Impact of the Core Components of the Phosphoenolpyruvate

Article pubs.acs.org/jpr

Impact of the Core Components of the PhosphoenolpyruvateCarbohydrate Phosphotransferase System, HPr and EI, on Differential Protein Expression in Ralstonia eutropha H16 Chlud Kaddor,† Birgit Voigt,‡ Michael Hecker,‡ and Alexander Steinbüchel*,†,§ †

Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, D-48149 Münster, Germany ‡ Institut für Mikrobiologie, Ernst-Moritz-Arndt Universität, Friedrich-Ludwig-Jahn-Straße 15, D-17489 Greifswald, Germany § King Abdul Aziz University, Jeddah 22254, Saudi Arabia S Supporting Information *

ABSTRACT: In Ralstonia eutropha H16, seven genes encoding proteins being involved in the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS) were identified. In order to provide more insights into the poly(3-hydroxybutyrate) (PHB)-leaky phenotype of the HPr/ EI deletion mutants H16ΔptsH, H16ΔptsI, and H16ΔptsHI when grown on the non-PTS substrate gluconate, parallel fermentations for comparison of their growth behavior were performed. Samples from the exponential, the early stationary, and late stationary growth phases were investigated by microscopy, gas chromatography and (phospho-) proteome analysis. A total of 71 differentially expressed proteins were identified using 2D-PAGE, Pro-Q Diamond and Coomassie staining, and MALDI-TOF analysis. Detected proteins were classified into five major functional groups: carbon metabolism, energy metabolism, amino acid metabolism, translation, and membrane transport/outer membrane proteins. Proteome analyses revealed enhanced expression of proteins involved in the Entner−Doudoroff pathway and in subsequent reactions in cells of strain H16 compared to the mutant H16ΔptsHI. Furthermore, proteins involved in PHB accumulation showed increased abundance in the wild-type. This expression pattern allowed us to identify proteins affecting carbon metabolism/PHB biosynthesis in strain H16 and translation/amino acid metabolism in strain H16ΔptsHI, and to gain insight into the molecular response of R. eutropha to the deletion of HPr/EI. KEYWORDS: Ralstonia eutropha, H16ΔptsH, H16ΔptsI, H16ΔptsHI, PHB, carbohydrate phosphotransferase system (PTS), fermentation, proteome, phosphoproteome, 2D-PAGE, Pro-Q Diamond, microscopy

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INTRODUCTION Ralstonia eutropha H16, a Gram-negative and facultative chemolithoautotrophic hydrogen-oxidizing β-proteobacterium, accumulates poly(3-hydroxybutyrate), PHB, and other polyesters as insoluble granules inside the cell. This bacterium oxidizes molecular H2 during lithoautotrophic growth by two energy-conserving [NiFe] hydrogenases (one membranebound hydrogenase and one soluble hydrogenase). CO2 is assimilated via the Calvin−Benson−Basham (CBB) cycle.1,2 Furthermore, formate is utilized as an alternative one-carbon energy source for organoautotrophic growth.3 R. eutropha utilizes also a wide range of organic carbon and energy sources for heterotrophic growth, including tricarboxylic acid cycle (TCC) intermediates, fatty acids, sugar acids, amino acids, and others.4 The utilization of sugars is, however, restricted to fructose and N-acetylglucosamine.5 R. eutropha H16 lacks the key enzymes of the Embden− Meyerhoff−Parnas (EMP) and the oxidative pentose phosphate © 2012 American Chemical Society

pathways, phosphofructokinase, and 6-phosphogluconate dehydrogenase, respectively.6−8 Fructose and sodium gluconate, both preferential carbon sources of R. eutropha, are therefore metabolized via the Entner−Doudoroff (ED) pathway leading to pyruvate and glyceraldehyde 3-phosphate. Pyruvate is further decarboxylated to the central intermediate acetyl-CoA thereby replenishing the TCC and various anabolic pathways. In contrast, an anabolic EMP pathway (gluconeogenesis) is operating in R. eutropha H16.8 In R. eutropha H16, N-acetylglucosamine is taken up by a sugar-specific phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTSNag) consisting of the two proteins EINag-HPrNag-EIIANag (NagF) and EIIBCNag (NagE). The bacterial PEP-PTS is a sugar transport system comprising the two general components, histidine phosphocarrier protein Received: January 14, 2012 Published: May 25, 2012 3624

dx.doi.org/10.1021/pr300042f | J. Proteome Res. 2012, 11, 3624−3636

Journal of Proteome Research

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(HPr, ptsH) and enzyme I component (EI, ptsI) that are also present in R. eutropha H16. Initial phosphorylation of EI using PEP as substrate is followed by transfer of the phosphoryl group from EI to the catalytic site His-15 of HPr. A range of carbohydrate-specific enzymes II (EII) catalyze the following translocation and phosphorylation cascade from HPr via EIIABC to the incoming sugar.9−11 In total, seven proteins were identified to encode homologous PEP-PTS proteins in the genome of R. eutropha H16.12,13 The functions of the remaining three proteins (EIIAMan, EIIANtr, and H16_A2203) are not yet unraveled in R. eutropha. Except for the above-mentioned independent PTSNag, a complete PTS including the EIIB and EIIC proteins/domains (permease components) is lacking in strain H16. For that reason, the functions of the key enzymes HPr and EI in strain H16 are still unknown. R. eutropha H16 has been extensively studied because of its ability to accumulate biodegradable polyesters. PHB is synthesized from acetyl-CoA, which is formed by the pyruvate dehydrogenase complex from pyruvate, via acetoacetyl-CoA and 3-hydroxybutyryl-CoA using enzymes encoded by the constitutively expressed phaC1AB1 operon of strain H16.14−18 To maximize the production of PHB, R. eutropha is typically grown in nitrogen-limited medium with an excess of available carbon source such as gluconate. PHB serves as a reserve of carbon and reducing equivalents for anabolism; however, granule formation, reutilization, and intracellular PHB degradation are not fully elucidated yet. PHB granules are surrounded by a thin membrane layer that consists mainly of the PHB synthases (PhaC), phospholipids, intracellular PHB depolymerases (PhaZ), phasins (PhaP), and a transcriptional regulator of phasin expression (PhaR).19 Since the PHB synthesizing enzymes are constitutively expressed in the wildtype and to avoid a futile cycle of PHB production and degradation, regulation of biosynthesis as well as mobilizing enzymes is essential. The involvement of phasins in regulation processes has been proposed by Pötter et al.20 Furthermore, Pries et al.21 suggested an exclusively regulatory function of HPr and EI in PHB metabolism and proposed a hypothetical model of a PHB mobilizing enzyme system. Besides the seven homologous PTS proteins, four phasins, seven PHB depolymerases, and two oligomer hydrolases (PhaY) were identified in the genome of R. eutropha H16 that contribute to PHB degradation.8,22−25 We have now cultivated several R. eutropha strains in a parallel cultivation system to directly compare (i) their growth characteristics, (ii) the quantification and analysis of accumulated PHB, and (iii) their differential protein expression pattern. Using 2D-PAGE and MALDI-TOF analysis, the cytosolic protein expression patterns of the wild-type H16 and of the double mutant H16ΔptsHI during growth on gluconate were also investigated. To gain a deeper insight into both proteomes, we evaluated the phosphorylation status of all detected protein spots. Therefore, all generated 2D gels were initially subjected to fluorescence staining with Pro-Q Diamond phosphoprotein dye and were afterward stained with Coomassie Brilliant Blue for detection of total protein. Protein identification and metabolic pathway predictions done in this study became possible due to the availability of the genome sequence of R. eutropha H16.8 Therefore, it was possible to use a proteomic approach to search for proteins that are affected by the loss of HPr and EI and for proteins that could be involved in regulatory mechanisms of PHB metabolism.

