Abstract
BackgroundIn 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 α-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/SignificanceThe 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.
Highlights
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 phosphoryl transfer chain of this system is composed of three proteins, EINtr, NPr and EIIANtr that are the respective paralogs of enzyme I (EI), HPr, and EIIA of the fructose phosphotransferase system (PTS) family; they are not associated with PTS permeases [4,5,6,7] but carry out multiple regulatory functions [8]
Proteins and HPr kinase/phosphorylase The genome of Brucella melitensis 16M [22] contains three genes encoding homologues of the proteins composing the PTSNtr (EINtr, NPr and EIIANtr, respectively) and ptsM/BMEI2032 encoding a homologue of an EIIA of the mannose PTS family (EIIAManlike)
Summary
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. 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 called the nitrogen PTS (PTSNtr) [4,5,6,7]. The phosphoryl transfer chain of this system is composed of three proteins, EINtr (encoded by ptsP), NPr (encoded by ptsO) and EIIANtr (encoded by ptsN) that are the respective paralogs of EI, HPr, and EIIA of the fructose PTS family; they are not associated with PTS permeases [4,5,6,7] but carry out multiple regulatory functions [8]. 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
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