Coping with Anoxia: A Comprehensive Proteomic and Transcriptomic

Article pubs.acs.org/jpr

Coping with Anoxia: A Comprehensive Proteomic and Transcriptomic Survey of Denitrification Yvonne Kohlmann,† Anne Pohlmann,†,⊥ Edward Schwartz,† Daniela Zühlke,‡ Andreas Otto,‡ Dirk Albrecht,‡ Christina Grimmler,∥ Armin Ehrenreich,§ Birgit Voigt,‡ Dörte Becher,‡ Michael Hecker,‡ Bar̈ bel Friedrich,† and Rainer Cramm*,†,# †

Institut für Biologie, Humboldt-Universität zu Berlin, Chausseestraße 117, 10115 Berlin, Germany Institut für Mikrobiologie, Ernst-Moritz-Arndt-Universität Greifswald, Friedrich-Ludwig-Jahn-Straße 15, 17489 Greifswald, Germany § Lehrstuhl für Mikrobiologie, Technische Universität München, Emil-Ramann-Straße 4, 85354 Freising, Germany ∥ Forschungsstelle für Nahrungsmittelqualität der Universität Bayreuth, 95326 Kulmbach, Germany ‡

S Supporting Information *

ABSTRACT: Ralstonia eutropha H16 is a denitrifying microorganism able to use nitrate and nitrite as terminal electron acceptors under oxygen deprivation. To identify proteins showing an altered expression pattern in response to oxygen supply, R. eutropha cells grown aerobically and anaerobically were compared in a comprehensive proteome and transcriptome approach. Nearly 700 proteins involved in several processes including respiration, formation of cell appendages, and DNA and cofactor biosynthesis were found to be differentially expressed. A combination of 1D gel-LC and conventional 2D gel analysis of six consecutive sample points covering the entire denitrification sequence revealed a detailed view on the shifting abundance of the key proteins of denitrification. Denitrification- or anaerobiosis-induced alterations of the respiratory chain included a distinct expression pattern for multiple terminal oxidases. Alterations in the central carbon metabolism were restricted to a few key functions including the isoenzymes for aconitase and isocitrate dehydrogenase. Although R. eutropha is a strictly respiratory bacterium, the abundance of certain fermentation enzymes was increased. This work represents a comprehensive survey of denitrification on the proteomic and transcriptomic levels and provides unique insight into how R. eutropha adapts its metabolism to low oxygen conditions. KEYWORDS: Ralstonia eutropha H16, spectral counting, membrane proteins, denitrification, anaerobiosis

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INTRODUCTION The use of nitrogen oxides as alternative electron acceptors enables facultative anaerobic microorganisms to cope with anoxic conditions. Complete denitrification involves the stepwise reduction of nitrate to molecular nitrogen, driven by four specific oxidoreductases, namely, nitrate reductase (NAR), nitrite reductase (NIR), nitric oxide reductase (NOR), and nitrous oxide reductase (NOS).1,2 The product of each enzymatic reduction step can be released into the environment. Therefore, denitrification contributes to emission of potentially toxic nitrite, nitric oxide, and the global warming gas nitrous oxide.3 Denitrification is of industrial and ecological importance. For example, denitrifiers are widely used for nitrate elimination in wastewater treatment plants and are responsible for the loss of nitrate in farmlands, where nitrate is used as the major nitrogen fertilizer.4,5 Moreover, persistence under anaerobic conditions and the reduction of nitrogen oxides is an important strategy of pathogenic microorganisms to withstand certain host defense mechanisms.6 The Gram-negative β-proteobacterium Ralstonia eutropha H16 was first described as a denitrifier in the early 1970s.7 Since © 2014 American Chemical Society

then, the genetic and biochemical basis for this has been the subject of investigations.8−10 The comparatively large genome (7.5 Mbp), with at least 6626 coding sequences (CDS), consists of two chromosomes and the megaplasmid pHG1.11 Gene clusters for the oxidoreductases of denitrification are located on chromosome 2 and pHG1.12 Apart from the key enzymes of denitrification, several more functions suspected to be included in adaptation to anaerobic conditions can be found in R. eutropha. Among them are the flavohemoprotein Fhp,13 the oxygen-independent coproporphyrinogen III oxidase HemN, 14 and the anaerobic ribonucleotide reductase NrdDG.15 Heterotrophic growth of R. eutropha is based on the use of a wide range of organic substrates, such as sugar acids, fatty acids, amino acids, or alcohols; however, sugar utilization is restricted to fructose and N-acetyl-glucosamine.16 In the absence of organic carbon sources, carbon dioxide is assimilated via the Calvin−Benson−Bassham cycle. Energy is obtained from the oxidation of molecular hydrogen by two Received: May 20, 2014 Published: September 8, 2014 4325

dx.doi.org/10.1021/pr500491r | J. Proteome Res. 2014, 13, 4325−4338

Journal of Proteome Research

Article

oxygen-tolerant hydrogenases.17−19 R. eutropha is one of the best-studied facultative hydrogen oxidizers20−22 and serves as a model organism for the production of biopolymers.23 Both features attracted biotechnological interest, for instance, in the autotrophic production of isotope-labeled biomolecules or isobutanol, energy conversion with biofuel cells, and lightdriven hydrogen production.24−26 In recent proteomic studies, the lithoautotrophic lifestyle of R. eutropha H16 was subjected to a detailed investigation.27,28 In this study, we report a comprehensive transcriptomic and proteomic survey to elucidate how this organism copes with oxygen depletion. Label-free spectral counting was applied for quantitative proteomics using separate 1D gel-LC−MS/MS data sets from soluble and membrane extracts. In addition, selected proteins were investigated by conventional 2D gel analysis. Furthermore, gene expression was monitored using microarray analysis of denitrifying and aerobically grown cells.

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Figure 1. Sample points in the course of denitrification. Arrows with letters indicate the targeted sample points for proteomic (A−F) and transcriptomic (A, D) analysis in the context of the appearance and disappearance of nitrogen compounds. Phases of denitrification are not drawn to scale. Note that, contrary to this schematic representation, most of the nitrous oxide was already consumed while some nitrite was still present at sample point D in the real experiment (see Supporting Information Figure 1).

METHODS

sampling, pressure within the fermenter was kept constant by adding helium. Additional investigations on the viability of the strains H16 and HF413 were done by monitoring growth on agar plates (1.5% Bacto-Agar) and in glass flasks under the culture settings described above. Hydroxyurea (HU) and coenzyme B12 were added in final concentrations of 5 mM and 200 nM, respectively.

