The Doubly Phosphorylated Form of HPr, HPr(Ser-P)(HisP), Is ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2005, p. 1364–1372 0099-2240/05/$08.00⫹0 doi:10.1128/AEM.71.3.1364–1372.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 71, No. 3

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) Armelle Cochu, Denis Roy, Katy Vaillancourt, Jean-Dominique LeMay, Israe¨l Casabon, Michel Frenette, Sylvain Moineau, and Christian Vadeboncoeur* ´ cologie Buccale, Faculte´ de Me´decine Dentaire, and De´partement de Biochimie et Groupe de Recherche en E de Microbiologie, Faculte´ des Sciences et de Geńie, Universite´ Laval, Que´bec, Que´bec, Canada Received 6 May 2004/Accepted 28 September 2004

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(SerP)(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(SerP)(His⬃P) as well as by HPr(His⬃P). 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(His⬃P) (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 (His15) by EI at the expense of PEP to generate HPr(His⬃P). 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(His⬃P) 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 (Ser46) by an ATP-dependent

* Corresponding author. Mailing address: Groupe de Recherche en ´ cologie Buccale, Faculte´ de Me´decine Dentaire, and De´partement E de Biochimie et de Microbiologie, Faculte´ des Sciences et de Geńie, Universite´ Laval, Que´bec, Que´bec, Canada, G1K 7P4. Phone: (418) 656-2319. Fax: (418) 656-2861. E-mail: Christian.Vadeboncoeur@bcm .ulaval.ca. 1364

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TABLE 1. Strains and plasmids Relevant characteristicsa

Strain or plasmid

Escherichia coli BL21(DE3) LMG194 S. salivarius ATCC 25975 S. thermophilus ATCC 19258 SMQ-301 Plasmids pBAD-HisB pET28a pET29a pST118 pST121 pST124 pETI-16 pHPW18 pLacS11A pHPK229 a

Source or reference

F⫺ ompT hsdSB(rB⫺ mB⫺) gal dcm (DE3) F⫺ ⌬lacX74 galE thi rpsL ⌬phoA (pvuII) ⌬ara714 leu::Tn10 Wild type

Novagen Invitrogen 18

Wild type Wild type

American Type Culture Collection 45

Expression vector, Ampr Expression vector, Kanr Expression vector, Kanr S. thermophilus His6-HPr fusion in pBAD-HisB S. thermophilus His6-EI fusion in pET28a S. thermophilus His6-IIALacS fusion in pET29a S. salivarius His6-EI fusion in pET28a S. salivarius His6-HPr fusion in pBAD-HisB S. salivarius His6-IIALacS fusion in pET29a S. salivarius HPrK/P in pET28a

Invitrogen Novagen Novagen 5 5 This work 24 11 24 3

Ampr, ampicillin resistance; Kanr, kanamycin resistance.

protein kinase/phosphorylase called HPrK/P, generating HPr(Ser-P) (8, 30). This phosphorylated form of HPr is not involved in sugar transport but plays a key role in inducer exclusion and expulsion mechanisms and in carbon catabolite repression in association with the catabolic control protein CcpA (4). The presence of functional EI and HPrK/P was demonstrated in S. thermophilus (5, 16). It was also established that S. thermophilus HPr possesses the His15 and Ser46 phosphorylation sites common to all gram-positive bacteria. However, S. thermophilus HPr possesses a proline at position 68, whereas HPrs from other gram-positive bacteria have an alanine, a serine, or an aspartate at this position (5). Interestingly, amino acid substitutions in this region of the protein interfere with HPr functions (21, 44). Nevertheless, this distinctive feature does not prevent phosphorylation of S. thermophilus HPr by EI (5, 16) or by HPrK/P (16). Indeed, determination of the cellular levels of the different forms of HPr indicates that HPr and HPr(Ser-P) dominate in exponentially growing cells of S. thermophilus ST11, while HPr and HPr(His⬃P) take over at the end of the exponential growth phase and in the stationary phase (16). From these results, a model describing how the phosphorylation of LacS fluctuates during growth on lactose has been proposed. During rapid growth, LacS would be mostly unphosphorylated since HPr(Ser-P) is unable to transfer its phosphate group. Conversely, the increase in HPr(His⬃P) at the end of the exponential growth phase would enhance LacS phosphorylation (16, 17). This model, however, does not take into account the presence of another potential phosphorylated form of HPr, HPr(Ser-P)(His⬃P). This doubly phosphorylated form of HPr is abundant in rapidly growing Streptococcus salivarius and Streptococcus mutans cells and may account for up to 70% of total cellular HPr (31, 44, 46). In S. mutans cells growing in a chemostat at a low rate (doubling time of 7 h) under conditions of glucose excess (200 mM), the doubly phosphorylated form of HPr represents nearly 23% of total HPr, indicating that the synthesis of HPr(Ser-P)(His⬃P) may also occur in very slowly

