A clear DNaseI protection site was observed when RG7112 His-PhbF was present in the assay. The protected site covers

positions 181 to 204 upstream from the translation start site indicating that His-PhbF binds to a 24 bp region of its own promoter which includes the conserved TG[N]TGC[N]3GCAA motif indicated by the MEME program, reinforcing the suggestion that it is the DNA site recognized by the H. seropedicae SmR1 PhbF. Furthermore, a putative sigma 70-dependent promoter was also identified upstream from the PhbF DNA-binding site (position 208 to 212 from the translation start site) (Figure 2C). The proximity of both sites also suggests that H. seropedicae SmR1 PhbF may repress its own expression. We verified the potential GSK923295 in vitro repressor activity of PhbF in E. coli ET8000 by using a gene reporter expression click here assay with phaP1

and phbF promoters fused to the lacZ gene. These genes were chosen because they have the putative PhbF-binding sequence highly similar to the consensus sequence, and also because EMSA assay showed clear interaction with these promoters. The β-galactosidase activities indicated that both phaP1 and phbF promoters were functional in E. coli (Figure 3). However, a clear decrease in β-galactosidase activity is observed if H. seropedicae SmR1 PhbF is present (expressed upon plasmid pMMS31), indicating that PhbF represses the expression of the phasin gene (phaP1) and also of its own gene promoter (phbF). Expression of an unrelated protein (NifH) did not affect β-galactosidase activity of E. coli bearing the phbF::lacZ and phaP1::lacZ fusions (data not shown), reinforcing the repressor effect of PhbF. Figure 3 β-galactosidase activity Bay 11-7085 of E. coli strain ET8000 carrying phbF::lacZ or phbP1::lacZ fusion (plasmids pKADO5 and pMMS35, respectively). Assays were performed as described in Material and Methods. The His-PhbF protein was expressed by the tac promoter from the plasmid pMMS31. Data represents the average ± standard deviation of at least three independent determinations. Background activity of cells carrying pMP220 (control vector)

in the presence of pMMS31 was less than 6 Miller units. Protein domain analysis indicated that PhbF contains a DNA-binding motif and a domain possibly involved in binding PHB. Therefore, we tested if H. seropedicae SmR1 PhbF was able to interact with PHB granules in vitro. The purified His-PhbF was incubated with PHB granules extracted from H. seropedicae SmR1 and the protein remaining in solution was visualized by SDS-PAGE (Figure 4). When His-PhbF was incubated with PHB granules most of the protein was extracted from solution (Figure 4, lane 2). The protein remained bound to the granule even after two washing steps (lanes 3 and 4), and was released only after heating in the presence of SDS, indicating a strong interaction between His-PhbF and PHB. Figure 4 Binding of His-PhbF to PHB granules.