Department of Biology, Washington University, St Louis, MO 63130, USA1
Department of Medical Microbiology and Immunology, University of South Florida College of Medicine, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612, USA2
Author for correspondence: Allen L. Honeyman. Tel: +1 813 974 2363. Fax: +1 813 974 4151. e-mail: ahoneyma{at}com1.med.usf.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: Streptococcus mutans, phosphoenolypruvate-dependent phosphotransferase system, mannitol
Abbreviations: EII, Enzyme II; PTS, phosphoenolpyruvate-dependent phosphotransferase system
The GenBank accession number for the sequence reported in this paper is AF210133.
a Present address: Department of Medical Microbiology, University of South Florida, Tampa, FL 33612, USA.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The PTS consists of two non-specific components, the Enzyme I and the HPr proteins, which are involved in the initial steps of carbohydrate transport by the PTS (Saier & Reizer, 1992 ). After the formation of phospho-HPr, sugar-specific components of the PTS, the Enzyme IIs (EIIs), vectorially transport and phosphorylate the metabolizable carbohydrate. The EII components of the PTS consist of three highly conserved functional domains, EIIA, EIIB and EIIC (Saier & Reizer, 1992
). These domains may exist on one single molecule or as separate domains on different molecules but all act together to transport the carbohydrate of interest. The mannitol PTS in E. coli utilizes a single sugar-specific Enzyme IICBA molecule. In other organisms, mannitol transport may utilize an EIICB-EIIA pair as depicted for glucose transport in E. coli (Fischer et al., 1989
, 1991
; Reiche et al., 1988
). We have recently demonstrated that mannitol transport in S. mutans has an EIIA component (Honeyman & Curtiss, 1992
). This is similar to the mannitol PTSs found in other Gram-positive organisms (Fischer et al., 1989
, 1991
; Reiche et al., 1988
; Henstra et al., 1996
).
In this report, we describe the isolation, characterization and nucleotide sequence of the mannitol-specific EIICB gene (mtlA) and an adjacent gene, mtlR. The mtlR gene is located within the same operon as mtlA, and the mtlR gene product is not necessary for mannitol utilization. The mtlR gene product may be involved with regulation of this operon, but the exact function of this locus is unknown.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
S. mutans strains were grown in Brain-Heart Infusion (BHI) broth or on BHI agar plates (Difco). E. coli strains were grown in Tryptic Soy Broth (TSB) or on Tryptic Soy Agar (TSA) plates (Difco). Antibiotics used for selection, as necessary, with E. coli were ampicillin, 100 µg ml-1, and chloramphenicol, 20 µg ml-1, and for selection with S. mutans, erythromycin, 10 µg ml-1 and kanamycin, 500 µg ml-1. All chemicals were reagent grade and were obtained from Sigma.
DNA isolation and manipulation.
Streptococcal chromosomal DNA was isolated in the following manner. A 5 ml starter culture was grown overnight in BHI broth in a 15 ml plastic screw-cap tube. This was used to inoculate 45 ml pre-warmed BHI broth in a 50 ml screw-cap tube. This culture was grown stationary overnight at 37 °C. The cells were harvested by centrifugation and washed in TE buffer (10 mM Tris, 1 mM EDTA, pH 8·0). Following the wash step, the cells were resuspended in 6 ml TE buffer and incubated for 1 h at 65 °C. The cells were then cooled on ice prior to the addition of 1 ml of 10 mg lysozyme ml-1 and 200 µl of 5000 units mutanolysin (Sigma) ml-1. The cell suspension was incubated for 1 h at 37 °C. Pronase (1 ml of 10 mg ml-1 stock solution) was added and incubation continued for 1 h. The cells were lysed by the addition of 1 ml of 20% Sarkosyl and 6·8 g guanidine hydrochloride was added and dissolved thoroughly. The DNA solution was incubated at 55 °C for 1·5 h prior to being layered onto a two-step caesium chloride gradient. The top layer (2 ml) of the gradient was 38% w/v caesium chloride while the bottom layer (2 ml) was 60%. Each layer contained 20 µl of 10 mg ml-1 ethidium bromide. The gradient was centrifuged at 30000 r.p.m. for 24 h at 4 °C in a Beckman SW41 rotor. The DNA band which formed at the interface between the two layers was removed with a Pasteur pipette. The ethidium bromide was extracted from the sample with an equal volume of water-saturated butanol and the sample dialysed overnight against TE buffer prior to use.
