Brucella PTS Figure 9. The EIIAMan-like protein interacts with SucA. DIC and corresponding fluorescent images were taken from E.coli (pSKoriTcat –pBad- divIVA-sucA and pMR10kan-ptsM-gfp) grown in different conditions: (A) without IPTG and arabinose (no synthesis of EIIAMan-GFP and SucA-DivIVA). (B) with IPTG and no arabinose (synthesis of EIIAMan-GFP) and (C) with both IPTG and arabinose (EIIAMan-GFP and SucA-DivIVA are synthesized). Only in the latter case EIIAMan-GFP co-localizes with SucA-DivIVA at the cell poles. doi:10.1371/journal.pone.0012679.g009 pBad-divIVA-sucA and pMR10kan-ptsM-gfp) showed a bipolar fluorescence pattern only when arabinose was present (Fig. 9C). This illustrates that SucA was targeted to the pole by DivIVA and able to recruit the EIIAMan-GFP fusion at the same location, thus confirming the interaction detected in the Y2H experiments. Discussion Since PTS permeases are lacking in a-proteobacteria, their soluble PTS proteins are not involved in carbohydrate transport and phosphorylation, but probably participate only in a regulatory phosphorelay (Fig. 10) [25,26,27]. In this paper, we present the first extensive biochemical and genetic characterization of the PTS components in an organism that lacks PTS permeases. A few studies on similar systems have previously been carried out, but were either limited to biochemical studies of PTS protein phosphorylation [60] or to genetic studies of mutants [61]. First we established that in B. melitensis the phosphoryl group transfer from PEP to the EIIAs is fully functional. Second, several pieces of evidence allow us to propose a link between the PTS and the virulence of B. melitensis. Finally, we report a connection between the PTS and systems likely to maintain the N/C balance. These three points are discussed in detail hereunder. 1- The PTS phosphorelay of B. melitensis is fully functional and senses the metabolic state B. melitensis EINtr autophosphorylates with PEP and transfers the phosphoryl group to the conserved His30 of NPr before it is passed on to either EIIANtr or the EIIAMan-like protein (Fig. 10). EINtr probably senses the PEP availability (PEP/pyruvate ratio), that is translated into relative levels of phosphorylated vs. non-phosphor- ylated forms of NPr and EIIAs [62]. In addition to His30, NPr is also phosphorylated on a serine residue. Similar to firmicutes, B. melitensis possesses an HprK/P using ATP or PPi as phosphoryl donor. Only phosphorylation of NPr with ATP is stimulated by FBP (Fig. 2B). Identical observations were made for L. casei HprK/ P [45]. However, Brucella spp lack a 6-phosphofructokinase and consequently hexose catabolism does not occur via the Embden- Meyerhof-Parnas pathway, but is redirected through the pentose phosphate and perhaps the Entner-Doudorof pathway. Accord- ingly, the only pathway that is expected to produce FBP is gluconeogenesis [63]. We therefore propose that, in contrast to firmicutes, the FBP signal sensed by B. melitensis HprK/P reflects gluconeogenetic instead of glycolytic activity. High gluconeoge- netic flux will probably activate HprK/P (via FBP) (Fig. 2B), which in turn will slow the PEP-dependent phosphoryl transfer from P- Ser-NPr to the EIIAs (Fig. 4) [1,64,65]. Interestingly, an inorganic pyrophosphatase named PPa inter- acts with NPr and HprK/P in Y2H tests (Fig. 8). PPi serves as substrate for the kinase reaction and is formed during P-Ser-HPr dephosphorylation [45]. Hydrolysis of PPi by PPa not only lowers the PPi concentration, but also produces inorganic phosphate (Pi), which inhibits both, ATP- and PPi-dependent kinase activities of B. melitensis HprK/P (Fig. 2C). Elevated PPa activity might therefore reduce phosphorylation on Ser61 of NPr. Similarly, Mijakovic [45] proposed that the B. subtilis pyrophosphatase YvoE (the yvoE gene is located in the hprK operon) indirectly decreases the kinase activity of HprK/P, and stimulates P-Ser-HPr dephosphorylation by HprK/P. The physical interaction of PPa with NPr and HprK/P might allow efficient regulation of HprK/P activity in B. melitensis. Alternatively, a link might exist between the PTS and the ppGpp production/degradation system (also called stringent response) as was demonstrated in E. coli [66]. NPr might affect the PPa-catalyzed conversion of PPi to Pi and thus modulate the PPi-producing ppGpp degrading activity of Rsh (RelA/SpoT homologue). Purified B. melitensis HprK/P barely dephosphorylated P-Ser- NPr under in vitro conditions (Fig. 3). Similar observations were PLoS ONE | www.plosone.org 9 September 2010 | Volume 5 | Issue 9 | e12679