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MATERIALS AND METHODS

Bacterial Strains, Media, and Cultivation Conditions

The following Ralstonia eutropha strains were used in this study: the wild-type R. eutropha H16 (DSM 428), the PHBnegative mutant PHB−4 (DSM 541), the PHB-leaky defined deletion mutants R. eutropha H16ΔptsH, H16ΔptsI, and H16ΔptsHI,13 as well as the transposon-induced mutant R. eutropha HF39 ptsI::Tn5::mob.21,26 For comparative analysis, R. eutropha was cultivated in parallel fermenters. Cells were grown for 72 h in six 2 L vessels of a parallel bioreactor systems (Biostat Bplus, Twin 2 L MO, Sartorius) each with 1.8 L of mineral salts medium (MSM)27 containing 0.05% (w/v) NH4Cl and 1.0% (w/v) sodium gluconate to provide conditions permissive for PHB accumulation. In general, PHB synthesis is observed under unbalanced growth conditions if a carbon source is present in excess, while another macroelement (N, O, P, S) is limiting growth and the yield of fermentation at the same time.28,29 The bioreactors were inoculated with 5% (v/v) of a well-grown preculture (cultivated aerobically in Erlenmeyer flasks with MSM plus 0.1%, w/v, NH4Cl and 1.0%, w/v, gluconate for 20 h at 30 °C), agitated at 200 rpm, and aerated at 1.5 L min−1. Aeration and agitation were adjusted according to the oxygen demand of the cultures. To avoid growth limitation, the dissolved oxygen concentration (pO2) was measured during fermentation. A decrease in the pO2 pressure was countervailed by an increase in the airflow and stirring rate. The temperature was set at 30 °C. The pH was maintained at 7.0 and was adjusted automatically by the addition of 2 M HCl or 2 M NaOH. Growth of cells was determined offline by measuring the optical density photometrically at 600 nm. Fifty milliliters of culture samples was withdrawn after 12, 29, 35, 48, and 72 h of fermentation, the cells sedimented by centrifugation (15 min, 5467g, 4 °C), and washed once with 0.9% (w/v) NaCl before lyophilization. Samples were quantified for their polyester contents by gas chromatography (GC) analysis. After samples had been withdrawn in the early stationary growth phase (29 h), NH4Cl was added to the cultures to a final concentration of 0.05% (w/v). When the limiting macroelement that caused polyester accumulation is supplied again to the culture, PHB degradation is induced, and the storage compound is used as a carbon and energy source.30 Additionally, culture samples withdrawn after 12, 29, and 35 h of cultivation were subjected to proteome analysis. Fluorescence Microscopy

The presence of cytoplasmic PHB inclusions was evidenced by microscopic analysis. To apply the viable colony staining method,31 samples of R. eutropha H16 and mutants were collected from the cultures and mixed with Nile red (0.25 mg dissolved in 1 mL of dimethyl sulfoxide [DMSO]) to a final concentration of 0.5 μg mL−1 and incubated for 30 min at 4 °C in the dark. Imaging was performed with a Zeiss Axio Imager.M1. PHB Analysis

Lyophilized cell material of R. eutropha (5−10 mg) was subjected to methanolysis in the presence of 85% (v/v) methanol and 15% (v/v) sulfuric acid for 3 h at 100 °C. The resulting methyl esters of 3-hydroxybutyrate were characterized by gas chromatography as described previously32,33 by using an Agilent 6850 GC (Agilent Technologies) equipped with a BP21 3625



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capillary column (50 m by 0.22 mm; film thickness, 250 nm; SGE) and a flame ionization detector (Agilent Technologies).

Pro-Q Diamond Phosphoprotein Dye. Fixation, staining, and destaining of gels were performed following the protocol of the manufacturer (Molecular Probes). The incubation and wash times were carefully determined to provide optimal staining. Following the manufacturer instructions, gels were imaged using a Molecular Imager PharosFX Plus System (BioRad) with 532 nm excitation and 605 nm bandpass emission filter (filter wheel B). Data were collected as 100 μm resolution, 16 bit gray-scale files using the Quantity One software (BioRad). With this software, fluorescent protein signals in 2D gels were displayed as dark spots on a light background. The dye is compatible with other staining methods and modern mass spectrometry. Prior to 2D-PAGE, we included a positive control using the PeppermintStick phosphoprotein molecular weight standard (Molecular Probes) on a one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel. Because of the high cost of the Pro-Q Diamond dye, it was reused several times in our study lowering its sensitivity. Therefore, we focused on the qualitative analysis of the Pro-Q Diamondstained phosphoproteome. Coomassie Brilliant Blue Total Protein Dye. After imaging the stained phosphoproteome gel, Coomassie staining was performed according to Raberg et al.34 The 2D gel images were again documented on a Molecular Imager PharosFX Plus System (Bio-Rad) under the same filter adjustment. A qualitative as well as quantitative analysis of the Coomassiestained total proteome was performed.

