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

摘要

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.