Versatile Metabolic Adaptations of Ralstonia eutropha H16 to a Loss of PdhL, the E3 Component of the Pyruvate Dehydrogenase Complex

Abstract

A previous study reported that the Tn5-induced poly(3-hydroxybutyric acid) (PHB)-leaky mutant Ralstonia eutropha H1482 showed a reduced PHB synthesis rate and significantly lower dihydrolipoamide dehydrogenase (DHLDH) activity than the wild-type R. eutropha H16 but similar growth behavior. Insertion of Tn5 was localized in the pdhL gene encoding the DHLDH (E3 component) of the pyruvate dehydrogenase complex (PDHC). Taking advantage of the available genome sequence of R. eutropha H16, observations were verified and further detailed analyses and experiments were done. In silico genome analysis revealed that R. eutropha possesses all five known types of 2-oxoacid multienzyme complexes and five DHLDH-coding genes. Of these DHLDHs, only PdhL harbors an amino-terminal lipoyl domain. Furthermore, insertion of Tn5 in pdhL of mutant H1482 disrupted the carboxy-terminal dimerization domain, thereby causing synthesis of a truncated PdhL lacking this essential region, obviously leading to an inactive enzyme. The defined ΔpdhL deletion mutant of R. eutropha exhibited the same phenotype as the Tn5 mutant H1482; this excludes polar effects as the cause of the phenotype of the Tn5 mutant H1482. However, insertion of Tn5 or deletion of pdhL decreases DHLDH activity, probably negatively affecting PDHC activity, causing the mutant phenotype. Moreover, complementation experiments showed that different plasmid-encoded E3 components of R. eutropha H16 or of other bacteria, like Burkholderia cepacia, were able to restore the wild-type phenotype at least partially. Interestingly, the E3 component of B. cepacia possesses an amino-terminal lipoyl domain, like the wild-type H16. A comparison of the proteomes of the wild-type H16 and of the mutant H1482 revealed striking differences and allowed us to reconstruct at least partially the impressive adaptations of R. eutropha H1482 to the loss of PdhL on the cellular level.

Ralstonia eutropha H16 is a Gram-negative, rod-shaped, and facultative chemolithoautotrophic hydrogen-oxidizing bacterium and has served as a model organism for polyhydroxyalkanoate (PHA) metabolism and hydrogen-based chemolithoautotrophy for nearly 50 years (12, 51). PHAs are accumulated as granules in the cytoplasm and serve the cells as storage compounds for carbon and energy. PHAs are synthesized under unbalanced growth conditions if a carbon source is present in excess and if another macroelement (N, O, P, or S) is depleted at the same time (3, 63). PHAs are synthesized and accumulated by a large variety of prokaryotes and may represent the major cell constituent, contributing up to about 90% of the cell dry weight (4). Although R. eutropha H16 is able to synthesize different PHAs with short carbon chain lengths (65), poly(3-hydroxybutyric acid) (PHB) is usually the predominant PHA in the bacterium (15, 28). When the limiting macroelement that caused PHB accumulation is supplied again, degradation (mobilization) of PHB is induced, and the storage compound is used as a carbon and energy source (67).

In addition to the interest of academia, the bacterium has been used in industry for large-scale PHA production (29). These polyesters exhibit thermoplastic and/or elastomeric properties, like synthetic polymers produced from petrochemicals, such as polypropylene (23, 33). Due to their biodegradability and origin from renewable resources, PHAs have attracted much interest for technical and medical applications (3, 23, 70).

Synthesis of PHB proceeds in three steps via the enzymes β-ketothiolase (PhaA), acetoacetyl-coenzyme A (CoA) reductase (PhaB), and PHA synthase (PhaC) (18, 19, 20, 38). The genes for these three enzymes are located in the PHA operon phaCAB (41, 42, 59). Furthermore, several additional genes coding for proteins participating in PHA metabolism are known. Whereas PhaC is essential for PHA biosynthesis in R. eutropha H16, PhaA and PhaB can be replaced by isoenzymes (62). In R. eutropha H16, PHAs are intracellularly degraded by PHA depolymerases (PhaZ) through hydrolytic or thiolytic cleavage (67), and seven genes putatively encoding intracellular PHA depolymerases plus two genes putatively encoding hydroxybutyrate oligomer hydrolases were detected (1, 26, 54, 71). At least three of these genes contribute to the intracellular degradation of PHB in R. eutropha H16 (71).

During heterotrophic growth, fructose or gluconate is exclusively metabolized via the Entner-Doudoroff (KDPG) pathway in R. eutropha, finally yielding pyruvate. Pyruvate is further metabolized by oxidative decarboxylation by the pyruvate dehydrogenase complex (PDHC) (21) to yield the central intermediate acetyl-CoA, which is then oxidized in the Krebs cycle or used in anabolic pathways, like PHB synthesis or synthesis of essential cell constituents. The PDHC belongs to the family of 2-oxoacid dehydrogenase multienzyme complexes, which irreversibly convert 2-oxoacids to the corresponding acyl-CoA derivates with concomitant formation of CO₂ and reduction of NAD⁺ to NADH. Currently, three different complexes of this family are known: (i) The PDHC, (ii) the 2-oxglutarate dehydrogenase complex (OGDHC), and (iii) the branched-chain dehydrogenase complex (BCDHC) for degradation of branched-chained amino acids (5). In addition, the glycin dehydrogenase complex (GDHC) (16) and the acetoin dehydrogenase complex (ADHC) (39) have related structures or catalyze related reactions, respectively. Except for the GDHC, these multienzyme complexes share architectural features and consist of three principal enzyme components: a substrate-specific decarboxylase/dehydrogenase (E1), a complex-specific dihydrolipoamide acetyltransferase (E2), and a nonspecific dihydrolipoamide dehydrogenase (E3) (2, 25). Aggregation of these three components results in the formation of a core complex. While the subunit conformations of E1 and E2 admittedly differ (58), the nonspecific E3 component is always arranged as a homodimer (45).

