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Journal of Bacteriology, July 2001, p. 4217-4226, Vol. 183, No. 14
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.14.4217-4226.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Accumulation of the PhaP Phasin of Ralstonia eutropha Is Dependent on Production of Polyhydroxybutyrate in Cells

Gregory M. York,1 Björn H. Junker,1,dagger JoAnne Stubbe,1,2 and Anthony J. Sinskey1,*

Department of Biology1 and Department of Chemistry,2 Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received 1 March 2001/Accepted 17 April 2001

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are granule-associated proteins that accumulate to high levels in strains that are producing PHAs. The accumulation of phasins has been proposed to be dependent on PHA production, a model which is now rigorously tested for the phasin PhaP of Ralstonia eutropha. R. eutropha phaC PHA synthase and phaP phasin gene replacement strains were constructed. The strains were engineered to express heterologous and/or mutant PHA synthase alleles and a phaP-gfp translational fusion in place of the wild-type alleles of phaC and phaP. The strains were analyzed with respect to production of polyhydroxybutyrate (PHB), accumulation of PhaP, and expression of the phaP-gfp fusion. The results suggest that accumulation of PhaP is strictly dependent on the genetic capacity of strains to produce PHB, that PhaP accumulation is regulated at the level of both PhaP synthesis and PhaP degradation, and that, within mixed populations of cells, PhaP accumulation within cells of a given strain is not influenced by PHB production in cells of other strains. Interestingly, either the synthesis of PHB or the presence of relatively large amounts of PHB in cells (>50% of cell dry weight) is sufficient to enable PhaP synthesis. The results suggest that R. eutropha has evolved a regulatory mechanism that can detect the synthesis and presence of PHB in cells and that PhaP expression can be used as a marker for the production of PHB in individual cells.

* Corresponding author. Mailing address: Bldg. 68-370, Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139. Phone: (617) 253-6721. Fax: (617) 253-8550. E-mail: asinskey{at}mit.edu.

dagger Present address: Max-Planck-Institute of Molecular Plant Physiology, 14476 Golm, Germany.

Journal of Bacteriology, July 2001, p. 4217-4226, Vol. 183, No. 14
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.14.4217-4226.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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