Bacterial Strains and Growth Conditions

Wild-type R. eutropha H16 (DSM428, ATCC 17699) and the nrdDG mutant HF413 were used in this study. HF413 was derived from H16 by deletion of the ∼3 kb nrdD−nrdG region.15 For proteome and transcriptome analysis, R. eutropha H16 was heterotrophically grown at 30 °C and 180 rpm in a 10 L bioreactor filled with minimal medium as previously described.28 Fructose 0.4% (w/v) served as the sole energy and carbon source. Three aerobically grown cultures served as reference for comparison with denitrifying cultures. One culture was grown in the presence of ammonium chloride containing heavy nitrogen (15N) instead of light nitrogen (14N) to allow for peptide quantification by an inverse metabolic labeling strategy. However, this strategy was not pursued further since, due to an increased number of sampling points for denitrifying cells, we moved to peptide quantification by spectral counting. Nevertheless, the cell material obtained with the 15N-grown culture was maintained as a biological replicate in the present investigation since the biological variation of R. eutropha cultures grown heterotrophically and aerobically in the presence of light or heavy nitrogen is neglectable, as shown previously.27,28 Aerobically grown cells were harvested at OD436 ∼ 1 and a partial oxygen pressure above 15% (v/v). For anaerobic growth under denitrifying conditions, minimal medium was supplemented with 0.1% (w/v) potassium nitrate, and the headspace was filled with helium with a minimal overpressure of 50 mbar. In general, minimal medium was inoculated to initial OD436 of 0.05. Aerobically growing cells in the exponential growth phase (OD436 around two) served as starting culture. During anaerobic cultivation, nitrite was assayed in the supernatant according the method of Lowe and Evans.29 N2O and N2 were determined in the headspace of the bioreactor by gas chromatography as previously described.13 Nitric oxide gas was determined by ozonechemiluminescence using a Sievers nitric oxide analyzer (NOA 280i) as described by Kay et al.30 The device was also used to measure nitrate + nitrite in culture supernatants after reduction to nitric oxide with vanadium(III) chloride in hydrochloric acid at 90 °C.31 Nitrate concentrations were then calculated by nitrite subtraction. Throughout anaerobic growth, six different stages of denitrification served as sample points (SPs) referred to as SP-A to SP-F (see Figure 1 and Supporting Information Figure 1). For the duration of

Microarray-Based Transcriptional Profiling

Ten milliliter samples of culture medium were removed from the fermenter at sample points A and D (see Supporting Information Figure 1) and pipetted immediately into 20 mL of RNAprotect solution (Qiagen) in 50 mL Falcon tubes and incubated for 5 min at room temperature. Cells were pelleted, frozen in liquid nitrogen, and stored at −80 °C. RNA was isolated by a modified hot-phenol procedure. Each pellet was thawed by adding 1.6 mL of TE (pH 8.0) containing 2 μL of Lysozyme (10 mg/mL, Novagen) and incubating for 1 min at room temperature. The thawed suspensions were transferred to glass tubes with screw caps. After adding 20 μL of 2mercaptoethanol and 160 μL of 10% (w/v) SDS, the samples were incubated for 2 min at 64 °C. One-hundred seventy six microliters of 1 M sodium acetate (pH 5.2) and 2 mL of watersaturated acid phenol were added to each sample, and incubation was continued for another 6 min with vigorous mixing using glass pestles. After chilling on ice, the homogenate was transferred to two 2 mL reaction tubes and spun at 13 000g in a precooled centrifuge. The aqueous phase of each sample was extracted once with 1 mL of a mixture of phenol/ chloroform/isoamyl alcohol (25:24:1, v/v/v) and once with 600 μL of chloroform. After combining the respective aqueous phases, RNA was precipitated by adding 1 volume of 4 M LiCl and incubating for 2 h on ice. Samples were spun at 13 000g for 15 min. The precipitates were washed with ice-cold 70% ethanol, dried briefly, and resuspended in RNase-free water. Contaminating DNA was removed from the samples by treatment with DNA-free DNase following the manufacturer’s instructions (Ambion). The RNA preparations were routinely checked for the absence of DNA by polymerase chain reaction (PCR) of 25 cycles using primers specific for the R. eutropha H16 rpoA gene (H16-rpoA-f: 5′-atgcaaacagcactcctcaagc-3′ and H16-rpoA-r: 5′-ttacttctccagacctgcggg-3′) and H16 genomic DNA as a positive control. RNA integrity was checked on an Agilent 2100 Bioanalyzer (Agilent Technologies). cDNA was prepared from the RNA using PrimeScript reverse transcriptase 4326



Article

(TaKaRa). After degrading the RNA by treatment with NaOH for 30 min at 70 °C, cDNA samples were purified on illustra GFX columns (GE Healthcare). Five microliter samples of cDNA were labeled with Cy3-ULS or Cy5-ULS (Kreatech Biotechnology) according to the manufacturer’s recommendations. Microarrays carrying 6676 different 5′ amino-6-modified oligonucleotides (representing all of the annotated ORFs of the R. eutropha H16 genome including all putative pseudogenes and a few controls) spotted in duplicate on CodeLink microarray slides (SurModics) were used in this study. Details of the production of microarrays have been described elsewhere.32 Labeled cDNAs were mixed (approximately 80 pmols each of the Cy3- and Cy5-labeled samples), and the mixture was added to hybridization buffer.33 Hybridization to the R. eutropha array was carried out in a Lucidea slide processor (GE Healthcare) for 15 h at 58 °C. Slides were washed twice with 1× SSC buffer containing 0.2% SDS and once with 0.1× SSC, rinsed with 2-propanol, and dried. Each hybridization was repeated on a second slide using the reverse combination of dye labels. The processed arrays were scanned on a GenePix 4000B scanner using GenePix Pro 6.0 software. Median intensities in both channels were corrected by subtracting median local background intensities. The ratios of background-corrected median intensities were normalized for total intensity. For cases in which one of the two median intensities was zero or negative, the latter was arbitrarily set to 1. Only ratios of 3-fold or greater were considered to be significant.

using an Ettan spot picker (GE Healthcare, Little Chalfont, UK) with a picker head of 2 mm and transferred into 96-well microtiter plates. In-gel digestion and extraction of peptides were accomplished with the Ettan spot handling workstation (GE Healthcare, Little Chalfont, UK) using a protocol according to Eymann and co-workers.35 Identification of R. eutropha proteins by MALDI-TOF-MS and MALDI-TOFTOF-MS was done as described by Fuchs and co-workers.36 For 1D gel-LC−MS/MS, cytosolic samples or enriched membrane samples were resolved on 1D SDS-PAGE. Lanes were cut in equidistant pieces and subjected to trypsin digestion. Tryptic peptides were eluted and subjected to LC− MS/MS measurement using an LTQ Orbitrap coupled online to a nano Acquity HPLC. Peptides were loaded and desalted on a reversed-phase C18 column, and a binary gradient was used for elution of the peptides in to the mass spectrometer. Samples were acquired at a resolution of 30 000 for the top 5 precursors, with the lock mass option enabled and leaving out masses with unassigned charge state or singly charged ions. Bioinformatic Data Analysis

Scaffold (version Scaffold_3.6.1, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Scaffold is based on the PeptideProphet and ProteinProphet algorithms.37,38 Peptide identifications were accepted if they exceeded specific database search engine thresholds. Identifications required at least ΔCn scores of greater than 0.10 and XCorr scores of greater than 2.2, 3.3, and 3.8 for doubly, triply, and quadruply charged peptides, respectively. Proteins identified by at least two distinct peptide matches with a probability of 0.95 or above are assumed to be significant. However, even if a protein missed the identification criteria in one experiment, then we assumed an identification anyway if the criteria were fulfilled in at least one other replicate experiment. Appropriate spectral counts were reported as Excel files by means of the Scaffold option “show lower scoring matches”. The spectral counts reported here are unweighted in the sense that MS/MS spectra matching a peptide that contributes to more than one protein are counted in each protein containing that peptide. Unweighted spectral counts were normalized by dividing them by the sum of all spectral counts in the corresponding experiment and multiplying the result by the average of the sums of all experiments. In the case of replicates, protein identification was deemed to be correct if at least two replicates led to normalized spectral counts (NSCs). If so, then NSC means were calculated, and ratios of the NSCs corresponding to anaerobic and aerobic cultivations were transformed to Log2. For proteins that were detectable under only one of the two culture conditions (so-called on/off proteins), Log2 values could not be calculated. However, to give a rough estimate for comparisons, we provide a pseudo-Log2 (indicated by a “+O2” or “−O2” addendum) by setting such zero NSC values arbitrarily to 0.5, representing half of the minimum of one detected spectrum. For statistical analysis, the one-sided, unpaired Student’s t-test (heteroscedastic) was applied.39 A cutoff of 0.05 (p value ≤ 0.05) was chosen for significance. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://www. proteomexchange.org) via the PRIDE partner repository40 with the data set identifier PXD000888. The number of transmembrane helices (TMH) in a protein was predicted by the program TMHMM, which is based on a hidden Markov model.41,42

