Phosphotransferase Transport System from

Transcription

1 INFECTION AND IMMUNITY, Apr. 1994, p Vol. 62, No /94/$ Copyright 1994, American Society for Microbiology Sequence and Expression of the Genes for HPr (ptsh) and Enzyme I (ptsi) of the Phosphoenolpyruvate-Dependent Phosphotransferase Transport System from Streptococcus mutans DAVID A. BOYD, DENNIS G. CVITKOVITCH, AND IAN R. HAMILTON* Department of Oral Biology, University of Manitoba, Winnipeg, Manitoba R3E 0W2, Canada Received 22 September 1993/Returned for modification 16 November 1993/Accepted 9 January 1994 We report the sequencing of a 2,242-bp region of the Streptococcus mutans NG5 genome containing the genes for ptsh and ptsi, which encode HPr and enzyme I (El), respectively, of the phosphoenolpyruvate-dependent phosphotransferase transport system. The sequence was obtained from two cloned overlapping genomic fragments; one expresses HPr and a truncated El, while the other expresses a full-length El in Escherichia coli, as determined by Western immunoblotting. The ptsl gene appeared to be expressed from a region located in the ptsh gene. The S. mutans NG5 pts operon does not appear to be linked to other phosphotransferase transport system proteins as has been found in other bacteria. A positive fermentation pattern on MacConkey-glucose plates by an E. coliptsi mutant harboring the S. mutans NG5ptsI gene on a plasmid indicated that the S. mutans NG5 El can complement a defect in the E. coli gene. This was confirmed by protein phosphorylation experiments with 32P-labeled phosphoenolpyruvate indicating phosphotransfer from the S. mutans NG5 El to the E. coli HPr. Two forms of the cloned El, both truncated to varying degrees in the C-terminal region, were inefficiently phosphorylated and unable to complement fully the ptsi defect in the E. coli mutant. The deduced amino acid sequence of HPr shows a high degree of homology, particularly around the active site, to the same protein from other gram-positive bacteria, notably, S. salivarius, and to a lesser extent with those of gram-negative bacteria. The deduced amino acid sequence of S. mutans NG5 El also shares several regions of homology with other sequenced Els, notably, with the region around the active site, a region that contains the only conserved cystidyl residue among the various proteins and which may be involved in substrate binding. Dental caries is caused by the dissolution of tooth enamel by acid end products generated during carbohydrate metabolism by acidogenic bacteria in the biofilms on the tooth surface (dental plaque). A principal etiological agent of this disease is Streptococcus mutans (5, 30), an organism capable of high levels of glycolytic activity in the presence of mono- and disaccharides. S. mutans transports such sugars via the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) (38, 46). The PTS is a group translocation process which utilizes PEP as an energy source and substrate and catalyzes the sequential phosphorylation of the general, non-sugarspecific proteins enzyme I (El) and HPr required for the uptake of all PTS sugars. In many cases, phospho-hpr, generated from phospho-ei, transfers the phosphoryl group directly to carbohydrate-specific, membrane-bound enzyme II (EIl), which in turn phosphorylates the incoming sugar. In other cases, phospho-hpr transfers the phosphoryl group to a third soluble protein called enzyme III (EIII) prior to interaction with EIl (4, 15, 49, 51). Earlier continuous-culture studies with S. mutans Ingbritt, using decryptified cells to measure phosphotransferase activity, indicated that the growth environment regulates PEP-dependent PTS sugar transport (21). Subsequently, this regulation was shown to be associated with repression of the membraneassociated, sugar-specific EIl component. For example, EIl activity for glucose (EIIgiC) and mannose (ElIman), measured * Corresponding author. Mailing address: Department of Oral Biology, Faculty of Dentistry, University of Manitoba, Room D33, 780 Bannatyne Ave., Winnipeg, Manitoba, Canada R3E 0W2. Phone: (204) Fax: (204) in isolated membrane preparations in the presence of excess El and HPr, was repressed by as much as 62-fold under conditions of low ph, high growth rates, and excess glucose compared with optimal PTS conditions (i.e., 5 mm glucose, D = 0.1 h- l, and ph 7.0) (50). The cellular concentration of HPr varied twofold under the same conditions. Similar repression of the ElI activities for glucose, mannose, and 2-deoxyglucose of the PTS was recently reported for a fresh strain of S. mutans growing in continuous culture (22); however, the cellular concentrations of El, but not HPr, also varied significantly with the growth conditions. Concentration-dependent glucose repression (33-fold) of EIIg'c in membrane preparations of S. mutans Ingbritt occurs during growth in continuous culture at glucose levels above 3 mm (ph 7.0 and D = 0.1 h'-) even during conditions of glucose limitation (22). El and HPr cellular levels varied fourfold under the same conditions. More recently, the effect of growth ph on ElI activity for glucose, mannose, and 2-deoxyglucose was examined with cells of S. mutans Ingbritt grown in continuous culture at D = 0.1 h-' with 10 mm glucose (glucose limited) (53). In this study, progressively lower ElI activity was observed as the growth ph was reduced from ph 8.0, with cells grown at ph 5.0 completely devoid of ElI activity. Similarly, cells of the organism grown at high growth rates (e.g., 0.5 and 1.0 h-l) also exhibited little EIlgic and Ellman activity. In the same study, it was observed that EIIIfman synthesis was repressed at conditions above and below ph 7.0 and was particularly sensitive at high growth rates. These studies indicate that the regulation of the PTS in S. mutans is a complex matter undoubtedly involving a variety of mechanisms. As a consequence, we have undertaken to study

2 VOL. 62? 1994 SEQUENCE OF HPr AND ENZYME I GENES FROM S. MUTANS 1157 the regulation of the glucose-pts in S. mutans and, as a first step, have begun to isolate and characterize the genes for the various components of the PTS. We report here the cloning and sequencing of the genes for HPr (ptsh) and El (ptsi) from S. mutans NG5 and their expression in Escherichia coli. MATERIALS AND METHODS Bacterial strains, plasmids, bacteriophage, and media. S. mutans NG5 was maintained on blood plates (Oxoid blood base agar no. 2, 5% sheep blood, % hemin) and grown for DNA isolation in TYE-glucose broth (1% tryptone, 0.5% yeast extract, 20 mm glucose, 17 mm K2P04). For routine subcloning, E. coli XLI-blue or SURE (Stratagene) was used as the host for plasmids constructed with vector pbluescript II (Stratagene) or puc9 (55). E. coli strains were maintained on LB plates (35) supplemented when needed with the appropriate drugs and/or chemicals at the following concentrations: 100,ug of ampicillin, 13,ug of tetracycline, 80,ug of isopropyl-3- D-thiogalactopyranoside (IPTG), or 33 pg of 5-bromo-4- chloro-3-indolyl-3-d-galactopyranoside (X-Gal) per ml. S. mutans NG5 genomic libraries were constructed in Xgtl 1 (25), using E. coli Y1090r- (25) as a host, and in XEMBL3 (13), using E. coli KW25 1 (Promega) as a host. The ptsi mutant E. coli CHE11 (18) was used as a host for a recombinant phage and plasmid harboring cloned S. mutans NG5 ptsh gene regions. Two plasmids, peip8 containing a 1.1-kb EcoRI fragment from the C-terminal region of the S. salivarius ptsi gene and peip14-ii containing a 0.45-kb HintdIII-XbaI fragment from the N-terminal region of the gene, both cloned into puc18 (14), were a generous gift from Michel Frenette, Laval University, Ste-Foy, Quebec, Canada. DNA isolation. For the isolation of DNA, S. mutans was inoculated into 500 ml of TYE broth and incubated anaerobically at 37 C. The cells were centrifuged and resuspended in 5 ml of TE buffer (10 mm Tris [ph 8.0], 1 mm EDTA), and the genomic DNA was isolated