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).
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(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 [32P]PEP and purified recombinant S. thermophilus ATCC 19258 proteins (EI, HPr, and IIALacS) showed that IIALacS was reversibly phosphorylated by HPr(Ser-P)(His~P) at a rate similar to that measured with HPr(His~P). 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(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 IIALacS very similar to group II S. thermophilus IIALacS. The results indicated that S. salivarius IIALacS 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 IIALacS in S. thermophilus is accomplished by HPr(Ser-P)(His~P) as well as by HPr(His~P).
Cochu
Armelle
A
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.
Roy
Denis
D
Vaillancourt
Katy
K
Lemay
Jean-Dominique
JD
Casabon
Israël
I
Frenette
Michel
M
Moineau
Sylvain
S
Vadeboncoeur
Christian
C
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D048249:Streptococcus thermophilus / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D026901:Membrane Transport Proteins; D012694:Serine; D006639:Histidine; C021499:lactose permease; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr