Two Anabaena sp. strain PCC 7120 DNA-binding factors interact with vegetative cell- and heterocyst-specific genes

Abstract

The DNA-binding factor BifA (previously called VF1) binds upstream of the developmentally regulated site-specific recombinase gene xisA in the cyanobacterium Anabaena sp. strain PCC 7120. Besides binding xisA, BifA also binds the glnA, rbcL, and nifH promoter regions. DNase I footprint analysis of BifA binding to glnA showed a protected region -125 to -148 bp upstream of the translation start site. The binding site is between the major glnA transcription start site used in vegetative cells (RNAII) and the major transcription start site used under nitrogen-deficient conditions (RNAI). The two BifA-binding sites on the rbcL promoter were localized to a 24-bp region from +12 to -12 nucleotides and to a 12-bp region from -43 to -54 nucleotides with respect to the transcription start site. Comparison of the BifA binding sites on the glnA, xisA, and rbcL upstream regions revealed the consensus recognition sequence TGT(N9 or 10) ACA. We have identified a second DNA-binding activity (factor 2) that interacts with rbcL and xisA upstream regions. Factor 2 can be resolved from BifA by heparin-Sepharose chromatography and was present in a bifA mutant. Analysis of partially purified vegetative cell and heterocyst extracts showed that whereas BifA was present in both cell types, factor 2 was present only in vegetative cells. DNase I footprint analysis of factor 2 binding to rbcL showed protection of a 63-bp region between positions -15 and -77 with respect to the transcription start site. The factor 2 binding site on xisA was localized to a 68-bp region that showed considerable overlap with the BifA binding sites.

Decoupling of ammonium regulation and ntcA transcription in the diazotrophic marine cyanobacterium Trichodesmium sp. IMS101

“…Two ORFs (ORF5 and ORF6) immediately downstream of ntcA were in the same orientation as ntcA (Figure 4). ORF5 was first identified in Anabaena PCC 7120 (Ramasubramanian et al, 1994(Ramasubramanian et al, , 1996 and ntcA-ORF5 form a conserved gene pair in nearly all cyanobacteria (AF Post, unpublished data). As no clear promoter elements were identifiable upstream of ORF5, we studied the transcription of ntcA and ORF5 by RT-PCR.…”

“…Two putative binding sites were identified upstream of ntcA in Anabaena PCC 7120 (Muro-Pastor et al, 2002), but they were not positioned at 40.5 nt as in most NtcA-activated promoters (Luque et al, 1994;Herrero et al, 2001). NtcA is required for heterocyst formation but its requirement for expression of the nif genes is not well established (Frias et al, 1994;Ramasubramanian et al, 1994;Muro-Pastor et al, 1999). Studies of the nifHDK operon transcription in Anabaena PCC 7120 and Trichodesmium IMS101 do not identify NtcA recognition sequences (Ramasubramanian et al, 1994;Chen et al, 1998).…”

“…Studies of the nifHDK operon transcription in Anabaena PCC 7120 and Trichodesmium IMS101 do not identify NtcA recognition sequences (Ramasubramanian et al, 1994;Chen et al, 1998). NtcA binds only weakly to these promoter regions (Ramasubramanian et al, 1994), but it is required for nif gene expression in Anabaena (Frias et al, 1994). Transcript levels of ntcA and nif genes were inversely proportional in the unicellular diazotroph Cyanothece sp.…”

“…NtcA is required for heterocyst formation but its requirement for expression of the nif genes is not well established (Frias et al, 1994;Ramasubramanian et al, 1994;Muro-Pastor et al, 1999). Studies of the nifHDK operon transcription in Anabaena PCC 7120 and Trichodesmium IMS101 do not identify NtcA recognition sequences (Ramasubramanian et al, 1994;Chen et al, 1998). NtcA binds only weakly to these promoter regions (Ramasubramanian et al, 1994), but it is required for nif gene expression in Anabaena (Frias et al, 1994).…”

Cloning, sequencing, and regulation of the global nitrogen regulator gene ntcA in the unicellular diazotrophic cyanobacterium Cyanothece sp. strain BH68K

“…1). It has been demonstrated in PCC 7942 and PCC 7120 that NtcA binds to the promoter region of and activates genes involved in nitrogen control such as glnA, nir, and ntcA itself (3,9,12,13). Given its similarity to the NtcA proteins from PCC 7942 and PCC 7120, it is possible that the Cyanothece NtcA protein may bind similar target sequences.…”