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

doi:10.1073/pnas.0502337102

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

^*Laboratoire de Chimie Bactérienne, UPR9043, Centre National de la Recherche Scientifique, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France; ^‡College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072, People's Republic of China; ^§Spectropole, Faculté de Saint-Jérôme, Case 511, Marseille Cedex 20, France; ^**National Key Laboratory of Agricultural Microbiology, Huazhong Agriculture University, Wuhan 430070, People's Republic of China; and ^¶Département de Chimie, UMR6114, Centre National de la Recherche Scientifique, 163 Avenue de Luminy, 13288 Marseille Cedex, France

Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved May 19, 2005 (received for review March 22, 2005)

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 ≈0.1 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 ¹⁹F 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.

Footnotes

↵ ∥ To whom correspondence regarding chemistry should be addressed. E-mail: ling{at}afmb.cnrs-mrs.fr. †† To whom correspondence regarding biology should be addressed. E-mail: cczhang{at}ibsm.cnrs-mrs.fr.
↵ † S.L. and H.C. contributed equally to this work.
Author contributions: S.L., H.C., S.B., and F.Z. performed research; S.L., L.P., S.B., and C.-C.Z. analyzed data; S.B., L.P., and C.-C.Z. designed research; S.B., L.P., and C.-C.Z. wrote the paper; and F.Z. and L.P. contributed new reagents/analytic tools.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: 2-OG, 2-oxoglutarate; DFPA, 2,2-difluoropentanedioic acid; MAS, magic angle spinning.
Data deposition: The atomic coordinates have been deposited in the Cambridge Structural Database, Cambridge Crystallographic Data Centre, Cambridge CB2 1EZ, United Kingdom (CSD reference no. 261398).