Transcriptional activation of NtcA-dependent promoters of Synechococcus sp. PCC 7942 by 2-oxoglutarate in vitro

Abstract

The transcription factor NtcA is a global regulator of nitrogen homeostasis in cyanobacteria. It thus positively regulates the expression of genes related to nitrogen assimilation such as glnA (which encodes glutamine synthetase) and ntcA itself in response to nitrogen shortage or depletion. The binding of NtcA to the glnA and ntcA promoters of Synechococcus sp. PCC 7942 in vitro now has been shown to be enhanced by 2-oxoglutarate. In vitro analysis of gene transcription also revealed that the interaction of NtcA with its promoter element was not sufficient for activation of transcription, and 2-oxoglutarate was required for transcriptional initiation by NtcA. Given that the intracellular concentration of 2-oxoglutarate is inversely related to nitrogen availability, it is proposed that this metabolite functions as a signaling molecule that transmits information on cellular nitrogen status to NtcA and thereby regulates the transcription of genes related to nitrogen assimilation in cyanobacteria.regulatory factor ͉ cyanobacteria ͉ RNA polymerase ͉ nitrogen assimilation

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

“…The use of the glutamine synthetase inhibitor MSX indicated that metabolized (as opposed to ambient) ammonium suppressed the transcription of N genes in the Trichodesmium filaments, similar to our findings in marine Synechococcus (Lindell and Post, 2001;Lindell et al, 2005). This is consistent with the concept that a-oxoglutarate accumulates and acts as the NtcA activator molecule when ammonium supplies are limiting Tanigawa et al, 2002). NtcA-binding affinity to its DNA targets is enhanced by a-oxoglutarate (Vazquez-Bermudez et al, 2002).…”

“…However, enhanced transcription of napA overcame ammonium repression in the presence of the glutamine synthetase inhibitor MSX, consistent with NtcA regulation in Synechococcus spp. (Suzuki et al, 1993;Lindell and Post, 2001;Tanigawa et al, 2002;Lindell et al, 2005). Not all regulatory aspects of NtcA are known in diazotrophic cyanobacteria.…”

SPR analysis of promoter binding of Synechocystis PCC6803 transcription factors NtcA and CRP suggests cross‐talk and sheds light on regulation by effector molecules

“…Crystal structures for the PII-PipX [15,16] Abbreviations: NtcA, nitrogen control factor of cyanobacteria; CRP, cAMP receptor protein, also called CAP, catabolite gene activator protein; PipX, PIIinteracting protein X; SYCRP1, Synechocystis CRP; 2OG, 2-oxoglutarate; RU, relative unit; SPR, surface plasmon resonance; FC1 and FC2, flow cells 1 and 2, respectively of the Biacore chip; IPTG, isopropyl-b-D-thiogalactoside only binds to PII if MgATP also binds), three PipX molecules are sequestered by one PII trimer in such a way that they become unavailable for activating NtcA. 2OG activates NtcA-dependent gene expression regulation [10,11] and also triggers (when ATP is also bound) the release of PipX from PII [13], making PipX available for coactivation of NtcA. The structures of NtcA bound to 2OG alone [15,17] or together with PipX [15] (Fig.…”

“…While CRP is activated by cAMP [5,8,9], NtcA is activated by 2-oxoglutatare (2OG) [10,11], an indicator of the cellular nitrogen level [12], and it is coactivated by a small (10 kDa) monomeric protein called PII-interacting protein X (PipX) [13,14] (Fig. 1).…”

Cyanobacterial Heterocysts

“…During nitrogen deprivation, binding of NtcA to its own promoter leads to an enhanced transcription of ntcA. In addition to being autoregulated at the level of gene expression, transcriptional regulation exerted by NtcA is modulated in response to the C-to-N balance of the cells and 2-oxoglutarate has been shown to exert a positive effect on NtcA function both in vivo and in vitro (Tanigawa et al, 2002;Valladares et al, 2008;Va´zquez-Bermu´dez et al, 2003). 2-Oxoglutarate is the substrate of GOGAT and thus the acceptor molecule for fixed nitrogen.…”