Protein Sample Preparation, 2D Gel- and 1D Gel-LC-Based Analysis

For proteome analysis, culture samples of 500 mL were harvested directly on ice, centrifuged for 20 min at 6000g and 4 °C, washed with H16P buffer (25 mM Na2HPO4/11 mM KH2PO4, pH 7.0, 0.3 mg/mL phenylmethylsulfonyl fluoride), and stored at −80 °C. Soluble and membrane fractions were prepared as described previously.28 Briefly, cells were disrupted by French press. Three rounds of centrifugation at 6000g followed by centrifugation of the appropriate supernatant at 88 000g yielded the soluble fraction. Pellets were washed with H16P buffer and sodium carbonate solution (0.1 M Na2CO3, pH 11). Membrane proteins were solubilized by incubating with lithium dodecyl sulfate. Protein concentration was determined using a bicinchoninic acid protein assay (Thermo Scientific, Rockford, IL) with bovine serum albumin as standard. Proteins were analyzed by 1D gel-LC−MS/MS and by 2D SDS-PAGE combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as described previously.27,28 Two-dimensional SDS-PAGE was carried out as described earlier using commercially available IPG strips in the pI range of 3−10.34 Briefly, the IPG strips were loaded with 100 μg protein extract by rehydratization for 18 h in a solution containing 8 M urea, 2 M thiourea, 1% Chaps (w/v), 20 mM DTT, and 0.1% Pharmalyte 3−10 (v/v). Isoelectric focusing was performed using MultiPhor II unit (Amersham Biosciences). After 2D PAGE, gels were fixed with 40% (v/v) ethanol and 10% (v/v) acetic acid and stained with the fluorescent dye Krypton according to the manufacturer’s instructions (Thermo Scientific). Stained gels were scanned (Typhoon 9400, GE Healthcare), and their images were analyzed employing Delta2D 4.0 software (Decodon GmbH, Germany). For identification, proteins of interest were cut from the 2D gels 4327



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Article

RESULTS AND DISCUSSION

that a number of adaptation processes are required for the switch from aerobic to anaerobic growth. Quantitative LC−MS/MS data were obtained by spectral counting. This method is based on the observation that the protein amount correlates with the number of MS spectra recorded for a protein.43 Comparability across all experiments was obtained by normalization resulting in the normalized spectral count (NSC). This strategy provides a simple and costefficient alternative to labeling experiments, thus allowing a quantitative analysis of a relatively large set of experimental conditions. For each detected protein, the Log2 value based on the ratio of NSC for the anaerobic and aerobic sample was calculated. Proteins with an average NSCs for the replicated experiments SP-A and SP-D showing a p value smaller than or equal to 0.05 and an at least 4-fold change were considered as significantly differentially expressed: 324 proteins met these requirements, in which 174 proteins were preferentially expressed in aerobically grown cells, whereas 150 proteins were highly produced under oxygen deficiency. A 16-fold overexpression was observed for 20 and 55 proteins under aerobic and anaerobic conditions, respectively. A top 30 list of proteins highly abundant under anaerobic conditions is given in Supporting Information Table 2. Most of them are associated with metabolic functions and are included in the adaptations of the electron transport chain, carbon metabolism, or synthesis of nucleotides and cofactors. Their regulation pattern is discussed in detail below.

Rationale and Design of the Study

Under low oxygen conditions and in the presence of nitrate or nitrite, R. eutropha switches from aerobic respiration to denitrification. Since the conversion of nitrate to dinitrogen is a four-step reduction process involving specific enzymes, we asked whether these phase transitions might be accompanied by fluctuations in the protein pattern in the time course of denitrification. Sample points (SPs) were set according to the course of nitrogen compound production and consumption distributed throughout the denitrification pathway (Figure 1): at SP-A and SP-B, approximately half of nitrate and nitrite, respectively, was consumed. SP-C was chosen close the maximum of NO accumulation. The time points SP-D and SP-E covered the early and late phase, respectively, of nitrous oxide reduction. SP-F reflects the final state of denitrification where all substrates where consumed. Aerobically grown cells (+O2) in the exponential phase served as a reference sample. Growth of the cultures is shown in Supporting Information Figure 2. We considered SP-A and SP-D as two phases of major importance for studying denitrification since they represent the adaption to low oxygen (SP-A) and the switch to the final reduction step (SP-D), respectively. Therefore, these two SPs were covered with biological replicates from three independent fermentations. Meeting the challenge of detecting both soluble and membrane proteins, a detailed proteome analysis was performed by 1D gel-LC−MS/MS. An additional 2D gel-based proteome study focused especially on soluble proteins presumed to be relevant for denitrification. Moreover, extracts from SP-A and SP-D were subjected to microarray analysis.

Anaerobic Respiration

Upon transition to low oxygen conditions, almost all proteins known to be involved in denitrification showed an increase in protein abundance during the first three sample points (SP-A to SP-C), as visualized for the catalytic subunits of the terminal oxidoreductases in Figure 2. In some cases, e.g., the nitrous

Classification and Quantification of the Proteome and Transcriptome Data Set

A detailed overview summarizing the results of quantitative proteome and microarray analyses is given in Supporting Information Table 1. Overall, there was a good correlation between microarray and proteome data. In total, 2261 distinct proteins were identified. The number of identified proteins corresponds to 34% of the whole genome capacity. While 344 proteins were exclusively detected in aerobically grown cells, anaerobic cultivation yielded 286 proteins that were not detected in the aerobic reference. A similar regulation pattern can be concluded by the results of microarray analysis. In general, significantly more genes were exclusively expressed on oxygen (SP-A: 284 genes; SP-D: 336 genes) than on nitrogen oxides as terminal electron acceptor (SP-A: 188 genes; SP-D: 206 genes). A classification of identified proteins by functional categories is summarized in Supporting Information Figure 3. For most of the identified proteins (592), no specific functions were assigned in the published R. eutropha H16 genome annotation.11 Proteins of unknown function showed the least coverage (19%) of all theoretically encoded proteins, whereas up to 70% coverage was obtained for proteins involved in major cellular functions, such as translation, ribosomal structure, biogenesis, cell division, and chromosome partitioning. Even though cell growth based on nitrogen oxide reduction was lower than under aerobic conditions (see Supporting Information Figure 2), we did not recognize significantly elevated abundances for the majority of growth-related proteins. Under anaerobic conditions, proteins involved in energy production and conversion (43 out of 195 identified proteins, 22%) and signal transduction (19 out of 84 identified proteins, 23%) were overrepresented. These results underline

