J. Schoen, A. G. Lawrence, J. Tian, A. He, P. Liu, J. Stubbe, and A. J. Sinskey
Department of Biology and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, 02139, USA
In order to better understand the interplay of PHB synthesis and degradation with the four-dimensional process of granule assembly and disassembly, time-resolved studies of the regulation of expression of PHB genes in Wautersia (formerly Ralstonia) eutropha H16 were undertaken. We examined mRNA transcript levels by RT-PCR and protein concentrations by quantitative Western blotting for PhaA, PhaB, PhaC, PhaP, PhaR, PhaZ1, PhaZ2, and PhaZ3 during growth on defined media, when PHB is accumulated to high levels and subsequently degraded. mRNA and protein levels were assayed at several timepoints as cells underwent nitrogen limitation and began to accumulate PHB, during PHB production, and after cells were switched to a carbon-deficient, nitrogen-sufficient PHB utilization medium. Our results show that the expression of both PhaP and PhaZ2 are upregulated during PHB production and downregulated during utilization. PhaC, PhaR, and PhaZ1 are all shown to be present at constitutive levels. Neither PhaZ3 mRNA transcript nor protein were detected under these culture conditions. Implications for the models of phaP regulation, phaR autoregulation and constitutive activity of PhaZ1 will be discussed, in addition to speculation on the role of PhaZ2.
論文來源：International Symposium on Biological Polyesters ,Auguest 22-27, 2004