In the PDHC, pyruvate is first decarboxylated by the pyruvate decarboxylase (E1) with formation of the intermediate hydroxyethyl thiopyrophosphate. Its hydroxyethyl moiety is then transferred to lipoamide, with its concomitant oxidation yielding acetyl dihydrolipoamide. This second step is also mediated by E1. The acetyl moiety is transferred in a third step to CoA by the dihydrolipoyl transacetylase (E2) component, and acetyl-CoA is released. The reoxidation of dihydrolipoamide formed in the second step is secured in the fourth and fifth steps by the dihydrolipoamide dehydrogenase (E3), which reduces NAD⁺ via flavin adenine dinucleotide (FAD) (40).

Dihydrolipoamide dehydrogenases (DHLDHs) belong to the class of flavoenzymes and the family of pyridine nucleotide-disulfide oxidoreductases and show a typical domain structure. Each subunit of a DHLDH consists of four domains: the FAD-binding domain, the NAD-binding domain, the central domain, and the interface domain. In the homodimer, the domains of both subunits partially interact with each other; therefore, dimerization is essential for an active enzyme (34, 49).

Pries et al. reported in 1992 on a Tn5-induced PHB-leaky mutant (H1482) showing reduced PHB accumulation (46). This mutant grew similarly to the wild type; however, the DHLDH activity was significantly lower, exhibiting only 29% of the activity of the wild type. Consequently, the PHB accumulated in the cells amounted to only about 30% of the cell dry mass. The H1482 mutant, as well as the other PHB-leaky subclass B mutants, H1474, H1479, and H1485, could be complemented by plasmid pVK6300, which harbored a 6.3-kbp EcoRI restriction fragment of the wild type. Sequence analysis mapped the Tn5 insertion in the pdhL gene (46). On the protein level, the insertion locus corresponded to the carboxyl terminus of the DHLDH, which is part of the PDHC (21, 46). The available genome sequence of R. eutropha H16 (43) encouraged the elucidation of this interesting phenomenon and an attempt to unravel the unknown linkage between phenotype and genotype.

MATERIALS AND METHODS

Bacterial strains, media, and growth conditions.

The bacterial strains used in this study are listed in Table S1 in the supplemental material. Cultivations in liquid media were done in Erlenmeyer or Klett flasks with baffles on a rotary shaker at 125 rpm. Cells of Escherichia coli were cultivated in Luria-Bertani (LB) medium (55) at 37°C. When cells were transformed with hybrid plasmids of pCR2.1-TOPO or pBBR1MCS-1 (27), 75 μg ml ampicillin⁻¹ or 50 μg ml chloramphenicol⁻¹, respectively, was added to the medium to maintain the plasmids.

Cells of R. eutropha were grown in 2-liter Klett flasks equipped with baffles at 30°C in 300 ml mineral salts medium (MSM) supplemented with 1% (wt/vol) sodium gluconate (56). To promote extensive accumulation of PHB, the concentration of NH₄Cl in the MSM was reduced from 0.1% (wt/vol) to 0.05% (wt/vol).

For growth experiments (see Fig. 2 and 3), precultures were grown in 100 ml MSM containing 1% (wt/vol) sodium gluconate and 0.05% (wt/vol) NH₄Cl (PHB storage conditions) for 48 h. In the case of cells harboring hybrids of plasmid pBBR1MCS-1, chloramphenicol (400 μg/ml) was added to precultures. The main cultures were done in 300 ml MSM (the same composition as preculture but without chloramphenicol) in 2-liter Klett flasks with baffles and were inoculated with 5% (vol/vol) of the preculture. To promote PHB degradation during cultivation (see Fig. 2), another 0.05% (wt/vol) NH₄Cl was added in the stationary phase after 30 h of cultivation. Plasmid loss in the main cultures could be excluded by determination of equal numbers of CFU after culture aliquots were spread on selective (chloramphenicol) or nonselective medium.

Determination of DHLDH activity in R. eutropha H16 and Tn5 mutant H1482.

DHLDH activity in crude extracts was determined by a spectrophotometric assay measuring the decrease of absorption at 365 nm due to consumption of NADH (diameter [d] = 1 cm; ɛ = 6.3 cm²/μmol) according to the method of Bergmeyer (6). The analysis was done in a mixture containing 0.85 ml 100 mM potassium phosphate buffer (pH 7.2), 0.02 ml 10 mM NADH, and 0.02 ml sample and was started by the addition of 0.1 ml lipoamide solution (20 mM in dimethyl sulfoxide [DMSO]).

Generation of a defined ΔpdhL deletion mutant.

