Cochu Armelle, Roy Denis, Vaillancourt Katy, Lemay Jean-Dominique, Casabon Israël, Frenette Michel, Moineau Sylvain, Vadeboncoeur Christian
Groupe de Recherche en Ecologie Buccale, Faculté de Médecine Dentaire, and Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, Université Laval, Québec, Québec, Canada, G1K 7P4.
Appl Environ Microbiol. 2005 Mar;71(3):1364-72. doi: 10.1128/AEM.71.3.1364-1372.2005.
In Streptococcus thermophilus, lactose is taken up by LacS, a transporter that comprises a membrane translocator domain and a hydrophilic regulatory domain homologous to the IIA proteins and protein domains of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The IIA domain of LacS (IIALacS) possesses a histidine residue that can be phosphorylated by HPr(HisP), a protein component of the PTS. However, determination of the cellular levels of the different forms of HPr, namely, HPr, HPr(HisP), HPr(Ser-P), and HPr(Ser-P)(HisP), in exponentially lactose-growing cells revealed that the doubly phosphorylated form of HPr represented 75% and 25% of the total HPr in S. thermophilus ATCC 19258 and S. thermophilus SMQ-301, respectively. Experiments conducted with [32P]PEP and purified recombinant S. thermophilus ATCC 19258 proteins (EI, HPr, and IIALacS) showed that IIALacS was reversibly phosphorylated by HPr(Ser-P)(HisP) at a rate similar to that measured with HPr(HisP). Sequence analysis of the IIALacS protein domains from several S. thermophilus strains indicated that they can be divided into two groups on the basis of their amino acid sequences. The amino acid sequence of IIALacS from group I, to which strain 19258 belongs, differed from that of group II at 11 to 12 positions. To ascertain whether IIALacS from group II could also be phosphorylated by HPr(HisP) and HPr(Ser-P)(HisP), in vitro phosphorylation experiments were conducted with purified proteins from Streptococcus salivarius ATCC 25975, which possesses a IIALacS very similar to group II S. thermophilus IIALacS. The results indicated that S. salivarius IIALacS was phosphorylated by HPr(Ser-P)(HisP) at a higher rate than that observed with HPr(HisP). Our results suggest that the reversible phosphorylation of IIALacS in S. thermophilus is accomplished by HPr(Ser-P)(HisP) as well as by HPr(His~P).
在嗜热链球菌中，乳糖由LacS摄取，LacS是一种转运蛋白，它包含一个膜转运结构域和一个与磷酸烯醇丙酮酸：糖磷酸转移酶系统（PTS）的IIA蛋白及蛋白结构域同源的亲水性调节结构域。LacS的IIA结构域（IIALacS）含有一个组氨酸残基，该残基可被PTS的一种蛋白质成分HPr(HisP)磷酸化。然而，对处于乳糖指数生长期的细胞中不同形式的HPr（即HPr、HPr(HisP)、HPr(Ser-P)和HPr(Ser-P)(HisP)）的细胞水平测定表明，在嗜热链球菌ATCC 19258和嗜热链球菌SMQ - 301中，HPr的双磷酸化形式分别占总HPr的75%和25%。用[32P]PEP和纯化的重组嗜热链球菌ATCC 19258蛋白（EI、HPr和IIALacS）进行的实验表明，IIALacS被HPr(Ser-P)(HisP)可逆磷酸化的速率与用HPr(HisP)测得的速率相似。对几种嗜热链球菌菌株的IIALacS蛋白结构域进行序列分析表明，根据其氨基酸序列可将它们分为两组。19258菌株所属的I组IIALacS的氨基酸序列与II组在11至12个位置上不同。为了确定II组的IIALacS是否也能被HPr(HisP)和HPr(Ser-P)(HisP)磷酸化，用唾液链球菌ATCC 25975的纯化蛋白进行了体外磷酸化实验，该菌株的IIALacS与II组嗜热链球菌的IIALacS非常相似。结果表明，唾液链球菌IIALacS被HPr(Ser-P)(HisP)磷酸化的速率高于用HPr(HisP)观察到的速率。我们的结果表明，嗜热链球菌中IIALacS的可逆磷酸化是由HPr(Ser-P)(HisP)以及HPr(His~P)完成的。