Plasmid DNA isolations for either large-scale or miniprep protocols were done as described by Sambrook et al. (1989) using the alkaline lysis procedure with a PEG precipitation step. Quick-lysis preparations of plasmid DNA for initial screening procedures were done according to a protocol provided by Stratagene with their mung bean nuclease digestion procedure. This involves pelleting 0·1 ml of bacterial cells from an overnight culture in a microcentrifuge tube. The pellet is resuspended in 30 µl TES (10 mM Tris, 1 mM EDTA, 150 mM NaCl, pH 8·0). An equal amount of water-saturated phenol is then added to the cells and the tube vortexed vigorously. The emulsion was allowed to set at room temperature for 10 min prior to centrifugation at 13000 r.p.m. for 10 min. Twenty microlitres of the aqueous phase was removed to a new microfuge tube and 1 µl RNase (200 µg ml-1) added. After incubation at room temperature for 5 min, 5 µl gel loading dye was added and the entire sample electrophoresed on a gel with supercoiled DNA standards. Plasmid DNA was subsequently isolated as described by Sambrook et al. (1989)
from the cultures of subclones that displayed the appropriate size of plasmid and was screened further by restriction endonuclease digestion. All DNA manipulations and enzyme procedures were done according to the manufacturers instructions and under NIH recombinant DNA guidelines.
Isolation of the S. mutans mtlA gene.
Isolation of the mtlA gene was accomplished by using a chromosomal walking technique using the integrative suicide vector pVA891 (Macrina et al., 1983 ). This plasmid will not replicate in S. mutans and streptococcal DNA fragments cloned into pVA891 can facilitate recombination with the homologous site on the streptococcal chromosome. Selection for erythromycin-resistant S. mutans transformants allows the isolation of colonies in which Campbell insertions into the chromosome have occurred.
The 1476 bp PstIHindIII fragment located in the PstI fragment contained in pYA3121 was cloned into pVA891 as a HindIII fragment and the resultant plasmid was designated pYA3122 (Fig. 1). This plasmid was transformed into S. mutans strain UA130 (Murchison et al., 1986
) and cells containing chromosomal integrations of pYA3122 selected for by growth on erythromycin. Chromosomal DNAs were isolated from several transformants and screened by Southern hybridization (Southern, 1975
) to determine that only one copy of pYA3122 had integrated into the chromosome at the correct site (data not shown). One such S. mutans isolate was designated as SMS207 and was used for further study.
|
DNA sequence analysis.
The complete nucleotide sequence of the 4065 bp AvaIPstI fragment contained in pYA3123 (this study, Fig. 1) was determined on both DNA strands. Several sequencing strategies were used. A series of random Tn5seq1 transposon insertions were used as the sequencing primer site as described by Nag et al. (1988)
. Also, a nested deletion series was generated by the procedure of Henikoff (1984)
from either end of the cloned fragment. In addition, the nucleotide sequence of various regions was determined using synthetic oligonucleotides from Midland Certified Reagents as sequencing primers. In all cases, Sequenase-modified T7 DNA polymerase (Tabor & Richardson, 1987
) from USB was used in the double-stranded sequencing technique of Chen & Seeburg (1985)
. Nucleotide sequence analysis was done using the following computer programs: Genepro (Riverside Scientific), Seqnce (Access Biosystems), GCG (Devereux et al., 1984
) and BLAST (Altschul et al., 1997
).
Transcriptiontranslation assays.
These assays were done on selected plasmid constructs using a transcriptiontranslation reaction kit from Promega. Aliquots of the labelled proteins were analysed using SDS-denaturing polyacrylamide gel electrophoresis (Laemmli, 1970 ) on a 10% gel. Following electrophoresis, the gel was treated with Enhance (Amersham) prior to drying. The dried gel was subjected to autoradiography and/or fluorography.
Characterization of growth of mannitol mutants.