Figure 2. Expression of key enzymes of denitrification. Normalized spectral counts (NSCs) are taken as a measure for the abundance of denitrification enzymes (represented by the respective catalytic subunit for heteromeric enzymes) determined from the mid log phase of aerobic cultures (+O2) and progressing growth phases of denitrifying cultures (A−F).

oxide reductase NosZ, the abundance appears to decrease at later sample points. There are also examples for a steady increase in abundance up to SP-F (e.g., the nitrite reductase NirS and the cobalamin-dependent ribonucleotide reductase NrdJ). Note, however, that only SP-A and SP-D are substantiated by biological replicates and thus care should be taken in the interpretation of these curves. Denitrificationspecific functions were found to be among the most heavily upregulated transcripts (T) and proteins (P). The most 4328



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Table 1. Regulation of Selected Denitrification Relevant Proteinsa 1D gel-LC−MS/MS microarray locus tag

gene name

MW, kDa

pI

TMH

PHG268 PHG269 PHG270 PHG271 PHG272 PHG273 PHG265 PHG266 H16_B0780 H16_B2263 H16_B2264 H16_B2265 H16_B2266 H16_B2267 H16_B2268 H16_B2333 H16_B2334 H16_B2277 H16_B2278 H16_B2279 H16_B2280 H16_B2281 H16_B2282 H16_B2283 H16_B2284 H16_B2285 PHG244 PHG245 H16_B2323 H16_B2324 PHG246 H16_B2325 PHG247 PHG248 PHG249 PHG250 PHG251 PHG252 PHG253 PHG254

narK1 narK2 narG1 narH1 narJ1 narI1 narX1 narL1 narK5 narK3 narK4 narG2 narH2 narJ2 narI2 narX2 narL2 nirS nirC nirF nirD nirG nirH nirJ nirN nirE norB1 norA1 norB2 norA2 norR1 norR2 nosL nosY nosF nosD nosR nosZ nosC nosX

46.6 49.5 140.1 58.1 24.6 25.1 72.2 23.9 46.3 46.4 49.3 139.3 58.1 25.6 25.1 73.4 23.6 60.8 12.4 44.0 35.8 16.7 18.2 42.3 54.1 26.2 84.4 25.4 84.4 25.8 56.3 56.5 19.2 29.5 33.5 47.5 96.4 70.1 13.3 36.4

9.8 9.6 7.2 6.5 4.6 10.1 6.8 5.5 9.5 9.9 9.6 7.2 6.4 4.6 10.2 6.6 6.2 8.7 9.2 9.6 10.0 7.6 9.9 8.0 8.5 8.4 8.8 6.1 9.0 6.4 7.4 7.0 8.2 9.4 8.8 8.8 9.4 7.4 8.6 10.6

TMH TMH

TMH TMH TMH TMH TMH

TMH TMH

TMH

TMH TMH

TMH

TMH

soluble fraction

membrane fraction

Log2

Log2

Log2

Log2

Log2

Log2

(A/+O2)

(D/+O2)

(A/+O2)

(D/+O2)

(A/+O2)

(D/+O2)

−O2 6.5 6.1 6.2 6.3 4.0 4.5

−O2 −O2 7.3 6.4 6.3 5.8 7.1 4.4

6.6 (−O2) 5.6 (−O2) 4.8 (−O2)

3.2 (−O2) 8.5 (−O2) 7.4 (−O2) 2.1

4.0 2.4 −O2 6.0 5.9 4.6 1.9 1.7 7.4 7.2 −O2 6.5 5.5 6.1 4.9 4.1 6.6 −O2 −O2 7.2 7.8

5.7 2.8 6.1 5.9 6.4 4.7 2.5 1.8 7.3 7.7 5.3 5.7 6.0 5.9 5.2 3.7 5.2 −O2 −O2 7.3 7.3

−0.1 7.0 6.5 5.5 5.3 −O2 6.0 6.2 5.2

5.9 6.4 6.1 5.4 −O2 6.4 6.2 4.3

6.7 (−O2) 5.4 (−O2) 3 (−O2)

6.3 (−O2)

6.2 (−O2)

2.7 4.1 8.7 7.8

(−O2) (−O2) (−O2) (−O2)

5.5 (−O2)

6.6 (−O2) 1

2.5 (−O2) 8.1 (−O2) 7.3 (−O2)

3.1 (−O2) 8.3 (−O2) 7.7 (−O2)

4.5 (−O2)

4.8 (−O2) 4.7 (−O2) 1.5 (−O2) 4.5

3.8 (−O2)

9 (−O2)

9.3 (−O2)

4.3

3

6.2 (−O2) 1.6 2.6

6.4 (−O2) 2.7

3.5 (−O2)

4.1 3.8 2.5 7.5 5.2 6.6 5.4

1.8

5.6 3.7 2.9 6.9 3.2 7.1

(−O2) (−O2) (−O2) (−O2)

5.9 (−O2) 2.3 2.6 6.8 (−O2) 3.2 6.3 (−O2)

2.5

1.5 (−O2) 7.3 (−O2) 4.7 6.3 4.8

3.9 1.2

6.7 (−O2) 4

1.2 6.3 (−O2)

4.8 (−O2) 5.3 (−O2) 6.4 (−O2) 5.8 2.8

(−O2) (−O2) (−O2) (−O2)

4 3.5 (−O2) 5 (−O2) 5.5 (−O2) 6.2 (−O2) 6.3 3.5

a

For microarray and 1D gel-LC−MS/MS analysis, Log2 values of the ratio comparing anaerobic (SP-A and SP-D) and aerobic growth samples (+O2) are reported. A Log2 value of 2 corresponds to 4-fold abundance. The term −O2 is used if a protein or expression of the corresponding gene was not detectable in aerobic cultures. The molecular weight (MW), the approximate isoelectric point (pI), and a note on the prediction of transmembrane helices (TMH) are given. Empty cells indicate that it was not detected.

are believed to be transporters for nitrate and/or nitrite, whereas NarG, NarH, and NarI are subunits of NAR. Both nar regions are flanked by additional genes that are potentially involved in assembly of NAR and/or insertion of the molybdenum cofactor. Regulation of nar genes in the denitrifier Pseudomonas aeruginosa involves the NarXL two-component regulatory system.37 Two sets of genes, narX1, -L1 and narX2, -L2, are present in R. eutropha on pHG1 and chromosome 2, respectively. In SP-D, the sensor components were found to be 97-fold (NarX1) and 26-fold (NarX2) upregulated. The corresponding gene expression values were 137-fold and 6fold, respectively. Taken together, it is likely that both

prominent examples were NarG1 (P|SP-D: 416-fold; T|SP-D: 158-fold) and NirS (P|SP-A: 512-fold; T|SP-A: 169-fold). A summary of the results for SP-A and SP-D is given in Table 1. R. eutropha H16 contains two sets of genes for the formation of NAR. The genes narK1, -K2, -G1, -H1, -J1, and -I1 on pHG1 are paralleled by the cluster narK3K4G2H2J2I2 on chromosome 2. Peptides for NarK3 and NarI1 have not been unequivocally detected by MS. However, a number of peptides were detected that could not be assigned because of the high degree of similarity between NarI1/I2 and NarK1/K3, respectively. Thus, we cannot exclude that NarK3 and NarI1 are present in denitrifying cells of R. eutropha. NarK proteins 4329



