Transcription Activation by NtcA in the Absence of Consensus NtcA-Binding Sites in an Anabaena Heterocyst Differentiation Gene Promoter

Abstract

Heterocyst differentiation is orchestrated by the N control transcriptional regulator NtcA and the differentiation-specific factor HetR. In Anabaena sp. strain PCC 7120, the devBCA operon is expressed from two different promoters activated upon N stepdown. The distal devB promoter (transcription start point [TSP] located at position −704) represents a canonical class II NtcA-activated promoter, including a consensus NtcA-binding site centered 39.5 nucleotides upstream from the TSP. Transcription activation from a second TSP (−454) requires NtcA and is impaired in hetR mutants. In a wild-type background, three different DNA fragments, including both or each individual promoter, directed gfp expression localized mainly to proheterocysts and heterocysts. Expression was undetectable in an ntcA background and, for the fragment including the proximal promoter alone, also in a hetR background. In spite of the absence of consensus NtcA-binding sequences between the two TSPs, NtcA was shown to interact with this DNA region, and NtcA and its effector, 2-oxoglutarate, were necessary and sufficient for in vitro transcription from the −454 TSP. No HetR binding to the DNA or in vitro transcription from the proximal devB TSP promoted by HetR alone were detected. However, a moderate positive effect of HetR on NtcA binding to the DNA between the two devB TSPs was observed. The proximal devB promoter appears to represent a suboptimal NtcA-activated promoter for which HetR may act as a coactivator, with the physiological effect of restricting gene activation to conditions of prevalence of high NtcA and HetR levels, such as those taking place during heterocyst differentiation.

Dynamics of Cylindrospermopsin Production and Toxin Gene Expression in <i>Aphanizomenon ovalisporum</i>

“…Similarly to those described in C. raciborskii CS-505 and R. brookii D9 by Stuken et al [14]. The putative NtcA sequences are non-canonical, but that is not necessarily an impediment to recognize NtcA, since some other sequences have been suggested for NtcA recognition [25] [33], which differ from the NtcA sequence GTAN 8 TAC reported as the optimal for NtcA binding. As far as we know, only one canonical intragenic NtcA box has been described in cyanobacteria [34], moreover 1762 intragenic NtcA binding region have been found in the Anabaena PCC7120 genome, presenting the abundance of NtcA targets and function in cyanobacteria [35].…”

Regulation of Internal Promoters in a Zinc-Responsive Operon Is Influenced by Transcription from Upstream Promoters

“…Runoff transcription assays were performed by using recombinant Anabaena RNA polymerase (RNAP), reconstituted and purified as previously described (17). Reactions were set up in a total volume of 15 l containing 40 mM Tris-HCl (pH 8.0), 50 mM KCl, 10 mM MgCl 2 , 0.1 mM EDTA, 0.5 mg/ml bovine serum albumin (BSA), 5% glycerol, 30 to 60 fmol DNA fragment, and 150 nM Anabaena RNA polymerase.…”

PipX, the coactivator of NtcA, is a global regulator in cyanobacteria

“…Although direct regulation and binding in the absence of predictable NtcA binding sites has recently been suggested (27,28), the rather attenuated NtcA-dependent pattern of Synpcc7942_1363 could be explained Table S2) relative to the TSS with activation (green) and repression (red) functions (details are provided in SI Appendix, Fig. S3 A and B).…”