Research Unit in Molecular Biology (URBM), University of Namur, Namur, Belgium.
PLoS One. 2010 Sep 10;5(9):e12679. doi: 10.1371/journal.pone.0012679.
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 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/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.
在许多细菌中，磷酸转移酶系统（PTS）是调节替代碳源同化的关键因素，特别是通过分解代谢抑制。胞内病原体布鲁氏菌属拥有四种 PTS 蛋白（EINtr、NPr、EIIANtr 和甘露糖家族的 EIIA），但没有 PTS 透性酶，这表明该 PTS 可能仅具有调节功能。
方法/主要发现：体外生化分析和两种形式的 EIIANtr（磷酸化或非磷酸化）的体内检测证实，布鲁氏菌 melitensis 的四种 PTS 蛋白形成了一个功能性磷酸传递系统。此外，在体外，蛋白激酶 HprK/P 在保守丝氨酸残基上磷酸化 NPr，为布鲁氏菌 PTS 提供了额外的调节水平。这种激酶活性被无机磷酸盐抑制，被果糖-1,6-二磷酸刺激。编码 HprK/P、EIIAMan 样蛋白和 NPr 的基因簇在α-变形菌中保守的一个基因座中，还包含关键的双组分系统 BvrR-BvrS 的基因。RT-PCR 显示这些基因之间存在转录联系，表明 PTS 和 BvrR-BvrS 之间存在相互作用。导致 EINtr 或 NPr 失活的突变显著降低了 VirB 蛋白的合成，VirB 蛋白形成一种 IV 型分泌系统。这两个突变体在固体培养基上也表现出小菌落表型。最后，使用酵母双杂交筛选整个布鲁氏菌 ORFeome，鉴定了 PTS 蛋白的相互作用伙伴。NPr 和 HprK/P 均被证明与无机焦磷酸酶相互作用，而 EIIAMan 样蛋白与 2-氧戊二酸脱氢酶的 E1 组分（SucA）相互作用。
结论/意义：布鲁氏菌能够将磷酸基团从 PEP 转移到 EIIAs 上，并且可以建立 PTS 与该生物体毒力之间的联系。基于蛋白质相互作用数据，提出了一个初步的模型，其中这个调节 PTS 还协调 C 和 N 代谢。