S. mutans strains were grown overnight in chemically defined medium (CDM) (van de Rijn & Kessler, 1980 ) containing 0·5% glucose and 0·5% mannitol. Three millilitres of an overnight culture was added to 100 ml pre-warmed medium containing the appropriate carbohydrate in a 250 ml flask plugged with a stopper surrounding an inverted 16x100 mm glass tube. At various time points, the flask was inverted to fill the test tube, which was inserted into a Spectronic 20 colorimeter. The optical density was read at 600 nm.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nucleotide sequence of the S. mutans mannitol genes
The nucleotide sequence of the entire AvaIPstI fragment contained in pYA3123 was determined (GenBank accession no. AF210133). Nucleotide sequence analysis of this region revealed the presence of two ORFs. The deduced protein from the first ORF exhibits extensive homology to the mannitol-specific EIIs of various organisms. This confirmed our hypothesis about the overall operon structure of the mannitol genes in S. mutans. This operon structure is depicted in Fig. 1.
The nucleotide sequence reported here extends from the MaeIII site shown in Fig. 1 to the PstI site contained in pYA3123. We have previously reported the nucleotide sequence of the 2·3 kb PstI fragment contained in pYA3121 (Honeyman & Curtiss, 1992
). Because the second ORF of the operon reported here crosses the previously described PstI site, we have included the nucleotide sequence data obtained previously which is necessary to extend the ORF to a termination codon so that a complete analysis of the deduced protein can be done.
The first ORF spans 1770 nucleotides, from base-pair 39 to base-pair 1808. The deduced protein from this ORF would consist of 589 amino acids with a molecular mass of 62·0 kDa. This ORF is immediately preceded by the nucleotide sequence AAGGAGG (bases 2329) which resembles the ribosome-binding site of Bacillus subtilis (Moran et al., 1982 ) and the putative ribosome-binding sites described in S. mutans (Honeyman & Curtiss, 1992
, 1993
; Rosey & Stewart, 1992
). These sequences are used for reference because the 3' end of the S. mutans 16s rRNA has not been described at this time.
The second ORF starts 18 bp after the end of the first ORF and continues for 1967 bp until 1 bp 5' to the start of the mtlF gene. This ORF is not preceded by an apparent ribosome-binding site. Other S. mutans genes have sequences which are complementary to the 3' end of the 16S rRNA molecule of B. subtilis and which may serve as ribosome-binding sites (Honeyman & Curtiss, 1992 , 1993
; Rosey & Stewart, 1992
). The first sequences 3' to the first ORF which resemble potential ribosome-binding sites are located at nucleotides 20292033 (AAGAG) and 21502156 (AGGAGG). These potential ribosome-binding sites are located 221 bp and 349 bp, respectively, from the end of the first ORF. The ORF following these potential ribosome-binding sites does not contain a methionine initiation codon for another 83 or 280 nucleotides, respectively. Generally, the spacing between the ribosome-binding site and the start site of translation is 810 bases (Moran et al., 1982
). Therefore, based upon the DNA sequence analysis, it is not possible to determine the potential start site of translation of the second ORF. The possibility exists that a codon other than methionine is used to initiate translation of this protein. It is also possible that this ORF is not preceded by a strong ribosome-binding site as generally found with Gram-positive genes (Vellanoweth & Rabinowitz, 1992
).
Analysis of the nucleotide sequence from the start of the first ORF to the end of the mtlD gene contained in pYA3121 for the presence of other transcriptional control elements such as other promoter elements or transcriptional terminators does not reveal any other potential regulatory elements. Thus, it appears that the ORFs reported here are part of an operon structure which includes the mtlF and mtlD genes as previously described.
Homology analysis of the S. mutans proteins
To determine the potential functions of the deduced proteins from these ORFs, the deduced proteins were compared to all GenBank entries using the BLAST program (Altschul et al., 1997 ). The deduced protein from the first ORF exhibited significant similarity only with the mtlA gene products from various other organisms (Table 1a
). For example, the deduced protein from S. mutans has 55% identity and 73% similarity with the mannitol EIICB gene product from Bacillus stearothermophilus. Based upon the similarity of the deduced protein from S. mutans with the proteins from other mtlA loci, we conclude that this locus is the S. mutans mtlA gene.