The upstream and downstream regions of pdhL were amplified using Taq polymerase and the primers pdhL_UP_ fw_SacI/pdhL_UP_rv_XbaI and pdhL_DN_fw_XbaI/pdhL_DN_rv_PstI (see Table S2 in the supplemental material). Taq polymerase has a non-template-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3′ ends of PCR products. The linearized pCR2.1-TOPO vector has single overhanging 3′ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector. In this way, the upstream fragment (800 bp) and the downstream fragment (625 bp) were subcloned into pCR2.1-TOPO. These fragments were prepared by digestion with SacI/XbaI or XbaI/PstI, respectively, and ligated to a single fragment. This fragment contained both flanking regions and was then cloned in the vector pJQ200mp18Tc (44). The resulting plasmid, pJQΔpdhL, was transferred by conjugation into R. eutropha using E. coli S17-1 (61) as a donor, and transconjugants were selected in a first step on MSM agar plates containing 1.5% (wt/vol) sodium gluconate as a carbon source plus 25 μg/ml tetracycline. Putative remaining E. coli contaminants were removed by spreading single colonies on the same plates, and heterogenotes were then propagated overnight on 0.8% (wt/vol) nutrient broth (NB) agar plates to enable the second crossover event. Dilutions of overnight cultures were again spread on NB agar plates containing 10% (wt/vol) sucrose to select for strains lacking the sacB gene. For clones that had lost the ability to grow on tetracycline-containing plates, deletion of pdhL was controlled by PCR using pdhL_UP_fw_SacI and pdhL_DN_rv_PstI as primers. Furthermore, the internal primers pdhL_intern-fw and pdhL_intern-rv binding inside pdhL were applied to exclude a putative nonspecific secondary reintegration of pdhL.

Vectors, oligonucleotides, and constructed plasmids for complementation experiments are listed in Tables S3, S4, and S5 in the supplemental material, respectively. Genes were amplified using Taq polymerase, and the primer pairs are listed in Table S4 in the supplemental material. Fragments were subcloned into pCR2.1-TOPO and subsequently prepared by digestion with XhoI and HindIII. The resulting fragments were cloned in the vector pBBRMCS-1.

Preparation of protein samples for proteome analysis.

To obtain crude extracts from cells that were cultivated in MSM, cells were harvested by centrifugation (15 min; 3,500 × g; 4°C), and the resulting cell pellets were then treated with crack solution (8 M urea, 2% [vol/vol] Triton X-114). The volume of crack solution corresponded to the volume of the centrifuged cell suspension, i.e., a pellet that resulted from 50 ml culture broth was resuspended in 50 ml crack solution. The mixture was then incubated for 1.5 h at room temperature on a gyratory shaker to break the cells. PHB and cell debris were then separated from the crude extract by centrifugation (1 h; 60,000 to 70,000 × g; 4°C), and proteins were subsequently extracted from the supernatant by phenol extraction as described previously (47). After acetone precipitation, the washed protein pellet was air dried by incubation at room temperature to evaporate the acetone and was then stored at −20°C.

Rehydration of proteins.

To the dry protein pellets, an adequate volume of rehydration buffer A (9 M urea, 4% [wt/vol] CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 100 mM dithiothreitol [DTT], distilled H₂O [H₂O dest.] ad 10 ml), depending on the pellet size, was added, and the mixture was incubated at room temperature for 2 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; 11,000 × g). The protein concentrations in the supernatants were subsequently measured.

Determination of protein concentration.

The protein concentration was determined by the method of Bradford (8). To reduce the disturbing influences of DTT and urea (48), 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 (70 mg Serva Blue G, 50 ml 96% [vol/vol] ethanol, 100 mg 85% [vol/vol] phosphoric acid, H₂O dest. ad 1,000 ml). After 10 min of incubation in the absence of light, absorption at 595 nm was measured against the reagent blank value. A calibration was done with bovine serum albumin (BSA) in a range of 1 to 1,000 μg.

2D PAGE: first-dimension IEF.

The equilibration of isoelectric focusing (IEF) strips, a two-dimensional (2D) gel run in a Dodeca cell (Bio-Rad), and staining and destaining procedures were done exactly as described by Raberg et al. (47).

Software-based analysis of 2D gel images.

Images from 2D PAGE were analyzed using the Decodon Delta 2D software (Decodon GmbH, Greifswald, Germany). From replicates comprising various samples of identical stages of cultivation (exponential and stationary growth phases of the wild-type R. eutropha H16 and mutant H1482, respectively; 4 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 comparison of the protein patterns during the different stages, spot quantities were likewise determined with Delta 2D software. For this, a proteome map comprising all gel images of each of the four groups was created using the union fusion approach of the software. Spot boundaries on the proteome map were detected and transferred to the original images, and the spots were automatically quantified by the software. The given spot quantities represent the relative portion (percent volume) of an individual spot in the total protein present on the respective average fusion image. For normalization, the 10 quantitatively most predominant spots were excluded from the normalization set. Normalization aimed at mitigating systematic differences between gel images, which may occur by variation in protein loading, imaging exposure times, and dye/staining efficiencies. It is a common procedure to exclude some of the quantitatively predominant spots from normalization because these spots disturb the quantification process of weaker spots (see reference 7 for further details).

Protein preparation, mass spectrometry, and data analysis.

Spots were cut from the 2D gels and transferred to 1.5-ml plastic tubes. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis was performed by employing the method of Shevchenko et al. (60) at the Institut für Mikrobiologie, Ernst-Moritz-Arndt University, Greifswald, Germany. For this, proteins were tryptically digested, and the mass spectra of the protein fragments were revealed by MALDI-TOF (Proteome Analyzer 4800; Applied Biosystems, Foster City, CA). The parameters for the measurements were set as described previously (68), except that the signal-to-noise ratio for the TOF-TOF measurements was raised to 10. These spectra were compared with hypothetical spectra of the R. eutropha H16 data bank, and proteins were identified by using the computer software GPMAW (Lighthouse Data, Denmark) as well as Mascot, a program associated with MALDI-TOF (search parameters were as in reference 68).

