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

Abstract

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 Polyhydroxybutyrates (PHBs) are biodegradable polymers with properties of thermoplastics synthesized by PHB synthases (PhaC) using (R)3-hydroxybutyryl coenzyme A as a substrate. In times of nutrient limitation, many bacteria generate these polymers when an appropriate carbon source is available (1). The soluble (R)3-hydroxybutyryl coenzyme A is transformed into insoluble polymers, called granules, in a nontemplate-dependent polymerization process (18). When the bacteria find themselves in a more hospitable environment, they degrade this polymer to generate building blocks and reducing equivalents for anabolism. However, bacteria in a nutrient-rich medium can also make and degrade PHB. The biology of PHB production and utilization under nutrient-rich conditions, however, remains to be elucidated.The widespread detection of genes in many microorganisms that carry out PHB synthesis and degradation suggests that these pathways have evolved to be a general mechanism for bacterial cell survival in times of stress. The organism that we have chosen to study PHB homeostasis is Wautersia eutropha H16. This organism contains a class I synthase to generate PHB (15).In this paper, we describe studies of PHB production and utilization by W. eutropha H16 in a nutrient-rich medium (dextrose-free tryptic soy broth [TSB]). Using antibodies (Abs) to most of the proteins identified as being involved in PHB homeostasis thus far, we carried out Western blotting as a function of growth time to measure the rates of appearance and disappearance of PhaC, PhaP (a phasin protein), and PhaR (a putative transcription factor), as well as PhaZ1 a , PhaZ1 b , PhaZ1 c , and PhaZ2 (formerly known as PhaZ1, PhaZ2, PhaZ3, and oligomer hydrolase, respectively); all of these proteins are thought to be involved in PHB degradation (10, 23). The average cell volume (20) and the average number of granules per cell at 4 and 24 h in TSB were determined by stereology analyses of transmission electron microscopy (TEM) images. These results and knowledge concerning the M w of PHB allowed us to estimate the amount of each protein per cell, the percentage of the granule surface that is covered by PhaC and PhaP, and the number of PhaC molecules per PHB polymer. A comparison of these results with similar studies performed under nitrogen-limited growth conditions (unpublished data) provided insight int...

PhaP Is Involved in the Formation of a Network on the Surface of Polyhydroxyalkanoate Inclusions in Cupriavidus necator H16

“…A structural function is also suggested for the imaged network by the fact that removal of the network results in a conformation change in the inclusion from rounded to conical. Finally, the calculated radius of PhaP, 2.1 nm, is consistent with the height of the network (32).…”

“…This implies that it cannot be a lipid bilayer, which has been measured at 7 to 13 nm for C. necator H16 (21). Similarly, it does not seem possible that it is a monolayer of PhaP, because quantitative Western blot analyses have determined that there is only enough PhaP present to cover 27 to 54% of the total inclusion surface (31,32). With these two possibilities seemingly ruled out, a newer model of the PHA inclusion structure was proposed in which the outer layer of the inclusion consists of a phospholipid monolayer, thus conforming to the 4-nm thickness previously determined for the outermost layer (29).…”

Kinetic Studies of Polyhydroxybutyrate Granule Formation in Wautersia eutropha H16 by Transmission Electron Microscopy

“…The growth conditions used in this study have been widely used in the PHB field, and therefore, these new parameters of cells should be useful to other workers interested in understanding the PHB homeostasis. Recently, our laboratory has carried out a series of studies to determine the amount of proteins involved in PHB homeostasis in W. eutropha under these growth conditions using quantitative Western analyses (34). The results of the Western analyses, together with the cell volume and granule surface area calculated in this study, have provided new information concerning the homeostasis of PHB.…”

“…Using this analysis method, we report for the first time the average volume of W. eutropha H16 cells and the total surface area of granules per cell at different stages of granule formation. These parameters, along with the results of quantitative Western analyses described in the accompanying paper (34), identified the proteins involved during PHB biosynthesis and degradation and the changes in concentration of these proteins as a function of time. The kinetic information is essential for proposing functional roles of the proteins in PHB homeostasis.…”

Structure of the Catalytic Domain of the Class I Polyhydroxybutyrate Synthase from Cupriavidus necator

“…Despite a number of studies investigating the process of chain termination in PhaCs, this event remains poorly understood. PhaC is thought to catalyze termination itself, although a phasin protein, PhaP, has been implicated in promoting the termination event (25,45,46). Proposals for the mechanism of termination include hydrolysis by a base-activated water molecule; transfer of the PHB chain to a nucleophilic residue on PhaC followed by hydrolysis; or chain transfer to an exogenous thiol-or hydroxyl-containing molecule (22,(47)(48)(49).…”