growing cells (43). Moreover, it was recently demonstrated that HPr(Ser-P)(His⬃P) is able to transfer a phosphate group to S. salivarius IIALacS (24). The abundance of HPr(Ser-P)(His⬃P) in some streptococcal species and its capacity to transfer a phosphate molecule to a IIA domain raised the question of whether this form of HPr can be synthesized at significant levels in growing S. thermophilus cells and, if so, whether it is involved in the control of LacS. In the work reported here, we showed that HPr(SerP)(His⬃P) is abundant in exponentially growing S. thermophilus cells. We also unequivocally demonstrated that HPr(SerP)(His⬃P) is involved in the reversible phosphorylation of LacS. MATERIALS AND METHODS Strains, plasmids, and growth conditions. The strains and plasmids used in this study are listed in Table 1. S. salivarius was grown at 37°C and S. thermophilus at 42°C in M17 broth (Difco Laboratories) supplemented with 0.4% lactose. Escherichia coli strains were grown aerobically at 37°C with agitation. Strain BL21(DE3) was grown in Luria-Bertani medium while strain LMG194 was grown in RM medium containing (per liter) 2% (wt/vol) Casamino Acids, 0.2% (wt/vol) glucose, 1 mM MgCl2, 6 g of Na2HPO4, 3 g of KH2PO4, 0.5 g of NaCl, and 1 g of NH4Cl, adjusted to pH 7.4 with NaOH. When necessary, 50 ␮g of ampicillin/ml and 30 ␮g of kanamycin/ml were added. DNA purification and manipulations. Chromosomal DNA was isolated from streptococci as described previously (13). DNA manipulations were performed with standard procedures (1). E. coli BL21(DE3) cells were made competent and transformed with plasmid DNA by electroporation (41). DNA fragments used for sequencing and subcloning were recovered from agarose gels with a QIAquick purification kit (Qiagen Inc.). The PCRs were performed with a DNA Thermal Cycler 480 (Perkin Elmer) as described previously (24). HPr determination. The levels of the different forms of HPr in exponentially growing cells were determined by crossed immunoelectrophoresis with anti-S. salivarius HPr rabbit polyclonal antibodies (46). The cell extracts were prepared from exponential-phase cells grown on 0.4% lactose as described previously (31). A standard curve was obtained with purified S. salivarius HPr whose concentration was determined by the BCA assay (Pierce Chemical Co.). The gels were analyzed with the Image Master 2D program (Amersham Bioscience). Protein purification. His tag-free EI and HPr were purified from S. salivarius as described previously (47). S. thermophilus ATCC 19258 and S. salivarius His6-HPrs were purified from E. coli LMG194 bearing pST118 and pHPW18, respectively (5, 11). S. thermophilus ATCC 19258 and S. salivarius His6-EIs were