PHA quantification.

Samples were subjected to methanolysis in the presence of 85% (vol/vol) methanol and 15% (wt/vol) sulfuric acid. The resulting 3-hydroxybutyric acid methyl esters were analyzed by gas chromatography (9, 66).

Measurement of pyruvate.

Pyruvate concentrations in the medium were determined by a spectrophotometric assay measuring the decrease of absorption at 365 nm due to consumption of NADH (d = 1 cm; ɛ = 3.5 cm²/μmol) according to the method of Czok and Lamprecht (13). The analysis was done in a mixture containing 2.8 ml 100 mM potassium phosphate buffer (pH 7.2), 0.1 ml 15 mM NADH, and 0.05 ml sample and was started by addition of 0.02 ml l-(+)-lactate dehydrogenase.

RESULTS AND DISCUSSION

For a more sophisticated analysis based on the first results of Pries et al. (46), (i) extensive in silico analyses were carried out and (ii) a defined deletion mutant, ΔpdhL, was generated to investigate putative differences in growth and PHB accumulation in comparison to the Tn5 mutant. In addition, (iii) complementation experiments were done using pBBR-MCS1 hybrid plasmids harboring E3 components of other bacteria, and (iv) a proteome analysis was done to identify the proteins of the wild type and the mutants, which were differentially expressed depending on the growth stage and state of PHB metabolism. Determination of pyruvate excretion and measurement of total DHLDH activity completed the investigations.

In silico analyses.

The organization of the genes of the multienzyme complexes PDHC and OGDHC of R. eutropha H16 had been investigated previously (21, 22). The entire genome sequence of the strain is known (43). When the Basic Local Alignment Search Tool (BLAST) was applied to verify these data and to screen R. eutropha H16 genomewide for additional 2-oxoacid multienzyme complex components, it was found that strain H16 is one of only a few organisms possessing homologues of all five types of 2-oxoacid multienzyme complexes mentioned in the introduction. These analyses also attested that strain H16 possesses five distinct DHLDH-coding genes. Two of them (pdhL of PDHC and odhL of OGDHC) are clustered with the corresponding E1 and E2 genes. The other three DHLDH genes (lpdaA, B1765, and B1098) are spread throughout the genome.

The DHLDH sequences of strain H16 were analyzed for the presence of conserved domains, as shown for PdhL using the PFAM (http://pfam.sanger.ac.uk/) and PROSITE (http://expasy.org/prosite/) databases (Fig. 1). Like all other E3 components, each of them possesses a dimerization domain (37, 45), which is typically found in pyridine nucleotide-disulfide oxidoreductases (class I and II oxidoreductases and NADH oxidases and peroxidases). Furthermore, all five DHLDHs harbor a domain mediating the pyridine nucleotide disulfide oxidoreductase function. This motif is conserved in class I and II pyridine nucleotide disulfate oxidoreductases and consists of a small NADH binding domain that is embedded in a larger FAD-binding region. An additional highly conserved motif (11, 36, 53), which was assigned as the active center comprising two cysteines, attracts attention in PdhL, OdhL, LpdaA, and B1765 and B1098. These essential cysteine residues form disulfide bridges that are involved in the transfer of reducing equivalents to NAD⁺ in class I pyridine nucleotide disulfide oxidoreductases. The in silico analysis revealed that the second glycine of the conserved motif of B1098 is replaced by threonine (see Table S6 in the supplemental material).

FIG. 1.

Schematic overview of PdhL of R. eutropha H16 and the truncated PdhL of R. eutropha H1482.

While the catalytically essential cysteines are highly conserved, the replacement of a glycine by a threonine may induce structural aberrations. Since preservation of the extended α-helix motif is essential for the steric orientation and consequently for the catalytic functionality of the disulfide bond (34, 35), such a mutation may result in loss of catalytic activity of B1098. Moreover, an N-terminal signal peptide comprising 35 amino acids (aa) (69) was identified for LpdaA in R. eutropha. Furthermore, it must be emphasized that, interestingly, the PdhL protein of R. eutropha possesses a lipoyl domain (52), like the lipoamide dehydrogenase of Burkholderia cepacia (BamB_2172). Lipoyl domains are usually conserved domains in E2 components of the dehydrogenase multienzyme complexes.

The published data for the PHB-leaky Tn5 mutants exhibiting a reduced PHB accumulation rate (21, 46) were verified and completed. Our investigations revealed that mutants H1474 and H1479 are most probably siblings of mutant H1482 because the Tn5 insertions had occurred at identical positions. Insertion of Tn5 had occurred in the dimerization domain in the carboxy-terminal region of pdhL, thereby disrupting the dimerization domain (Fig. 1 shows a schematic overview). As a consequence of this Tn5 insertion, a carboxy-terminally truncated PdhL of 499 aa (wild type, 595 aa) is synthesized by mutant H1482 lacking 46 aa of the total 72 aa of the essential dimerization domain, most probably leading to an inactive enzyme. It is postulated that the essential dimerization of two PdhL molecules is prevented in H1482 by the loss of this dimerization domain. The catalytic activity of the DHLDH is therefore lost, because a cluster of two monomers together with the incorporated cofactor FAD is essential for interaction with the E2 component of the PDHC. This assumption corresponds to observations for the E3 component of E. coli by Lindsay et al. (31), who clearly showed that dimerization of two E3 monomers with insertion of FAD is absolutely necessary for E3 activity. Indeed, significantly lower total DHLDH enzyme activity in mutant H1482 than in the wild-type H16 was observed by a spectrophotometric assay, which probably also indirectly decreased PDHC activity. While crude extracts of the wild type exhibited a specific activity of 1.2 U/mg protein, only 0.8 U/mg protein was measured in H1482. Both, the complete deletion of PdhL and expression of a carboxy-terminally truncated PdhL protein apparently cause a strong negative effect on PHB accumulation and explain the PHB-leaky phenotype. A total loss of DHLDH activity is probably prevented by compensation by at least one of the other four E3 homologues in R. eutropha. Otherwise, the loss of PdhL would be expected to cause even greater impairments of cells if they are cultivated in the presence of carbon sources that are metabolized via PDHC. However, genome-encoded expression of the other four homologous E3 components is obviously not sufficient to restore the wild-type phenotype in mutants.