Preparation of Soluble Protein Samples for Proteome Analysis

To obtain crude extracts from cells that were cultivated in MSM, 50 mL of cell suspensions was harvested by centrifugation (15 min, 3345g, 4 °C), and the resulting cell pellets were then treated each with 50 mL of crack solution (8 M urea, 2% [vol/vol] Triton X-114). To block any phosphatase activity during lysis, sodium orthovanadate (1 mM Na3VO4) was used as a general inhibitor for protein phosphotyrosyl phosphatases. The mixture was then incubated for 1.5−2.0 h at room temperature on a gyratory shaker to break the cells. PHB and cell debris were then separated from the crude extract by ultracentrifugation (1 h, 29.500 rpm, 70 000g, 4 °C), and proteins were subsequently extracted from the supernatant by phenol extraction as described previously by Raberg et al.34 After acetone precipitation, the washed protein pellet was airdried by incubation at room temperature to evaporate the acetone and was then stored at −20 °C. Rehydration of Proteins

Rehydration buffer A (250 μL; 9 M urea, 4%, w/v, 3-[(3cholamidopropyl)-dimethylammonio]-1-propanesulfonate [CHAPS], 100 mM dithiothreitol [DTT], and distilled H2O [dH2O] to a total volume of 10 mL) were added to the dry protein pellets, and the mixture was then incubated at room temperature for 3 h. To ensure effective rehydration, the samples were stirred several times during this period. Protein solutions were then transferred to 1.5 mL plastic tubes and centrifuged (5 min, 11 000g). The protein concentration in the supernatants was subsequently measured.

Software-Based Analysis of 2D Gel Images

Images from 2D-PAGE were analyzed using the Delta2D version 3.4 (DECODON GmbH). From replicates comprising various samples of identical stages of cultivation (exponential [12 h] and stationary growth phases [29 h, 35 h] of the wildtype R. eutropha H16 and the deletion mutant H16ΔptsHI; four replicates for each sample), average fusion images were generated for each group after the necessary warping steps were performed. Spots were color coded according to their expression profiles in the dual-channel images of these fusions. For further comparisons of the protein patterns during the different stages, spot quantities were likewise densitometrically determined by the Delta2D version 3.4. For this, a proteome map comprising all gel images of each of the four replicate groups was created using the software’s union fusion approach. Spot boundaries on the proteome map were detected and transferred to the original images, and spots were automatically quantified by the software. The given spot quantities represent the relative portion (% volume) of an individual spot of the total protein present on the respective average fusion image. Normalization aimed at mitigating systematic differences between gel images, which may occur by variations in protein loading, imaging exposure times, and dye/staining efficiencies. Using analysis of variance (ANOVA), differentially expressed proteins between replicate groups were identified. To ensure significance, a negative filter (0.5 to 2.0) was set such that protein spots were chosen for matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry analysis only if their expression levels increased or decreased by a factor of 2.

Determination of Protein Concentration

The protein concentration was determined by the method of Bradford.35 To reduce the disturbing influences of DTT and urea,36 which are part of rehydration buffer A, only 5 μL of highly concentrated protein solution was mixed with a volume of 5 mL of Bradford reagent. After 10 min of incubation in the dark, absorption at 595 nm was measured against the reagent blank value. A calibration was done with bovine serum albumin in a range of 1 to 1000 μg. Two-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE)

First-Dimension Isoelectric Focusing (IEF). An aliquot of protein solution that contained 2 mg of protein was mixed with 100 μL of rehydration buffer B (2.5 mL rehydration buffer A, 125 μL ampholyte solution [pH 3 to 10; Serva], 125 μL Triton X-100, trace amount of bromophenol blue) and was filled up to 300 μL with rehydration buffer A. The isoelectric focusing (IEF) strips (pH 3 to 10 nonlinear, 18 cm; Serva) were passively rehydrated overnight at room temperature with the prepared protein solution (max. 340 μL) while overlaid with 2 mL of mineral oil. After rehydration of the strips with the protein solutions, strips (while overlaid with mineral oil) were focused in a focusing tray by a series of voltage increases according to the manufacturer’s instructions (Serva). Second-Dimension and Gel Staining. The equilibration of IEF strips as well as the 2D gel run in a Dodeca cell (BioRad) was done as described by Raberg et al.34 Proteins separated on 2D gels were first stained with Pro-Q Diamond and subsequently with Coomassie Brilliant Blue.

Protein Preparation, Mass Spectrometry, and Data Analysis

Spots were cut from the 2D gels and transferred to 1.5 mL plastic tubes. MALDI-TOF analysis was performed by employing the method of Shevchenko et al.37 For this, proteins 3626



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were tryptically digested, and the mass spectra of the protein fragments were revealed by MALDI-TOF (Proteomics Analyzer 4800; Applied Biosystems). The parameters for the measurements were set as described by Voigt et al.,38 except that the signal-to-noise ratio for the TOF/TOF measurements was raised to 10. For database search, the Mascot search engine (Matrix Science Ltd., version 2.1.04)39 with a specific R. eutropha database was used. Search parameters were as described by Voigt et al.38 One missed cleavage was allowed. The precursor-ion mass tolerance was 0.55 Da, and the fragment-ion mass tolerance was 50 ppm. Oxidation of methionine and carbamidomethylation of cysteine were allowed as modifications. Most proteins were not only identified by peptide mass fingerprinting (PMF) but by tandem mass spectrometry. Proteins were regarded as identified when the overall protein score was higher than 51 (≥98%) or when the ion score was higher than 26 (≥98%; Supplemental Table 2 in Supporting Information).

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Figure 2. PHB accumulation by R. eutropha H16, PHB−4, and PTS mutants. PHB contents (wt % of CDW) were determined by GC analysis at different time points of each cultivation: in the exponential (12 h), early stationary (29 h), and stationary (35 h, 48 h, 72 h) growth phases. After taking the sample in the early stationary growth phase, NH4Cl was added to the cultures to a final concentration of 0.05% (w/v) to induce polymer degradation. Data are mean values of two independent experiments ± standard deviations.