as follows. The cells were heated at 60 C for 20 min, the suspension was cooled to 37 C, and lysozyme (20 mg/ml) and mutanolysin (200 U/ml) were added; the mixture was incubated for I h. Proteinase K (100,ug/ml) was then added, the mixture was incubated for a further I h, and then the cells were lysed by the addition of sodium dodecyl sulfate (SDS) to 4% with gentle inversion of the mixture. The NaCl concentration was adjusted to 0.7 M, and then 1/10 volume of 0.7 M NaCl-10% cetyltrimethylammonium bromide was added; the mixture was incubated at 65 C for 20 min and then extracted with an equal volume of chloroform. The upper aqueous phase was collected and extracted once with phenolchloroform-isoamyl alcohol (25:24:1, vol/vol/vol) and once with chloroform-isoamyl alcohol (24:1, vol/vol). The DNA was precipitated from the aqueous phase with 2 volumes of ethanol, washed once with 70% ethanol, and resuspended in TE buffer at I mg/ml. Rapid screening of plasmids was done from 1.5-ml E. coli cultures by using alkaline lysis (3). Plasmid DNA to be sequenced or for use in creating unidirectional deletions was isolated from 3- to 10-mi E. coli cultures, using the Magic Minipreps Kit (Promega). Phage DNA was isolated from liquid lysates by the method of Kaslow (26). Recombinant DNA methodology. Agarose gel electrophoresis carried out in 0.04 M Tris-acetate M EDTA (ph 8.0) and plaque and Southern hybridizations were as described in Maniatis et al. (33). All hybridizations were carried out in aqueous solutions. Transfer of DNA was to Hybond N+ (Amersham) for detection by radiolabelled probes and to Photogene Nylon (GIBCO/BRL) for detection by biotinlabelled probes. Radiolabelled DNA probes were prepared by using the Nick Translation or Random Primers Labelling kit, and biotin-labelled DNA probes were prepared with the Bio-Nick Labelling Kit (GIBCO/BRL). Recombinant plasmids used for sequencing were produced by subcloning specific restriction fragments after purification from agarose gels, using the Geneclean kit (Bio 101), by restriction digestion deletion and religation to produce specific subclones, or by unidirectional exonuclease III digestion (23) with the Erase-a-Base system (Promega). E. coli was transformed by electroporation using a Gene Pulser apparatus (Bio-Rad) according to the method of Dower et al. (11). Sequencing was carried out on double-stranded DNA by the dideoxy termination method (44) using [Io-35S]dATP and the Sequenase version 2.0 kit (U.S. Biochemicals). The sequence was confirmed by sequencing overlapping clones and determining the sequence on both strands. For sequencing the N-terminal region of the ptsh gene, we used a 16-mer sequencing primer we had synthesized (University of Manitoba DNA Laboratory) on the basis of a previously sequenced region of the ptsh gene. The sequence of the primer was 5'-TGTAATATCTGAGGCA-3'. Preparation of a cytoplasmic fraction from S. mutans. The preparation of an S. mutans cytoplasmic fraction containing HPr and El for use as a positive control in Western immunoblotting has previously been described (54). Protein electrophoresis and Western immunoblotting. Crude protein preparations from E. coli harboring plasmids were prepared by emulsifying a 1- to 2-cm streak from an LB plate containing the appropriate antibiotics in 500 VLI of 50 mm Na2HPO4 (ph 7.4)-150 mm NaCi-10 mm EDTA, adding 250 [LI of mm Tris (ph 6.8), 6% SDS, 15% 3-mercaptoethanol, 0.006% bromophenol blue, and 30% glycerol and boiling for 2 min. Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) according to the method of Laemmli (29) and transferred to Immobilon-P membranes (Millipore) according to the method of Towbin et al. (48). After incubation of membranes with the appropriate primary antibody (1:750 dilution in Tris-saline buffer), El- and HPr-specific bands were detected by incubation with goat anti-rabbit immunoglobulin G alkaline phosphate conjugate followed by detection with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate p-toluidine. In vivo complementation of the ptsi mutant E. coli CHE1. To determine whether the cloned S. mutans NG5 ptsl gene or truncated forms of it could complement the ptsi mutation in E. coli CHE1, pdb102, pdb105, and pdb201 were transformed into CHEI1 and streaked onto MacConkey plates containing 1% glucose. The positive control was E. coli XLl-Blue with or without pbluescript, and the negative control was E. coli CHE 1I with or without pbluescript. The colors of the streaks were compared to determine whether the glucose was fermented. In vitro phosphorylation of PTS proteins. Membrane-free cytoplasmic extracts were made from E. coli CHE 1 harboring the appropriate plasmids, as follows: cells were grown overnight at 37 C in 2 liters of LB medium containing 50 ig of ampicillin per ml. The cells were harvested by centrifugation and washed once in 20 ml of Tris-HCI (ph 7.5)-l mm EDTA-1 mm dithiothreitol, and the final cell pellet was suspended in the same buffer at a 1:1 ratio (wt/vol). Cell lysis was accomplished by two passages through a French pressure cell at 10,000 lb/in2, and the whole cells were removed by centrifugation at 10,000 x g for 15 min. Membranes were removed by centrifugation at 200,000 x g for 16 h; this was followed by centrifugation of the cytoplasmic fraction at

3 1158 BOYD ET AL. 200,000 x g for 4 h. The supernatant was then dialyzed at 4 C against 4 liters of PEMPP buffer (10 mm KPO4 [ph 7.5], 1 mm EDTA, 14 mm 2-mercaptoethanol, 0.1 FLM pepstatin A, 0.1 mm phenylmethylsulfonyl fluoride) with one change. Protein concentrations of the cytoplasmic fractions were determined by the method of Lowry et al. (31). Phosphorylation of PTS proteins with 32P-PEP and subsequent autoradiography were done according to the method of Bourassa et al. (4) with slight modification. 32P-PEP was synthesized by the method of Mattoo and Waygood (34), using purified carboxykinase from E. coli, kindly provided by H. Goldie, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. Since relative amounts of El and the truncated El proteins varied in the E. coli extracts, the ratios of El to total protein were determined by an enzyme-linked immunosorbent assay (ELISA), using anti-el antibody with an extract from E. coli harboring pbluescript as a background control. The El-toprotein ratios were used to determine the amounts of total protein added to the phosphorylation reactions and were as follows: 12 plg for pdb102, 108,ug for pdb105, and 216,ig for pdb201. Positive and negative controls contained 20 jig of protein from membrane-free extract of S. mutans NG5 and 216,ug of E. coli CHE11 harboring pbluescript II, respectively. Samples were incubated at room temperature in a 25-,ul reaction mixture containing 0.1 mm 32P-PEP (4.0 x 103 dpm nmol-'), 5 mm MgCl2, 12.5 mm NaF, and 10 mm HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (ph 7.5). The reactions were stopped after 5 min by the addition of 10,lA of 188 mm Tris-HCl (ph 8.0), which contained 6% (wt/vol) SDS, 30% (vol/vol) glycerol, 6% (vol/vol) 2-mercaptoethanol, and 0.005% bromophenol blue. Samples were boiled for 1 min and loaded onto a 12.5%, 1.5-mm-thick SDS-polyacrylamide gel which was electrophoresed for 1 h at 100 V in a Bio-Rad Mini Protean II apparatus (Bio-Rad Laboratories, Richmond, Calif.). The dried gel was placed on X-ray film with an intensifying screen at - 70 C for 22 h. Chemicals and enzymes. Restriction enzymes, T4 DNA ligase, goat anti-rabbit immunoglobulin G alkaline phosphatase conjugate, DNA, and protein molecular weight standards were from GIBCO/BRL and were used as directed by the supplier. Radiolabelled nucleotides ([-y-32p]datp, [<x_32p] datp, [ot-32p]dctp, and a-35s-datp) were obtained from Dupont New England Nuclear. All other chemicals were obtained from Sigma Chemical Co., Fisher ientific Limited, or Difco. Rabbit anti-el (S. salivarius) and anti-hpr (S. mutans) polyclonal antibodies were a generous