The genomic region of the PDHC was screened for the existence of promoter sequences (data not shown) by the method of Reese (50). Directly downstream (46 bp) of pdhL, the last gene of the PDHC gene cluster, a putative promoter sequence in correct orientation for the genes located downstream was detected. Therefore, putative polar effects of the Tn5 insertion could be excluded with significant certainty.

The in silico analysis also confirmed the presence of a genomic EcoRI fragment comprising all three genes of the PDHC, in agreement with the 6.3-kbp EcoRI fragment, which restored the wild-type phenotype in the PHB-leaky Tn5 mutant (46).

Complementation experiments.

Two genes of R. eutropha were chosen for complementation experiments: pdhL and the pdhL homologue odhL. In addition, two homologous genes from other bacteria were used: lpd of E. coli K-12 and pdhL of B. cepacia (pdhL_BC); interestingly, the latter also encodes an N-terminal lipoyl domain. Growth and PHB accumulation by the wild-type R. eutropha H16 were compared with those of the Tn5 mutant H1482 and H1482 harboring pBBR1MCS-1::pdhL, pBBR1MCS-1::odhL, pBBR1MCS-1::lpd, or pBBR1MCS-1:: pdhL_BC. In addition, R. eutropha H16 transformed with pBBR1MCS-1::pdhL was investigated. For this, cells were cultivated under conditions permissive for PHB synthesis (MSM containing 1.0% [wt/vol] sodium gluconate and 0.05% [wt/vol] NH₄Cl). After 30 h of cultivation, an additional 0.05% (wt/vol) NH₄Cl was added to induce PHB degradation; one sample at the exponential growth phase and several samples at the stationary growth phase were withdrawn (Fig. 2A) and analyzed for PHB content (Fig. 2B). Five different groups could be distinguished. (i) Cells of the wild type and of the wild type harboring pBBR1MCS-1::pdhL showed almost identical growth behavior and PHB accumulation. While minor amounts of PHB were synthesized in the exponential growth phase, maximal accumulation of PHB occurred in the stationary phase; when degradation began after provision of NH₄Cl, the PHB contents of the cells decreased, as in all strains investigated in this experiment. The finding that expression of additional PdhL molecules by transformation of R. eutropha H16 with pBBR1MCS-1::pdhL does not affect growth or result in a significant increase of PHB accumulation demonstrates that PdhL is not the limiting element of the PDHC complex components. The components of the PDHC assemble in defined stoichiometric amounts; therefore, overexpression of only one component of the complex does not show any influence. (ii) Mutant H1482 showed delayed growth in comparison to the wild type and entered the stationary phase at a lower optical density (OD); accumulation of PHB was also significantly reduced. (iii) Plasmid pBBR1MCS-1::pdhL nearly restored the wild-type phenotype in R. eutropha H1482. (iv) pBBR1MCS-1::pdhL_Bc, however, complemented the mutant only partially. (v) In contrast, hybrid plasmids harboring the gene for OdhL (pBBR1MCS-1::odhL) or Lpd (pBBR1MCS-1::lpd) did not restore growth and PHB accumulation to the wild-type level.

FIG. 2.

Growth experiment to characterize the complemented Tn5 mutant H1482 in comparison to the wild-type H16 and the parent strain, H1482, of R. eutropha. For complementation, hybrid plasmids of pBBR1MCS-1 harboring pdhL, odhL, lpd (of E. coli), or pdhL_Bc (of B. cepacia) were transferred into H1482 or H16. Additionally, pBBR1MCS-1::pdhL was transferred into H16 as a control. (A) Cells were grown under conditions permissive for PHB synthesis. After 30 h cultivation, PHB degradation was induced (see Materials and Methods for details). OD was monitored with a Klett-Summerson photometer. Samples for PHB quantification were withdrawn in the exponential growth phase when the density had reached 400 KU and in the stationary growth phase after 18.5, 30, 33.5, and 37 h. wt, wild type. (B) PHB quantification (percentage [wt/wt] of cell dry weight [CDW]) in cells by gas chromatography (GC) analysis (as described in Materials and Methods) of the complemented Tn5 mutant H1482 in comparison to the wild-type H16 and the parent strain, H1482, of R. eutropha. Samples were taken from the growth experiments shown in panel A. exp., exponential growth phase; stat., stationary growth phase.

