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

Abstract

In Streptococcus thermophilus, a lactic acid bacterium widely used by the dairy industry, lactose is transported via a secondary symporter-type transport system consisting of a single membrane protein, LacS, that belongs to the glycoside-pentoside-hexuronide:cation symporter family (34), a subgroup of the major facilitator superfamily (39). In most bacteria that use this mode of transport, internalized lactose is hydrolyzed by ␤-galactosidase into glucose and galactose, which are metabolized via the Embden-Meyerhof-Parnas and Leloir pathways, respectively (15,20). However, most strains of S. thermophilus are unable to metabolize galactose due to insufficient expression levels of the galactokinase-encoding galK gene (29,48,49) and release the hexose into the external medium (19). The galactose expulsion phenomenon is closely associated with S. thermophilus LacS, which is able to catalyze two modes of transport: a ⌬p-driven lactose uptake in symport with protons and a lactose-galactose exchange (32-34). The exchange mode is stimulated by phosphorylation of a histidine residue at the C-terminal end of LacS. The phosphate donor has been identified as HPr(HisϳP) (16), and the target histidine is part of a hydrophilic domain homologous to IIA proteins (35). Both HPr and IIA proteins are components of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS).The PTS sequentially catalyzes the transport and PEP-dependent phosphorylation of mono-and disaccharides in a group translocation process involving the non-sugar-specific proteins enzyme I (EI) and HPr and sugar-specific EII proteins or domains called IIA, IIB, IIC, and IID (36,40). Sugar transport by the PTS is initiated by phosphorylation of HPr on a histidine residue at position 15 (His 15 ) by EI at the expense of PEP to generate HPr(HisϳP). The phosphate molecule is then sequentially transferred to the IIA and IIB domains or proteins. Sugar substrates of the PTS pass through the membrane by pores made up of IIC or IIC/IID proteins and are phosphorylated by phospho-IIBs. In addition to its pivotal role in sugar transport and its involvement in the control of S. thermophilus LacS, the HPr(HisϳP) of gram-positive bacteria is also involved, via phosphotransfer reactions, in the regulation of gene transcription and enzyme activity (7,42).HPrs of gram-positive bacteria can also be phosphorylated on a serine residue at position 46 (Ser 46 ) by an ATP-dependent

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

“…The ptsI gene located upstream from hprR ( Fig. 4) therefore remains the only presumed target for the 54 -dependent regulator in this organism.…”

“…There are also numerous proteins of unknown function that contain a PTS domain (6). The PTS-mediated regulation of the lactose transporters from Streptococcus thermophilus (53) and Streptococcus salivarius (54) and of several transcription activators from firmicutes (1) and some Enterobacteriaceae, such as FrzR of E. coli (55), has been studied in detail.…”

“…In alpha-, beta-, and gammaproteobacteria, the ptsN gene, which encodes EIIA Ntr , is frequently located downstream from rpoN (111). The RpoN protein, also known as 54 , is required for the expression of several genes encoding enzymes for the utilization of nitrogen sources, but RpoN also regulates the expression of many other genes and operons. Inactivation of the ptsN gene from Klebsiella pneumoniae lowered the expression of several RpoN-controlled transcription units (112) Ntr .…”

The Activity of the Lactose Transporter from Streptococcus thermophilus Is Increased by Phosphorylated IIA and the Action of β-Galactosidase

“…Previous studies on the role of the IIA domain of LacS had already shown that (i) the LacS-IIA domain is not essential for translocation (10); (ii) LacS is phosphorylated on His-552 by either HPr(His∼P) or HPr(Ser-P)(His∼P) (33,35,36), whereas LacS is not affected by HPr(Ser-P) (12); and (iii) the LacS-IIA domain modulates the activity of the carrier domain. However, it was not established whether regulation took place via interaction of the carrier domain with phosphorylated LacS-IIA, dephosphorylated LacS-IIA, or both LacS-IIA species.…”

“…The stimulation of lactose counterflow upon phosphorylation of LacS-IIA by HPr(His∼P) is most likely caused by a direct interaction of the phosphorylated IIA domain with the carrier domain. As the levels of the different HPr species depend on the strain and growth phase of the organism (11,36), these parameters will determine which of the two histidinephosphorylated HPr species [HPr(His∼P) or HPr(Ser-P)-(His∼P)] will be most involved in the phosphorylation of LacS.…”