Functions of multiple phasins and interactions with enzymes of PHB synthesis an d degradation and regulatory proteins in Ralstonia eutropha
Zusammenfassung der Projektergebnisse
The recently revealed genome sequence of Ralstonia eutropha H16 provides a chance for detailed analyses of the metabolism and the cellular structure of this bacterium. Knowledge of the genome sequence provides the basis for techniques such as proteomic investigations by 2D PAGE due to the ability to easily and efficiently detect differentially expressed proteins by MALDI-TOF analysis. Within the scope of this work a protein extraction protocol applicable to PHB harboring cells of Ralstonia was developed and established at the beginning. Moreover, to ensure state of the art analysis of 2D gels in the course of proteome analyses, the Delta2D analysis software (DECODON) was purchased. Thereupon several 2D based proteome analyses were completed or initiated, respectively, to gain knowledge of unknown aspects of carbon metabolism and regulation of PHB synthesis. In a first approach the proteome of R. eutropha HI 6 was portrayed and analyzed at characteristic cultivation stages with regard to poly(3-hydroxybutyric acid) (PHB) metabolism to detect and identify proteins that are differentially expressed during different phases of PHB metabolism. Furthermore, the investigation comprised analysis of several mutants harboring different mutations in genes relevant for PHB metabolism. Therefore, samples from (i) the exponential growth phase, (ii) the stationary growth phase permissive for PHB biosynthesis, and (iii) a phase permissive for PHB mobilization were analyzed. Among several proteins exhibiting quantitative changes during the time course of a cultivation experiment, flagellin, which is the main protein of bacterial flagella, was identified. Initial investigations that report on changes of flagellation for R. eutropha were done, but 2D PAGE and electron microscopic examinations of cells revealed clear evidence that R. eutropha exhibited further significant changes in flagellation depending on the life cycle, nutritional supply, and, in particular, PHB metabolism. The results of our study suggest that R. eutropha is strongly flagellated in the exponential growth phase and loses a certain number of flagella in transition to the stationary phase. In the stationary phase under conditions permissive for PHB biosynthesis, flagellation of cells admittedly stagnated. However, after the addition of a nitrogen source to cells that are carbon deprived but had accumulated PHB, thereby providing conditions permissive for intracellular PHB mobilization, flagella were lost. This might be due to a degradation of flagella; at least, the cells stopped flagellin synthesis while normal degradation continued. In contrast, under nutrient limitation or the loss of phasins, cells retained their flagella. The results were published and represent the first extensive proteomic investigation of R. eutropha H16. A sophisticated analysis of the Tn5-induced PHB-leaky mutant Ralstonia eutropha H1482 showing a reduced PHB synthesis rate and a significantly lower dihydrolipoamide dehydrogenase (DHLDH) activity but similar growth behaviour as the wild type R. eutropha H16 was done taking advantage of the available genome sequence of this organism. In silico analyses were performed to detennine genomic localization and organization of all complex components of 2-oxoacid multienzyme complexes of R. eutropha and showed that R. eutropha possesses all five known types of 2-oxoacid multienzyme complexes and owns five DHLDH (E3 components) coding genes. Moreover it was revealed, that insertion of Tn5 in pdhL (encoding E3 component of the pyruvate dehydrogenase complex) of mutant HI 482 disrupted the carboxy terminal dimerization domain thereby causing synthesis of a truncated PdhL lacking this essential region. Furthermore, (i) a defined deletion mutant ApdhL was generated to investigate putative differences in growth and PHB accumulation in comparison to the Tn5 mutant, and (ii) complementation experiments were performed