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Flavin-Dependent Thymidylate Synthase ThyX Activity: Implications for the Folate Cycle in Bacteria ^,

Damien Leduc ,^1,2,, Frédéric Escartin,^3,4, H. Frederik Nijhout,⁵ Michael C. Reed,⁶ Ursula Liebl,^3,4 Stéphane Skouloubris,^1,2 and Hannu Myllykallio^1,2*

INSERM Avenir group, Institut de Génétique et de Microbiologie, CNRS UMR8621, F-91405 Orsay, France,¹ Université Paris-Sud, F-91405 Orsay, France,² Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS UMR7645, F-91128 Palaiseau, France,³ INSERM U696, F-91128 Palaiseau, France,⁴ Department of Biology, Duke University, Durham, North Carolina 27708,⁵ Department of Mathematics, Duke University, Durham, North Carolina 27708⁶

Received 24 August 2007/ Accepted 10 September 2007

Although flavin-dependent ThyX proteins show thymidylate synthase activity in vitro and functionally complement thyA defects in heterologous systems, direct proof of their cellular functions is missing. Using insertional mutagenesis of Rhodobacter capsulatus thyX, we constructed the first defined thyX inactivation mutant. Phenotypic analyses of the obtained mutant strain confirmed that R. capsulatus ThyX is required for de novo thymidylate synthesis. Full complementation of the R. capsulatus thyX::spec strain to thymidine prototrophy required not only the canonical thymidylate synthase ThyA but also the dihydrofolate reductase FolA. Strikingly, we also found that addition of exogenous methylenetetrahydrofolate transiently inhibited the growth of the different Rhodobacter strains used in this work. To rationalize these experimental results, we used a mathematical model of bacterial folate metabolism. This model suggests that a very low dihydrofolate reductase activity is enough to rescue significant thymidylate synthesis in the presence of ThyX proteins and is in agreement with the notion that intracellular accumulation of folates results in growth inhibition. In addition, our observations suggest that the presence of flavin-dependent thymidylate synthase X provides growth benefits under conditions in which the level of reduced folate derivatives is compromised.

Published ahead of print on 21 September 2007.

Supplemental material for this article may be found at http://jb.asm.org/.

D.L. and F.E. contributed equally to this work.

Present address: Unité de Pathogénie Bactérienne des Muqueuses, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.

This article has been cited by other articles:

• Escartin, F., Skouloubris, S., Liebl, U., Myllykallio, H. (2008). Flavin-dependent thymidylate synthase X limits chromosomal DNA replication. Proc. Natl. Acad. Sci. USA 105: 9948-9952 [Abstract] [Full Text]