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purified from E. coli BL21(DE3) bearing pST121 and pETI-16, respectively (5, 24). The portion of S. thermophilus ATCC 19258 lacS coding for IIALacS was cloned into the overexpression plasmid pET-29a(⫹), giving plasmid pST124, with the approach used to clone the DNA fragment coding for S. salivarius IIALacS (24). The protein IIALacS coded for by pST124 possessed two additional amino acids (LE) and a His6 tag at the C terminus. S. thermophilus and S. salivarius His6-IIAsLacS was purified from E. coli BL21(DE3) bearing pST124 or pLacSIIA as described elsewhere (24). After separation on Ni-nitrilotriacetic acid columns (11), His6-HPrs and His6EIs were further purified on a Superdex 200 HR column (Pharmacia), whereas His6-IIAsLacS was further purified by chromatography on a MonoQ HR 5/5 column (Pharmacia). S. salivarius HPrK/P was purified without a His tag from E. coli bearing pHPK229 as described previously (3). Recombinant S. thermophilus His6-EI (ε280 nm ⫽ 30,640 cm⫺1/M), S. salivarius His6-EI (ε280 nm ⫽ 31,920 cm⫺1 M⫺1), S. thermophilus and S. salivarius His6-HPrs (ε280 nm ⫽ 2,560 cm⫺1 M⫺1), as well as S. thermophilus and S. salivarius His6-IIAsLacS (ε280 nm ⫽ 6,970 cm⫺1 M⫺1) were quantified by spectrophotometry at 280 nm. Synthesis of His6-HPr(Ser-P). S. thermophilus and S. salivarius His6HPr(Ser-P) were synthesized with purified S. salivarius HPrK/P (5 ␮g) and His6-HPr (500 ␮g), as described previously (24). The purity of the His6HPr(Ser-P) was verified by polyacrylamide gel electrophoresis (PAGE) under native conditions (22) and quantified as described above for recombinant His6HPr. Phosphorylation of His6-IIALacS by His6-HPr(His⬃32P) and His6-HPr(SerP)(His⬃32P). [32P]PEP was prepared according to the method of Mattoo and Waygood (26) with purified PEP carboxykinase from E. coli K-12 HFr 3000, which was kindly provided by A. H. Goldie (University of Saskatchewan). Phosphorylation of His6-IIALacS by His6-HPr(His⬃P) was performed as described previously (24) with the following modifications. Phosphorylation was carried out in 50 mM Tris-acetate (pH 7.5) containing 1 mM dithiothreitol, 2 mM MgCl2, 0.8 or 1.5 ␮M His6-EI, 2 to 20 ␮M His6-HPr, 4.5 or 10 ␮M His6-IIALacS, and 1 mM [32P]PEP (15 ␮Ci/␮mol). The proteins were separated by sodium dodecyl sulfate (SDS)-PAGE on a 12% or 15% polyacrylamide gel, and 32P-labeled proteins were revealed by exposure to a Phosphoimager (Fuji imaging plate for bio imaging analyzer, type BAS-IIIS). Intensities were quantified with Image Gauge version 3.0 software (Fujifilm). The synthesis of His6-HPr(Ser-P)(His⬃32P) was carried out in 50 mM Trisacetate (pH 7.5) containing 1 mM dithiothreitol, 2 mM MgCl2, 0.8 or 1.5 ␮M His6-EI, and 12 ␮M (S. salivarius) or 35 ␮M (S. thermophilus) His6-HPr(Ser-P). After incubation of the mixture at 37°C for 10 min, 1 mM [32P]PEP (15 ␮Ci/ ␮mol) was added, and the solution was incubated at 37°C for an additional 10 min. The solution was then placed at 10°C, and His6-IIALacS was added at a final concentration of 4.5 or 10 ␮M. Analysis of the reaction products was carried out as described for the phosphorylation of His6-IIALacS by His6-HPr(His⬃P). Dephosphorylation of 32P⬃His6-IIALacS by HPr and HPr(Ser-P). The methods used for the synthesis and purification of S. thermophilus 32P⬃His6-IIALacS with His tag-free S. salivarius EI and HPr and those used to study the dephosphorylation of 32P⬃His6-IIALacS by HPr(Ser-P) are described elsewhere (24). The reaction products were analyzed as described for the phosphorylation of His6-IIALacS. Computer analysis of sequence data. Computer-assisted DNA and protein sequence data analyses were performed with the Genetics Computer Group sequence analysis software package, version 10.3 (Genetics Computer Group, Inc.) (10). Nucleotide sequence accession number. The nucleotide sequence of the 3⬘ end of S. thermophilus ATCC 19258 lacS coding for the IIALacS domain has been entered in the GenBank database under accession number AY601651.

RESULTS Cellular levels of different forms of HPr in S. thermophilus. S. thermophilus ATCC 19258 cells were grown on lactose and harvested during the exponential growth phase under conditions that prevented significant changes in the ratios of the various forms of HPr (46). The cellular levels of the different forms of HPr were determined by subjecting membrane-free cellular extracts to crossed immunoelectrophoresis with anti-S. salivarius HPr rabbit polyclonal antibodies. Although HPr (His⬃P) and HPr(Ser-P) migrate together during electrophoresis, they can be distinguished by boiling a portion of the

FIG. 1. Determination of intracellular levels of the different forms of HPr in S. thermophilus ATCC 19258 by crossed immunoelectrophoresis. Each sample contained 5 ␮g of cytoplasmic proteins obtained from lactose-grown cells and was probed with polyclonal anti-HPr rabbit antibodies. Panel A: proteins from cells harvested at mid-log phase; Panel B; same as panel A, but incubated 3 min at 100°C before electrophoresis. The numbers indicate immunoprecipitates corresponding to (1) HPr(Ser-P)(His⬃P); (2) HPr(His⬃P) and HPr(Ser-P); (3) HPr(Ser-P); and (4) unphosphorylated HPr. The long arrows indicate the directions of the first and second dimensions. The black dot corresponds to the initiation point of the first-dimension electrophoresis.