gift from Christian Vadeboncoeur, Laval University, Ste-Foy, Quebec, Canada. Computer-aided analysis. The similarity of the S. mutans NG5 El and HPr proteins to proteins in other organisms was assessed by using the ALIGN program (6). Multiple sequence alignment of the various HPr and El proteins was carried out with the CLUSTALV program (24). Nucleotide sequence accession number. The sequence shown in Fig. 3 has been submitted to the GenBank data base under accession number L RESULTS Cloning of the ptsi gene. Gagnon et al. (14) have cloned and sequenced the ptsi gene from a genomic DNA library of S. salivarius. Two plasmids from that study, peip8 and peip14-il, clones carrying portions of the C-terminal and N-terminal regions, respectively, of the ptsi gene of S. salivarius, were used in this study. Both peip8 and peip14-ii were used as probes to screen various digests of S. mutans NG5 DNA (data not A Nc s D E X RV Nc D ND I I 1 Il I RVS D E X RV NC 1I I 11 III 11 I I ptai region B Plasuid Enzyme I c E expression pdblol I + (68) E N pdb102 I + (68) E pdb103 + (68) E D pdb104 I + (68) E RV pdb105 + (56) X N pdb112 NC IpDB2O1 NC INFECT. IMMUN. + (59) 500 b FIG. 1. (A) Restriction map of the S. mutans NG5 genomic ptsi region as determined by Southern blot hybridization of genomic digests with peip8 and peip14-ii, plasmids carrying portions of the ptsi gene from S. salivarius (14). (B) Restriction fragments subcloned into plasmids and used for sequencing and expression studies. Expression data are from Western immunoblot analysis of plasmids, as shown in Fig. 2. Number in parentheses is the size (in kilodaltons) of the expressed El. (C) Precise location and orientation of the ptsh and ptsi genes as determined from sequence analysis (Fig. 3). Restriction sites: D, DraI; E, EcoRI; Nc, NcoI; N, NspV; RV, EcoRV; S, SstI;, al; X, XbaI. shown) in order to construct a crude restriction map of the ptsi region (Fig. 1A). Since both plasmids hybridized to a 4.8-kb EcoRI fragment, we constructed a genomic DNA library in Agtl 1, using a complete EcoRI digest of S. mutans NG5 DNA as a first step in cloning of the ptsi gene. reening of this library with peip8 produced three to five positive plaques for every 1,500 plaques screened. We purified the DNA from four of these plaques, digested them with EcoRI, and found that all four contained a 4.8-kb EcoRI fragment as an insert to which peip8 hybridized (data not shown). Since the initial probing of S. mutans NG5 genomic DNA with peip8 indicated that the ptsi gene is localized on a 3.6-kb EcoRI-SstI fragment, we subcloned this fragment from one of the positive Xgtll clones into pbluescript II SK and named this clone pdb101 (Fig. 1B). Location, orientation, and expression of the ptsi gene. To determine whether the complete ptsi gene is expressed from pdb1l1, we performed a Western immunoblot of a crude protein preparation from E. coli harboring pdb1l1, using anti-el antibody raised against S. salivarius El. Vadeboncoeur et al. (52) had previously shown that these antibodies crossreact with S. mutans El. A 68-kDa band was detected in both the lane containing a sample from pdb1o1 and the positive control lane containing a sample of a supernatant fraction from S. mutans (see Fig. 2, lanes 1 and 2), indicating that a complete full-length ptsi gene was contained on the cloned 3.6-kb EcoRI-SstI fragment. To determine more precisely the location and orientation of the ptsi gene on pdb1o1, a series of deletion subclones were constructed (Fig. 1B). Crude protein preparations from E. coli harboring the subclones were subjected to Western immunoblotting, using anti-el antibody, and the sizes of the El bands were analyzed (Fig. 2). All plasmids, with the exception of pdb112, expressed the ptsi gene. In the case of pdb105, however, the El band was truncated, showing a size of 56 kda (Fig. 2, lane 6). These results indicated that the ptsi gene is oriented on pdb101 with its 5' end located in the vicinity of the XbaI site and the 3' end

4 VOL. 62, 1994 SEQUENCE OF HPr AND ENZYME I GENES FROM S. MUTANS ft. A.i I dm 4N'NMP 4wow 7 8 of S. salivarius (12a) and >35Cc identity with the HPr of E. coli (8). As the full length of the S. salih'arius HPr is 87 amino acids, it appeared that the pdbio insert was probably missing the first 13 N-terminal residues of the ptsh gene. This would explain why we could not detect any expression of HPr in Western immunoblots of pdb101 using anti-hpr antibody. In order to clone the complete ptsh gene, we constructed a library in XEMBL3, using 15- to 20-kb fragments from a partial Sau3A digest of S. mutans NG5 genomic DNA. The library was screened with pdb101; positive plaques were isolated, and their DNA was purified. The phage DNA yields were low in the E. coli KW251 background, and the phage genome appeared to be subject to deletions and/or rearrangements. Overexpression of the cloned S. salivarilus ptsh gene was found to be toxic to E. coli hosts with an intact PTS (12a). Consequently, with a ptsi mutant (E. coli CHEl 1 [18]) as a host, in E. coli as phage DNA yields increased, and restriction analysis revealed FIG. 2. Expression of S. mutans NG5 ptsi gene determined by Western immunoblot analysis. Anti-E I antibody was that the one phage genome contained a stable 13- to 15-kb reacted with crude protein preparations from E. coli harboring plas- insert. Southern hybridization with pdb1o1 as a probe remids carrying various portions of the ptsl region from. S. mutans NG5 vealed that the phage insert included the 3.5-kb NcoI fragment (see Fig. 1). Lane 1, NG5 supernatant; lane 2, pe)b101; pdb102; lane 4, pdbi03; lane 5, pdbi04; lane 6, pl lane 3, that included part of the ptsl gene and continued upstream of pdbi 12; lane 8, pbluescript. Size standards (in kiilodaltons) are the ptsh gene for approximately 1.9 kb (Fig. 1). To overcome indicated on the left. the potential toxic effect of ptsh gene overexpression, we subcloned this fragment into the unique NcoI site of the streptococcal integration vector pva891 (32), which has a of the gene between the first EcoRV site and fiirst DraI site. lower copy number than puc-type vectors, and used E. coli The promoter-acting sequences are located between the CHE-11 as the host. The plasmid, pdb201, was stably maintained. EcoRI and XbaI sites (Fig. IA). The ptsh gene encoding HPr is located imrnediately up- Expression of the ptsh gene from pdb201 was examined by stream of the ptsi gene in S. salivarius (14), as it is in other Western immunoblotting a crude protein preparation from E. gram-positive (8, 17, 27) and in gram-negative t)acteria (7, 8, coli CHE11 harboring pdb201, using anti-hpr antibody raised 39, 43). Western immunoblotting of proteins from E. coli against S. mutans HPr. Two bands of approximately 14 kda in harboring pdbio1, using anti-hpr antibodies, fa iled to detect size were detected in both the lane containing a sample from any HPr protein (data not shown). pdb201 and the positive control lane containing a sample of a Sequence of the ptsi gene. Once the relative Ic)cation of the supernatant fraction from S. mlutans (Fig. 4, lanes 1 and 2). ptsi gene on pdb1o1 was ascertained from expre:ssion studies, Though various streptococcal species contain two forms of HPr the sequence of this region was determined. IFhe sequence differing only in the presence or absence of the N-terminal including 140 bp upstream of the EcoRI site ( see below) is methionine (41, 50), the additional band observed probably presented in Fig. 3. Analysis of the sequence revealed the represents a degradation product, as we would not expect E. presence of two open reading frames (ORFs) between the coli to process the streptococcal HPr. The higher-molecular- longer one weight band seen in the pdb201 extract and pbluescript EcoRI site and the first downstream DraI site. TIhe of 1,734 nucleotides begins with an AUG codon ait position 372 extract (Fig. 4, lanes 2 and 3) must