Each of the resulting 19 recombinant plasmids was then introduced into both the wild type (FJ1) and the phaR mutant (FJR1) of R. sphaeroides and analyzed for luciferase activity (Table 2). Results showed that the luciferase activity derived from the wild-type (FJ1) R. sphaeroides harboring recombinant plasmids that carried any of the ZD1839 concentration DNA fragment (FP1, FP1-5, FP1-6, FP1-12, FP1-13, FP1-14, FP1-15, FP1-16, and FP1-17) shown to be able to bind the PhaR protein was approximately 50% (ranging from 2.0 ± 0.1 to 2.3 ± 0.4 RLU) of those (ranging from 4.1 ± 0.3 to 4.8 ± 0.2 RLU) containing the mutated PhaR-binding site to which the PhaR protein could not bind. However, all of the
19 luxAB fusion constructs yielded similar levels of luciferase activity in the phaR mutant (FJRI) of R. sphaeroides. These results strongly suggest that PhaR represses phaP expression. We have previously found that the PhaR protein regulates the expression of the phaP gene, which encodes phasin in R.
Ku0059436 sphaeroides FJ1. While investigating how PhaR regulates phaP expression, we found two 11-bp motifs (CTGCGGC(T)GCAG) present in the promoter region of the phaP gene. Because extensive searches of the GenBank failed to detect the presence of this sequence in the genomes of other bacteria, we characterized the sequence and determined its nucleotide residues that are important for the binding of PhaR. Results showed that the spacer region of this motif was not critical and could be replaced by any three or four bases. However, any base deletion or substitution in the two dyad regions of the palindrome rendered the motif unable to bind PhaR. As oxyclozanide mentioned above, two copies of the PhaR-binding motif exist in the promoter region of phaP. Multiple copies of such motif are also found in
other bacteria. For example, six 18-bp motifs of TGTCACCAACGGGCACTA that have been shown to be the binding site of the PhaR protein of Azotobacter vinelandii are present in the phbR–phbB intergenic region of the organism (Peralta-Gil et al., 2002). Similarly, three PhaR-binding sites with the sequence GCAMMAAWTMMD, where M, W, and D represent A or C, A or G, and A, G, or C, respectively, are found in the promoter region of the phaP gene of Ralstonia eutropha (Potter et al., 2005). In Paracoccus denitrificans, two TGC-rich sequences (TGC1 and TGAII) in the promoter region of the phaP gene were identified as the PhaR-binding sequences (Kojima et al., 2006). The sequences of TGCI and TGCII are CTGCACCGCAGCAA and TGCAATGCTGCGGTGCAG, respectively. These two sequences are similar to the consensus PhaR-binding sequence (CTGCN3−4GCAG) of R. sphaeroides, which we have determined in this study. The significance of the existence of multiple copies of the PhaR-binding site in the genomes of various bacteria remains to be determined. The consensus PhaR-binding sequence (CTGCN3−4GCAG) of R.