Similarly, the defined deletion mutant R. eutropha ΔpdhL transformed with the various pBBR1MCS-1 constructs harboring the different E3 component genes described above, the Tn5 mutant H1482, and the wild-type H16 were cultivated under the conditions described above. Growth was recorded, and samples for PHB quantification were taken during (i) the exponential phase at an OD of 400 Klett units (KU), (ii) the stationary growth phase after 23.5 h cultivation, and (iii) the later stationary growth phase after 31.25 h cultivation (Fig. 3A and B). The defined R. eutropha ΔpdhL deletion mutant exhibited the same phenotype regarding growth and PHB synthesis as the Tn5 mutant H1482. In addition, complementation experiments using the five pBBR1MCS-1 constructs gave results analogues to those for H1482, except in the case of pBBR1MCS-1::odhL: while the plasmid coding for OdhL did not restore growth and PHB accumulation to the wild-type level in H1482, the defined ΔpdhL mutant was fully complemented by it. Therefore, an essential function of a lipoyl domain found in PdhL of R. eutropha, and also in PdhL of B. cepacia, could not be verified for PdhL of the PDHC of R. eutropha H16. However, Lpd of E. coli also lacking a lipoyl domain was not able to restore the wild-type phenotype in any way.

FIG. 3.

Growth experiment to characterize the complemented ΔpdhL deletion mutant in comparison to the wild-type H16 and the Tn5 mutant H1482 of R. eutropha. For complementation, hybrid plasmids of pBBR1MCS-1 harboring pdhL, odhL, lpd (of E. coli), or pdhL_Bc (of B. cepacia) were transferred into the strains as indicated. (A) Cells were grown under conditions permissive for PHB synthesis (see Materials and Methods for details). OD was monitored with a Klett-Summerson photometer. Samples for PHB quantification were withdrawn in the exponential growth phase when the density had reached 400 KU and in the stationary growth phase after 23.5 h and 31.25 h. (B) PHB quantification (percentage [wt/wt] of CDW) of cells by GC analysis (as described in Materials and Methods) of a complemented ΔpdhL deletion mutant in comparison to the wild-type H16 and the parent strain, H1482, of R. eutropha. Samples were taken from the growth experiments shown in panel A.

Proteome analysis.

The wild-type H16 and mutant H1482 were again cultivated under conditions permissive for PHB accumulation in MSM. Samples for PHB and proteome analysis were withdrawn in the exponential growth phase at an OD of 400 KU and in the stationary growth phase after 31 h of cultivation. In agreement with the previous growth experiments, mutant H1482 accumulated significantly less PHB (8%, [wt/wt] PHB in the exponential phase and 36% [wt/wt] in the stationary phase) than the wild type (28% [wt/wt] in the exponential phase and 54% [wt/wt] in the stationary phase). A minimum of four 2D gel replicates of each sample were created and scanned after being stained, and the resulting gel images were analyzed with the Delta 2D software of DECODON. After building virtual average fusion gel images of the replicates of each group, a proteome map was generated by fusing all images of the project. Based on this proteome map, which contained the spots for all gels, in a first step, software-assisted spot detection took place. In a second step, spot marks were transferred to all other gels to allow densitometric spot quantification for each single gel. Statistical analysis of spot quantities between replicate gel groups identified several differentially expressed proteins. To ensure significance, only those spots whose quantities increased or decreased by a factor of 2 were further analyzed. Such proteins were subjected to MALDI-TOF analysis.

Four different classes of spots of interest could be distinguished: class A spot proteins were increasingly expressed by mutant H1482 in the exponential and/or in the stationary phase, whereas class B spot proteins were decreasingly expressed by mutant H1482 in the exponential and/or in the stationary phase in comparison to the wild type. Class C spot proteins were increasingly synthesized by both strains in the exponential phase, whereas class D spot proteins were increasingly expressed in the stationary phase in both the wild type and mutant H1482. (Dual views of average fusions of the four replicate 2D gel groups and classified differentially expressed proteins are shown in Fig. S1 and Table S7 in the supplemental material; spot quantification data for all protein classes are shown in Fig. 4.)

FIG. 4.

Quantification of protein spots in image fusions of 2D PAGE gels shown in Fig. S1 in the supplemental material. Spot quantities are given as percent volume (representing the relative portion of an individual spot in the total protein present on the respective average fusion image). Quantification was done with Delta 2D software.

Class A spots.

Among those proteins of the mutant that were increasingly expressed in the exponential and/or in the stationary phase at least at a 2-fold-higher level than the wild type were d-3-phosphoglycerate dehydrogenase (SerA2, forming three spots with differing isoelectric points [IEPs]), gluconolactonase (Gln2), succinyl-CoA:3-ketoacid-coenzyme A transferase (H16_A1331, subunit a), threonine-tRNA ligase (ThrS), demethylmenaquinone methyltransferase 2 (MenG2), acetyl-CoA acetyltransferase (BktB), branched-chain aminotransferase (H16_A0561, forming two spots), and malate dehydrogenase (Mdh1, forming two spots). Demethylmenaquinone methyltransferase 3 (MenG3), tyrosine aminotransferase (TyrB2), aconitate hydratase 2 (AcnB), and a zinc-containing alcohol dehydrogenase (H16_B1699, forming two spots) were increasingly expressed solely in the exponential growth phase. The S-succinyltransferase (OdhB; E2 of ODHC) was about 10-fold more strongly expressed by H1482. An upregulation in the stationary phase was observed for the dihydrolipoamide dehydrogenase OdhL (E3 of ODHC).

Class B spots.

