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Journal of Bacteriology, April 2008, p. 2911-2919, Vol. 190, No. 8
0021-9193/08/$08.00+0     doi:10.1128/JB.01486-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Binding of the Major Phasin, PhaP1, from Ralstonia eutropha H16 to Poly(3-Hydroxybutyrate) Granules{triangledown}

Liv Neumann,1 Francesco Spinozzi,2 Raffaele Sinibaldi,2 Franco Rustichelli,2 Markus Pötter,1 and Alexander Steinbüchel1*

Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität, D-48149 Münster, Germany,1 Dipartimento di Scienze Applicate ai Sistemi Complessi (SASC), Sezione di Scienze Fisiche, Università Politecnica delle Marche, Via Ranieri 6, 60131 Ancona, Italy2

Received 14 September 2007/ Accepted 11 January 2008

The surface of polyhydroxybutyrate (PHB) storage granules in bacteria is covered mainly by proteins referred to as phasins. The layer of phasins stabilizes the granules and prevents coalescence of separated granules in the cytoplasm and nonspecific binding of other proteins to the hydrophobic surfaces of the granules. Phasin PhaP1Reu is the major surface protein of PHB granules in Ralstonia eutropha H16 and occurs along with three homologues (PhaP2, PhaP3, and PhaP4) that have the capacity to bind to PHB granules but are present at minor levels. All four phasins lack a highly conserved domain but share homologous hydrophobic regions. To identify the region of PhaP1Reu which is responsible for the binding of the protein to the granules, N-terminal and C-terminal fusions of enhanced green fluorescent protein with PhaP1Reu or various regions of PhaP1Reu were generated by recombinant techniques. The fusions were localized in the cells of various recombinant strains by fluorescence microscopy, and their presence in different subcellular protein fractions was determined by immunodetection of blotted proteins. The fusions were also analyzed to determine their capacities to bind to isolated PHB granules in vitro. The results of these studies indicated that unlike the phasin of Rhodococcus ruber, there is no discrete binding motif; instead, several regions of PhaP1Reu contribute to the binding of this protein to the surface of the granules. The conclusions are supported by the results of a small-angle X-ray scattering analysis of purified PhaP1Reu, which revealed that PhaP1Reu is a planar, triangular protein that occurs as trimer. This study provides new insights into the structure of the PHB granule surface, and the results should also have an impact on potential biotechnological applications of phasin fusion proteins and PHB granules in nanobiotechnology.

* Corresponding author. Mailing address: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstraße 3, D-48149 Münster, Germany. Phone: 49-251-833 9821. Fax: 49-251-833 8388. E-mail: steinbu{at}uni-muenster.de

{triangledown} Published ahead of print on 25 January 2008.

Journal of Bacteriology, April 2008, p. 2911-2919, Vol. 190, No. 8
0021-9193/08/$08.00+0     doi:10.1128/JB.01486-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Jendrossek, D. (2009). Polyhydroxyalkanoate Granules Are Complex Subcellular Organelles (Carbonosomes). J. Bacteriol. 191: 3195-3202 [Full Text]  


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