The Doubly Phosphorylated Form of HPr, HPr(Ser-P)(His∼P), Is Abundant in Exponentially Growing Cells of Streptococcus thermophilus and Phosphorylates the Lactose Transporter LacS as Efficiently as HPr(His∼P)

Abstract

ABSTRACT 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 (IIA LacS ) possesses a histidine residue that can be phosphorylated by HPr(His∼P), a protein component of the PTS. However, determination of the cellular levels of the different forms of HPr, namely, HPr, HPr(His∼P), HPr(Ser-P), and HPr(Ser-P)(His∼P), 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 [ 32 P]PEP and purified recombinant S. thermophilus ATCC 19258 proteins (EI, HPr, and IIA LacS ) showed that IIA LacS was reversibly phosphorylated by HPr(Ser-P)(His∼P) at a rate similar to that measured with HPr(His∼P). Sequence analysis of the IIA LacS 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 IIA LacS from group I, to which strain 19258 belongs, differed from that of group II at 11 to 12 positions. To ascertain whether IIA LacS from group II could also be phosphorylated by HPr(His∼P) and HPr(Ser-P)(His∼P), in vitro phosphorylation experiments were conducted with purified proteins from Streptococcus salivarius ATCC 25975, which possesses a IIA LacS very similar to group II S. thermophilus IIA LacS . The results indicated that S. salivarius IIA LacS was phosphorylated by HPr(Ser-P)(His∼P) at a higher rate than that observed with HPr(His∼P). Our results suggest that the reversible phosphorylation of IIA LacS in S. thermophilus is accomplished by HPr(Ser-P)(His∼P) as well as by HPr(His∼P).