Several proteins that were expressed at a level at least 2-fold higher in the wild type than in mutant H1482 in the exponential and/or the stationary phase were identified (see Table S7 in the supplemental material). These proteins are marked in blue in Fig. S1 in the supplemental material, and the spot quantities are shown in Fig. 4. The proteins comprise ribulose bisphosphate carboxylase (CbbS2, small chain), a pirin-like protein (H16_A0355), ADP-d-beta-d-heptose 6-epimerase (HldD), PHG093/HoxI, and fructose-1,6-/sedoheptulose-1,7-bisphospate aldolase (CbbA2; two different IEP). Glyceraldehyde 3-phosphate dehydrogenase (CbbG2) was decreasingly expressed by mutant H1482 solely in the exponential phase.

Class C and D spots.

Class C spot proteins, i.e., proteins increasingly expressed in the exponential growth phase in both strains, were marked with green (see Fig. S1A in the supplemental material), whereas class D spot proteins, i.e., proteins increasingly expressed in the stationary growth phase in both strains, were marked with purple (see Fig. S1B in the supplemental material). Identified proteins are listed in Table S7 in the supplemental material. Most proteins assigned as class C spots, which were increasingly expressed in the exponential growth phase, play a role in catabolism, anabolism, or nutrient uptake. Pyruvate kinase (Pyk2) and 3-phosphoglycerate kinase (CbbK1), involved in the Entner-Doudoroff pathway, were identified, as well as dihydrodipicolinate synthase (DapA1), which is involved in lysine biosynthesis. Among those proteins that were increasingly expressed in the stationary growth phase in both strains (class D spot proteins), two proteins of the E1 component of the ADHC (AcoA and AcoB) were found. Furthermore, several proteins that ensure the degradation of toxic substances, like a chloroperoxidase (H16_B1943), haloperoxidase (H16_A2213), and superoxide dismutase (H16_B1110), were detected.

Excess of pyruvate due to defectve PdhL and adaptation of the proteome of R. eutropha mutant H1482.

The adaptations observed for the mutant H1482 predominantly concern central catabolism. In particular, enzymes involved in the Entner-Doudoroff pathway and the tricarboxylic acid cycle (TCC) and in PHB biosynthesis were expressed differently than the wild type (Fig. 4 and 5; see Fig. S1 and Table S7 in the supplemental material). Furthermore, PHB-negative mutants of R. eutropha, like PHB⁻4 (57), excrete large amounts of pyruvate under cultivation conditions permissive for PHB accumulation in the wild type (64). To discover if the PHB-leaky mutant H1482 also excretes pyruvate under such conditions, the wild type, H1482, and PHB⁻4 were grown in MSM containing 1.0% (wt/vol) sodium gluconate and 0.05% (wt/vol) NH₄Cl, and the amounts of pyruvate in the supernatant were determined. Three samples of each organism were taken in (i) the exponential growth phase, (ii) the early stationary growth phase, and (iii) the later stationary growth phase. Samples from the exponential growth phase contained only a little pyruvate in the wild type (2.2 mM) and either mutant H1482 (2.0 mM) or PHB⁻4 (1.2 mM). In the early stationary growth phase, the amounts of pyruvate slightly increased in the wild-type H16 (3.4 mM) and mutants H1482 (4.1 mM) and PHB⁻4 (6.7 mM). They further drastically increased in the later stationary phase only in the mutants H1482 (18.4 mM) and PHB⁻4 (28.9 mM), but not in the wild type (2.9 mM).

FIG. 5.

Central metabolism of R. eutropha H16 with special regard to proteins differentially expressed in the wild type and mutant H1482 of R. eutropha. The red arrows and numbers mark proteins that are expressed in H1482 at a lower level than in the wild type, whereas the green arrows and numbers mark proteins that are expressed at a higher level in H1482 than in the wild type. The numbers in the scheme indicate the following involved enzymes: 1, hexokinase; 2, glucose-6-phosphate dehydrogenase; 3, phosphogluconate dehydratase; 4, phospho-2-keto-3-desoxygluconate aldolase; 5, glyceraldehyde-3-phosphate dehydrogenase; 6, phosphoglycerate dehydrogenase; 7, phosphoglyceromutase; 8, enolase; 9, pyruvate kinase; 10, glucokinase; 11, gluconolactonase; 12, glucose dehydrogenase; 13, fructose-bisphosphate aldolase; 14, 3-phosphoglycerate dehydrogenase; 15, pyruvate dehydrogenase/decarboxylase (E1 of PDHC); 16, dihydrolipoamide acetyltransferase (E1 of PDHC); 17, dihydrolipoamide dehydrogenase (E3 of PDHC); 18, aldehyde dehydrogenase; 19, acetyl-CoA ligase; 20, acetoin dehydrogenase enzyme system; 21, spontaneous reaction in the presence of O₂; 22, alcohol dehydrogenase (ADH); 23, acetyl-CoA acetyltransferase; 24, acetoacetyl-CoA reductase; 25, PHB synthase; 26, 3-oxoacid-CoA transferase; 27, 3-hydroxybutyrate dehydrogenase; 28, alcohol dehydrogenase (B1699); 29, citrate synthase; 30, aconitase; 31, isocitrate dehydrogenase; 32, 2-oxoacid dehydrogenase multienzyme complex; 33, succinyl-CoA synthetase; 34, succinate dehydrogenase; 35, fumarase; 36, malate dehydrogenase; 37, malic enzyme; 38, pyruvate kinase.

