Gene products from Bacillus megaterium involved in the metabolism of polyhydroxyalkanoic acid (PHA) and the biogenesis of PHA inclusion -bodies
Polyhydroxyalkanoates (PHAs) comprise a family of macromolecules produced by many bacteria as a carbon and energy reserve and are perceived to have commercial potential as biodegradable thermoplastics. To investigate the biogenesis of PHA inclusion-bodies and the functions of inclusion-body proteins from Bacillus megaterium strain 11561, we identified and cloned a 7.9 kb DNA fragment harboring five genes, phaP, -Q, -R, -B, and -C specifying proteins having known or putative functions in PHA metabolism and/or inclusion-body biogenesis. Sequence similarities to known pha genes identified phaB and -C as specifying acetoacetyl-CoA reductase and PHA synthase, respectively. Putative proteins encoded by phaP, -Q, and -R were not ascribed functions due to lack of significant similarities to known proteins. Both the functionality of the pha gene cluster with respect to PHA accumulation and the transcriptional organization of the genes were determined. Subsequent studies were carried out to further investigate functions of phaP, -Q, and -R.
PhaP was established as a major PHA inclusion-body associated protein and was shown to localize to inclusion-bodies in living cells. Further, we demonstrated a phasin-like role for this protein due to its affect on the formation of PHA inclusion-bodies. In addition, our data is consistent with PhaP functioning as a storage protein, implying that the role of PHA inclusion-bodies may be that of a reserve of amino acids in addition to reduced carbon. Regulation of phaP was influenced by PhaQ We showed that PhaQ is a transcriptional repressor of phaP.
Moreover, we demonstrated the binding of PhaQ to inclusion-bodies, suggesting that its mode of regulation may involve its localization. Similarly, we showed that PhaR is bound to PHA inclusion-bodies. Our data demonstrated the requirement of phaR for PHA accumulation in vivo and that both PhaC and PhaR were necessary for PHA synthase activity in vitro. Evidence suggests that PHA synthase from strain 11561 can exist in an active or inactive state and that this state is either directly or indirectly influenced by PhaR. A working model is proposed to describe the roles of PhaP, -Q and -R in the metabolism of PHA and biogenesis of PHA inclusion-bodies.