cell extract: the phosphoamidate bond of HPr(His⬃P) is heat labile, while the phosphoester bond of HPr(Ser-P) is heat stable. Quantitative comparison of the peaks of the boiled and unboiled samples of the same cell extract made it possible to estimate the cellular concentrations of each of the four forms of HPr (46). The results shown in Fig. 1 indicate that S. thermophilus ATCC 19258 possessed large amounts of HPr(Ser-P)(His⬃P). As expected, the peak corresponding to this intermediate was not observed when samples were heated for 3 min before electrophoresis, while the peak corresponding to HPr(Ser-P) increased after this treatment. A standard curve obtained with purified HPr from S. salivarius was used to quantify the in vivo amounts of the different forms of HPr in exponentially growing cells (Table 2). The cellular levels of the various HPr intermediates were also determined in the industrial S. thermophilus strain SMQ-301 and, for comparison, in S. salivarius grown in M17 medium containing lactose. Although the relative proportions of the different forms of HPr varied from one strain to the other, the three strains contained substantial amounts of HPr(Ser-P)(His⬃P), as reported previously for S. salivarius


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TABLE 2. Cellular levels of the different forms of HPr in S. thermophilus and S. salivariusa Organism

S. thermophilus S. salivarius

Mean % of total HPrb ⫾ SE HPr

HPr(His⬃P)

HPr(Ser-P)

HPr (Ser-P)(His-P)

Mean total HPr (␮g/mg of protein) ⫾ SE

N.D. 5⫾4 3⫾3

10 ⫾ 4 56 ⫾ 7 3⫾3

15 ⫾ 2 14 ⫾ 3 41 ⫾ 4

75 ⫾ 6 25 ⫾ 0 53 ⫾ 4

39.4 ⫾ 6.7 75 ⫾ 1.2 46.0 ⫾ 6.2

Strain

ATCC 19258 SMQ-301 ATCC 25975

a

Cells were grown in M17 broth supplemented with 0.4% lactose and harvested at mid-log phase. The levels of the different forms of HPr are expressed as percentages of total HPr. The values (⫾ standard errors) are the means of three determinations carried out with samples from two cultures (six samples in all). N.D., not detected. b

(31, 44, 46), Thus, the presence of a proline at position 68 of S. thermophilus HPr (5) did not prevent the synthesis of the doubly phosphorylated product in vivo. Unphosphorylated HPr made up an insignificant proportion of the total HPr in all species, while the relative proportions of HPr(Ser-P) were almost threefold lower in S. thermophilus strains than in S. salivarius. Unlike S. salivarius and S. thermophilus ATCC 19258, exponentially growing cells of S. thermophilus SMQ-301 possessed significant amounts of HPr(His⬃P). Synthesis of HPr(Ser-P) and HPr(Ser-P)(His⬃P) in vitro. HPr from S. thermophilus was purified from E. coli with a His6 tag added at the N terminus. The protein was over 95% pure, as determined by SDS-PAGE (Fig. 2). To synthesize the doubly phosphorylated form of HPr, His6-HPr was first incubated with ATP and HPrK/P to produce HPr(Ser-P). Native PAGE and staining with silver nitrate were used to verify whether all the free HPr was transformed into HPr(Ser-P) (data not shown). The absence of free HPr in the HPr(Ser-P) solution was also verified by analyzing the reaction products of a PEPdependent phosphorylation experiment carried out with [32P]PEP, EI, and HPr(Ser-P). After separation of the proteins by native PAGE and detection of the phosphoproteins by autoradiography, we found that all of the labeled proteins generated migrated to positions corresponding to those of His6-HPr(Ser-P)(His⬃32P) and His6-EI(His⬃32P) (Fig. 3). Experiments conducted with unlabeled PEP revealed that 15% of

FIG. 2. Analysis of purified S. thermophilus His6-HPr and His6IIALacS by SDS-PAGE. Proteins (2.5 ␮g for HPr and 5 ␮g for IIALacS) were separated on 15% polyacrylamide gels and revealed by staining with Coomassie blue. Molecular mass markers are shown in the right lane of each gel.

the HPr(Ser-P) was transformed into the doubly phosphorylated form of HPr (data not shown). Phosphorylation of S. thermophilus IIALacS by HPr(SerP)(His⬃P). The 3⬘ end of S. thermophilus ATCC 19258 lacS, which codes for IIALacS, was purified from E. coli BL21(DE3). The recombinant S. thermophilus His6-IIALacS was over 95% pure, as estimated by SDS-PAGE (Fig. 2). The purified protein migrated electrophoretically as a protein with a molecular mass of ⬃21,500 Da, which was close to the molecular mass calculated from the translated amino acid sequence (19,689 Da). To determine whether IIALacS could be phosphorylated by HPr(Ser-P)(His⬃P), we first incubated His6-EI, His6-HPr(SerP), and [32P]PEP together at 37°C for 10 min to synthesize HPr(Ser-P)(His⬃32P). The reaction mixture was then placed at 10°C for 5 min. His6-IIALacS was then added, and the reaction was allowed to continue for a further 2 min. A similar experiment was conducted in parallel with free HPr as a control. The reaction products were then separated by SDS-PAGE and analyzed by autoradiography (Fig. 4A). The results unequivocally demonstrated that S. thermophilus IIALacS could be phosphorylated by the doubly phosphorylated form of HPr (Fig. 4A, lane 1). To determine whether this reaction was reversible, we incubated purified His6-IIALacS(His⬃32P) with His6-HPr(Ser-P) under the conditions described previously (24). Analysis of the products by SDS-PAGE and autoradiography revealed the presence of HPr(Ser-P)(His⬃32P), indicating that His6-IIALacS(His⬃32P) was able to transfer a phosphate group to HPr(Ser-P) (Fig. 4B). Phosphorylation rates of S. thermophilus IIALacS by HPr(His⬃P) and HPr(Ser-P)(His⬃P). To determine whether

FIG. 3. PEP-dependent phosphorylation of HPr and HPr(Ser-P). The reactions were carried out with purified S. thermophilus proteins in 50 mM Tris-acetate (pH 7.5) containing 1 mM dithiothreitol, 2 mM MgCl2, 1 mM [32P]PEP (15 ␮Ci/␮mol), 1.5 ␮M His6-EI, and 2 ␮M HPr (lane 1) or 35 ␮M HPr(Ser-P) (lane 2). The proteins were separated by native PAGE, and phosphoproteins were revealed by autoradiography.

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FIG. 4. Reversible phosphorylation of His6-IIALacS by His6-HPr(Ser-P)(His⬃P) and His6-HPr(His⬃P). A. Phosphorylation of His6-IIALacS. The reactions were carried out under the conditions described in the legend to Fig. 3B except that the EI concentration was 0.8 ␮M. Dephosphorylation of P⬃IIALacS. 32P⬃IIALacS was dephosphorylated by HPr and HPr(Ser-P) in 50 mM Tris-acetate (pH 7.5) containing 1 mM dithiothreitol, 2 mM MgCl2, and HPr or HPr(Ser-P) in a total volume of 15 ␮l. After the mixture was incubated for 10 min at 37°C, 32P⬃IIALacS was added, and the incubation was extended for 5 min. The proteins were separated by SDS-PAGE and revealed by autoradiography. The signal observed under the band corresponding to 32P⬃IIALacS is a denatured form of phospho-IIALacS that arose during the purification of 32P⬃IIALacS.

His6-HPr(Ser-P)(His⬃P) could transfer its phosphate group to His6-IIALacS as efficiently as His6-HPr(His⬃P), we compared the phosphorylation rates of His6-IIALacS by both forms of HPr under identical experimental conditions. Since only 15% of the His6-HPr(Ser-P) was transformed into the doubly phosphorylated form of HPr under the conditions used, the concentration of His6-HPr(Ser-P) in the reaction medium was corrected so that the concentrations of HPr(His⬃P) and HPr(Ser-P)(His⬃P) were similar. The experiments were conducted at 10°C to slow down the reaction, which allowed the phosphorylation rates to be determined. As shown in Fig. 5A, His6-HPr(Ser-P)(His⬃P) and His6-HPr(His⬃P) phosphorylated IIALacS at similar rates. Moreover, similar rates were obtained when S. thermophilus HPr was replaced with S. salivarius HPr (Fig. 5A and 5B), indicating that the Pro68 in S. thermophilus HPr did not interfere with the phosphotransfer activity of the protein. Phosphorylation of IIALacS by HPr(Ser-P)(His⬃P): a widespread phenomenon among S. thermophilus strains? Phosphorylation of IIALacS by HPr(Ser-P)(His⬃P) requires two conditions: the ability of the cells to synthesize the doubly phosphorylated form of HPr, and the ability of this doubly phosphorylated form to transfer its phosphate group to IIALacS. To determine whether IIALacS phosphorylation by HPr(Ser-P)(His⬃P) is common among S. thermophilus strains, we first compared the amino acid sequences of three proteins involved in the process, HPr, EI, and IIALacS, with the sequences available in the databases. The HPr amino acid sequences have so far been determined for seven S. thermophilus strains: ATCC 19258, SMQ-119, SMQ-173, and SMQ-301 (5), CNRZ302 (accession number AAL47557), LMD-9 (http://genome.jgi-psf.org/draft_microbes /strth.info.html), and LMG18311 (http://www.biol.ucl.ac.be /gene/genome/). All of these HPrs have the same amino acid