RESULTS

Growth and PHB Accumulation by R. eutropha H16, PHB−4, and PTS Mutants during the Time Course of Cultivation

described in the materials and methods section. Our results employing six parallel fermentations are in good agreement with previous growth and PHB accumulation experiments performed in Erlenmeyer flasks.13 As expected, the wild-type H16 and the mutant PHB−4 reached the highest and lowest optical density (maximal OD600 nm = 15.2 and 4.5, respectively) when grown in MSM containing gluconate. Both, the lag phases and growth rates of the mutants, are similar to the wildtype. The growth rates of all strains were in the range of 1.0 h−1 to 1.5 h−1 under these conditions. All strains entered the stationary growth phase after 10 h of cultivation. Growth ceased due to exhaustion of the nitrogen source in the medium. The surplus of the carbon source allowed an increase in cell mass due to the accumulation of intracellular PHB. This is generally observed in a difference of the optical density between the wildtype and the mutants in the stationary growth phase. The PHB contents of the cells reflected the results of the growth experiment. Generally, minor amounts of PHB were accumulated in the exponential growth phase, whereas the maximal production of polyester occurred in the stationary growth phase. In the case of the wild-type, this was up to 82.6% (w/w) PHB of cell dry weight (CDW) (Figure 2). The PHB contents of mutants H16ΔptsH, H16ΔptsI, and H16ΔptsHI, as well as the Tn5 mutant HF39 ptsI::Tn5::mob were significantly lower, which is in accordance with their PHB-leaky phenotype. As shown in Figure 2, the PHB contents of mutant cells were similar to each other and increased up to 44.9% (w/w, PHB of CDW) in the case of H16ΔptsI. After the addition of NH4Cl to the cultures, the optical densities of the cultures of all strains increased, and PHB was degraded and used as intracellular carbon and energy source at the same time. Mutants defective in ptsI showed the strongest net mobilization of PHB as described previously by Pries et al.21 This Tn5-induced mutant exhibited a similar phenotype regarding growth and PHB production as the deletion mutant H16ΔptsI but with a significantly lower optical density during the stationary phase.

Growth and PHB accumulation of the wild-type R. eutropha H16 and the PHB-negative mutant PHB−4 were compared with those of the defined deletion mutants H16ΔptsH, H16ΔptsI, and H16ΔptsHI, respectively. Additionally, the Tn5 mutant HF39 ptsI::Tn5::mob was investigated. For this, cells were cultivated in MSM under conditions permissive for PHB accumulation for 72 h. The time course of fermentation is shown in Figure 1. To determine the PHB contents at different stages of growth, samples were withdrawn during the cultivation after 12, 29, 35, 48, and 72 h, and the polymer contents were quantified (Figure 2). After 29 h of cultivation and having withdrawn the sample of the early stationary growth phase, NH4Cl as limiting macroelement was added to the medium to induce PHB degradation (Figure 1, arrow) as

Figure 1. Growth behavior of R. eutropha H16, PHB−4, and PTS mutants. The wild-type H16 (red square), H16ΔptsH (blue diamond), H16ΔptsI (orange circle), H16ΔptsHI (green triangle), HF39 ptsI::Tn5::mob (brown asterisk), and PHB−4 (pink open triangle) were cultivated in MSM under storage conditions containing 1.0% (w/ v) sodium gluconate as the sole carbon source and 0.05% (w/w) NH4Cl as the limiting nitrogen source. Samples were withdrawn in the exponential (12 h), early stationary (29 h), and stationary growth phases (35, 48, and 72 h) to analyze the PHB contents of the cells. After taking the sample in the early stationary growth phase, NH4Cl was added to the cultures (arrow) to a final concentration of 0.05% (w/v) to induce polymer degradation.

Occurrence and Distribution of Intracellular PHB Inclusions

During fermentation of R. eutropha in MSM under conditions permissive for PHB biosynthesis, samples were withdrawn from the cultures at the different growth stages to investigate the occurrence and distribution of PHB granules in the cells. 3627



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in the granules. Using the light optical microscope scale, we measured the average number and size of PHB granules as well as the gap between these inclusions. Cells contained between eight and 20 well-separated granules with diameters between 0.3 and 0.6 μm. Some of the granules were too small and were not sufficiently defined to be quantified. The distances between granules were estimated to average between 0.3 and 1.1 μm (Supplemental Table 1 in Supporting Information).

Micrographs taken in the early stationary growth phase after 29 h of fermentation revealed a large number of inclusions in the rod-shaped wild-type cells exhibiting no coalescence and occupying the major part of the cytoplasm (Figure 3A). This

General Characteristics and Statistical Analysis of the (Phospho-) Proteome

The protein profiles of cells grown in MSM plus gluconate in the exponential (12 h), early stationary (29 h), and stationary (35 h) growth phases were studied by 2D gel-based proteome analysis. Therefore, a minimum set of four 2D gel replicates of each sample was prepared and scanned after staining with ProQ-Diamond phosphoprotein stain and Coomassie Brilliant Blue total protein stain. To provide a view of the phosphoproteome of R. eutropha H16 with possible modifications in mutant H16ΔptsHI, we performed a qualitative analysis. For each strain, we selected a gel of the early stationary growth phase with good protein resolution, low distortion, and best protein stain and matched the phosphoprotein image from Pro-Q Diamond staining to the total protein image from Coomassie staining (Figure 4). The demonstrated results and comparison of further images confirmed the specific staining of phosphoproteins with Pro-Q Diamond. We were able to identify a couple of proteins that were strongly visible on the Pro-Q Diamond stained image but not on the Coomassie stained gel. As a key feature, the staining intensity of the Pro-Q Diamond dye correlates with the number of phosphate groups attached to the protein. On the basis of an isoelectric point (pI) shift and unchanged molecular weight (MW), several phosphoproteins may represent isoforms of the same protein with a varying phosphorylation status. It is remarkable that distinct phosphoproteins were upregulated in the wild-type and in the mutant. Three of the highly phosphorylated proteins were also detected on the Coomassie-stained gel. They were identified in the subsequent MALDI-TOF analysis (phosphoenolpyruvate synthase [PpsA], outer membrane protein/porin [H16_A3402], and translation elongation factor P [Efp2]) and are mentioned in the total proteome analysis below. For qualitative and quantitative evaluation of the total proteome of both investigated strains, average fusion gel images of the replicates of each sample were generated in a first step. After software-assisted spot detection, densitometric spot quantification was performed. A number of differentially expressed proteins were recognized after statistical analysis of spot quantities between the replicate gel groups. Only spots with significantly differing amounts of protein by a factor of at least 2 were subjected to MALDI-TOF analysis. In total, 77 spots exhibiting differential protein expression were detected; six of them could not be identified. Seven spots were identified to be composed of different proteins, and 19 proteins occurred in different isoforms of the same protein species. Therefore, 81% of all proteins identified in this study are encoded on chromosome 1, 12% on chromosome 2, and 7% on megaplasmid pHG1, which is consistent with recent findings.40,41 Identified proteins were classified into functional pathway groups using annotations from the KEGG (Kyoto Encyclopedia of Genes and Genomes) database.42−44 Proteins identified in the 2D gels were largely organized into the following categories: carbon metabolism, membrane transport/

