Journal: Journal of Bacteriology
Article Title: cis-Acting Sequences Required for NtcB-Dependent, Nitrite-Responsive Positive Regulation of the Nitrate Assimilation Operon in the Cyanobacterium Synechococcus sp. Strain PCC 7942
Figure Lengend Snippet: (A) Schematic representation of the insertion of transcriptional luxAB fusions into the cmp locus of the Synechococcus chromosome. The recombinational plasmid pYK5, which cannot replicate in the cyanobacterium, contains a promoterless luxAB gene cluster, a spectinomycin resistance cassette ( spe r ), and a PlacI q :: ntcA transcriptional fusion, which are flanked by Synechococcus cmpB and cmpC genes. Various fragments of the nirA-nirB intergenic region were cloned between the Spe I (Sp) and Bgl II (Bg) sites preceding the luxAB gene cluster. Transformation of Synechococcus with the plasmid resulted in spectinomycin-resistant strains in which homologous recombination had transferred the reporter construct to the cyanobacterial chromosome. (B) The nirA-nirB intergenic region of Synechococcus sp. strain PCC 7942, showing the nucleotide sequences of both DNA strands. Numbers above the sequences indicate positions of the nucleotides with respect to the translation start site of nirA . The transcription start sites of the nirA and nirB operons are indicated by filled triangles. The putative −10 elements of the promoters of the nirA and nirB operons are underlined. The three NtcA-binding sites ( nir I, nir II, and nir III) and the two potential binding sites for LysR-type DNA-binding proteins (L1 and L2) are boxed. The letters above and below the sequences represent the base replacements created in strains YKA4, YKA5, YKA6, YKA7, YKA9, and YKB4. Sites of the luxAB fusion are also indicated.
Article Snippet: The PlacI q :: ntcA transcriptional fusion, the promoterless luxAB gene cluster, and the spectinomycin resistance cassette were sequentially cloned into pT7Blue T-Vector (Novagen) to form a 5.0-kbp insert flanked by Nhe I recognition sites, which was subsequently excised from the plasmid and ligated into the Xba I site in the cmpC fragment in pG23 to form pYK5.
Techniques: Plasmid Preparation, Clone Assay, Transformation Assay, Homologous Recombination, Construct, Periodic Counter-current Chromatography, Binding Assay, DNA Binding Assay