sequences. The complete amino acid sequences of EI are available for four strains, ATCC 19258, CNRZ302, LMD-9, and LMG18311, and exhibit over 99% identity. Clearly, these strains should produce significant amounts of HPr(SerP)(His⬃P), as found in strains ATCC 19258 and SMQ-301, assuming that they produce adequate amounts of functional HPrK/P. Interestingly, a survey of the amino acid sequences of IIALacS from various S. thermophilus strains suggests that these protein domains can be separated into two groups (Fig. 6). The amino acid sequence of IIALacS from group I, to which IIALacS from strain ATCC 19258 belongs, differs from the amino acid sequence of IIALacS from group II, to which strain SMQ-301 belongs, at 11 to 12 positions (indicated in bold in Fig. 6). Some of these substitutions are located near the histidine residue that can be phosphorylated (shaded in grey in Fig. 6). Analysis of the amino acid sequence of S. salivarius IIALacS indicated that most S. thermophilus IIALacS from group II differed from S. salivarius IIALacS at only three positions (V532I, K561N, and E616K) and that the sequence near the histidine residue that can be phosphorylated is almost totally conserved (EDGVIVLIHVGIGTVKLN), the only modification being the substitution K561N in S. salivarius IIALacS (Fig. 6). As we have already purified S. salivarius IIALacS and showed that it can be phosphorylated by HPr(Ser-P)(His⬃P) (24), we used it as the representative S. thermophilus group II IIALacS. To determine whether HPr(Ser-P)(His⬃P) was as efficient as HPr(His⬃P) in transferring its phosphate group to a IIALacS from group II, we determined phosphorylation rates with purified EI, HPr, and IIALacS from S. salivarius. The results indicated that S. salivarius IIALacS was phosphorylated by HPr(Ser-P)(His⬃P) more rapidly than by HPr(His⬃P) (Fig. 7).


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FIG. 5. Rates of S. thermophilus IIALacS phosphorylation by HPr(His⬃P) and HPr(Ser-P)(His⬃P). The reactions were carried out as described in the legend to Fig. 3 except that the concentration of EI was 0.8 ␮M and that of HPr was 5 ␮M. After the mixtures were incubated for 10 min to allow the synthesis of HPr(His⬃P) or HPr(Ser-P)(His⬃P), IIALacS was added to a final concentration of 4.5 ␮M. Samples were removed at intervals, and the proteins were separated by SDS-PAGE (12% polyacrylamide). The dried gels were exposed for 18 h on a phosphoimager. Results from two independent experiments are expressed in units of phosphostimulated luminescence (PSL). The bars indicate the standard error. Panel A: (‚) phosphorylation of S. thermophilus IIALacS by S. salivarius HPr(Ser-P)(His⬃P); (E) phosphorylation of S. thermophilus IIALacS by S. thermophilus HPr(Ser-P)(His⬃P); (F) phosphorylation of S. thermophilus IIALacS by S. thermophilus HPr(His⬃P). Panel B: (Œ) phosphorylation of S. thermophilus IIALacS by S. salivarius HPr(His⬃P); (F) phosphorylation of S. thermophilus IIALacS by S. thermophilus HPr(His⬃P). The results presented in panels A and B were carried out with different preparations of [32P]PEP.