be a host protein reacting and codes for a putative protein of 577 amino acids with a with anti-e. coli antibodies contained in the anti-hpr antibody molecular weight of 63,282. This ORF could be identified as preparation. To complete the sequence of the ptsh gene ptsi as the amino acid sequence of its putativre translation through and upstream of the EcoRI site, we used a custom- S. salivarius made primer we had synthesized on the basis of a previously product exhibited 89.4% identity with the El frorn (14), 62.4% identity with the El of Staphyloco ccus carnosus sequenced region of the ptsh gene and pdb201 as a template. (27), 61% identity with the El of Bacillus subotilis (16, 40), The complete ptsh ORF is 264 nucleotides, beginning with an 47.4% identity with the El of E. coli (7, 43), 39.5% o identity with AUG codon at position 104 (Fig. 3), and codes for a putative the El domain of the multiphosphoryl transfer pirotein (MTP) protein of 87 amino acids with a molecular weight of 8,926. of Rhodobacter capsulatus (56), and 37.8% iderntity with the The S. mutans HPr amino acid sequence exhibits 97% identity El-like phbi gene product protein I of Alcalige nes eutrophus with the HPr from S. sa/ii'arius (12a), 78% identity with the (39). Located 33 bp downstream of the ptsi gene stop codon is HPr of Enterococcuisfaecalis (9), 65% identity with the HPrs of the beginning of a region of dyad symmetry, posiitions 2138 to Staphylococclus aureus (2) and Staphylococcius carnosuls (12), 2160, which could play a role in transcriptionai termination 62% identity with the HPr of B. subtilis (17), 38% identity with (Fig. 3). However, because of the relative instability of the the FPr domain of the MTP of R. capsll/atius (56), 37% identity putative hairpin with a AXG of -8.5 kcal/mol ( kj/mol) with the HPr of E. coli (8), and 34Cc identity with the HPr-like - (47), the possibility of significant readthrough iin this region phbh gene product protein H from A. elutrophlus (39). cannot be ruled out. Base composition. The base composition of the overall Cloning, expression, and sequence of the ptesh gene. Se- sequence is 60.2Cc A+T, with ptsh being 60.6c A+T and ptsi quence analysis upstream of the ptsi gene revea led a shorter being 58.4% A+T. This is consistent with data showing that ORF in the same orientation and separated from it by 4 bp that the S. mlutanis A+T content is between 60 and 64% (20). As continues to the EcoRI site defining the insert-v( ector junction might be expected from the base composition, the wobble in pdb101. Translation of this ORF revealed a putative positions of the codons are occupied by an A or T in 82.8% of protein of 74 amino acids that had >80% identityiwith the HPr the ptsh codons and 72.8% of the ptsi codons (data not

5 1160 BOYD ET AL. INFECr. IMMUN RBS ptsh AAAGATTGAAAATAAAAAAGCATTGTGCTAlTAATCTCTAATTGTAAGGGTTATCATTTGATAATTCAAAR AACACATACTAAAAAAGGAGAACCATACT G GCT TCA AAA 115 M A S K 4 GAT TTT CAC ATT GTT GCA GAA ACA GGA ATT CAT GCA CGC CCA GCT ACT TTG CTT GTT CAA ACA GCA AGT AAG TTT GCC TCA GAT ATT ACA 205 D F H I V A E T G I H* A R P A T L L V 0 T A S K F A S D I T 34 CTT GAT TAC AAA GGA AAA GCA GTA AAC CTT AAG TCA ATT ATG GGT GTT ATG AGC CTT GGT GTT GGT CAA GGT GCT GAT GTT ACA ATC ACT 295 L D Y K G K A V N L K S I M G V M S L G V G Q G A D V T I T 64 RBS ptsi GCT GAA GGT GCA GAT GCA GAT GAT GCA ATT GCA GCT ATT AMT GAA ACG ATG ACT AAA GAA GGA TTG GCT TAA AGAT ATG ACA GAA ATG CTT 386 A E G A D A D D A I A A I N E T M T K E G L A * MT E M L 87,5 AAA GGA ATT GCA GCA TCT GAT GGT GTT GCT GTT GCT AAG GCA TAT CTA CTT GTT CAA CCG GAC TTA ACA TTT GAG ACT GTT TCA GTC ACA 476 K G I A A S D G V A V A K A Y L L V Q P D L T F E T V S V T 35 GAT ACA CAA GCA GAA GAG GCT CGT TTG GAT GCA GCT CTA GAA GCT TCT CAA AAC GAG CTT TCT CTT ATC CGC CAA AAA GCA GTA GAT ACC 566 D T Q A E E A R L D A A L E A S Q N E L S L I R Q K A V D T 65 CTT GGT GAA GAA GCC GCA GCT GTA TTT GAT GCT CAT TTG ATG GTT CTT GCT GAC CCA GAG ATG ATT GGG CAG ATT AAA GAG ACA ATT CGT 656 L G E E A A A V F D A H L M V L A D P E M I G Q I K E T I R 95 ACA AAA GAA GTT AAT GCA GAA AGT GCT CTC AAA GAA GTG ACA GAT ATG TmT GTT ACT TTG TIT GAA AAT ATG GAA GAC AAT CCC TAT ATG 746 T K E V N A E S A L K E V T D M F V T L F E N M E D N P Y M 125 CAA GAG CGC GCT GCA GAT ATT CGT GAC GTT GCC AAG CGT GTT TTG GCA CAC CTT CTG GGT GTT GAA TTG CC AAC CCA GCA ACG ATT AGT 836 Q E R A A D I R D V A K R V L A H L L G V E L P N P A T I S 155 GAA GAA TCT ATT GTA ATT GCT CAT GAC TTA ACG CCA TCT GAT ACG GCT CAA TTG GAT GCT AAC TAT GTT AAA GCT TTT GTG ACG AAT ATT 926 E E S I V I A H D L T P S D T A Q L D A N Y V K A F V T N I 185 GGT GGA CGC ACA AGT CAT TCT GCT ATT ATG GCA CGT ACA CTT GAA ATC GCT GCT GTA CTT GGT AC AAT GAT ATT ACA GAG CGT GTG AAA 1016 G G R T S H* S A I M A R T L E I A A V L G T N D I T E R V K 215 AAT GGT GAT ATT GTT GCT GTC AAT GGA ATT ACT GGT CAA GTG ATT ATT AAT CCA ACT GAA GAT CAA ATT GCA GAG TTC AAA GCA GCT GGT 1106 N G D I V A V N G I T G Q V I I N P T E D Q I A E F K A A G 245 GAG ACT TAT GCT AAG CAA AAA GCA GAA TGG GCT CTC CTT AAG GAT GCT GAA ACG GTT ACT GCT GAT GGT AAG CAC ITT GAA TTG GCA GCC 1196 E T Y A K Q K A E W A L L K D A E T V T A D G K H F E L A A 275 AAT ATT GGT ACA CCT AAA GAC GTr GAG GGT GTC AAT AAC AAT GGT GCT GAA GCA GTT GGC CTT TAT CGT ACA GAA mtt CTA TAC ATG GAT 1286 N I G T P K D V E G V N N N G A E A V G L Y R T E F L Y M D 305 TCG CAA GAT ITT CCA ACT GAA GAT GAA CAA TAT GAA GCT TAT AAA GCT GTT CTT GAA GGT ATG AAT GGC AAG CCA GTT GTT GTC CGC ACG 1376 S Q D F P T E D E Q Y E A Y K A V L E G M N G K P V V V R T 335 ATG GAT ATT GGT GGT GAT AAG GAA CTT CCT TAC TTT GAT CTT CCA AAA GAA ATG AAC CCA TTC CTT GGT TIT CGT GCG CTT CGT ATT TCA 1466 M D I G G D K E L P Y F D L P K E M N P F L G F R A L R I S 365 ATC TCA GAA ACA GGT AAT CAG ATG TTC CGC ACA CAG TTG CGT GCC CTT TTG CGT GCT TCT GTT CAT GGT CAG TTG CGC ATC ATG TTC CCA 1556 I S E T G N Q M F R T Q L R A L L R A S V H G Q L R I M F P 395 ATG GTA GCT TTG CTC AAT GAG TTC CGT AAA GCT AAA GGT ATT CTG GAA GAA GAA AAG GCT AAT CTT AM GCT GAA GGC GTT GCT GTT TCA 1646 M V A L L N E F R K A K G I L E E E K A N L K A E G V A V S 425 GAT GAT ATC CAA GTT GGT ATC ATG ATT GAA ATT CCT GCA GCT GCA ATG CTG GCT GAC CAA TT GCT AAG GAA GTT GAT TTC TTC TCA ATC 1736 D D I Q V G I M I E I P A A A M L A D Q F A K E V D F F S I 455 GGT ACA AAT GAT CTT ATT CAA TAC ACC ATG GCT GCT GAC CGT ATG AAT GAA CAA GTA TCA TAC CTC TAC CAA CCT TAT AAT CCA TCC ATT 1826 G T N D L I Q Y T M A A D R M N E Q V S Y L Y Q P Y N P S I 485 CTT CGC TTG GTT GAT CAT GTG GTT AAG GCT GCT CAT GCT GAA GGT AAA TGG GCT GGT ATG TGC GGT GAG ATG GCT GGC GAT CAG ACA GCA 1916 L R L V D H V V K A A H A E G K W A G M C G E M A G D Q T A 515 GTT CCC CTT CTT GTT GGT ATT GGA CTT GAT GAG TTC TCT ATG TCT GCA ACA TCG GTT CTC CGT ACA CGC AGT CTT ATG AAG AAA TTA GAT 2006 V P L L V G I G L D E F S M S A T S V L R T R S L M K K L D 545 ACT GCT AAA ATG CAA GAA CTT GCT CAG CGT GCT CTG ACA GAA TGT GCA ACG ATG GAA GAA GIT CTT GAG TTA GAA AAA GAA TAT ATT GAT 2096 T A K M Q E L A Q R A L T E C A T M E E V L E L E K E Y I D 575 ITT GAC TAA TTCGITTATAAGAGAGAGTAGGTTGAGGTCTGTGATTGITGAGCMTCATAGGMGCCACITACTICTTCITTTTGTITGCTT GAGACAAATCTAT 2212 F D * 577 TATTTGCTGTTTGACAATAMATGMAIT 2242 FIG. 3. Nucleotide sequence of the S. mutans NG5 ptsh and ptsi genes and their deduced amino acid sequences. Putative - 35 and - 10 boxes and RBS are overlined. A putative transcriptional terminator-like region is indicated by inverted arrows. The HPr active-site histidyl residue (residue 15) and the El active-site residue (residue 191) are indicated by asterisks. shown). The above observations are similar to those found for 56- and 59-kDa El proteins, respectively. Transformants were the ptsh and ptsi genes of S. salivarius (14). streaked on MacConkey plates containing 1% glucose and In vivo complementation. E. coli CHEl1 was transformed compared with CHEl 1, with or without pbluescript, and with with pdb102, which expresses a full-length El, and with E. coli XL1-Blue, with or without pbluescript (data not pdb105 and pdb201, which express C-terminally truncated shown). The streaks of E. coli CHEl1, with or without

