Polyhydroxyalkanoic acids (PHAs) are a class of polyesters stored in inclusion bodies and found in many bacteria and in some archaea. The terminal step in the synthesis of PHA is catalyzed by PHA synthase. Genes encoding this enzyme have been cloned, and the primary sequence of the protein, PhaC, is deduced from the nucleotide sequences of more than 30 organisms. PHA synthases are grouped into three classes based on substrate range, molecular mass, and whether or not there is a requirement for phaE in addition to the phaC gene product. Here we report the results of an analysis of a PHA synthase that does not fit any of the described classes. This novel PHA synthase from Bacillus megaterium required PhaC (PhaC Bm ) and PhaR (PhaR Bm ) for activity in vivo and in vitro. PhaC Bm showed greatest similarity to the PhaCs of class III in both size and sequence. Unlike those in class III, the 40-kDa PhaE was not required, and furthermore, the 22-kDa PhaR Bm had no obvious homology to PhaE. Previously we showed that PhaC Bm , and here we show that PhaR Bm , is localized to inclusion bodies in living cells. We show that two forms of PHA synthase exist, an active form in PHA-accumulating cells and an inactive form in nonaccumulating cells. PhaC was constitutively produced in both cell types but was more susceptible to protease degradation in the latter type. Our data show that the role of PhaR is posttranscriptional and that it functions directly or indirectly with PhaC Bm to produce an active PHA synthase.

Polyhydroxyalkanoate (PHA) accumulation and the morphology of PHA inclusion bodies were examined in Bacillus megaterium, strain 11561. Our results show a pattern of PHA degradation and synthesis, and of inclusion body growth and proliferation not previously reported. Degradation of PHA in the lag phase was followed by synthesis of PHA at an accelerating rate during exponential growth. PHA accumulation reached a maximum rate at late exponential/early stationary phase and the rate declined to a lower steady state in the stationary phase. During exponential and early stationary phase growth, PHA had a faster doubling rate than that of total cell biomass (w/w). Results of the morphology studies suggest that PHA inclusion bodies proliferated by budding and reached maximum size by early stationary phase growth. This pattern was observed in minimal and in rich media.

Polyhydroxyalkanoic acids (PHA) are carbon and energy storage polymers that accumulate in inclusion bodies in many bacteria and archaea in response to environmental conditions. This work presents the results of a study of PHA inclusion body-associated proteins and an analysis of their coding region in Bacillus megaterium 11561. A 7,917-bp fragment of DNA was cloned and shown to carry a 4,104-bp cluster of 5 pha genes,phaP, -Q, -R, -B, and -C. The phaP and -Q genes were shown to be transcribed in one orientation, each from a separate promoter, while immediately upstream, phaR, -B, and -C were divergently transcribed as a tricistronic operon. Transfer of this gene cluster to Escherichia coliand to a PhaC− mutant of Pseudomonas putidagave a Pha+ phenotype in both strains. Translational fusions to the green fluorescent protein localized PhaP and PhaC to the PHA inclusion bodies in living cells. The data presented are consistent with the hypothesis that the extremely hydrophilic protein PhaP is a storage protein and suggests that PHA inclusion bodies are not only a source of carbon, energy, and reducing equivalents but are also a source of amino acids.

Polyhydroxyalkanoate (PHA) accumulation and the morphology of PHA inclusion bodies were examined in Bacillus megaterium, strain 11561. Our results show a pattern of PHA degradation and synthesis, and of inclusion body growth and proliferation not previously reported. Degradation of PHA in the lag phase was followed by synthesis of PHA at an accelerating rate during exponential growth. PHA accumulation reached a maximum rate at late exponential/early stationary phase and the rate declined to a lower steady state in the stationary phase. During exponential and early stationary phase growth, PHA had a faster doubling rate than that of total cell biomass (w/w). Results of the morphology studies suggest that PHA inclusion bodies proliferated by budding and reached maximum size by early stationary phase growth. This pattern was observed in minimal and in rich media.