Fig. 3

Analysis of SpdR binding motifs. a SpdR-binding assays to CC0517 and CC1746. DNA fragments containing the regions upstream of genes CC0517 and CC1746 were ³²P-labeled and incubated with increasing concentrations of His₆-SpdR (25, 50, 100, 250 and 500 nM) in an electrophoretic mobility shift assay (EMSA). As negative control, a reaction was carried out without His₆-SpdR (−). In a competition assay, His₆-SpdR was utilized at a 250 nM concentration and a 30x excess of unlabeled competitor fragment was added as follows: S, unlabeled specific fragment; N, unlabeled non-specific fragment. b Sequences recognized by the SpdR protein. An in silico search in C. crescentus NA1000 genome was performed with the consensus CTGCGAC-N₅-GTCGCAG derived by the EMSA experiments. The ‘DNA pattern’ tool of RSA website [29] was used in the search, and one substitution was allowed. The position indicated refers to the first nucleotide of the sequence shown relative to the putative start codon in the NA1000 strain (+1). The same sequence is proposed to control expression of CC2151 and CC2152, which are divergently transcribed. The position of this sequence with respect to each gene is shown; for CC2152, the position refers to the nucleotide at the position 3’ of the sequence shown, which corresponds to the 5’ end of the reverse complementary sequence. Gene numbers refer to the CB15 strain (CC) and the correspondent number in NA1000 strain (CCNA)