Nonmetabolizable analogue of 2-oxoglutarate elicits heterocyst differentiation under repressive conditions in Anabaena sp. PCC 7120

Abstract

In response to combined nitrogen starvation in the growth medium, the filamentous cyanobacterium Anabaena sp. PCC 7120 is able to develop a particular cell type, called a heterocyst, specialized in molecular nitrogen fixation. Heterocysts are regularly intercalated among vegetative cells and represent 5-10% of all cells along each filament. In unicellular cyanobacteria, the key Krebs cycle intermediate, 2-oxoglutarate (2-OG), has been suggested as a nitrogen status signal, but in vivo evidence is still lacking. In this study we show that nitrogen starvation causes 2-OG to accumulate transiently within cells of Anabaena PCC 7120, reaching a maximal intracellular concentration of Ϸ0.1 mM 1 h after combined nitrogen starvation. A nonmetabolizable fluorinated 2-OG derivative, 2,2-difluoropentanedioic acid (DFPA), was synthesized and used to demonstrate the signaling function of 2-OG in vivo. DFPA is shown to be a structural analogue of 2-OG and the process of its uptake and accumulation in vivo can be followed by 19 F magic angle spinning NMR because of the presence of the fluorine atom and its chemical stability. DFPA at a threshold concentration of 0.3 mM triggers heterocyst differentiation under repressing conditions. The multidisciplinary approaches using synthetic fluorinated analogues, magic angle spinning NMR for their analysis in vivo, and techniques of molecular biology provide a powerful means to identify the nature of the signals that remain unknown or poorly defined in many signaling pathways.cyanobacterium ͉ Krebs cycle ͉ nitrogen metabolism ͉ nutrient starvation ͉ signal transduction

ATPase as a switch in P II signal transduction

“…2-OG is a key intermediate of the Krebs cycle and a signal of nitrogen status (1,5,6). P II has been considered as a signaling protein that integrates the status of carbon and nitrogen availability, as well as the energy charge.…”

The nitrogen interaction network in Synechococcus WH5701, a cyanobacterium with two PipX and two PII-like proteins

“…Due to the lack of 2-oxoglutarate dehydrogenase in cyanobacteria, synthesis of 2-oxoglutarate represents the final step in the oxidative branch of the TCA cycle and directly links 2-oxoglutarate levels to nitrogen assimilation . Thus, 2-oxoglutarate accumulates during nitrogen starvation, making this metabolite an excellent indicator of the intracellular carbon to nitrogen balance (Forchhammer, 2004;Laurent et al, 2005). In line with this, interactions of the nitrogen regulators P II and NtcA are modulated by this metabolite.…”

Regulation of intracellular free calcium concentration during heterocyst differentiation by HetR and NtcA in Anabaena sp. PCC 7120

“…5). These results suggest that 2-OG also is involved in the regulation of [Ca 2ϩ ] i , enforcing its signaling in the initiation of heterocyst differentiation (8). The difference of ccbP expression after nitrogen deprivation between the wild type and ntcA Ϫ (Fig.…”

“…One of the signals that triggers the differentiation from a vegetative cell to a heterocyst in response to nitrogen deprivation is the increase of the intracellular concentration of ␣-ketoglutarate (2-OG) (7,8). Another important signal in heterocyst differentiation is the intracellular concentration of free calcium, [Ca 2ϩ ] i (9,10).…”