ANABAENA SP STRAIN PCC-7120 BIFA GENE ENCODING A SEQUENCE-SPECIFIC DNA-BINDING PROTEIN CLONED BY IN-VIVO TRANSCRIPTIONAL INTERFERENCE SELECTION

VF1 is a DNA-binding protein from the cyanobacterium Anabaena sp. stra in PCC 7120. VF1 was originally identified on the basis of its binding affinity to the upstream region of xisA, which encodes a heterocyst-s pecific site-specific recombinase. VF1 also binds to the glnA, rbcL, a nd nifH promoters in vitro, suggesting that VF1 interacts with genes e xpressed in both vegetative cells and heterocysts. The role of VF1 in regulating gene expression in PCC 7120 is unknown. As a step towards t he goal of understanding the role of VF1 in regulating gene expression , we have cloned the bifA gene by using a genetic selection strategy. bifA encodes a protein, BifA, that has chromatographic and DNA-binding properties indistinguishable from those of VF1. The cloning strategy was based on a transcriptional interference assay in which a strong sy nthetic promoter, conII, interferes with the expression of an aadA gen e, which provides resistance to spectinomycin and streptomycin (S. J. Elledge, P. Sugiono, L. Guarente, and R. W. Davis, Proc. Natl. Acad. S ci. USA 86:3689-3693, 1989). A selection plasmid, pAM994, which has th e conII promoter negatively regulated by a VF1-binding site, was used to enrich for VF1-producing clones from an expression library containi ng PCC 7120 DNA fragments. Mobility shift assays were used to identify a 672-bp open reading frame that encoded VF1-like binding activity. T he deduced BifA amino acid sequence shows 77% identity to NtcA, which is a global regulator involved in nitrogen control in Synechococcus sp . strain PCC 7942. Both BifA and NtcA belong to the cyclic AMP recepto r protein (CRP) family of prokaryotic regulatory proteins. Genes simil ar to envM, hisB, and ORF60-S were found near the bifA gene.