Functional Characterization of the Incomplete Phosphotransferase System (PTS) of the Intracellular Pathogen Brucella melitensis Marie Dozot1, Sandrine Poncet2, Ce´ cile Nicolas1, Richard Copin1, Houda Bouraoui2, Alain Maze´ 2, Josef Deutscher2, Xavier De Bolle1, Jean-Jacques Letesson 1 * 1 Research Unit in Molecular Biology (URBM), University of Namur, Namur, Belgium, 2 Laboratoire de Microbiologie et Ge´ne´tique Mole´culaire (INRA-CNRS-AgroParisTech), Thiverval-Grignon, France Abstract Background: In many bacteria, the phosphotransferase system (PTS) is a key player in the regulation of the assimilation of alternative carbon sources notably through catabolic repression. The intracellular pathogens Brucella spp. possess four PTS proteins (EINtr, NPr, EIIANtr and an EIIA of the mannose family) but no PTS permease suggesting that this PTS might serve only regulatory functions. Methodology/Principal Findings: In vitro biochemical analyses and in vivo detection of two forms of EIIANtr (phosphorylated or not) established that the four PTS proteins of Brucella melitensis form a functional phosphorelay. Moreover, in vitro the protein kinase HprK/P phosphorylates NPr on a conserved serine residue, providing an additional level of regulation to the B. melitensis PTS. This kinase activity was inhibited by inorganic phosphate and stimulated by fructose-1,6 bisphosphate. The genes encoding HprK/P, an EIIAMan-like protein and NPr are clustered in a locus conserved among a-proteobacteria and also contain the genes for the crucial two-component system BvrR-BvrS. RT-PCR revealed a transcriptional link between these genes suggesting an interaction between PTS and BvrR-BvrS. Mutations leading to the inactivation of EINtr or NPr significantly lowered the synthesis of VirB proteins, which form a type IV secretion system. These two mutants also exhibit a small colony phenotype on solid media. Finally, interaction partners of PTS proteins were identified using a yeast two hybrid screen against the whole B. melitensis ORFeome. Both NPr and HprK/P were shown to interact with an inorganic pyrophosphatase and the EIIAMan-like protein with the E1 component (SucA) of 2-oxoglutarate dehydrogenase. Conclusions/Significance: The B. melitensis can transfer the phosphoryl group from PEP to the EIIAs and a link between the PTS and the virulence of this organism could be established. Based on the protein interaction data a preliminary model is proposed in which this regulatory PTS coordinates also C and N metabolism. Citation: Dozot M, Poncet S, Nicolas C, Copin R, Bouraoui H, et al. (2010) Functional Characterization of the Incomplete Phosphotransferase System (PTS) of the Intracellular Pathogen Brucella melitensis. PLoS ONE 5(9): e12679. doi:10.1371/journal.pone.0012679 Editor: Edgardo Moreno, Universidad Nacional, Costa Rica Received April 12, 2010; Accepted August 15, 2010; Published September 10, 2010 Copyright: 2010 Dozot et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Fonds de la Recherche Fondamentale Collective (FRFC, Belgium, convention 2.4521.04), by Action de recherche concertee (ARC, No. 04/09-325 and 08/13-015) and by an agreement with Commisariat general aux Relations internationales de la Communaute franc¸aise de Belgique - Fonds National pour la Recherche Scientifique - Centre National de la Recherche Scientifique (CGRI-FNRS-CNRS, reference PVB/ADK/FR/ad/2681). M. Dozot held a PhD fellowship from the Fonds pour la formation a` la Recherche dans l’Industrie et dans l’Agriculture (FRIA). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jean-jacques.letesson@fundp.ac.be Introduction In order to successfully colonize an ecological niche, bacteria have to integrate different signals indicating environmental changes, and subsequently trigger an adequate adaptative response by modulating their cellular activities. The appropriate response to changes in nutrient availability, for example, relies on diversified mechanisms, including global regulation systems such as the phosphoenolpyruvate (PEP): carbohydrate phosphotrans- ferase system (PTS). The PTS catalyzes the uptake and con- comitant phosphorylation of carbohydrates and is composed of several proteins forming a phosphorelay transferring the phos- phoryl group from PEP to the incoming sugar: (i) the general PTS proteins enzyme I (EI) and HPr are cytoplasmic components usually common to all PTS carbohydrates; (ii) the enzyme II complex is specific for one or several sugars and is generally composed of at least three domains (or distinct proteins) including the cytoplasmic EIIA and EIIB, and the membrane-crossing EIIC (sometimes also EIID) that constitutes the permease of the system [1,2]. PTS proteins are usually phosphorylated on a conserved histidine, with the exception of most EIIB components that are phosphorylated on a cysteine. Besides its function in the transport and phosphorylation of carbon sources, the PTS plays a key role in the regulation of many aspects of bacterial physiology, including carbon catabolite repression (CCR) (for reviews see [1,2,3]). Interestingly, a paralog of the classical PTS was proposed to function as a regulatory link between carbon and nitrogen metabolism. This system was first identified in Escherichia coli and PLoS ONE | www.plosone.org 1 September 2010 | Volume 5 | Issue 9 | e12679