6 VOL. 62, 1994 SEQUENCE OF HPr AND ENZYME I GENES FROM S. MUTANS FIG. 4. Expression of S. mutans NG5 ptsh gene in E. coli as determined by Western immunoblot analysis. Anti-HPr antibody was reacted with crude protein preparation from E. coli harboring pdb201 (see Fig. 1B). Lane 1, NG5 supernatant; lane 2, pdb201; lane 3, pva891. pbluescript, were pale pink, and there was no coloration of the surrounding medium. On the other hand, streaks of E. coli XL1-Blue, with or without pbluescript, and of E. coli CHEll harboring pdb102 were dark red, and there was dark red coloration of the medium surrounding the streaks after overnight incubation at 37 C. The streaks of the transformants harboring either pdb105 or pdb201 were a very light red, and there was no coloration of the surrounding medium. Thus, it appeared that the full-length S. mutans ptsi gene complemented the ptsi mutation in E. coli CHEll, whereas two truncated forms missing 20 or 26% of the wild-type protein C terminus showed little, if any, complementation. Phosphorylation of PTS proteins in E. coli CHEll harboring cloned S. mutans NG5 ptsi genes. To examine directly whether the cloned S. mutans NG5 El protein could function in the phosphotransfer reactions of the PTS in E. coli CHE11, protein phosphorylation experiments were carried out with exogenously supplied PEP and membrane-free cell extracts of E. coli CHEll harboring various recombinant plasmids (Fig. 5). With membrane-free cell extracts from S. mutans NG5 as a positive control, three proteins became phosphorylated; EI, HPr, and a 34-kDa protein which was most likely an EIII (Fig. 5, lane 1). No phosphorylated proteins were observed in the negative control, E. coli CHEl 1 harboring pbluescript (Fig. 5, lane 5). An extract, prepared from E. coli CHEll harboring the full-length S. mutans El expressed from pdb102, exhibited El phosphorylation, as did the E. coli HPr (Fig. 5, lane 2). When extracts prepared from E. coli CHEl1 harboring pdb105 or pdb201 were used, very faint bands corresponding in size to the truncated Els appeared (Fig. 5, lanes 3 and 4, arrowheads), indicating that the mutant Els may be capable of very limited phosphorylation under conditions in which the EI levels, as determined by ELISA, were the same in each extract. Less tenuous evidence that the truncated Els may retain some limited function is the presence in these reactions of phospho- HPr. Intermediate-sized phosphoproteins seen in the reactions with extracts from pdb105 and pdb201 are likely EIII proteins and/or proteolytic degradation products of EI (Fig. 5, lanes 3 and 4). No intermediate-sized phosphoproteins were seen in the reaction with the extract from pdb102, probably due to the lesser total protein, and thus lower levels of E. coli PTS components, in the reactions. The appearance of phospho-hpr in the reactions would indicate that the S. mutans El can function in the phosphotransfer function of the E. coli PTS El- Eli- HPr- i-14.3 FIG. 5. PAGE of phosphorylated PTS-related proteins in reactions using 32P-PEP and extracts prepared from E. coli CHEll harboring various forms of the cloned S. mutans NG5 ptsi gene. Extracts in each reaction were from: lane 1, S. mutans; lane 2, E. coli CHE11 harboring pdb102; lane 3, E. coli CHEl1 harboring pdb105; lane 4, E. coli CHEll harboring pdb201; lane 5, E. coli CHEl1 harboring pbluescript. The identities of the major phosphorylated proteins are indicated on the left, and size standards (in kilodaltons) are indicated on the right. The arrowheads indicate truncated S. mutans Els of 56 (lane 3) and 59 (lane 4) kda as expressed from pdb105 and pdb201, respectively. and that the truncated Els on pdb105 and pdb201 retain limited phosphotransfer capabilities. DISCUSSION The principal aim of this study was to gain insight into the arrangement and sequence of the region of the S. mutans NG5 chromosome that harbors the genes coding for the general proteins, El and HPr, of the PEP:sugar PTS. This information is intended to initiate further research on the mechanisms controlling the expression of these genes under various environmental conditions (53). The cloning of the ptsl gene proceeded via selection of ptsi-hybridizing clones from a Agtll genomic library and the eventual subcloning of a 3.6-kb EcoRI-SstI fragment (pdb101) that expressed El but did not express HPr. Sequence analysis of this fragment revealed the presence of two ORFs, a truncated ptsh followed 4 bp downstream by ptsi. Another construct with the complete ptsh gene on a 3.5-kb NcoI fragment (pdb201) was isolated and used as a template to complete the sequence of ptsh. The order of these two genes is typical of the pts operons of both gram-positive and gramnegative organisms (7, 8, 12, 14, 17, 27, 39, 43, 56). Analysis of the sequence upstream of ptsh revealed a putative ribosome binding site (RBS) AAGGAGA at positions 89 to 95 and a putative promoter region with a -10 box TATAAT at positions 32 to 37 and a - 35 box TTGAAA at positions 9 to 14 (Fig. 3). This structure is very similar to a gram-positive consensus promoter (19). Analysis of the 3' end of ptsh identified a putative RBS AGAAGGA at positions 352 to 358 (Fig. 3). Sequence analysis revealed that the inserts from the plasmids used in this study were cloned in the opposite orientation to the lacz gene of the vector, yet all of them except one (pdb112) expressed the ptsi gene in E. coli (Fig. 1 and 2). The fact that the subclone in which all of the ptsh sequences had been deleted showed no EI expression would indicate that the ptsh gene contains promoter-acting sequences allowing expression of the ptsi gene. Similar results were found with S.

7 A) HPrs NG5 MASKDFHIVAETGIHARPATLLVQTASKFASDITDYKGKAVNLKSIkVMSLGVGQGADVTITAEGADADDAIAAINETMTKEGIA 87 Ss MASKDFHIVAETGIHARPATLLVQTASKFASDITLDYKGKAVNLKSIM3VMSLGVGQGADVTISAEGADADDAIVAIAETMTKEGLA 87 Ef MEKKEFHIVAETGIHARPATLLVQTASKFNSDINLEYKGKSVNLKSITVMSLGVGQGSDVTITVDGADEAEGMAAIVETLQKEGLAEQ 89 Sa MEQNSYVIIDETGIHARPATMLVQTASKFDSDIQLEYNGKKVNLKSIMGVMSLGVGKDAEITIYADGSDESDAIQAISDVLSKEGLT 87 MEQQSYTIIDETGIHARPATMLVQTASKFDSDIQLEYNGKKVNLKSIMGVMSLGVGKDAEITIYADGSDEADAIQAITDVLSKEGLTE 88 Bs MAQKTFKVTADSGIHARPATVLVQTASKYDADVNLEYNGKTVNLKSIMGVMSLGIAKGAEITISASGADENDALNALEETMKSEGLGE 88 Ae MLQRDTTIINKLGLHARASAKLTQIAGNFVSQVKMSRNGRQVDAKSIMSVMMLAAGIGSTVTLETDGPDEQEAMDALLALIANRFGEGE 89 Ec MFQQEVTITAPNGLHTRPAAQFVKEAKGFTSEITVTSNGKSASAKSLFKLQTLGLTQGTVVTISAEGEDEQKAVEHLVK-----LMAELE 85 Rc AQGIDVVVTGAHGLHARPATTLVDLAKGFAAEIRIRNGAKVANGKSLISLLNLGAAQGAALRISAEGADATAALAAIAAAFEAGLEDEED 90 **** * ** **... B) Enzyme Is NG5 1 MTEMLKGIAASDGVAVAKAYLLVQPDLTFETVSVT--DTQAEEARLDAALEASQNELSLIRQKAVDTLGEEAAAVFDAHIMIVADPEMIG 88 Ss 1 MTEMLKGIAASDGVAVAKAYLLVQPDLSFETVTVE--DTSAEEARLDAALAASQDELSVIREKAVESLGEEAAAVFDAHIUVIADPEMTG 88 1 MAKQIKGIAASDGVAIAKMY LLVEPDLSFDNESVT--DTDAVAKFNGANKSKVELT KIRNNAEKQLGADKAAIFDAHLLVLEDPELIQ 88 Bs 1 M-QELKGIGASAGIAIAKAYRLEEPDLTVEKKNIS--DSEAEVSRFDEAIARSKEELEKIKEHALKELGQDKADIFSAHLLVLSDPELLN 87 Ec 1 M---ISGILASPGIAFGKALILKEDEIVIDRKIaSADQVDQEVERFLSGRAKASAQLETIKTKAGETFGEEKEAIFEGHIMLLEDEELEQ 87 Rc TLVGISSSPGYAIAPVFRFARDEVVFDT---DAADAAFETDRLDTALQTAWHELEELHDEVWKTSGPARAAIFRAHQEFLHDPEMVA 356 Ae 1 MPFALHGIPVSRGVAIGRAHLLAPAALDVSHYLVDEDQLDAEVERIRAARAAVRAELAAIKRDLPRDAPEELGAFLDVHAMILDDEALAR 90 ** ** ** * ****-*** * * * * * NG5 QIKETIRTKEVNAESALKEVTDMFVTLFENMEDNPYMQERA-ADIRDVAKRVLAHLLGVELPNPATI SEESIVIAHDLTPS 168 Ss QIKETIRAKQVNAEAALTEVTDMFIAIFEGEDNPYMQERA-ADIRDVTKVILLGKKLPNPATI NEESIVVAHDLTPS 168 PIEDKIKNESVNAAQALTDVSNQFITIFESMDNE-YIAERAKADIRDVSKRVLAHILGVELPNPSIV DESVVIIGNDLTPS 168 Bs PVKEKISTDSVNAEFALKETSSMFVTMFESMDNE-YMKERA-ADIRDVTKRVTGHLLGVEIPNPSMI SEEVIIVAEDLTPS 166 Ec EIIALIKDKHMTADAAAHEVIEGQASALEELDDE-YLKERA-ADVRDIGIRLLRNILGLKIIDLSAI QDEVILVAADLTPS 166 Rc