FIGURE 2.
Transcription of NsrR1 is regulated by NtcA. (A) Nitrogen-responsive expression of NsrR1 in Nostoc sp. PCC 7120 and its ntcA mutant derivative CSE2. Expression was analyzed in cells grown in the presence of ammonium and transferred to medium containing no source of combined nitrogen (N2) for the number of hours indicated. The upper panel shows hybridization to the NsrR1 probe. The lower panel shows hybridization to a probe for 5S RNA used as loading and transfer control. Size markers are indicated on the left. (B) Comparison of promoter sequences upstream of nsrR1 homologs encoded in cyanobacterial genomes. The verified TSS in Nostoc sp. PCC 7120 (Mitschke et al., 2011) is marked with a red circle. Putative NtcA binding sites (highlighted in gray) and –10 boxes (framed) are indicated. (C) Electrophoretic mobility shift assays showing binding of purified His-tagged NtcA protein to a DNA fragment containing the wild type promoter of NsrR1 from Nostoc sp. PCC 7120 or a mutated version altered in the positions indicated in red. The position of retarded DNA fragments is indicated with a black triangle, whereas the free fragments are indicated with empty triangles.