DISCUSSION Previous experiments conducted in vitro showed that phosphorylation of HPr on Ser46 by HPrK/P severely reduces the rate of phosphorylation on His15 by EI and, conversely, the phosphorylation of HPr by EI inhibits the phosphorylation on Ser46 by HPrK/P (9, 37). The fact that Bacillus subtilis contains barely detectable amounts of HPr(Ser-P)(His⬃P) is consistent with these findings (25, 28). However, the doubly phosphorylated form of HPr is present in large amounts in S. mutans and

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S. salivarius (12, 31, 43, 46) and has also been detected, although in lower quantities, in Lactococcus lactis (27) and Enterococcus faecalis (23). In this paper, we showed that HPr(SerP)(His⬃P) was also present in substantial amounts in exponentially lactose-growing S. thermophilus cells, suggesting that this doubly phosphorylated form of HPr is widespread among streptococci. This indicates that phosphorylation of HPr(Ser-P) by EI and/or of HPr(His⬃P) by HPrK/P occurs at a substantial rate in vivo in these lactic acid bacteria. Our results contrast with those of Gunnewijk and Poolman (16), who reported that HPr(Ser-P)(His⬃P) represents at most 5% of total HPr in S. thermophilus ST11. These authors also detected large amounts of free HPr in exponentially growing cells, while this form was not detected in strains ATCC 19258 and SMQ-301 or in S. salivarius or S. mutans (12, 31, 43, 46). We have no explanation for these differences, but it is noteworthy that the method used by Gunnewijk and Poolman (16) to determine the cellular concentrations of the various forms of HPr and the growth conditions were different. On the other hand, our results indicated that the relative proportions of HPr(Ser-P)(His⬃P) varied from strain to strain. Therefore, it cannot be ruled out that the low levels of HPr(Ser-P)(His⬃P) found in strain ST11 was merely strain specific. HPr(Ser-P) has several regulatory functions in gram-positive bacteria, including control of transporter activity and regulation of gene transcription (4). The regulation of sugar metabolism in low-G⫹C gram-positive bacteria is thus largely dependent on the cellular levels of HPr(Ser-P). A threefold decrease in the levels of HPr(Ser-P) in S. salivarius caused by a mutation in the pts promoter abolishes lactose and galactose exclusion by PTS sugars and results in derepression of galactokinase, ␤-galactosidase, and ␣-galactosidase activities (44). Interestingly, we found that the relative proportion of HPr(Ser-P) was threefold lower in S. thermophilus than in S. salivarius. These low levels of HPr(Ser-P) may explain the uncommon behaviors of S. thermophilus. First, it has been observed that S. thermophilus cometabolizes sucrose and lactose, PTS and non-PTS sugars, respectively (17), whereas several lactic acid bacteria metabolize PTS sugars preferentially to non-PTS sugars via an inducer exclusion mechanism involving HPr(Ser-P) (27, 31, 52, 56). Second, the lac promoter of S. thermophilus possesses a cre sequence and is controlled by CcpA (50). If the association of CcpA with the lac promoter cre sequence requires HPr(Ser-P), as it is the case for many genes controlled by CcpA in B. subtilis (8), then one would expect that repression of the lac genes would be less stringent in S. thermophilus than in species possessing higher levels of HPr(Ser-P). This is consistent with the findings that S. salivarius ␤-galactosidase is repressed 24- to 75-fold by PTS sugars (44), while the S. thermophilus enzyme is repressed less than twofold (51). Lastly, the lower levels of HPr(Ser-P) in S. thermophilus also agree with the observation that the lac operon is expressed at higher levels in lactose-grown S. thermophilus cells than in S. salivarius cells grown under the same conditions even though the lac promoter regions of both strains are virtually the same (48, 49). In S. thermophilus, lactose uptake is mediated by LacS, a transporter that possesses a hydrophilic intracellular IIA domain. Based on their amino acid sequences, S. thermophilus IIALacS domains can be separated into two groups. It has

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FIG. 6. Multiple alignments of IIALacS. The residues are numbered based on the S. thermophilus LacS sequence. Residues that distinguish group I IIALacS from group II are indicated in bold in the IIALacS group I sequences. The histidine residue that can be phosphorylated is shaded in gray. The sequence of IIALacS from strain LMD-9 (group II) was obtained from http://genome.jgi-psf.org/draft_microbes/strth/strth.info.html, scaffold 4 gene 1324. St, Streptococcus thermophilus; Ss, Streptococcus salivarius. The GCG programs Pileup and Pretty were used for optimal alignments.

