysis and regulation of CO- fixation gene expression in a ribulose 1,5bisphosphate carboxylase-oxygenase deletion strain of Rhodospirillum rubrum. J. Bacteriol. 1993, 175, 5066–77. (53) Paoli, G. C.; Vichivanives, P.; Tabita, F. R. Physiological control and regulation of the Rhodobacter capsulatus cbb operons. J. Bacteriol. 1998, 180, 4258–4269. (54) Dubbs, J. M.; Bird, T. H.; Bauer, C. E.; Tabita, F. R. Interaction of CbbR and RegA transcription regulators with the Rhodobacter sphaeroides cbb1 promoter-operator region. J. Biol. Chem. 2000, 275, 19224–19230. (55) Vichivanives, P.; Bird, T. H.; Bauer, C. E.; Tabita, F. R. Multiple regulators and their interactions in vivo and in vitro with the cbb regulons of Rhodobacter capsulatus. J. Mol. Biol. 2000, 300, 1079– 1099. (56) Badger, M. R.; Price, G. D. CO2 concentrating mechanisms in cyanobacteria: molecular components, their diversity and evolution. J. Exp. Bot. 2003, 54, 609–622. (57) Tichi, M. A.; Tabita, F. R. Maintenance and control of redox poise in Rhodobacter capsulatus strains deficient in the Calvin-BensonBassham pathway. Arch. Microbiol. 2000, 174, 322–33. (58) Edgren, T.; Nordlund, S. The fixABCX genes in Rhodospirillum rubrum encode a putative membrane complex participating in electron transfer to nitrogenase. J. Bacteriol. 2004, 186, 2052–60. (59) Schirch, L. Folate in serine and glycine metabolism; John Wiley and Sons, Inc.: New York, 1984; Vol. 1, pp 399-431. (60) Wood, H. G. Life with CO or CO2 and H2 as a source of carbon and energy. FASEB J. 1991, 5, 156–163. (61) Seravalli, J.; Kumar, M.; Lu, W. P.; Ragsdale, S. W. Mechanism of carbon monoxide oxidation by the carbon monoxide dehydrogenase/acetyl-CoA synthase from Clostridium thermoaceticum: kinetic characterization of the intermediates. Biochemistry 1997, 36, 11241–11251. (62) Rossbach, S.; Hennecke, H. Identification of glyA as a symbiotically essential gene in Bradyrhizobium japonicum. Mol. Microbiol. 1991, 5, 39–47. (63) Emanuelsson, O.; Brunak, S.; von Heijne, G.; Nielsen, H. Locating proteins in the cell using TargetP, SignalP, and related tools. Nat. Protoc. 2007, 2, 953–971. (64) Marchler-Bauer, A.; Bryant, S. H. CD-Search: protein domain annotations on the fly. Nucleic Acids Res. 2004, 32, 327–331. (65) Olson, E. R.; Dunyak, D. S.; Jurss, L. M.; Poorman, R. Identification and characterization of dppA, an Escherichia coli gene encoding a periplasmic dipeptide transport protein. Bacteriology 1991, 173, 234–244. (66) Smith, M. W.; Payne, J. W. Expression of periplasmic binding proteins for peptide transport is subject to negative regulation by phosphate limitation in Escherichia coli. FEMS Microbiol. Lett. 1992, 79, 183–190. (67) Navarro, C.; Wu, L. F.; Mandrand-Berthelot, M. A. The nik operon of Escherichia coli encodes a periplasmic binding-protein-dependent transport system for nickel. Mol. Microbiol. 1993, 9, 1181–1191.
PR700771U
Journal of Proteome Research • Vol. 7, No. 8, 2008 3275