Figure 3. Micrographs of cells of R. eutropha cultivated in MSM under conditions permissive for PHB accumulation for 29 h at 30 °C. R. eutropha H16 (A); H16ΔptsHI (B); H16ΔptsH (C); H16ΔptsI (D); and HF39 ptsI::Tn5::mob (E). The corresponding phase contrast light microscopy show cells containing cytoplasmic inclusions (left). The differential interference contrast micrographs are mimicking cells stereo vision (middle). Fluorescence images after staining with Nile red (right). Scale bars: 1 μm.

observation was consistent with the high PHB content verified by GC analysis (Figure 2). The micrographs in Figure 3 showed single elongated cells and not chains of normally shaped rods. When compared to the cells of strain PHB−4, the cell sizes of PHB accumulating strains increased 2- to 3-fold in the stationary growth phase (data not shown). Cells of mutants H16ΔptsI, H16ΔptsHI, and the Tn5 mutant showed significant differences in their appearance; they seemed to contain much less although larger inclusions. Cells of the deletion mutant H16ΔptsH included obviously a higher number of intracellular granules in comparison to the other investigated mutants (Figure 3C). However, the PHB contents were similar in all mutants (Figure 2). After provision of NH4Cl to the cultures, the size of the cells as well as the number of granules decreased, which is in accordance with the quantitative determination obtained by GC analysis. All granules stained positive with the lipophilic fluorescent dye Nile red, which indicates the accumulation of abundant amounts of hydrophobic compounds 3628



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(C) growth phases are shown in Figure 6. Comparing the proteomes of strain H16 and H16ΔptsHI, 16 different proteins (without isoforms) involved in carbon metabolism, energy metabolism, PHB biosynthesis, and other pathways were identified that exhibited at least a 2-fold higher expression in the wild-type in all three growth phases (marked in blue in Figure 6 and labeled in Table 1): glyceraldehydes 3-phosphate dehydrogenase (CbbG1, CbbG2), phosphoribulokinase (CbbP1, CbbP2), a putative peptidoglycan binding domain (H16_A3701), acetoacetyl-CoA reductase (PhaB1), phasin (PHA-granule associated protein; PhaP3, PhaP1/PhaP3), ATP synthase F1 sectordelta-subunit (AtpH), HoxI (fifth subunit of soluble hydrogenase), a putative lactaldehyde dehydrogenase (H16_A1919), pyridoxamine 5-phosphate oxidase (PdxH), transmembrane protein (H16_A2362), a universal stress protein UspA family (H16_A0533), outer membrane protein and related peptidoglycan associated (lipo)proteins (H16_A0990), and acetoin dehydrogenase E1 component alpha-subunit (AcoA). In contrast, 16 different proteins (without isoforms) were at least 2-fold overexpressed in the double mutant in all three growth phases (marked in orange in Figure 6 and labeled in Table 1). A major fraction of these proteins is involved in protein translation and amino acid metabolism. These proteins comprised ATP synthase F1 sectorbeta-subunit (AtpD), translation elongation factor EF-TU (GTPase; TufB), Flagellin (FliC), ABC-type transporter periplasmic component (HAAT family; branched-chain amino acid transport system; LivK2), 3isopropylmalate dehydrogenase (LeuB3), protein translation elongation factor TS (EF-TS; Tsf), dihydrodipicolinate synthase (DapA1), translation elongation factor P (EF-P; Efp2), outer membrane protein and related peptidoglycanassociated (lipo)protein (OpcL), cochaperonin (HSP10; GroES), hydrogenase expression/formation protein (HypE1), peroxiredoxin (H16_A1460), ATP synthase F1 sectorepsilonsubunit (AtpC), phosphoglycerate mutase 2 protein (Pgam2), a putative intracellular protease/amidase (ThiJ), and ribulosephosphate 3-epimerase (Rpe). Some of the above-mentioned proteins occur in several isoforms with identical MWs but different pIs (Figure 7). Several proteins were identified that were more abundant in the exponential phase in both strains including LivK2, CbbG2/ CbbG1, DapA1, Efp2, TufB, AtpH, H16_A1919, H16_A1460, H16_A2362, 30S small subunit ribosomal protein S2 (RpsB), and 50S large subunit ribosomal protein L9 (RplI). Among those proteins that were increasingly expressed in the stationary growth phase in both strains are OpcL, HoxI, peptidyl-prolyl cis−trans isomerase cyclophilin family B (PpiB), and phosphoenolpyruvate synthase (PpsA). Additionally, several isoforms of the universal stress proteins UspA family (H16_A2220 and H16_A0533), cochaperonin (GroES), and antioxidant proteins (peroxiredoxin [H16_A1460] and thiol peroxidase [Tpx]) were detected under all growth conditions. The spot quantification data of proteins/isoforms belonging to the five major functional pathway groups of this study are shown in Figure 7.

Figure 4. Variations of phosphoproteome profiles of R. eutropha H16 and H16ΔptsHI. Expression pattern of the phosphoproteome stained with Pro-Q Diamond dye (green spots) versus the total proteome pattern stained with Coomassie Brilliant Blue (red spots) in R. eutropha H16 (A) and H16ΔptsHI (B), both from the early stationary growth phase (29 h). For better visualization, the gels were imaged in an inverse mode. White spots mark equally expressed proteins. Pro-Q Diamond staining indicates the phosphorylation status of the protein (highly/lightly/not phosphorylated) compared to the Coomassie staining. Arrows/selections indicate highly phosphorylated proteins that occur at a very low concentration (if at all) in the Coomassie stained gel.

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outer membrane proteins, translation, energy metabolism, and amino acid metabolism (Figure 5). Differential Protein Expression in R. eutropha H16 and H16ΔptsHI

DISCUSSION

Phosphoproteome Analysis of R. eutropha Cells Grown on Gluconate

The dual channel views of the average fusion gels of the wildtype H16 and the double mutant H16ΔptsHI in the exponential (A), the early stationary (B), and the stationary

The loss of the phosphoryl-transfer proteins of the PTS and its effect on carbon and PHB metabolism in R. eutropha H16 3629



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Figure 5. Distribution and classification of differentially expressed proteins identified in 2D gels of R. eutropha H16 and H16ΔptsHI into functional pathway groups based on the KEGG database.