EAKALI-GQGRSAGFAWHRVFSDRADMLGAMKD-AVLSGRA-IDLRDAGQRVLQHLGRVRTGETHLP TAPCILIADDLTPS 434 Ae EPEALIRGRRYNAEWALTTRLEELMHQFDEIEDE-YLRER-KTDIRQVVERILKAIAGAPVLVPAPVPALAADGEAATGVIVVAHDIAPA 178 * * **** * * *-* -* *--- * * - NG5 Ss Bs Ec Rc Ae NG5 Ss Bs Ec Rc Ae NG5 Ss Bs Ec Rc Ae NG5 Ss Bs Ec Rc Ae NG5 Ss Bs Ec Rc Ae DTAQLDANYVKAFV TNIGGRTSHSAIMARTLEIAAVLG TNDITERVKNGDIVAVNGITGQVIINPTEDQIAEFKAAGETYAKQKAEWALL DTAQINKKYVKAFV TNIGGRTSHSAIMARTLEIAAVLG TNNITELVKDGDIIAVSGITGEVVINPTEEQIAEFKAAGEAYAKQKAEWALL DTAQL1KEYVQGFV TNIGGRTSHSAIMSRSLEIPAWG TKSITEEVEAGDTIVVDGMTGDVLINPSDEVIAEYQEKRENFFKDKQELQKL DTAQLNREEVKGFT TDIGGRTSHSAIMARSLEIPAWG TKAATGTIQNGVTVIVDGINGDVIIDPSAETVKEYEEKHNAYIAQKAEWAKL ETAQLNLXKVLGFI TDAGGRTSHTSIMARSLELPAIVG TGSVTSQVKNDDYLILDAVNNQVYVNPTNEVIDKMRAVQEQVASEKAEIAKL DTARLDPALVRGIA TAQGGPTSHTSIIARALDIPAVAG VGPRLLDLATGTPVLGI TIVRAETAAAATTAOR E DMLQFRHTVFHGFV TDMGGRTSHTAIVARSLDIPAAVG VQSASELIRQDDWIIIDGDAGLVIVDPTAIILEEYRHRQSERALEKKRLQRL 1* ** *** * * * * *1 * KDAETVTADGKHFELAANIGTPKDVEGVNNNGAEAVGLYRTEFLYMDS-QDFPTEDEQYEAYKAVLEGMNGKPVVVRTMDIGGDKELPYF KDAQTVTADGKEIEIAANIGTPKDVEGVNDNGAEAVGLYRTEFLYMDS-QDFPTEDDQYEAYKAVLEGMNGKPVVVRTMDIGGDKELPYF RDAESVTADGHHVELAANIGTPNDLPGVIENGAEGIGLYRTEFLYMGR-DQMPTEEEQFEAYKAVLEAMKGKRVVVRTLDIGGDKELPYL VNEPTVSKDGHHVELAANIGTPDDVKGVLENGGEAVGLYRTEFLYMGR-DQLPTEDEQFDAYKTVLERMEGKSVVVRTLDIGGDKELPYL KDLPAITLDGHQVEVCANIGTVRDVEGAENGAEGVGLYRTEFLFDR-DALPTEEEQFAAYKAVAEACGSQAVIVRTMDIGGDKELPYM RYKPALTVDGARVEVVANISDVAEAIASVEAGAEGVGLRTEFILF-EAPPGEDEQIAIYAAMLSALNGLPIIIRTLDVGGDKEIPYL RHTPAVTLDGLEIDLLANIEKAEDAGALAGAVGVGLFRSEFLFHRRDELPGEDEQFQAYRGAVDAMHGLPVTIRTIDIGADKPLDAR **** ** *** * * **. ******** * **** * *. ******. --DLPKEMNPFLGFRALRISISETGNQMFRTQLRALLRASVHGQLRIMFPMVALLNEFRKAKGILEEEKANLKAEGVAVSDDIQV GIMIE --DLPKEMNPFIZYRALRSISETGNQTGRTLRRALRISVHGKLRIMFPV ALLTEFRTAKGILEEEAKLVAEGVAVADDIEV GIMIE --DLPEEMNPFGMYRAIRLCIDVP--EIFRPQLRALLRASVFGKLNIMFPMVATIEFRDAKAK LKEQlMLVADDIEL GIME --QLPKEMNPFIGYRAIRLCLEEQ--EIFRTQLRALLRASTYGNLKIMFPMIATVNEKEAKAILLEEKEKLVKAGQAVSDDIEV GMMVE --NFPKEENPFLGWRAIRIAMDRR--EILRDQLRAILRASAFGKLRIMFPMI ISVEEVRALRKEIEIYKQELRDEGKAFDESIEI GVMVE --RMPVEQNPFLGERGIRFCLSH--EDLFRTQLRAIYRASAGGQVRIMFPMIAMIEELETARRIAEEVRLEVG-----AAPVEI GIMIE GDEFETALNPALGLRAIRWSLSEPG--MFLTQLRALLRASAFGPVRLLVPMLAHASEIDQTIALIAIcAIRQLDERGEAYDPGMKV GAME ** ** * * **** *** * ** * *A*V* IPAAAMIADFAKEVDFFSIGTNDLIQYTMAADR MNEQVSYLYQPYNPSILRLVDHVVKAHAEGKWAGM CGEMAGDQTAVPLLVGMGLD IPAAATMTQFAKEVDFFSIGTNDLIQYTMAADR M-.NEQVSYLYQPYNPSILRLINNVITATT EGKWEGM CGEAMGDQTAVPLLVGT-3LD IPTA IIARVFA tatdifln DU<F.V.:TVr>DT.<N'CT.Vrf~TEVT1 JJL'n wa<wframwrn.r I T1~ir< T IPSTAVIADQFAKEVDFFSIGTNDLIQYTMAADR MNERVSYLYQPYNPAILRLITLVIEAAHKEGKWVGM CGEMAGDEIAIPILLGLGLD TPAAATIARHLAKEVDFFSIGTNDLTQYTLAVDR GNDMISHLYQPMSPSVLNLIKQVIDASHAEGKWTGM CGEIAGDERATLLLLGMGLD IPSAVMMAPEL1AKRVDFFSIGTNDLTQYALAMDR,MHPVLAKQADGLHPAVLRLIDSTVRAAEAARIWVGA CGGIAGDPVGAAVLSGLGVR IPAAVLLLPLFLRKMDFLSIGTNDLIQYTLAIDR ADNAVAHLFDPLHPAVLQLVARTIREANRAGVPVAV CGEMAGDPSMTRRLLGMGLR * -**.******* **..***.- * * *.* **,*** **.*. EFS MSATSVLRTRSLMKKLDTAKMQELAQRALTECATMEEVLELEK-EYIDFD 577 EFS MSATSVLRTRSLMKKLDTAKMEEYANRALTECSTMEEVLELSK-EYVNVD 577 EFS MSATSILKARVLIRSLNESEMKLESERAV-QCATSEEVVDLVE-EYTKNA 574 EFS MSATSILPARTQISKLSKQEAESFKEKIL-SMSTTEEVVAFVK-ETFK 570 EFS MSAISIPRIKKIIRNTNFEDAKVLAEQALAQ-PTTDELMTLVN-KFIEEKTIC 575 ELS VSIPAVAGIKAQLRHSAMA@ENRDLARRALA-CTTAAEVRGLK 827 EFS MHPAQLLRVKQEILHAHCERLEPLVDQVLQAFDPEEQARPLRSWHDPEAIRVF 592 *.*

8 VOL. 62? 1994 SEQUENCE OF HPr AND ENZYME I GENES FROM S. MUTANS 1163 salivarius in which a putative RBS, as well as putative - 10 and -35 boxes, were identified in the ptsh coding sequence (14). For Staphylococcus carnosus, a plasmid containing the 3' end ofptsh and the complete ptsi gene did not express El in E. coli when the insert was cloned in the antisense orientation to the lacz gene of the vector (27). With B. subtilis and E. coli, no active promoter for ptsi could be detected within the ptsh gene (7, 17, 43). The intracellular concentration of HPr and El in oral streptococci can vary according to growth conditions, and the level of HPr is higher than that of El (22, 45, 53, 54). It has been suggested (27) that ptsi gene expression may be attenuated by possession of its own weak RBS and by stem-loop structures near the beginning of its coding sequence, situations found for the ptsi genes of Staphlylococcus carnosus (27), B. subtilis (17), and E. coli (7, 43). Although we have identified a putative RBS, we could not identify any stem-loop structures near the 5' end of the S. mutans NG5 ptsi gene. While the ptsi genes of S. mutans NG5 and S. salivarius are probably cotranscribed with ptsh under certain conditions, as has been shown in the pts operons of B. subtilis and E. coli (7, 43), existence of their own promoters could provide an explanation for the differing expression of ptsh and ptsi in the oral streptococci in vivo. It should be noted, however, that because of the high A+T content of streptococcal DNA, it is highly likely that random sequences of DNA could resemble E. coli promoter sequences (36). It has been observed that S. pneumoniae sequences that do not normally function as promoters can do so when cloned in E. coli (10). Thus, it is possible that the expression of the cloned S. mutans NG5 ptsi gene could have been initiated fortuitously from an A+T-rich region in the ptsh sequence. Transcript analysis of this region of the genome will be needed to clarify these questions and whether the stem-loop structure identified following the ptsi gene functions in transcriptional termination. We have sequenced 103 bp upstream of ptsh and 137 bp downstream ofptsl and have not detected any other ORFS. In other bacteria, the pts operons have been found to be associated with genes coding for other PTS enzymes. In B. subtilis, the ptsg gene coding for Ellg'-Elllg is located 100 bp upstream of the ptsh gene (17), while in E. coli the ptsi gene is followed by the crr gene coding for EIIIgC (7, 43). In the fructose-pts operon of R. capsulatus, frub codes for an MTP in which the C-terminal El domain is preceded by domains for FPr and EIIIfru (56). Downstream of the frub gene is fruk, coding for fructose 1-phosphate kinase, and then frua, coding for EIIfru. The genes associated with the ptsh and ptsl genes in Staphylococcus carnosus and S. salivarius have yet to be identified, although large amounts of these sequences have been analyzed in S. salivarius (14, 27). Thus, it appears that the pts operons from gram-positive cocci may have distinct arrangements differing from other known pts operons, although further sequence analysis will be needed to clarify this. To determine whether the S. mutans NG5 El could complement a mutation in the ptsl gene of E. coli CHE II in vivo, we transformed it with pdb1o1. PTS function in the transformed CHE II was restored, as indicated by the appearance of dark red colonies and red coloration of the surrounding medium after growth on MacConkey-glucose plates. This finding is in agreement with the observation that the Els of the grampositive bacteria, Staphylococcus carnosus and B. subtilis, are able to complement an El defect in E. coli (16, 27). We were also interested in determining whether two truncated forms of El would complement the ptsi mutation in E. coli CHE1 1. Western blot analysis indicated that pdbi05 and pdb201 expressed C-terminally truncated forms of El 56 and 59 kda in size, respectively. Colonies of E. coli CHEI 1 transformed with either of these two plasmids, when grown on MacConkeyglucose medium, appeared as very light red but with no red coloration of the surrounding medium. Protein phosphorylation experiments were conducted with extracts from E. coli CHE 11 harboring the appropriate plasmids to determine whether these truncated Els had a limited capacity for phosphotransfer (Fig. 5). As expected, the wild-type El, as expressed from pdbl02, was efficiently phosphorylated and was able to transfer the phosphate to the E. coli HPr (Fig. 5, lane 2). However, when