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Figure 3. Two-dimensional gel analysis and NSC data for selected proteins. Letters A−F denote six different sample points (SPs). The figure depicts details of false-color images of 2D gels (green, proteins present under control conditions; red, proteins present under denitrifying conditions; yellow, equally present under both conditions). Filled arrows refer to spots that were analyzed by MALDI-TOF-MS. Open arrows denote the respective positions of the filled arrows in gels from other SPs. Panels A2 and B2 are taken from a second gel run with a different pH gradient to enhance resolution for NirS spots. Diagrams on the right show absolute values of normalized spectral counts (NSCs) in arbitrary units (y axis) for SP-A to SP-F (x axis) obtained by LC−MS/MS analysis. Yellow bars, NSC derived from the soluble fraction; brown bars, NSC derived from the membrane fraction.

genes are upregulated during denitrification. Except for NirC and NirH, all of the corresponding proteins were detected by MS. In contrast to NirS, which has been detected predominantly in the soluble fraction, NirF, NirD, and NirJ were almost exclusively found in the membrane fraction. While the proteins are overall hydrophilic and a membrane-associated location has not been reported, it is possible that they are part of a high-molecular-weight complex that sediments to the membrane fraction during preparation. In P. aeruginosa, NirF was found to be a lipoprotein that forms a transient membraneassociated maturation complex with NirS.46 Two gene regions, norR1A1B1 on pHG1 and norR2A2B2 on chromosome 2, respectively, encode functional NOR enzymes.47 The norR1 and norR2 genes encode transcriptional regulators that control expression of the respective norA1B1 and norA2B2 operons.48 High upregulation observed on both protein and transcript level was in line with the activation of NorR by the denitrification product NO.49 The products of norB1 and norB2 belong to the quinol-oxidizing qNOR

chromosome 2 and pHG1 encode sufficient information for formation and regulation of two independent NAR enzymes. Moreover, R. eutropha contains a periplasmic respiratory nitrate reductase NAP, encoded within a cluster of five genes on pHG1 (napE, -D, -A, -B, and -C). Microarray analysis showed that some of the nap genes were upregulated up to 7-fold (Log2 = 2.8). However, only the catalytic subunit NapA was detected in a single sample point (SP-D) in the proteome, indicating that NAP does not play a vital role in denitrification. This result is in line with previous investigations proposing a function for NAP in stationary phase metabolism in R. eutropha.44 In P. aeruginosa, NAP gene expression was shown to be repressed during denitrification.45 The NIR of R. eutropha belongs to the cytochrome cd1 group of respiratory nitrite reductases. The structural gene nirS is flanked by nirC encoding a c-type cytochrome that may act as the electron donor for NirS and a set of genes, nirF, -D, -G, -H, -J, -N, and -E, involved in biosynthesis of the d1 heme cofactor. Microarray analysis clearly showed that all of the accessory 4330



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subtype. 50 NorB1 and NorB2 peptides were detected considerably more (20- to 30-fold) in the membrane than in the soluble fraction. This is in accordance with the membraneintegral location of these enzymes. Interestingly, considerable amounts of the soluble NO-binding proteins, NorA1 and NorA2, were detected in the membrane fraction. Since NO is a hydrophobic molecule, binding of NO by NorA1 and NorA2 is probably facilitated close to the membrane. The NOS of R. eutropha is encoded by the nosZ gene on pHG1. The nosZ gene precedes a cluster of five additional genes nosRDFYL and is flanked by nosC and nosX. All genes were strongly upregulated. Products of nosR, -D, -F, -Y, and -L were detected exclusively during denitrification. The respective proteins are thought to be involved in assembly of a functional enzyme and maturation of the unusual CuZ copper center.51 NOS enzymes have been purified as soluble proteins from the periplasm of several bacteria. Interestingly, about 10-fold higher NSC values were obtained for NosZ from R. eutropha in the membrane fraction compared to that for the soluble fraction. As outlined above for NirF, NirD, and NirJ, this result may indicate that NOS forms high-molecular-weight complexes. On the other hand, a membrane association of NOS has been described for Flexibacter canadensis, Thiobacillus denitrificans, and Bacillus azotoformans.52 Thus, it is also possible that NosZ attaches to membrane proteins, e.g., NosY or NosR. Although denitrification is a respiratory process that happens at the cytoplasmic membrane, many of the subunits of denitrification enzymes are predicted to have a high degree of hydrophilicity and thus are expected to be accessible by 2D gel analysis. Therefore, soluble extracts of SP-A to SP-F were subjected to gels with a pH range of 3−10. Figure 3 shows the spot pattern of NarG1/G2, NirS, NosZ, NorA1/A2, and Fhp. The soluble NorA protein was taken as a reference to the formation of membrane-integral NorB, as both proteins are encoded in one operon. The flavohemoprotein Fhp was included in the analysis, since bacterial flavohemoglobins are supposed to be involved in adaption to anaerobiosis and to play a role in the stress response of cell damage induced by reactive nitrogen species including nitric oxide.53 In R. eutropha, Fhp is expressed under conditions of oxygen deprivation and to bind NO molecules.13,54 Moreover, flavohemoglobin has been shown to detoxify NO by turning it into nitrate.55 For the characterization of individual spots indicated in Figure 3, proteins were identified by MALDI-TOF-MS-based analysis. Spots for the highly similar proteins NarG1 and NarG2, as well as NorA1 and NorA2, could not be separated by this method. A line of spots appeared in SP-C, two of which were identified as a mixture of NarG1 and NarG2. The maximum of NarG1/G2 spot intensity was observed in SP-E, whereas no NarG1/G2 peptides were detected in SP-A and SP-B. Similarly, NirSderived spots were not detected in SP-A and SP-B but appeared in SP-C. Compared to NarG1/G2, NirS showed an even higher degree of variation with respect to molecular mass and charge. To substantiate this observation, the experiment was repeated for SP-A and SP-D with an independent culture. In total, 12 spots were identified as NirS. The reason for the existence of a heterogeneous NirS population in R. eutropha is not known. A similar behavior has not been described for NIR proteins from other denitrifiers. Regarding NosZ, only a single spot was identified. Like NarG1/G2 and NirS, NosZ became visible first in SP-C. In contrast, the intensity of the Fhp spot remained nearly constant during the interval sampled. Since the 2D gel shows only proteins of the soluble fraction, the variations in

spot intensity among SP-A to SP-D might be misleading if the dominant amount of a certain protein is present in the membrane fraction. We included diagrams in Figure 3 showing normalized spectral counts derived from the soluble and the membrane fraction. Nevertheless, 2D gel spot intensity and spectral counting does not always match perfectly. For example, the low number of spectral counts detected for Fhp at SP-F was not reflected by the respective spot in the 2D gel. Although spectral counts may not be taken as a basis for an absolute quantification, these values are nevertheless useful in comparisons with the intensity pattern of the gel spots. A peak in NarG1/G2 abundance at SP-E is shown by both the 2D gel and the NSC data. However, there was no perfect pattern correlation between spot intensities and NSCs. A number of reasons may account for these differences. For instance, it has to be considered that (i) NSC relate to the sum of all detected (and including potentially undetected) 2D gel spots of a given protein and (ii) 2D gel spots are derived from intact proteins, whereas normalized spectral counts are derived from peptides of fragmented proteins. In this context, we point out that the absolute values of NSC from the soluble fraction cannot be compared directly to those obtained from the membrane fraction, since the general loss of protein during the membrane preparation procedure was not quantified. Electron Transport Chain

