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J. Biol. Chem., Vol. 282, Issue 25, 18206-18211, June 22, 2007

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Growth-dependent Phosphorylation of the PtsN (EII^Ntr) Protein of Pseudomonas putida^*

From the Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Cientificas, Campus de Cantoblanco, Madrid 28049, Spain

The nitrogen-related branch of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) of Pseudomonas putida includes the ptsN gene encoding the EII^Ntr (PtsN) enzyme. Although the implication of this protein in a variety of cellular functions has been observed in diverse bacteria, the physiological signals that bring about phosphorylation/dephosphorylation of the PtsN protein are not understood. This work documents the phosphorylation status of the EII^Ntr enzyme of P. putida at various growth stages in distinct media. Culture conditions were chosen to include fructose (the uptake of which is controlled by the PTS) or glucose (a non-PTS sugar in P. putida) in minimal medium with casamino acids, ammonia, or nitrate as alternative nitrogen sources. To quantify the relative ratio of PtsN/PtsN P in live cells, we resorted to the in situ electrophoresis of whole bacteria expressing an E-epitope-tagged EII^Ntr followed by the fractionation of the thereby released native proteome in a non-denaturing gel. Although the PtsN species phosphorylated in amino acid His⁶⁸ was detected under virtually all growth scenarios, the relative levels of the non-phosphorylated form varied dramatically depending on the growth phase and the nutrients available in the medium. The share of phosphorylated PtsN increased along growth in a fashion apparently independent of any trafficking of sugars. The large variations of non-phosphorylated PtsN in different growth conditions, in contrast to the systematic excess of the phosphorylated PtsN form, suggested that the P-free PtsN is the predominant signaling species of the protein.

Received for publication, December 4, 2006 , and in revised form, May 2, 2007.

^* This work was supported in part by European Union grants of the Sixth Framework Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 34-91-585-4536; Fax: 34-91-585-4506; E-mail: vdlorenzo{at}cnb.uam.es.

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This article has been cited by other articles:

				K. Pfluger and V. de Lorenzo Evidence of In Vivo Cross Talk between the Nitrogen-Related and Fructose-Related Branches of the Carbohydrate Phosphotransferase System of Pseudomonas putida J. Bacteriol., May 1, 2008; 190(9): 3374 - 3380. [Abstract] [Full Text] [PDF]