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J. Bacteriol. doi:10.1128/JB.01380-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Flavin-dependent thymidylate synthase ThyX activity: Implications for the folate cycle in Bacteria

INSERM Avenir group, Institut de Génétique et de Microbiologie, CNRS UMR8621, Orsay, F-91405; Univ Paris-Sud, Orsay, F-91405; Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS UMR7645, Palaiseau, F-91128; INSERM U696, Palaiseau, F-91128; Department of Biology, Duke University, Durham, NC 27708, USA; Department of Mathematics, Duke University, Durham, NC 27708, USA

^* To whom correspondence should be addressed. Email: hannu.myllykallio{at}igmors.u-psud.fr.

	Abstract

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 have 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 requires not only canonical thymidylate synthase ThyA, but also dihydrofolate reductase FolA. Strikingly, we also found that addition of exogenous methylenetetrahydrofolate transiently inhibits the growth of the different Rhodobacter strains used in this work. To rationalise 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 when the level of reduced folate derivatives is compromised.

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• 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]