Genes for eight terminal oxidases are present in the genome of R. eutropha H16.12 Three of them are cytochrome oxidases, whereas the remaining five are quinol oxidases. The cta gene cluster encodes a typical aa3-type heme copper oxidase (HCO). The cco gene cluster codes for a cbb3-type oxidase, and the cox gene cluster represents a bb3-type oxidase. The quinol oxidases are genetically represented by three clusters for bo3-type oxidases (Cyo1, Cyo2, and Cyo3) and two gene clusters for bdtype oxidases (Cyd1 and Cyd2). The bd-type oxidases are not related to the HCO family and represent alternative quinol oxidases. The presence of the four oxidases, Cta, Cco, Cyo1, and Cyd1, could be confirmed by proteome analyses of denitrifying R. eutropha cells. A clear downregulation was detectable for the three subunits of the Cta enzyme on both the transcript and protein levels. In fact, aa3-type oxidase was previously described to show relatively low oxygen affinity, suggesting that this enzyme predominantly operates under high oxygen tension.56,57 The quinol oxidase Cyo1 was found to be slightly more abundant in denitrifying cells. In contrast, a clear upregulation on both the transcript and protein levels was observed for CydA1 (P|SP-D: 7-fold; T|SP-D: 2-fold) and CcoN (P|SP-D: 91-fold; T|SP-D: 5-fold). This points to a crucial function of Cco in adaption to anaerobic growth in R. eutropha. Indeed, cbb3-type oxidases are described to have a high affinity to oxygen and an important function in bacterial microaerobic metabolism.58 They are crucial for rhizobial adaption to microaerobic conditions during nitrogen fixation59 and can also be found in the respiratory chains of microaerophilic pathogens like Campylobacter jejuni or Helicobacter pylori.60 The switch of respiratory chains to anaerobiosis has been discussed as an adaption to microaerobic conditions during infection with the opportunistic pathogen P. aeruginosa, the cause of acute and chronic infections in patients with compromised immune defenses.61,62 The potential of Brucella suis, the agent of the zoonotic disease brucellosis, to persist or grow under anaerobic conditions was described as an advantage in long-term survival of the strain inside the host.63 Proteome 4331



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analysis of anaerobically grown B. suis cells revealed an induction of denitrification enzymes including the highly abundant nitrate reductase and induction of high-affinity cbb3type and bd-type terminal oxidases.64 B. suis contains all necessary genes for full denitrification pathway, including a nitric oxide reductase which has been shown to be essential for virulence.65 Switching to anaerobiosis is probably tightly controlled. Among the well-studied bacterial oxygen sensors for transcriptional control,66 members of the FNR-DNR-type transcriptional regulator family are known to be crucial for the response to changes in oxygen availability and functional in response to nitric oxide.67−69 Studies in Paracoccus denitrificans or P. aeruginosa also revealed that these regulators are necessary for the transcriptional shift to anaerobiosis. In R. eutropha, several genes for this type of regulator can be found. Peptides for one FNR-like protein (Fnr3) and one DNR-like protein (DnrD) were identified in this proteome study. The dnrD gene is integrated in the NIR/nor/nos gene cluster on pHG1, whereas the fnr3 gene is colocalized with the genes of the cco gene cluster coding for the high-affinity cbb3-type oxidase. As both operons have a possible role in anaerobiosis, it is tempting to speculate that these regulators play a key role in regulating the adaption of the respiratory chain during anaerobiosis. Gene expression and abundance of Fnr3 was slightly higher in aerobic cells, which might be taken as an indication for negative autoregulation. In contrast, DnrD was significantly upregulated (T|SP-D: 32-fold) during denitrification.

and 73% identical to the well-investigated AcnA and AcnB from Escherichia coli, revealed a change in expression. While the aerobically predominant AcnA showed no alterations in response to oxygen supply, AcnB was upregulated during denitrification (P|SP-A: 4-fold; T|SP-A: 11-fold). Induction of acnB transcripts were also observed for E. coli upon transition to microaerobic conditions.75 Similarly, a shift in expression was observed for the two isocitrate dehydrogenases isoenzymes Icd1 and Icd2. There were only minor changes seen for Icd2, but upregulation of Icd1, in particular at early phase of denitrification (P|SP-A: 8-fold; T|SP-A: 7-fold). In contrast to Icd1, however, AcnB showed a peak of abundance at SP-C, where NO accumulation was highest. Perhaps AcnB formation is enhanced in the midphase of denitrification to compensate for damage by NO, which is known to destroy the iron−sulfur cluster of aconitase.76 Downregulation of fumarate hydratase FumC (P|SP-A: 4-fold), a superoxide tolerant isoenzyme of FumA, may reflect another adaption of the citrate cycle to anaerobiosis, since less superoxide is expected to be produced during denitrification compared to aerobic respiration. In the context of metabolic adaption to oxygen supply, NAD(P)H and transhydrogenases appear to play a crucial role. Genes for four transhydrogenases are present in the genome of R. eutropha.12 Transcripts were detected for three of them, showing an overall pattern of downregulation. Only one of them, Pnt3, was detected on the protein level. The abundance of the subunits (encoded by h16_A3128, h16_A3130, and h16_A3131) decreased markedly during denitrification. For example, at SP-D, H16_A3130 was 16-fold downregulated on the protein level and 4-fold downregulated on the transcript level. A similar pattern of downregulation was observed for NAD+ kinase (H16_A1132) that forms the transhydrogenase substrate NADP+. Thus, the need for NADPH appears to be much higher under aerobic growth conditions, perhaps due to its role in providing reducing power for oxidative defense mechanisms.

Central Pathways

In general, only moderate changes were observed in central carbon metabolism upon transition to denitrification. There are a few exceptions that appeared to be related to redox balancing mechanisms. For an overview of expression changes concerning the central carbon metabolism, see Supporting Information Figure 4. The genes for alcohol dehydrogenase (ADH) adh and lactate dehydrogenase (LDH) ldh displayed a 26-fold and 12fold increases, respectively, and the corresponding proteins could be detected in strongly elevated amounts in denitrifying cells. Both genes have been characterized previously.70,71 The upstream regulatory regions of both genes share a high degree of identity, opening the possibility of a similar mode of regulation. An observed increase in abundance of the aldehyde dehydrogenase ExaC (up to 26-fold) and the acetyl-CoA synthetase H16_B1102 (up to 7-fold) is in line with the view that the formation of ethanol and probably also lactate serve as redox valves. Indeed, oxygen-limited cultures of a close relative of R. eutropha H16, strain N9A, were shown to excrete partially oxidized compounds, such as ethanol, acetate, and succinate.72,73 A functional ADH has been isolated from cells grown under oxygen limitation of this strain.74 Concerning the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate node, downregulation of the three pyruvate kinases (Pyk1, Pyk2, and Pyk3) and the anaplerotic pyruvate carboxylase Pyc were observed in denitrifying cells, whereas the pyruvate dehydrogenase complex was slightly increased (about 2-fold). In addition, isocitrate dehydrogenase Icd1 appeared to be upregulated, suggesting a somewhat enhanced conversion of citrate under anaerobic conditions. In competition for pyruvate, these adaptations may balance the metabolic flux of carbon toward the citrate cycle on the one hand and the fermentation enzymes LDH and ADH on the other hand. Furthermore, one of the two aconitase isoenzymes AcnA and AcnB, which are 54

DNA Synthesis

Ribonucleotide reductases (RNRs) are ubiquitous enzymes found in Bacteria, Archaea, and Eukarya. They catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, a step that is essential for DNA synthesis and repair.77 Three different types of RNRs are known: (i) the aerobic class I enzymes present in prokaryotes and eukaryotes (NrdAB); (ii) the bacterial and archaeal class II enzymes, which are coenzyme B12-dependent (NrdJ); and (iii) the anaerobic class III enzymes (NrdDG).77,78 The genome of R. eutropha contains genetic information for all three types of enzymes.11 NrdAB proteins were present in aerobic and denitrifying cells. Both protein and transcript amounts were slightly decreased under aerobic conditions. A similar behavior was previously reported for NrdAB in P. aeruginosa.79 For NrdJ, microarray and proteomic data showed a strong upregulation (P|SP-A: 39fold; T|SP-A: 32-fold) in denitrifying cells. Finally, denitrification led to more than 100-fold upregulation of nrdDG encoding a class III RNR (NrdD) and the corresponding activase (NrdG). Peptides for NrdD and NrdG were identified only in denitrifying cells. These results are in line with a characteristic FNR-type binding motif in the upstream region of the nrdDG operon, indicating an oxygen-dependent regulation.15 Since all three RNRs appear to be present during denitrification in R. eutropha, we investigated which of the enzymes contributed to deoxyribonucleotide synthesis under oxygen limitation. The 4332



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type cytochrome formation systems of type I (Ccm system). The regions are located close to the nap genes on pHG1 and on chromosome 2, respectively, and both of them include eight genes (PHG214−221 and h16_B0952−59). All of them showed either low or no signals in the microarray analysis, except for h16_B0956 (ccmF2), which showed about 1.5% of the maximum spot intensity. None of the encoded proteins could be detected by proteomic analysis. On the other hand, three genes, h16_A3449 (ccsA), h16_A3450 (ccsB), and h16_A3455 (dsbD), on chromosome 1 are likely to encode a type II cytochrome c maturation system. All of the corresponding proteins could be detected in the membrane fraction. In conclusion, it appears that a type II system is instrumental in c-type cytochrome biogenesis in R. eutropha H16.

tyrosyl radical regeneration of class I RNRs is believed to be strictly oxygen-dependent,80,81 and class II enzymes strictly depend on the presence of coenzyme B12,82,83 which was not present in our growth medium. Wild-type and a nrdDG mutant (strain HF413) were grown in the presence of the tyrosyl radical scavenger hydroxyurea (HU) that is known to inhibit class I RNR.84 As expected, the nrdDG mutant was not able to grow under these conditions (Supporting Information Figure 5), but growth could be restored to some extent by addition of cobalamin, a coenzyme B12 precursor (Supporting Information Figures 5 and 6). Denitrifying wild-type cells were sensitive to HU treatment, indicating that class I RNR contributes to deoxyribonucleotide synthesis even under oxygen limitation. However, in contrast to the findings for P. aeruginosa,85 cobalamin addition did not restore growth of HU-treated cells under aerobic conditions. This behavior may be due to the low abundance of NrdJ during aerobic growth rather than to a lack of cobalamin uptake, since the putative cobalamin transporter (encoded by the btu operon on chromosome 1) was found to be present in comparable amounts under both growth conditions (Supporting Information Table 3).

Surface Appendages and PHB Granules

R. eutropha contains several genes for flagella, fimbria, flp pili, and type IV pilus formation. Nearly all of them were downregulated under denitrifying conditions (Supporting Information Table 4), suggesting that denitrification goes along with a loss of cell appendages. Proteome analysis of anaerobic lifestyle of the human pathogen Vibrio cholerae revealed a similar decreased abundance of flagellin during anaerobic growth,90 which was assumed to be an adaptation to anaerobiosis during infection of the human intestine. In contrast, flagella-dependent motility, which is essential for biofilm formation and other pathogenic adaptations to anoxic conditions, is induced by nitrate in denitrifying P. aeruginosa cells.91−93 The reason for the regulation of cell appendages genes in R. eutropha H16 is unclear. A change in flagellation in response to nutrient supply and accumulation of poly(3hydroxybutyrate) (PHB) was previously reported by Raberg et al.94 PHB can be used as sustainable resource for biodegradable plastics. R. eutropha has been served as a model organism to understand the biosynthesis and regulation of PHB-producing and -degrading enzymes.23,95 During PHB accumulation, R. eutropha forms typical PHB granules consisting of lipids, enzymes, and structural proteins. The characteristic structural proteins in these PHB granules are phasins. Among several similar phasin genes present in the R. eutropha genome, phaP1 encodes the dominant one.96 The products of four different phasin genes (phaP1 ,P2, P3, and P4) were detected in this study. One of them (PhaP2) was found to be more abundant in response to oxygen deprivation (up to 24-fold). In contrast, PhaP3 and PhaP1 were up to 8-fold more abundant under aerobic conditions. Pötter et al. showed that single deletions of the phaP1, P2, P3, or P4 gene did not result in a significant difference in growth behavior, indicating that none of these genes are essential.97 In the same study, it was also shown that PhaP1 and P3 are regulated by a common mechanism via PhaR, whereas PhaP2 and P4 are regulated in a different way. Our findings support these results and suggest an upregulation of PhaP2 in response to anaerobiosis. Several other genes for PHB accumulation including two acetoacetyl-CoA reductases (PhaB2 and PhaB3), which reduce acetoacetyl-CoA to R-3hydroxybutyryl-CoA, were found to be differentially expressed. PhaB3 was more abundant under aerobic conditions, whereas PhaB2 was found exclusively under anaerobic conditions. In addition, a putative polyhydroxyalkanoate synthase PhaC2 was more abundant under anaerobic conditions. The expression pattern of the PHB-producing strains and several mutants was investigated in a previous microarray study.32 The expression of

Cofactor Synthesis

R. eutropha is a strictly respiratory organism. Respiratory processes require biosynthesis of heme for the production of cytochromes. The biosynthesis of tetrapyrrole requires an enzymatic oxidative decarboxylation step: the conversion of coproporphyrinogen III to protoporphyrinogen IX. Under aerobic conditions, this step is catalyzed by an oxygendependent coproporphyrinogen III oxidase (HemF). Under denitrifying anaerobic conditions, this enzymatic function is replaced by a member of the radical S-adenosylmethionine (SAM) protein family, the oxygen-independent coproporphyrinogen III oxidase (HemN), which uses SAM as the oxidizing agent.86−88 HemN from R. eutropha has been previously characterized, and transcript analysis in R. eutropha showed that hemN expression is nitrate-independent and strongly induced under anaerobic conditions.14 In the proteome of denitrifying cells, HemN was up to 9-fold more abundant than that under aerobic conditions. These results are confirmed by the microarray data, showing a more than 6-fold upregulation under anaerobic conditions. Radical SAM enzymes participate in numerous biosynthetic pathways. Besides HemN, several other family members were identified in the proteome and showed a similar regulation pattern. The majority of them are involved in cofactor biosynthesis. For example, the lipoate synthase LipA (H16_A0123) encoded within the lipAB operon was detected only in anaerobically grown cells. Other representatives are the biotin synthase BioB (2- to 13-fold upregulated) encoded by the bio operon and the anaerobic ribonucleotide reductase activase NrdG (119-fold upregulated). The SAM-dependent thiazole biosynthesis protein ThiH, an enzyme essential for thiamin synthesis,86 is not encoded in the R. eutropha genome. Instead, R. eutropha can draw on a glycine oxidase ThiO.89 ThiO and ThiC, a hydroxymethyl pyrimidine synthase also involved in thiamin biosynthesis, displayed high amounts in the membrane fraction of anaerobic cells. This underlines the importance of invoking oxygen-independent enzymatic mechanisms like SAM-dependent enzymes during anaerobic growth, even though the impact of increased cofactor biosynthesis in response to oxygen limitation remains unclear. In this context, it is interesting to note that the genome of R. eutropha contains two highly similar gene regions encoding c4333