extracts containing either of the truncated Els were used, it appeared that a small amount of each El form may have been phosphorylated (Fig. 5, lanes 3 and 4, arrowheads) and the phosphate might have been passed to the E. coli HPr. This small amount of PTS phosphotransfer could explain the weak MacConkey-glucose reactions with E. coli CHEI 1 harboring either pdb105 or pdb201. On the basis of sequence analysis of the S. mutans NG5 ptsl gene, the El expressed from pdb105 would be missing 149 C-terminal residues of the wild-type El, while the El expressed from pdb201 would be missing 112 C-terminal residues of the gene. Thus, the 112 C-terminal residues of El appear to be essential for optimal phosphoryl transfer function in the PTS. While the exact nature of the role of this sequence in El function in S. mutans is unknown, this segment contains the conserved cystidyl residue at position 506, which could be important to phosphotransfer coupling to HPr. Figure 6A shows an alignment of the amino acid sequences of 11 HPr proteins. The HPrs from the gram-positive bacteria exhibit high homology overall, especially around the PEPdependent phosphorylation site, histidine-15, and the site of ATP-dependent phosphorylation in gram-positive bacteria, serine-46 (2). The HPrs of the gram-negative bacteria are less homologous to the gram-positive HPr, although they still exhibit significant homology around histidine-15, with glycine- 13, arginine-17, and alanine-26 conserved in all HPrs. Arginine-17 has recently been shown to be essential for normal HPr activity in E. coli (1) and Staphylococcus carnosus (28). The sequence around serine-46 of the gram-negative HPrs, which does not function as a phosphorylation site in these orgarisms, is less conserved, although lysine-45 and leucine-53 are present in all HPrs. Interestingly, the A. eutrophus phbh product, protein H, has a serine-46 region more similar to that in the gram-positive HPrs, and one wonders whether the serine-46 residue can be phosphorylated by an ATP-dependent kinase. The functions of the phbh and phbi products from A. eutrophus are unknown, although they appear to be related to poly(p-hydroxybutyric acid) metabolism (39). A more com- FIG. 6. Alignments of the amino acid sequences of S. mutans NG5 HPr and El with other related proteins. Under the groups of sequences, - an asterisk (*) indicates identical residues and a dot ( ) indicates similar residues in the above group(s). Residue numbers are indicated; gaps are not counted in the numbering scheme. Abbreviations: NG5, S. mutans NG5; Ss, S. salivarius; Ef, Enterococcusfaccalis;, Staphylococcus carnosus; Sa, Staphylococcus aureus; Bs, B. subtilis; Ec, E. coli; Rc, R. capsulatus; Ae, A. eutrophus. (A) Alignment of HPrs. (Top group) Sequences from gram-positive organisms; (bottom group) sequences from gram-negative organisms. Rc is the FPr domain from its MTP, and Ae is protein H, the product of phbh. (B) Alignment of Els. Conserved regions discussed in the text are boxed. Rc is the El domain of its MTP, and Ae is protein I, the phbi product.

9 1164 BOYD ET AL. plete analysis of the relatedness of sequenced HPrs has recently been published (40). An alignment of seven El proteins revealed several regions of homology spread over most areas of the protein but showing that the 150 residues N terminal are generally less conserved than the rest of the protein (Fig. 6B). The end of this region in A. eutrophus is of interest as it has a putative proline-alaninerich linker (residues 152 to 165) occurring in what is a gap region in all of the other Els. This region may serve as a flexible hinge connecting two rigid domains (40). One homologous region of note is the region from residues 181 to 197 (S. mutans NG5 residues) containing the histidyl residue (residue 191), which is the site of PEP-dependent phosphorylation. Also of note is the conserved region, residues 431 to 469, in which there are 18 of 39 residues (46%) strictly conserved and 29 of 39 (74%) either identical or similar. Gagnon et al. (14) have postulated that this may be a domain important in PEP binding. Residues 504 to 529 are also of interest because of the location of the only conserved cystidyl residue (residue 506) among the El proteins. This residue may provide an essential catalytic thiol group to the active site (56). Recent reports have shown that the El proteins share these regions of homology, and at least one other (residues 354 to 363), with the pyruvate: orthophosphate dikinase from maize and Bacteroides symbiosus, as well as the PEP synthase from E. coli (37, 40, 56). Similarly to EI, the pyruvate:orthophosphate dikinase and PEP synthase enzymes also bind PEP and catalyze the transfer of a phosphoryl group via a phosphohistidine intermediate. Extensive analysis of their primary structures has indicated that all of the above proteins belong to an El family (40). Clearly, our results show that the El and HPr of S. mutans NG5, products of the ptsi and ptsh genes, are expressed in E. coli and that the El can complement a ptsi defect in E. coli. The ptsi gene can be expressed from a promoter-like sequence in the ptsh gene, and if this sequence is a true streptococcal promoter, this transcriptional organization would be unique among pts operons, having been described so far only in oral streptococci. The S. mutans NG5 ptsh and ptsi genes do not appear to be linked with either EIIs or EllIs, which is in contrast to the same genes in B. subtilis and E. coli. ACKNOWLEDGMENT This research was supported by grant MT from the Medical Research Council of Canada. REFERENCES 1. Anderson, J. W., K. Pullen, F. Georges, R. E. Klevit, and E. B. Waygood The involvement of the arginine-17 residue in the active site of the histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli. J. Biol. Chem. 268: Beyreuther, K., H. Raufuss, 0. hrecker, and W. Hengstenberg The phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus. I. Amino acid sequence of the phosphocarrier HPr. Eur. J. Biochem. 75: Birnboim, H. C A rapid alkaline extraction method for isolation of plasmid DNA. Methods Enzymol. 100: Bourassa, S., L. Gauthier, R. Giguere, and C. Vadeboncoeur A IIIman protein is involved in the transport of glucose, mannose and fructose by oral streptococci. Oral Microbiol. Immunol. 5: Bowden, G. H. W Which bacteria are cariogenic in humans?, p In N. W. Johnson (ed.), Dental caries, vol. 1. Markers of high and low risk groups and individuals. Cambridge University Press, Cambridge. 6. Dayhoff, M. O., W. C. Barker, and L. T. Hunt Establishing homologies in protein sequences. Methods Enzymol. 91: De Reuse, H., and A. Danchin The ptsh, ptsl, and crr genes INFECF. IMMUN. of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: a complex operon with several modes of transcription. J. Bacteriol. 170: De Reuse, H., A. Roy, and A. Danchin Analysis of the ptsh-ptsi region of the Escherichia coli K-12: nucleotide sequence of the ptsh gene. Gene 35: Deutscher, J., B. Pevec, K. Beyreuther, H. H. Kiltz, and W. Hengstenberg Streptococcal phosphoenolpyruvate-sugar phosphotransferase system: amino acid sequence and site of ATP-dependent phosphorylation of HPr. Biochemistry 25: Dillard, J. P., and J. Yother Analysis of Streptococcus pneumoniae sequences cloned into Escherichia coli: effect of promoter strength and transcription terminators. J. Bacteriol. 173: Dower, W. J., J. F. Miller, and C. W. Ragsdale High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 16: Eisermann, R., A. Fisher, A. Kessler, A. Neubauer, and W. Hengstenberg Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and protein sequencing of the Staphylococcus carnosus histidine-containing protein, and cloning and DNA sequencing of the ptsh gene. Eur. J. Biochem. 197: a.Frenette, M. Personal communication. 13. Frischauf, A.