Expanding the Direct HetR Regulon in Anabaena sp. Strain PCC 7120

Abstract

In response to a lack of environmental combined nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 differentiates nitrogen-fixing heterocyst cells in a periodic pattern. HetR is a transcription factor that coordinates the regulation of this developmental program. An inverted repeat-containing sequence in the hepA promoter required for proheterocyst-specific transcription was identified based on sequence similarity to a previously characterized binding site for HetR in the promoter of hetP.

The HetR-binding site that activates expression of patA in vegetative cells is required for normal heterocyst patterning in Anabaena sp. PCC 7120

“…2) causes great reduction in interaction with HetR. It is notable that although Videau et al [19] found no binding of recombinant HetR to the hetR site in EMSA and Flaherty et al [16] found no binding of GST-HetR to the patA site in vitro, we observed clear band shifts in EMSA, showing that EF-Ts-HetR bound to those sites. We suggest that the EF-Ts-HetR fusion that we used was likely more active than the refolded recombinant HetR or the GST-HetR fusion used by the other two laboratories, because EF-Ts greatly enhances the solubility of overexpressed proteins in E. coli [25].…”

“…In addition, the binding of HetR to another predicted site, the one upstream of hepA [10], was confirmed and shown to be much weaker than that to the hetZ site ( Fig. S1) as reported [19]. EF-Ts-HetR (2 or 4 lmol/L) was then used to bind 40-bp fragments P hetP -40, P hetZ -40, P hetR -40 and P patA -40, respectively, with or without RGSGR, the pentapeptide inhibitor of HetR [2,10] (Fig.…”

“…Deletion of HetRbinding sites up-regulated the expression of alr3234 and alr0202, respectively, in whole filaments and specifically in heterocysts. Inhibitory effects of HetR have been reported on other genes [16,19], but alr0202 is unique in that it is repressed by the HetR-binding site specifically in heterocysts. In C. crescentus and B. subtilis, the master (response) regulator can differentially control the expression of target genes by activation and inhibition [27,28].…”

“…HetR has been shown to directly up-regulate hetP and hetZ, two genes that are involved early in the differentiation of heterocysts [10,17], and hepA, a gene involved in formation of the heterocyst envelope polysaccharide layer [19].…”

“…The crystal structure of the HetR-DNA complex has been reported, showing the importance of GGG(n5)CCC and several flanking base pairs in the interaction [18]. More recently, HetR was shown to regulate hepA, a gene involved in formation of the polysaccharide layer of heterocyst envelope, via a lower affinity recognition site in its promoter [19].…”