already been demonstrated that IIALacS from strain A147, which belongs to IIALacS group II, can be phosphorylated by HPr(His⬃P) (16, 35). In the present study, we demonstrated that group I S. thermophilus IIALacS domains could also be phosphorylated by HPr(His⬃P), but also by HPr(SerP)(His⬃P), and that the rate of IIALacS phosphorylation by the doubly phosphorylated form of HPr was virtually the same as the rate observed with HPr(His⬃P). We also compared the rates of phosphorylation of S. salivarius IIALacS by HPr(His⬃P) and HPr(Ser-P)(His⬃P) and found that the doubly phosphorylated form of HPr transferred its phosphate group to IIALacS more rapidly than HPr(His⬃P) did. The amino acid sequence of S. salivarius IIALacS differs from the sequence of most group II S. thermophilus IIALacS at only three positions. Notably, one difference occurs nine amino acids downstream from the histidine residue that can be phosphorylated and consists of replacement of an Asn residue which is conserved in all group II IIALacS by a Lys residue, which is conserved in all IIALacS from group I. A Lys residue is also found in the homologous E. coli protein IIAGlc at the same position with respect to His-90, the residue phosphory-

lated by HPr(His⬃P) (38). This lysine, which is located at the surface of the protein, does not interact with His-90 (55) and is not involved in the interactions between IIAGlc and its substrate HPr (53). It is thus unlikely that the substitution K561N modifies the structure of IIALacS and affects its interactions with the different phosphorylated forms of HPr. Based on these data, S. salivarius IIALacS was considered a suitable representative of S. thermophilus group II IIALacS. Consequently, IIALacS domains from group II are, in all likelihood, also efficiently phosphorylated by the doubly phosphorylated form of HPr. Our results strongly suggest that the level of LacS phosphorylation in S. thermophilus does not depend solely on the amount of HPr(His⬃P), as previously suggested (16), but is also determined by the amount of HPr(Ser-P)(His⬃P). Since the doubly phosphorylated form of HPr is abundant in rapidly growing S. thermophilus cells (this work), whereas HPr(His⬃P) dominates at the end of the exponential and during the stationary phase of growth (16), it may be inferred that the phosphorylation state of LacS remains rather constant. Since the phosphorylation of LacS stimulates the lactose-galactose ex-


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The nucleotide sequence of lacS from S. thermophilus LMG18311 was obtained from the UCL Life Sciences Institute (ISV) website at http://www.biol.ucl.ac.be/gene/genome/.

FIG. 7. Rates of S. salivarius IIALacS phosphorylation by HPr (His⬃P) and HPr(Ser-P)(His⬃P). The experimental procedures were as described in the legend to Fig. 5 except for the concentration of HPr(Ser-P), which was adjusted to 12 ␮M because the amount of HPr(Ser-P)(His⬃P) that can be synthesized in vitro with S. salivarius EI and HPr(Ser-P) (24) is greater than the amount obtained with S. thermophilus proteins (15% versus 50%). Results from two independent experiments are expressed in units of phosphostimulated luminescence (PSL). The bars indicate the standard error.

change reaction, a mode of lactose transport that is more efficient than lactose-H⫹ symport (17), our results suggest that LacS functions as an antiporter under most growth conditions and not only at the late exponential and stationary phases of growth, as reported previously (16, 17). In gram-positive bacteria, HPr(His⬃P) is also involved in the regulation of gene transcription through the reversible phosphorylation of activators and antiterminators on one or several histidine residues in PTS regulation domains (PRDs) and IIA domains (14, 42). These regulators are phosphorylated by HPr(His⬃P) when the amount of carbon and/or energy source limits growth, allowing the induction of carbohydrate catabolic operons sensitive to carbon catabolite repression. Conversely, conditions of carbon excess and rapid growth stimulate the synthesis of HPr(Ser-P), maintaining the regulators in an unphosphorylated inactive state, thus preventing the induction of several sugar metabolic pathways (4, 8). PRD proteins have been found in S. mutans (2, 6, 54), suggesting that these proteins play an active role in gene regulation in streptococci. The finding that HPr(Ser-P)(His⬃P) can transfer its phosphate group to the histidine residue of IIALacS as efficiently as can HPr(His⬃P) raises the question of whether PRD proteins in streptococci are phosphorylated by both forms of HPr and, if so, whether the targeted residues are the same. ACKNOWLEDGMENTS We thank Gene Bourgeau for editorial assistance. We are grateful to FQRNT-NOVALAIT-MAPAQ in collaboration with Agriculture and Agri-Food Canada for financial support. A.C. is a recipient of a FQRNT studentship, and I.C. is a recipient of a CRSNG studentship.

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