ND: no specific PCR product was detected as in the negative contr

ND: no specific PCR product was detected as in the negative control experiment without reverse transcription, and thus was not taken into account for statistic analysis. (B) Expression of the four PhaP phasins. qRT-PCR analysis was performed and the results are presented as described for (A). The transcription profile of phaP and phaR involved in PHB accumulation was also examined using qRT-PCR (FigureĀ 4B). PF-02341066 supplier In contrast to

the PHB-metabolic genes, induction of some of the phaP encoding putative phasins correlated with PHB accumulation. Among the four phaP, phaP4 was most prominently induced under PHB-accumulating conditions in YEM medium reaching levels up to 40 times greater than that of the control, sigA, which encodes the house-keeping sigma factor. These results imply that phaP4 may play an important role in PHB accumulation.

When cultured in YEM, CX-4945 phaP1 and phaP2 were induced to levels up to 10 times greater than the control, implying that phaP1 and phaP2 may also have roles in PHB accumulation. In PSY medium, both phaP1 and phaP4 were induced to lower levels, which may be relevant to the lower PHB accumulation seen in this medium (FigureĀ 3B). On the other hand, expression of phaR was kept at a low level and only barely enhanced upon PHB accumulation, which is consistent with the self-regulation model proposed in R. eutropha[16]. Transcription of phaP3 was almost constant and as low as that of phaR, and thus this paralog might be irrelevant to PHB accumulation under

these conditions. When all these results are considered, it is conceivable that PHB accumulation in B. japonicum during free-living growth may not depend on either the redundancy or expression levels of the genes for PHB synthesis and degradation. Instead, it seems probable that the major mechanism allowing B. japonicum to accumulate large amounts of PHB may be the Progesterone formation of PHB granules stabilized by phasins. The four PhaP phasins and PhaR bound to PHB with different affinities phaP1, phaP2, phaP3, phaP4, and phaR were cloned individually into Escherichia coli and expressed as N-terminally His6-tagged fusion proteins. For unknown reason, the His6-tag fusions could not be purified by the conventional affinity chromatography. Therefore, the crude extracts of E. coli cells containing the fusions were used directly in the PHB binding experiment. Because the N-terminus of each fusion protein contained the same single His6-tag, we assumed that each His6-tag equally reacts with the anti-His6-tag antibody, presumably regardless of fusion partner, and the signal intensities on immunoblots probed for the His6-tag were used to represent the amounts of the phasin fusions contained in the extracts.

Comments are closed.