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Applied and Environmental Microbiology, July 2008, p. 4477-4490, Vol. 74, No. 14
0099-2240/08/$08.00+0     doi:10.1128/AEM.00440-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Ralstonia eutropha H16 Flagellation Changes According to Nutrient Supply and State of Poly(3-Hydroxybutyrate) Accumulation

Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität, D-48149 Münster, Germany,¹ Institut für Medizinische Physik und Biophysik, Elektronenmikroskopie und Analytik, Universitätsklinikum Münster, Westfälische Wilhelms- Universität, D-48149 Münster, Germany,² Institut für Integrierte Funktionelle Genomik (IFG), Zentrum für Interdisziplinäre Klinische Forschung (IZKF), Medizinische Fakultät der Universität Münster, D-48149 Münster, Germany,³ Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Grisebachstraße 8, D-37077 Göttingen, Germany,⁴ Göttingen Genomics Laboratory, Georg-August-Universität Göttingen, Grisebachstraße 8, D-37077 Göttingen, Germany,⁵ Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestraße 117, D-10115 Berlin, Germany,⁶ Institut für Mikrobiologie, Ernst-Moritz-Arndt Universität, Friedrich Ludwig Jahn Straße 15, D-17489 Greifswald, Germany⁷

Received 22 February 2008/ Accepted 10 May 2008

Two-dimensional polyacrylamide gel electrophoresis (2D PAGE), in combination with matrix-assisted laser desorption ionization-time of flight analysis, and the recently revealed genome sequence of Ralstonia eutropha H16 were employed to detect and identify proteins that are differentially expressed during different phases of poly(3-hydroxybutyric acid) (PHB) metabolism. For this, a modified protein extraction protocol applicable to PHB-harboring cells was developed to enable 2D PAGE-based proteome analysis of such cells. Subsequently, 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, under conditions permissive for intracellular PHB mobilization after a nitrogen source was added to cells that are carbon deprived but with full PHB accumulation, flagella are 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.

Published ahead of print on 23 May 2008.