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.

FIG. 1.

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

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.

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.

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.

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.