(EI, 64.3 kDa/pI 5.1) as regulatory enzymes that occur in very low concentration in the cell, both proteins were not detected on 2D gels of the wild-type. Instead, we searched for secondary effects in the cells that resulted from the loss of both components. Deletion of ptsHI induced obviously secondary effects on central metabolic pathways. The higher expression level of glyceraldehyde 3-phosphate dehydrogenase (CbbG1, CbbG2) in the wild-type in all three growth phases, as revealed by proteome analyses, probably led to an increased amount of pyruvate, a key intermediate in several metabolic pathways. Pyruvate can be decarboxylated to acetyl-CoA and further metabolized through the TCC. In the exponential growth phase, the TCC provides the cells with reduction equivalents in the form of NADH for the respiratory chain. Furthermore, precursors for the biosynthesis of amino acids and other necessary metabolites are generated. Two proteins of the TCC, malate dehydrogenase (Mdh1), and succinate dehydrogenase (SdhB) were upregulated in the wild-type in the exponential growth phase (Figure 7). Additionally, a strong expression of the E1 component (alpha-subunit) of the acetoin dehydrogenase (AcoA) was observed in the stationary growth phase of the wild-type. In R. eutropha H16, the formation of the acetoin dehydrogenase complex is induced in the presence of acetoin that is converted to acetaldehyde and acetyl-CoA.50,51 It was supposed that acetoin is synthesized in the stationary growth phase by a side reaction of the pyruvate decarboxylase.52 Furthermore, increased expression of the putative lactaldehyde dehydrogenase (H16_A1919) was detected in the wild-type. H16_A1919 is not characterized yet, and its actual function in R. eutropha remains unknown. Another enzyme that showed stronger expression in strain H16 compared to the mutant in all three growth phases was phosphoribulokinase (CbbP1, CbbP2). In R. eutropha H16 most genes required for CO2 assimilation are organized within two highly homologous cbb operons located on chromosome 2 and on megaplasmid pHG1.8,53 R. eutropha H16 synthesizes the key enzymes of the Calvin cycle, phosphoribulokinase, and ribulosebisphosphate carboxylase, even during heterotrophic growth with various organic substrates, although at a lower rate than during autotrophic growth with hydrogen and carbon dioxide or with formate as the carbon and energy source.54−56 Fructose, gluconate, and citrate allowed the production of intermediate enzyme activities of up to 20% of the autotrophic values.55 In

suggested that protein phosphorylation might be involved in the regulation of the storage compound synthesis. Posttranslational modifications such as reversible protein phosphorylation play a major regulatory role in many cellular processes. In general, it is possible to analyze changes of protein phosphorylation through a gel-based proteomic approach that may enable the identification of kinase/phosphatase substrate preferences in the investigated strain. Pro-Q Diamond stain is suitable for the fluorescent detection of phophoserine-, phosphothreonine-, and phosphotyrosine-containing proteins in 2D gels. Phosphohistidine and phosphoaspartate residues are very labile and often lost during electrophoresis or fixation step. Several studies reported on the successful 2D gel-based proteome and phosphoproteome analysis used Pro-Q Diamond.45−49 To our knowledge, this is the first use of multiplexed proteomics technology for investigation of phosphoproteins in 2D gels of R. eutropha H16. Findings obtained from Pro-Q Diamond staining indicate a differentiation of lightly phosphorylated, high-abundance proteins from strongly phosphorylated, low-abundance proteins compared to the Coomassie staining. A few phosphoproteins strongly visible on the Pro-Q Diamond stained gel were definitely detected. Some of them were also detected on the total proteome map, and three of these proteins were identified as PpsA, H16_A3402, and Efp2. PpsA is a phosphotransferase and converts pyruvate into PEP, an essential step in gluconeogenesis. Because of the catalyzed reaction of PpsA, it was identified as highly phosphorylated on the phosphoproteome images. The occurrence of PpsA and outer membrane/ transport proteins in R. eutropha will be discussed below. Efp2 is a prokaryotic protein required for translational elongation by synthesizing peptide bonds on 70S ribosomes. The elongation factor is essential for cell viability. The effect of phosphorylation on Efp2 activity is unclear; it may inhibit the elongation stage of translation and represent a possible mechanism of translational control and regulation of protein biosynthesis. Total Proteome Analysis of R. eutropha Cells Grown on Gluconate

Regarding the total proteome evaluation of R. eutropha, the observed differences may be due to the deletion of ptsHI, to the growth rate of the cultures, and to the PHB accumulation state of the cells. Because of the low molecular weight of HPr (HPr, 9.5 kDA/pI 5.7) and the hypothesized function of HPr and EI 3630



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Figure 6. Protein profiles of R. eutropha H16 and H16ΔptsHI during the time course of the experiment as revealed by 2D-PAGE. Expression patterns of R. eutropha H16 (blue spots) versus patterns of H16ΔptsHI (orange spots), from the exponential growth phase (12 h) (A), the early stationary growth phase (29 h) (B), and the stationary growth phase (35 h) (C). Black spots mark equally expressed proteins. Labeled proteins show at least a 2-fold overexpression in the wild-type (blue) or in the mutant strain (orange) in all three growth phases and were identified via MALDITOF analysis. Black labels indicate a significantly stronger protein expression only in one or two growth phases but not in all three. Superscript letters refer to isoforms of the same protein. The main characteristics of identified proteins are summarized in Table 1. The detected but not identified (n.i.) six proteins are also labeled.

contrast to the PTS mutants, increased amounts of Cbb proteins in the wild-type may mediate a recycling of intermediates providing additional acetyl-CoA and NADPH for PHB biosynthesis and other anabolic pathways. One further protein that attracted our attention in the 2D gels was HoxI, the fifth NAD-reducing hydrogenase subunit of R. eutropha. HoxI was found to be strongly upregulated in the wild-type in all growth phases and occurred in three isoforms in our study (Figures 6 and 7). All five subunits of the soluble hydrogenase (HoxF, HoxU, HoxY, HoxH, and HoxI) were identified in 2D gels of lithoautotrophically (H2−CO2) grown cells of R. eutropha, with HoxI representing the dominant protein species. 40,41 The reason for the significant differential expression of this protein in our study is unclear. Physiological and enzymatic results of previous experiments indicated a correlation between the formation of enzymes of H2 oxidation and CO2 assimilation in cells of R. eutropha.55

In contrast, only two overexpressed proteins involved in central carbon metabolism were identified in mutant H16ΔptsHI in all three growth phases: ribulose-phosphate 3epimerase (Rpe) and phosphoglycerate mutase 2 (Pgam2). Rpe catalyzes the reversible epimerization of ribulose 5-phosphate to xylulose 5-phosphate and is a component of the pentose phosphate pathway and the Calvin cycle leading to the interconversion of carbohydrates and the regeneration of ribulose-1,5-bisphosphate, respectively. Pgam2 catalyzes one of the last steps of the glycolysis/ED pathway in which a phosphate group is transferred from 3-phosphoglycerate to 2phosphoglycerate. Since this is a reversible reaction, Pgam2 acts also in the gluconeogenic pathway. Additionally, two isoforms of the PEP synthase (PpsA; above reaction) were overexpressed in the mutant in the early stationary and stationary growth phase. In contrast to the wild-type, enzymes of gluconeogenesis showed enhanced expression in the mutant leading to the opposite direction of PHB biosynthesis. This 3631