-M., H. Lehrach, A. Polstka, and N. M. Murray Lambda replacement vectors carrying polylinker sequences. J. Mol. Biol. 170: Gagnon, G., C. Vadeboncoeur, R. C. Levesque, and M. Frenette Cloning, sequencing and expression in Escherichia coli of the ptsi gene encoding enzyme I of the phosphoenolpyruvate phosphotransferase system transport system from Streptococcus salivarius. Gene 121: Gauthier, L., D. Mayrand, and C. Vadeboncoeur Isolation of a novel protein involved in the transport of fructose by an inducible phosphoenolpyruvate fructose phosphotransferase system in Streptococcus mutans. J. Bacteriol. 160: Gonzy-Treboul, G., and M. Steinmetz Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: cloning of the region containing the ptsh and ptsi genes and evidence for a crr-like gene. J. Bacteriol. 169: Gonzy-Treboul, G., M. Zagorec, M.-C. Rain-Guion, and M. Steinmetz Phosphoenolpyruvate:sugar phosphotransferase system in Bacillus subtilis: nucleotide sequence ofptsh, ptsh and the 5'-end of ptsl and evidence for the ptshl operon. Mol. Microbiol. 3: Gottesman, M., M. Hicks, and M. Gellert Genetics and function of DNA ligase in Escherichia coli. J. Mol. Biol. 77: Graves, M. C., and J. C. Rabinowitz In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene. Evidence for "extended" promoter elements. J. Biol. Chem. 261: Hamada, S., and H. D. Slade Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol. Rev. 44: Hamilton, I. R Effect of changing environment on sugar transport and metabolism by oral bacteria, p In J. Reizer and A. Peterkofsky (ed.), sugar transport and metabolism by Gram-positive bacteria. Ellis Horwood, Chichester, England. 22. Hamilton, I. R., L. Gauthier, B. Desjardins, and C. Vadeboncoeur Concentration-dependent repression of the soluble and membrane components of the phosphoenolpyruvate:sugar phosphotransferase system of Streptococcus mutans by glucose. J. Bacteriol. 171: Henikoff, S Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28: Higgins, D. G., A. J. Bleasby, and R. Fuchs CLUSTALV: improved software for multiple sequence alignment. Comp. Appl. Biosci. 8: Huynh, T. V., R. A. Young, and R. A. Davis Constructing and screening cdna libraries in XgtlO and Xgtll, p In D. M.

10 VOL. 62, 1994 SEQUENCE OF HPr AND ENZYME I GENES FROM S. MUTANS 1165 Glover (ed.), DNA cloning techniques: a practical approach. IRL Press, Oxford. 26. Kaslow, D. C A rapid biochemical method for purifying lambda DNA from phage lysates. Nucleic Acids Res. 14: Kohlbrecher, D., R. Eisermann, and W. Hengstenberg Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: molecular cloning and nucleotide sequence of the Staphylococcus carnosus ptsi gene and expression and complementation studies of the gene product. J. Bacteriol. 174: Kruse, R., W. Hengstenburg, W. Beneicke, and H. R. Kalbitzer Involvement of various amino-terminal and carboxy-terminal residues in the active site of the histidine-containing protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus carnosus-site-directed mutagenesis with the ptsh gene, biochemical characterization and NMR studies of the mutant proteins. Protein Eng. 6: Laemmli, U. K Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (London) 227: Loesche, W. J Role of Streptococcus mutans in human dental decay. Microbiol. Rev. 50: Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Macrina, F. L., R. P. Evans, J. A. Tobian, D. L. Hartley, D. B. Clewell, and K. R. Jones Novel shuttle plasmid vehicles for Escherichia-Streptococcus transgeneric cloning. Gene 25: Maniatis, T., E. F. Fritsch, and J. Sambrook Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 34. Mattoo, R. L., and E. B. Waygood An enzymatic method for [32P]phosphoenolpyruvate synthesis. Anal. Biochem. 128: Miller, J. H Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 36. Mulligan, M. E., and W. R. McClure Analysis of the occurrence of promoter sites in DNA. Nucleic Acids Res. 14: Pocalyko, D. J., L. J. Carroll, B. M. Martin, P. C. Babbitt, and D. Dunaway-Mariano Analysis of sequence homologies in plant and bacterial pyruvate phosphate dikinase, enzyme I of the bacterial phosphoenolpyruvate: sugar phosphotransferase system and other PEP-utilizing enzymes. Identification of potential catalytic and regulatory motifs. Biochemistry 29: Postma, P. W., and J. W. Lengeler Phosphoenolpyruvate: carbohydrate phosphotransferase system of bacteria. Microbiol. Rev. 49: Preis, A., H. Priefert, N. Kruger, and A. Steinbuchel Identification and characterization of two Alcaligenes eutrophus gene loci relevant to the poly(,b-hydroxybutyric acid)-leaky phenotype which exhibit homology to ptsh and ptsi of Escherichia coli. J. Bacteriol. 173: Reizer, J., C. Hoischen, A. Reizer, T. N. Pham, and M. H. Saier, Jr Sequence analyses and evolutionary relationships among the energy-coupling proteins Enzyme I and HPr of the bacterial phophoenolpyruvate:sugar phosphotransferase system. Protein i. 2: Robitaille, D., L. Gauthier, and C. Vadeboncoeur The presence of two forms of the phosphocarrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system in streptococci. Biochimie 73: Roeske, C. A., R. M. Kutny, R. A. Budde, and R. Chollet Sequence of the phosphothreonyl regulatory site peptide from inactive maize leaf pyruvate:orthophosphate dikinase. J. Biol. Chem. 263: Saffen, D. W., K. A. Presper, T. L. Doering, and S. Roseman Sugar transport by the bacterial phosphotransferase system: molecular cloning and structural analysis of the Escherichia coli ptsh, ptsi, and crr genes. J. Biol. Chem. 262: Sanger, F., S. Nicklen, and A. R. Coulson DNA sequencing with chain-terminating inhibitors. Proc. NatI. Acad. i. USA 74: Thibault, L., and C. Vadeboncoeur Phosphoenolpyruvatesugar phosphotransferase transport system of Streptococcus mutans: purification of HPr and enzyme I and determination of their intracellular concentrations by rocket immunoelectrophoresis. Infect. Immun. 51: Thompson, J Sugar transport in the lactic acid bacteria, p In J. Reizer and A. Peterkofsky (ed.), Sugar transport and metabolism by Gram-positive bacteria. Ellis Horwood, Chichester, England. 47. Tinoco, I., 0. C. Uhlenbeck, D. M. Crothers, and J. Gralla Improved estimation of secondary structure in ribonucleic acids. Nature (London) 246: Towbin, H., T. Staehelin, and J. Gordon Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. i. USA 76: Vadeboncoeur, C., and L. Gauthier The phosphoenolpyruvate:sugar phosphotransferase system in Streptococcus salivarius. Identification of a III"an protein. Can. J. Microbiol. 33: Vadeboncoeur, C., Y. Konishi, F. Dumas, L. Gauthier, and M. Frenette HPr polymorphism in oral streptococci is caused by partial removal of the N-terminal methionine. Biochimie 73: Vadeboncoeur, C., and M. Proulx Lactose transport in Streptococcus mutans: isolation and characterization of factor ll1lac, a specific component of the phosphoenolpyruvate-lactose phosphotransferase system. Infect. Immun. 46: Vadeboncouer, C., M. Proulx, and L. Trahan Purification of proteins similar to HPr and Enzyme I from the oral bacterium Streptococcus salivarius. Biochemical and immunological properties. Can. J. Microbiol. 29: Vadeboncoeur, C., S. St. Martin, D. Brochu, and I. R. Hamilton Effect of growth rate and ph on intracellular levels and activities of the components of the phosphoenolpyruvate:sugar phosphotransferase system in Streptococcus mutans Ingbritt. Infect. Immun. 59: Vadeboncoeur, C., L. Thibault, S. Neron, H. Halvorson, and I. R. Hamilton Effect of growth conditions on levels of components of the phosphoenolpyruvate:sugar phosphotransferase system in Streptococcus mutans and Streptococcus sobrinus grown in continuous culture. J. Bacteriol. 169: Vieria, J., and J. Messing The puc plasmids, an M13mp7- derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19: Wu, L.-F., J. M. Tomich, and M. M. Saier, Jr Structure and evolution of a multidomain multiphosphoryl transfer protein. Nucleotide sequence of the frub (HI) gene in Rhodobacter capsulatus and comparison with homologous genes from other organisms. J. Mol. Biol. 213: