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Journal of Bacteriology, June 2005, p. 3825-3832, Vol. 187, No. 11
0021-9193/05/$08.00+0     doi:10.1128/JB.187.11.3825-3832.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Analysis of Transient Polyhydroxybutyrate Production in Wautersia eutropha H16 by Quantitative Western Analysis and Transmission Electron Microscopy

Departments of Chemistry,¹ Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 ,² Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142³

Received 8 November 2004/ Accepted 12 January 2005

Polyhydroxybutyrates (PHBs) are polyoxoesters generated from (R)3-hydroxybutyryl coenzyme A by PHB synthase. During the polymerization reaction, the polymers undergo a phase transition and generate granules. Wautersia eutropha can transiently accumulate PHB when it is grown in a nutrient-rich medium (up to 23% of the cell dry weight in dextrose-free tryptic soy broth [TSB]). PHB homeostasis under these growth conditions was examined by quantitative Western analysis to monitor the proteins present, their levels, and changes in their levels over a 48-h growth period. The proteins examined include PhaC (the synthase), PhaP (a phasin), PhaR (a transcription factor), and PhaZ1_a, PhaZ1_b, and PhaZ1_c (putative intracellular depolymerases), as well as PhaZ2 (a hydroxybutyrate oligomer hydrolase). The results show that PhaC and PhaZ1_a were present simultaneously. No PhaZ1_b or PhaZ1_c was detected at any time throughout growth. PhaZ2 was observed and exhibited an expression pattern different from that of PhaZ1_a. The levels of PhaP changed dramatically and corresponded kinetically to the levels of PHB. Transmission electron microscopy (TEM) provided the dimensions of the average cell and the average granule at 4 h and 24 h of growth (J. Tian, A. J. Sinskey, and J. Stubbe, J. Bacteriol. 187:3814-3824, 2005). This information allowed us to calculate the amount of each protein and number of granules per cell and the granule surface coverage by proteins. The molecular mass of PHB (10⁶ Da) was determined by dynamic light scattering at 4 h, the time of maximum PHB accumulation. At this time, the surface area of the granules was maximally covered with PhaP (27 to 54%), and there were one or two PhaP molecules/PHB chain. The ratio of PHB chains to PhaC was 60, which required reinitiation of polymer formation on PhaC. The TEM studies of wild-type and phaR strains in TSB provided further support for an alternative mechanism of granule formation (Tian et al., J. Bacteriol. 187:3814-3824, 2005).

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