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Table 1. Occurrence of Proteins in R. eutropha H16 and H16ΔptsHI Cells Grown in Medium Containing Sodium Gluconate As Identified by 2D-PAGE and MALDI-TOF Analysisa locus tag

replicon

gene

protein

Proteins That Were at Least 2-Fold Overexpressed in the Wild-Type H16 in All Three Growth Phases H16_A0533 Chr1 universal stress protein UspA family H16_A0990 Chr1 outer membrane protein and related peptidoglycan-associated (lipo)proteins H16_A1381 Chr1 phaP1 phasin (PHA-granule associated protein) H16_A1439 Chr1 phaB1 acetoacetyl-CoA reductase H16_A1919 Chr1 putative lactaldehyde dehydrogenase H16_A2172 Chr1 phaP3 phasin (PHA-granule associated protein) H16_A2362 Chr1 transmembrane protein H16_A2802 Chr1 pdxH pyridoxamine-phosphate oxidase H16_A3640 Chr1 atpH membrane-bound ATP synthase F1 sectordelta-subunit H16_A3701 Chr1 putative peptidoglycan binding domain H16_B0144 Chr2 acoA acetoin dehydrogenase E1 component alpha-subunit H16_B1386 Chr2 cbbG2 glyceraldehyde 3-phosphate dehydrogenase H16_B1389 Chr2 cbbP2 phosphoribulokinase PHG093 PHG1 hoxI HoxI PHG418 PHG1 cbbG1 glyceraldehyde 3-phosphate dehydrogenase PHG421 PHG1 cbbP1 phosphoribulokinase Proteins That Were at Least 2-Fold Overexpressed in Mutant H16ΔptsHI in All Three Growth Phases H16_A0394 Chr1 thiJ putative intracellular protease/amidase/DJ-1/PfpI family H16_A0493 Chr1 pgam2 phosphoglycerate mutase 2 protein H16_A0705 Chr1 groES cochaperonin GroES (HSP10) H16_A1204 Chr1 dapA1 dihydrodipicolinate synthase H16_A1460 Chr1 peroxiredoxin H16_A2054 Chr1 tsf protein translation elongation factor TS (EF-TS) H16_A2549 Chr1 efp2 translation elongation factor P (EF-P) H16_A2619 Chr1 leuB3 3-Isopropylmalate dehydrogenase H16_A2828 Chr1 opcL outer membrane protein or related peptidoglycan-associated (lipo)protein H16_A3030 Chr1 livK2 ABC-type transporter periplasmic component: HAAT family H16_A3317 Chr1 rpe ribulose-phosphate 3-epimerase H16_A3505 Chr1 tuf B translation elongation factor EF-TU (GTPase) H16_A3636 Chr1 atpC membrane-bound ATP synthase F1 sectorepsilon-subunit H16_A3637 Chr1 atpD membrane-bound ATP synthase F1 sectorbeta-subunit H16_B2360 Chr2 f liC flagellin PHG017 PHG1 hypE1 HypE1 Other Identified Proteins H16_A0220 Chr1 argC1 N-acetyl-gamma-glutamyl-phosphate reductase H16_A0386 Chr1 Sigma54 (RpoN) modulation protein H16_A0918 Chr1 upp1 uracil phosphoribosyltransferase H16_A1090 Chr1 glutathione S-transferase H16_A1218 Chr1 ppiB peptidyl-prolyl cis−trans isomerase cyclophilin family B H16_A2038 Chr1 ppsA phosphoenolpyruvate synthase H16_A2055 Chr1 rpsB SSU ribosomal protein S2 H16_A2220 Chr1 universal stress protein UspA family H16_A2276 Chr1 rplI 50S LSU ribosomal protein L9 H16_A2368 Chr1 ndk nucleoside diphosphate kinase H16_A2568 Chr1 fabD (acyl-carrier-protein) S-malonyltransferase H16_A2629 Chr1 sdhB succinate dehydrogenase (Fe−S protein subunit) H16_A3077 Chr1 purM phosphoribosylformylglycinamidine cyclo-ligase H16_A3175 Chr1 tpx thiol peroxidase H16_A2634 Chr1 mdh1 malate dehydrogenase H16_A3395 Chr1 sspA stringent starvation protein A (glutathione S-transferase) H16_A3402 Chr1 outer membrane protein (porin) H16_A3492 Chr1 f usA1 translation elongation factor G (EF-G) H16_A3688 Chr1 TRAP-type transporter periplasmic component H16_B2202 Chr2 hypothetical protein

pI

MW (kDa)

6.64 5.81 5.96 6.74 5.45 6.13 5.38 6.86 4.88 9.12 5.48 6.41 6.09 5.65 6.41 6.33

17.0 32.9 20.0 26.4 50.3 19.6 23.7 24.1 19.1 42.0 35.4 35.9 33.3 18.6 35.9 33.1

5.48 5.8 5.8 5.78 5.08 5.26 4.81 5.34 7.79 8.13 5.77 5.49 5.06 5.18 5.12 5.14

20.4 24.8 10.5 31.3 20.2 30.9 21.0 38.4 17.7 41.4 25.2 43.0 14.5 50.9 44.8 36.6

6.01 6.71 6.3 6.38 5.72 5.12 6.33 5.31 7.96 5.91 5.31 6.21 5.09 5.47 6.14 6.45 8.97 5.24 8.69 9.35

34.0 13.6 24.1 26,0 18.2 87.0 27.3 15.7 15.8 15.3 31.6 26.8 37.2 17.2 35.1 23.8 41.3 77.3 41.6 20.5

a

Total proteins at least 2-fold upregulated in the wild-type or in the double mutant in all three growth phases are indicated; phosphoproteins were not quantitatively evaluated. Terms H16_A, H16_B, and PHG indicate the locus of genes on chromosome 1, chromosome 2, and on the megaplasmid PHG1, respectively. The values of pI and MW are theoretical data.

