Versatile Metabolic Adaptations ofRalstonia eutrophaH16 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 ⌬pdhL 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 inte...

Detection of phase-dependent transcriptomic changes and Rubisco-mediated CO2 fixation into poly (3-hydroxybutyrate) under heterotrophic condition in Ralstonia eutropha H16 based on RNA-seq and gene deletion analyses

“…In particular, pdhL exhibited an 18.5-fold increased expression in the growth phase compared with the PHA production phase, which was consistent with a previous observation that disruption of pdhL decreased the growth rate and PHA productivity on fructose [30]. pdhA2 (H16_A1753) and aceE (H16_B1300), which encode paralogs of PdhA1 and PdhL, respectively, were barely expressed throughout cultivation.…”

Data Driven Modelling of a Chemically Defined Growth Medium for Cupriavidus necator H16

“…It is perhaps therefore not surprising that glucose supported little or no growth yet it has been reported that prolonged cultivation (> 70 h) with glucose as the sole carbon source resulted in a mutant that was able to utilize glucose (30). The mutant acquired such ability by mutating the N-acetylglucoasmine phosphotransferase system, which when deleted led to inability to utilize glucose (30,31). Glycerol supported low growth of C. necator H16 especially in the medium forcing during the scoping trial.…”

Extracytoplasmic diaphorase activity of Streptomyces coelicolor A3(2)

“…This could be due to the activity of a paralogous enzyme compensating for the loss of SCO2180. Similar dispensability of DLDH is known for Ralstonia eutropha [14]. We also generated a recombinant S. coelicolor A3(2) strain harboring extra copies of SCO2180 on a high-copy-number plasmid, but the increase in the gene copy number neither affected the growth nor the phenotype of this strain (data not shown).…”