|
The S. mutans MtlA protein is approximately 120 amino acid residues longer than the mannitol EIICB proteins from Clostridium acetobutylicum (GenBank accession no. U53868), B. stearothermophilus (Henstra et al., 1996 ) and other organisms. These additional amino acids are located at the carboxyl-terminal end of the S. mutans MtlA protein, outside the previously described EIICB domains. This raises questions about the origin and function of this region of the MtlA molecule. When the amino acid sequence of this extended region of the S. mutans MtlA protein is compared to the GenBank database, similarity is found with the carboxyl-terminal end of various mannitol EIICB molecules (Table 1b
). Thus, S. mutans has two regions within the mtlA gene product that have similarity with various EIIB domains (Fig. 2
). At some point during evolution of the mannitol PTS in S. mutans, the EIIB domain must have undergone duplication. It is not known, at this time, which of the duplicated domains of the S. mutans MtlA protein is the functionally active part of the PTS machinery. However, the similarity displayed by the terminal 119 amino acids with various EIICB domains is less than that displayed by the preceding 119 amino acids of the S. mutans MtlA protein. When the preceding amino acids (residues 352470) are compared to the GenBank database, similarities with the carboxyl-terminus of several EIICB proteins are found. These similarity indexes are generally in the range of 44% identity with 65% similarity (data not shown). These values are greater than those displayed by the terminal 119 amino acids (Table 1b
). These data would tend to indicate, based upon similarity alone, that the terminal 119 amino acids of the MtlA protein may not be the functionally active region of the EIICB domain. However, the possiblity exists that both duplicated regions of the EIIB domain of this molecule are functional.
|
|
It is interesting to note that the S. mutans MtlR protein does not have similarity to the OrfX protein from Ent. faecalis. This is interesting in light of the overall operon structure and protein homology in other regions between the mannitol operons of S. mutans and Ent. faecalis (Fischer et al., 1991 ; Honeyman & Curtiss, 1992
). Fischer et al. (1991)
have described an orfX gene within the mannitol operon of Ent. faecalis similar to the second ORF described here. We have previously reported a high degree of homology (55·4 and 61·1% identity, 87·2 and 87·5% conserved amino acid substitutions) between the MtlF and MtlD proteins of these organisms (Honeyman & Curtiss, 1992
). Although the sequence data for the orfX gene from Ent. faecalis is limited (Fischer et al., 1991
), its deduced protein (104 amino acid residues) does not display any homology to the deduced MtlR protein of S. mutans, which is at the same location within the operon structure. These data seem incongruent with the existing homology data and the data on the overall structure of the mtl operons of these organisms.
Insertional inactivation of mtlR
To determine if mtlF and mtlD are part of a single transcriptional unit which includes mtlR, the mtlR locus was insertionally inactivated in vitro. The interposon -Kan (Perez-Casal et al., 1991
), which is flanked by transcriptional and translational terminators, was cloned into the BglII site of pYA3124 (Fig. 1
) to generate pYA3125. This plasmid was linearized by restriction endonuclease digestion and used to transform UA130 (Murchison et al., 1986
), with selection for kanamycin resistance. Kanamycin-resistant colonies would contain the interposon integrated into the chromosome interrupting the mtlR gene by means of a homologous double crossover. Kanamycin-resistant colonies were obtained and one transformant, ALH154, was used for characterization of the mtlR::
-Kan mutation. This in vitro-generated mutation should have a polar effect on the loci 3' to the insertion.
To determine the effect of the interposon insertion into mtlR, S. mutans was grown in CDM (van de Rijn & Kessler, 1980 ) supplemented with 0·2% glucose, with 0·2% mannitol, or (to determine the effects of diauxic growth on the mutant strain) with both carbohydrates at 0·2%. Wild-type UA130 cells grown with either glucose or glucose plus mannitol showed a quick increase in cell numbers as compared to cells grown with mannitol alone (Fig. 4a
). Wild-type cells grown with glucose plus mannitol exhibited diauxic growth, reaching higher final OD600 values than cells grown with either glucose or mannitol alone. In contrast, cells of the mtlR insertion mutant strain showed similar growth curves in either glucose or glucose plus mannitol, and there was no growth with mannitol as the sole carbon source (Fig. 4b
). Therefore, mannitol utilization is blocked in the mtlR::
-Kan mutant, ALH154.
|
To determine if the effect was due to the polar insertion of -Kan into mtlR, a mutation with a non-polar insertion element was also generated in vitro. The kanamycin-resistance gene from the
-Kan cassette was amplified by PCR. The primers for this PCR reaction were generated in such manner as to produce a promoter-less kanamycin cassette which does not have a transcriptional terminator following the antibiotic resistance gene. This cassette was positioned at the same site in the S. mutans chromosome and in a manner as previously described for the
-Kan cassette. Strains containing this non-polar insertion into the mtlR gene (exemplified by ALH124) grow normally on mannitol media and have the ability to ferment mannitol (data not shown). Therefore, the MtlR gene product is not necessary for growth on mannitol or for expression of the operon. Thus, based upon DNA sequence analysis and insertional mutagenesis data, mtlA and mtlR appear to be co-transcribed with mtlF and mtlD and polar insertions into mtlR block mannitol utilization.