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substrate in R. eutropha, and the respective phosphate acceptors, EI and HPr, are lacking in the investigated mutant. The wild-type H16 has been isolated from water and soil, and therefore, its genome encodes for a large number of membrane transport systems that facilitate the uptake of multiple compounds. Almost 30% of all proteins deduced from the genomic sequence of strain H16 are predicted to be transport proteins.8 We identified five outer membrane proteins/isoforms and six transport proteins/isoforms that were differentially expressed in both strains. Transporters were either ATPbinding cassettes involved in the uptake of amino acids (HAAT family) or tripartite ATP-independent periplasmic transporters (TRAP-T family). The overrepresentation of transport-related proteins is difficult to interpret and may be the result of the adaptation of the organism to the loss of PTS components. A set of proteins/isoforms involved in translation processes (Tsf, TufBa, TufBb, and Efp2; Figures 6 and 7) and amino acid metabolism (LeuB3, DapA1a, DapA1b, and ArgC1b; Figures 6 and 7) exhibited at least a 2-fold higher expression in mutant H16ΔptsHI compared to the wild-type in all three growth phases. Under nutrient poor growth conditions such as we have chosen, the metabolism of cells is oriented toward an economical usage of substrates and metabolites for the biosynthesis of proteins and nucleic acids.57 The enhanced expression of genes involved in translation and amino acid metabolism may be linked to the overexpression of transportrelated proteins. In particular, five isoforms of the ABC-type transporter LivK2 (perplasmic component; hydrophobic amino acid uptake transporter) were identified in the mutant that may explain the observed correlation. Additionally, an affected membrane permeability for gluconate and other carbon sources may exist in the mutant. According to this, the uptake of the carbon source from the medium would be hindered leading to a lower concentration of gluconate in the cells of the mutants. A lowered carbon-to-nitrogen ration would limit the biosynthesis of PHB as observed for all investigated PTS mutants. Effect on PHB Accumulation in R. eutropha

Because of the competition of TCC and PHB biosynthesis for acetyl-CoA, the amount of accumulated PHB changes in the course of time. In the exponential phase, acetyl-CoA is used in the TCC, and therefore, the PHB content in the cells is rather low. In the stationary growth phase and under unbalanced growth conditions, the excess of available carbon and energy is stored in the form of intracellular PHB inclusions. This observation was confirmed by GC analysis (Figure 2) and micrographs. Tian et al.58 studied the average volume of R. eutropha cells and the total surface area of PHB granules per cell at different stages of granule formation by TEM analysis. It was speculated that an increased cell size in the stationary phase may be a regulatory mechanism required to maximize the cells capacity for granule storage. The amount of PHB increased in the cells to the point where NH4Cl was added to the cultures and reutilization of PHB was induced. Our data obtained with the fluorescence microscope are in accordance with previous results obtained from TEM images.58 Moreover, three proteins involved in PHB biosynthesis were found to be strongly upregulated in the wild-type in all investigated growth phases. Their amounts increased from exponential phase to the late stationary phase. Besides the NADPH-dependent PhaB1 (acetoacetyl-CoA reductase; encoded by the phaC1AB1 operon),26,59,60 two granule-associated phasins, the predominant PhaP1 and PhaP3 were identified. A reduced production

Figure 7. Quantification of protein spots in 2D gels of R. eutropha belonging to the five major functional pathway groups. Spot quantities are given as percent volume (representing the relative portion of an individual spot of the total protein present on the respective average fusion image). Letter indexes refer to isoforms of the same protein.

tendency was confirmed by PHB quantification. PEP is typically utilized as the phosphate donor in a functional carbohydraterelated PTS. However, gluconate is not a PTS-mediated 3633



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Competence Network Gö ttingen “Genome Research on Bacteria”.

of PHB biosynthetic proteins such as in mutant H16ΔptsHI was also observed in PTS mutants of Azotobacter vinelandii lacking the EINtr/NPr proteins.61,62 Although PHB degradation was induced in the stationary growth phase, no depolymerase was detected in the proteomes of both investigated strains during the course of this study. The occurrences of stress-related proteins on 2D gels attracted attention. It is speculated that the stress-related proteins provide protection under oxidative stress and support the correct folding of nascent proteins in bacteria.63−65 Some of the stress-related proteins are also synthesized under other stress conditions like nutrient limitation. It has been previously reported that PHB-accumulating bacteria overexpress heat shock proteins (HspA) in response to stress.66 It was suggested that HspA can act like a PHA granule-associated protein such as phasins, which affect PHA granule coalescence.67 In summary, the enhancement of the central catabolism comprising enzymes involved in the ED pathway, the TCC, and PHB biosynthesis in the wild-type leads to an increased concentration of acetyl-CoA and therefore to a higher level of accumulated PHB in the cells. The up- and downregulation of catabolic and anabolic proteins is interpreted as an adaption to growth conditions and cell division during cultivation. Our findings support and contribute to previously published proteome data of R. eutropha H16.40,41,68,69 In contrast, overexpression of proteins involved in amino acid metabolism and protein translation in the mutant may rely on an enhanced expression of transport proteins and/or a modified membrane permeability. Additionally, enzymes of gluconeogenesis were found to be upregulated in the mutant. These effects were somehow responsible for the restricted cell growth of the double mutant with a significantly lower PHB content. Data obtained in this study provide a first insight into the metabolic changes and adaptations of R. eutropha cells, which were affected in the main components of PTS. Our 2D gel analysis was restricted to cytosolic proteins of the cells, membranebound proteins were not isolated by the applied method and are extremely underrepresented in 2D gels.70 For the future, transcriptome analyses would be of great value to gain a deeper insight into the metabolic processes and interactions induced by the deletion of ptsH/ptsI.

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ABBREVIATIONS USED ABC transporter, ATP binding cassette transporter; CBB, Calvin−Benson−Basham; CDW, cell dry weight; ED, Entner− Doudoroff; EMP, Embden−Meyerhoff−Parnas; MALDI-TOF, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry; PEP-PTS, phosphoenolpyruvate-carbohydrate phosphotransferase system; PHB, poly(3-hydroxybutyrate); PMF, peptide mass fingerprinting; PTSNag, N-acetylglucosamine-specific PTS; TCC, tricarboxylic acid cycle; 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis

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ASSOCIATED CONTENT

S Supporting Information *

Estimated average number and diameter of granules per R. eutropha cell; MS data for R. eutropha H16 proteins identified in 2D gels; MS spectra of proteins identified by peptide mass fingerprinting (PMF). This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49-251-8339821. Fax: +49-251-8338388. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was financially supported by the Bundesministerium für Bildung und Forschung (FKZ-0313751) within the 3634



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