Considering the enzymes of the Entner-Doudoroff pathway (Fig. 5), it is obvious that the expression of glyceraldehyde-3-phosphate dehydrogenase, which converts glyceraldehyde-3-phosphate into 1,3-diphosphoglycerate, is strongly downregulated in H1482. This might result in large amounts of glyceraldehyde-3-phosphate that cannot be metabolized to pyruvate. However, since mutant H1482 excretes significant amounts of pyruvate into the medium, especially in the stationary phase, the conversion catalyzed by the enzyme seems to continue yielding 1 mol of NADH⁺ H⁺ and 1 mol of ATP per mol of glyceraldehyde-3-phosphate converted. This indicates a diminished flux at the PDHC, which obviously metabolizes pyruvate at a lower rate. Consequently, pyruvate accumulates in the cells and is subsequently discharged. Accordingly, significant pyruvate excretion has also been observed for an lpdA mutant of E. coli (30). Protein biosynthesis obviously has to be adapted to this situation by downregulating the key enzymes of carbon metabolism leading to pyruvate. This explains the downregulation of the glyceraldehyde-3-phosphate dehydrogenase of the Entner-Doudoroff pathway.

Adaptation of proteins of the TCC in R. eutropha H1482.

The proteome of H1482 shows several significant changes in enzymes of the TCC in comparison to the wild type (Fig. 5). Several enzymes, like aconitase, the E3 component of the ODHC, and malate dehydrogenase, are upregulated in the mutant in the exponential growth phase, but most of the other enzymes also seem to be upregulated as a general reaction to the bottleneck for supply of energy and metabolites for the anabolism occurring due to the diminished supply of acetyl-CoA. As a consequence of increased activity of TCC for energy generation, other metabolic processes are obviously shifted down. PHB synthesis, especially, is negatively affected, resulting in reduced amounts of PHB in the mutant cells. In particular, the upregulated malate dehydrogenase may provide increased amounts of oxalacetate for condensation with acetyl-CoA to citrate. Subsequent increased isomerization of citrate to isocitrate by aconitase additionally raises the suction of acetyl-CoA into the TCC. Altogether, the irreversible condensation to citrate is promoted, and an increased fraction of the available acetyl-CoA is then further oxidized in the TCC.

Adaptation of enzymes of PHB synthesis in R. eutropha H1482.

Several enzymes involved in PHB biosynthesis or related to it, like β-ketothiolase (BktB) and 3-oxoacid-CoA transferase, are expressed at a higher level in mutant H1482 in the exponential and stationary growth phases (Fig. 5; see Fig. S1 in the supplemental material). Besides their roles in PHB metabolism, the two proteins together are able to convert acetoacetate via acetoacetyl-CoA to two molecules of acetyl-CoA (43, 62). Acetoacetate might be generated as an intermediate during degradation of branched-chained amino acids and could serve as an additional source for acetoacetyl-CoA in mutant cells under circumstances where mutant cells suffer from acetyl-CoA deficiency. The resulting acetyl-CoA can then be metabolized in the TCC for generation of energy.

General adaptations of proteomes at different growth stages.

The study concerning the class C and D proteins includes observations supplemental to the study of class A and B proteins. Therefore, only proteins that have not been covered in the previous discussion of classes A and B are discussed here. Class C proteins were expressed at a higher level in the exponential growth phase than in the stationary growth phase in both the mutant and the wild type. These proteins comprise (i) enzymes involved in catabolism, like pyruvate kinase and 3-phosphoglycerate kinase; (ii) enzymes involved in anabolism, like dihydrodipicolinate synthase; and (iii) proteins involved in nutrient uptake. The upregulation of these proteins is interpreted as an adaption to growth conditions and cell division in the exponential phase, with an enhanced need for nutrients as a substrate for energy metabolism and anabolism.

Among the class D proteins, which are increasingly expressed in the stationary phase in both strains, the two proteins of the E1 component of the ADHC (AcoA and AcoB) attract attention. It is speculated that acetoin is formed in the stationary phase by a side reaction of the pyruvate decarboxylase of the PDHC if the PDHC is not fully functioning due to the lack of an intact DHLDH (24). In R. eutropha, acetoin is metabolized by the ADHC to acetaldehyde and acetyl-CoA (39); the formation of the latter enzyme is induced only in the presence of acetoin (17). The resulting acetyl-CoA can then be used for PHB synthesis in the stationary phase.

Furthermore, increased expression of the response regulator of nitrogen regulation (NtrC) was detected. In R. eutropha H16, NtrC is not yet characterized in much detail; it shares high amino acid sequence homology with NtrC of Klebsiella pneumoniae (32). A role for NtrC in R. eutropha H16 as a master switch, as in K. pneumoniae, is therefore not unlikely during nitrogen deficiency in the stationary phase. Interestingly, recently published transcriptomic data support our observation. For R. eutropha grown under nitrogen limitation, increased transcription of the genes ntrC and ntrB, the latter encoding the corresponding signal transduction histidine kinase, was detected (10). The increased expression of several proteins observed to be necessary for degradation of toxic substances, like chloroperoxidase and superoxide dismutase, may be caused by toxic by-products accumulating in the stationary growth phase.

Conclusions.

The adaptations of the mutant H1482 predominantly concern the central catabolism and comprise especially enzymes involved in the Entner-Doudoroff pathway, the TCC, and PHB biosynthesis. The PDHC plays a key role in the provision of the central metabolite acetyl-CoA, which is required for generation of both energy and intermediates for anabolism (14). Mutant cells apparently try to counteract the lack of acetyl-CoA caused by an impaired PDHC by adjusted expression of the enzymes of the metabolic pathways mentioned above. Although adaptation obviously succeeds sufficiently to allow growth of the mutant, the amount of available acetyl-CoA for PHB synthesis appears to be much lower than in the wild type, resulting in significantly reduced accumulation of PHB in the mutant.