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Figure 4. Localization of expressed genes based on protein identification. The number of expressed genes in a sliding window of 50 consecutive CDS (both DNA strands) was plotted against genomic position. Blue, expressed in aerobically grown cells; red, expressed in anaerobically grown cells (SP-A and SP-D). The interval indicated by the concentric gray circles correlates to 10 expressed genes. Hot spots: I, flp pilus assembly proteins (CpaB3, CpaC3, CpaF3, TadB2, TadC2, and TadG2a); II, type VI protein secretion system (H16_B2430−34); III, chromosome 2-coded reductases of nitrate, nitrite, and nitric oxide; IV, Cbb-related proteins; V, hydrogenase- and Cbb-related proteins; VI, anaerobic ribonucleotide reductase, pHG1-coded reductases of nitrate, nitric oxide, and nitrous oxide.

in nonpathogenic bacteria may be involved in intracellular communication, influence cell proliferation, and facilitate bacterial fitness.101−103

phasin genes was found to be growth phase-dependent and most prominent in the stationary phase. Since stationary phase cultures are probably oxygen-limited, our findings may point to a functional relation between anaerobiosis and PHB synthesis.

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Hot Spots of Denitrification-Linked Genes 98

Similar to what was observed for Cupriavidus necator CH34, the R. eutropha genome revealed a distinct distribution of core functions and accessory functions among the three replicons.99 Chromosome 1 is assumed to be the ancestral replicon that carries mainly housekeeping functions, while chromosome 2 and pHG1 primarily harbor alternative traits that provide the organism with selective advantages under special growth conditions, i.e., the genetic information needed for lithoautotrophy and denitrification. Most of these genes are present in gene clusters that can be easily visualized as loop-out regions in the density map of expressed genes shown in Figure 4. The map was calculated by sliding-window analysis of genes encoding proteins that have been detected by LC−MS analysis. About 74% of the 2261 proteins identified in this study are encoded by chromosome 1, which equals 46% of the coding capacity on this replicon. Twenty-one and 4% of the proteins are encoded by chromosome 2 and pHG1, equating 19 and 22% of the respective coding capacities. Prominent loops are formed by the NAR2, NIR, NOR2 cluster (region III on chromosome 2) and NrdGD, NAR1, NOR1, and NOS (region VI on pHG1), respectively. While these two regions are examples for denitrification-specific upregulation, other gene clusters are downregulated during denitrification. These include two highly similar copies of the cbb operon present on chromosome 2 and pHG1 (regions IV and V, respectively) encoding enzymes of the Calvin cycle. Both operons are controlled by the negatively autoregulated transcriptional activator CbbR, which is encoded by a single functional cbbR gene located upstream of the chromosomal cbb operon.100 Further examples for proteins highly upregulated during aerobic growth are the flp pilus assembly proteins (CpaB3, CpaC3, CpaF3, TadB2, TadC2, and TadG2a; region I) and a putative type VI protein secretion system (h16_B2427−34; region II).28 The biological role of this system in R. eutropha is unknown. It has been proposed, that type VI secretion systems

CONCLUSIONS

By applying multiple proteomic and transcriptomic techniques, we obtained new insights into metabolic adaptions of denitrifying R. eutropha H16. On the basis of the identification of more than 2200 proteins, one-fourth of which were classified as membrane proteins, we present a comprehensive overview on the repertoire of functions involved in transition from aerobic to an oxygen-limited life style. Nearly 700 proteins participating in several processes, including respiration, cell appendage formation, and DNA and cofactor biosynthesis were found to be differentially expressed. Regulation patterns of proteins relevant for denitrification were addressed by analyzing six consecutive time points during denitrification. Using both a 2D gel-based proteomic screening and LC−MS peptide analysis, changes in the abundance pattern of the key enzymes of denitrification, namely, nitrate reductase, nitrite reductase, nitric oxide, and nitrous oxide reductase were detected. In addition, the switch from oxygen to nitrate respiration was found to be attended by redox-balancing functions. Decreased oxygen availability led to alterations in the electron transport chain, which is exemplified by upregulation of a putative highaffinity terminal oxidase. Furthermore, oxygen-dependent (ribonucleotide reductases, coproporphyrinogen III oxidases) or oxygen-insensitive functions (fumarate hydratase) were replaced or at least backed up by low-oxygen-adapted enzymatic counterparts. However, in general, alterations in central carbon metabolism appear to be moderate. A number of metabolic and structural adaptations of R. eutropha, including, for example, production of phasins and surface appendages, are not understood in detail. Moreover, more than one-third of all differentially expressed transcripts encode proteins with unknown or uncertain functions. The study of those mechanisms remains an interesting and challenging task for future work. 4334



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ASSOCIATED CONTENT

S Supporting Information *

Table 1: List of all R. eutropha locus tags along with the corresponding transcriptomic and proteomic data. Figure 1: Production and consumption of nitrogen compounds in denitrifying culture. Figure 2: Comparison of aerobic and anaerobic growth. Figure 3: Functional categorization of identified proteins. Figure 4: Expression of the enzymes involved in the central carbon metabolism. Figure 5: Growth of wild-type and class III ribonucleotide reductase negative mutant. Figure 6: Production of denitrification intermediates during anaerobic growth. Table 2: Top 30 proteins most abundant under anaerobic conditions. Table 3: Expression of genes for cobalamin uptake. Table 4: Expression of flagella, fimbria, flp pili, and type IV pili. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49 3834 8619021; Fax: +49 3834 8619015; E-mail: [email protected]. Present Addresses ⊥

(A.P.) Institut für Virusdiagnostik, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany. # (R.C.) Stiftung Alfried Krupp Kolleg Greifswald, 17487 Greifswald, Germany. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Bundesministerium für Bildung und Forschung (BMBF) as a project of the Competence Network Gö ttingen ‘‘Genome research on bacteria’’ (GenoMikPlus). The authors also thank Angelika Strack, Steffen Lütte, and Marcus Ludwig for technical assistance and advice.

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ABBREVIATIONS 1D gel-LC−MS/MS, one-dimensional gel-based liquid chromatography tandem mass spectrometry; 2D, two-dimensional; ADH, alcohol dehydrogenase; FNR, fumarate and nitrate reduction regulator; HCO, heme copper oxidase; HU, hydroxyurea; LDH, lactate dehydrogenase; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; SP, sample point; MBH, membrane-bound hydrogenase; NAR, nitrate reductase; NIR, nitrite reductase; NOR, nitric oxide reductase; NOS, nitrous reductase; NSC, normalized spectral count; P, protein level; PCR, polymerase chain reaction; PHB, poly(3-hydroxybutyrate); PEP, phosphoenolpyruvate; RNR, Ribonucleotide reductase; SAM, Sadenosylmethionine; T, transcript level; TMH, transmembrane helices; MW, molecular weight

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