The DNA-binding factor BIFA in Anabaena sp. strain PCC 7120

Abstract

BifA (previously named VF1) is a DNA-binding protein from the cyanobacterium Anabaena sp. strain PCC 7120. BifA was originally identified based on its binding activity to the upstream region of xisA, and the glnA, rbcL, and nifH promoter regions in vitro, suggesting that BifA interacts with genes expressed in both vegetative cells and heterocysts. DNase I footprint analysis of BifA binding to glnA showed a 24-bp protected region between the major glnA transcription start site used in vegetative cells (RNA$sb{rm II}$) and the major transcription start site used under nitrogen-deficient conditions (RNA$sb{rm I}$). The two BifA binding sites on the rbcL promoter were found to be centered around and upstream of the transcription start site. Comparison of the BifA binding sites on the glnA, xisA, and rbcL upstream regions revealed the consensus recognition sequence TGT(N$sb{rm 9 or 10}$)ACA. We have cloned the bifA gene (672 bp) using a genetic selection strategy. The cloning strategy was based on a transcriptional interference assay in which a strong synthetic promoter, conII, interferes with the expression of an aadA gene. BifA is related to the Crp family of prokaryotic regulatory proteins and is suggested to be a global regulator in PCC 7120. A bifA null mutant was constructed by interrupting the gene with an $Omega$ Sp$sp{rm r}$/Sm$sp{rm r}$ cassette. The bifA mutant was not able to grow with nitrate or atmospheric dinitrogen as the sole nitrogen source, but could be grown on medium containing ammonium ion. The bifA mutant was unable to form heterocysts after induction on a medium lacking combined nitrogen. The bifA mutant was complemented with a DNA fragment containing the bifA gene and 251-bp of upstream sequence but not with a similar construction containing 87-bp of upstream sequence. Northern analysis of bifA in the wild-type strain during nitrogen stepdown showed a peak of bifA message at an early stage (12 hours) of heterocyst induction. Ammonium-grown cultures of the bifA mutant showed reduced gLnA message and the absence of a 1.7-kb transcript, the major glnA transcript in the wild type after nitrogen stepdown.