using recombinant pBBRI-MCSI vectors harbouring different E3 components of other microorganisms. In addition, (iii) a proteome analysis was done to identify differentially expressed proteins ofthe wild type and the mutants in dependency on growth stage and state of PHB metabolism. Complementation experiments showed that different plasmid encoded E3 components of R. eutropha H16 or of other bacteria like Burkholderia cepacia (that possesses an amino terminal lipoyl domain like the wild type HI6) were able to restore the wild type phenotype at least partially. A comparison of the proteomes ofthe wild type and of mutant H1482 revealed striking differences and allowed to reconstruct at least partially the impressive adaptations of R. eutropha H1482 to a loss of PdhL on the cellular level. Investigations of protein-protein interactions with a bacterial two-hybrid system did not detect interacting partners so far. Further attempts promise to elucidate regulation of PHB metabolism and to identify still unknown mediators of PHB metabolism. Investigations concerning recombinant Escherichia coli strains heterologously expressing vector encoded combinations of defined phasins and depolymerases delivered first auspicious results. Sequencing, assembly and annotation of the genome of R. eutropha H16 and subsequent analyses regarding gene content and expression of genes relevant for PHB metabolism have been published. Among several interesting aspects unravelled by its analyses regarding PHA biosynthesis, the occurrence of multiple homologues of the ß-ketothlolase phaA awakes interest and the role of these homologues is subject of current investigations of another PhD student. First results promise to indicate which of these homologous enzymes are actually involved in PHB synthesis. Knowledge of the complete genome sequence also provides insights into a remarkable metabolic versatility of R. eutropha and offers the basis for exploiting the biotechnological potential of this bacterium by metabolic engineering and advanced investigations such as the analysis of the transcriptome as well as further proteomic studies.
Projektbezogene Publikationen (Auswahl)
- Ralstonia eutropha H16 changes flagellation according to extracellular nutrient supply and state of poly(3-hydroxybutyrate)-accumulation. ISBP 2006, Minneapolis (USA), 27.-31.08.2006
Raberg, M., M. Pötter, and A. Steinbüchel
- Ralstonia eutropha H16 changes flagellation according to extracellular nutrient supply and state of poly(3-hydroxybutyrate)-accumulation. VAAM Jahrestagung 2006, Jena (Germany),19.-22.03.2006
Raberg, M., M. Pötter, and A. Steinbüchel
- 2007. Studies on the influence of phasins on accumulation and degradation of PHB and naostructure of PHB granules in Ralstonia eutropha H16. Biomacromolecules 8:657-662
Kuchta, K., Chi, L., Fuchs, H., Pötter, M., and A. Steinbüchel
- Ralstonia eutropha H16 changes flagellation according to nutrient supply and state of poly(3-hydroxybutyrate)-accumulatlon. Prokagenomics 2007 - 3rd European Conference on prokaryotic genomics, Göttingen (Germany), 7.-10.10.2007
Raberg, M., F. Reinecke, R. Reichelt, U. Malkus, S. König, M. Pötter, W. F. Fricke, A. Pohlmann, B. Friedrich, B. Bowien, and A. Steinbüchel
- 2008. Ralstonia eutropha H16 flagellation changes according to nutrient supply and state of poly(3-hydroxybutyrate) accumulation. Appl. Environ. Microbiol. 62:2540-2546
Raberg, M., Reinecke, F., Reichelt, R., Malkus, U., König, S., Pötter, M., Fricke, W. F., Pohlmann, A., Voigt, B., Hecker, M., Friedrich, B., Bowien, B. and Steinbüchel, A.
- 2008. Ralstonia eutropha strain H16 as model organism for PHA metabolism and for biotechnological production of technically interesting biopolymers. J. Mol. Microbiol. Biotechnol. 16:91-108
Reinecke, F., and A. Steinbüchel
- 2009. Dihydrolipoamide dehydrogenases in Ralstonia eutropha H16 - metabolic adaptations to a loss of pdhL, encoding the E3 component of the pyruvate dehydrogenase complex (PDHC). Appl. Environ. Microbiol.
Raberg, M., Bechmann, J., Brandt, U., Schlüter, J., Uischner, B., Voigt, B., Hecker, M., and A. Steinbüchel