In this report, we have described the mtlA and mtlR genes of S. mutans. The mtlA gene encodes the EIICB domains of the PTS permease for mannitol transport. The MtlA protein is approximately 120 amino acids longer than several other EIICB molecules. The extended region of the MtlA protein also displays homology to the carboxyl-terminal end of the EIIB domain. Thus, a part of this domain was duplicated during evolution of the mannitol PTS in S. mutans. It is not currently known which region of the MtlA protein acts as the functional region of the EIIB domain. The mtlR locus, based upon deduced protein similarity, appears to encode a regulatory protein which has a helixturnhelix region located at its amino-terminal end. Based upon DNA sequence analysis and insertional mutagenesis data, mtlA and mtlR appear to be part of a multiple cistronic message which includes the mtlF and mtlD genes previously described. Insertion of a polar interposon into mtlR blocks mannitol utilization, while insertion of a non-polar cassette does not. This indicates that the MtlR protein is not neccessary for mannitol utilization or expression of the operon. Investigations into the exact function of the mtlR gene product are currently under way. Studies on the regulation of the mannitol PTS operon, in relation to the effects of catabolite repression, are also in progress.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Boyd, D. A., Cvitkovitch, D. G. & Hamilton, I. R. (1994). Sequence and expression of the genes for HPr (ptsH) and enzyme I (ptsI) of the phosphoenolpyruvate-dependent phosphotransferase transport system from Streptococcus mutans. Infect Immun 62, 1156-1165.[Abstract]
Bullock, W. O., Fernandez, J. M. & Short, J. M. (1987). XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. Biotechniques 5, 376-378.
Chen, E. Y. & Seeberg, P. H. (1985). Supercoil sequencing: a fast and simple method for sequencing plasmid DNA.DNA 4, 165-170.[Medline]
Devereux, J., Haeberli, P. & Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12, 387-395.[Abstract]
Dodd, I. B. & Egan, J. B. (1990). Improved detection of helixturnhelix DNA-binding motifs in protein sequences. Nucleic Acids Res 18, 5019-5026.[Abstract]
Drucker, D. B. & Melville, T. H. (1968). Fermentation end-products of cariogenic and non-cariogenic streptococci. Arch Oral Biol 13, 565-570.[Medline]
Fischer, R. & Hengstenberg, W. (1992). Mannitol-specific Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus carnosus. Sequence and expression in Escherichia coli and structural comparison with the Enzyme IImannitol of Escherichia coli. Eur J Biochem 204, 963-969.[Abstract]
Fischer, R., Eisermann, R., Reiche, B. & Hengstenberg, W. (1989). Cloning, sequencing and overexpression of the mannitol-specific Enzyme-III-encoding gene of Staphylococcus carnosus. Gene 82, 249-257.[Medline]
Fischer, R., von Strandmann, R. P. & Hengstenberg, W. (1991). Mannitol-specific phosphoenolpyruvate-dependent phosphotransferase system of Enterococcus faecalis: molecular cloning and nucleotide sequences of the Enzyme IIIMtl gene and the mannitol-1-phosphate dehydrogenase gene, expression in Escherichia coli, and comparison of the gene products with similar enzymes. J Bacteriol 173, 3709-3715.[Medline]
Henikoff, S. (1984). Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing.Gene 28, 351-359.[Medline]
Henstra, S. A., Tolner, B., ten Hoeve Duurkens, R. H., Konings, W. N. & Robillard, G. T. (1996). Cloning, expression, and isolation of the mannitol transport protein from the thermophilic bacterium Bacillus stearothermophilus. J Bacteriol 178, 5586-5591.
Henstra, S. A., Tuinhof, M., Duurkens, R. H. & Robillard, G. T. (1999). The Bacillus stearothermophilus mannitol regulator, MtlR, of the phosphotransferase system. A DNA-binding protein, regulated by HPr and IICBmtl-dependent phosphorylation. J Biol Chem 274, 4754-4763.
Hiratsuka, K., Wang, B., Sato, Y. & Kuramitsu, H. (1998). Regulation of sucrose-6-phosphate hydrolase activity in Streptococcus mutans: characterization of the scrR gene. Infect Immun 66, 3736-3743.
Honeyman, A. L. & Curtiss, R.III (1992). Isolation, characterization, and nucleotide sequence of the Streptococcus mutans mannitol-phosphate dehydrogenase gene and the mannitol-specific factor III gene of the phosphoenolpyruvate phosphotransferase system. Infect Immun 60, 3369-3375.[Abstract]
Honeyman, A. L. & Curtiss, R.III (1993). Isolation, characterization, and nucleotide sequence of the Streptococcus mutans lactose-specific enzyme II (lacE) gene of the PTS and the phospho-ß-galactosidase (lacG) gene. J Gen Microbiol 139, 2685-2694.[Medline]
Kunst, F., Ogasawara, N., Moszer, I. & 148 other authors (1997). The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390, 249256.[Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]
Lee, C. A. & Saier, M. H.Jr (1983). Mannitol-specific Enzyme II of the bacterial phosphotransferase system. III. The nucleotide sequence of the permease gene. J Biol Chem 258, 10761-10767.
Macrina, F. L., Evans, R. P., Tobian, J. A., Hartley, D. L., Clewell, D. B. & Jones, K. R. (1983). Novel shuttle plasmid vehicles for EscherichiaStreptococcus transgeneric cloning. Gene 25, 145-150.[Medline]
Manoil, C. & Beckwith, J. (1985). TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A 82, 8129-8133.[Abstract]
Moran, C. P.Jr, Lang, N., LeGrice, S. F., Lee, G., Stephens, M., Sonenshein, A. L., Pero, J. & Losick, R. (1982). Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol Gen Genet 186, 339-346.[Medline]
Murchison, H. H., Barrett, J. F., Cardineau, G. A. & Curtiss, R.III (1986). Transformation of Streptococcus mutans with chromosomal and shuttle plasmid (pYA629) DNAs. Infect Immun 54, 273-282.[Medline]
Nag, D. K., Huang, H. V. & Berg, D. E. (1988). Bidirectional chain-termination nucleotide sequencing: transposon Tn5seq1 as a mobile source of primer sites. Gene 64, 135-145.[Medline]
Perez-Casal, J., Caparon, M. G. & Scott, J. R. (1991). Mry, a trans-acting positive regulator of the M protein gene of Streptococcus pyogenes with similarity to the receptor proteins of two-component regulatory systems. J Bacteriol 173, 2617-2624.[Medline]
Postma, P. W. & Lengeler, J. W. (1985). Phosphoenolpyruvate:carbohydrate phosphotransferase system of bacteria. Microbiol Rev 49, 232-269.
Reiche, B., Frank, R., Deutscher, J., Meyer, N. & Hengstenberg, W. (1988). Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific Enzyme IIImtl of Staphylococcus aureus and Staphylococcus carnosus and homology with the Enzyme IImtl of Escherichia coli. Biochem 27, 6512-6516.[Medline]
van de Rijn, I. & Kessler, R. E. (1980). Growth characteristics of group A streptococci in a new chemically defined medium. Infect Immun 27, 444-448.[Medline]
Rosey, E. L. & Stewart, G. C. (1992). Nucleotide and deduced amino acid sequences of the lacR, lacABCD, and lacFE genes encoding the repressor, tagatose 6-phosphate gene cluster, and sugar-specific phosphotransferase system components of the lactose operon of Streptococcus mutans. J Bacteriol 174, 6159-6170.[Abstract]
Saier, M. H.Jr & Reizer, J. (1992). Proposed uniform nomenclature for the proteins and protein domains of the bacterial phosphoenolpyruvate:sugar phosphotransferase system. J Bacteriol 174, 1433-1438.[Medline]
Saier, M. H., Jr, Yamada, M., Erni, B. & 7 other authors (1988). Sugar permeases of the bacterial phosphoenolpyruvate-dependent phosphotransferase system: sequence comparisons. FASEB J 2, 199208.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). In Molecular Cloning: a Laboratory Manual, 2nd edn, pp. 140141. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sato, Y., Yamamoto, Y., Suzuki, R., Kizaki, H. & Kuramitsu, H. K. (1991). Construction of scrA::lacZ gene fusions to investigate regulation of the sucrose PTS of Streptococcus mutans. FEMS Microbiol Lett 63, 339-345.[Medline]
Schachtele, C. F. & Mayo, J. A. (1973). Phosphoenolpyruvate-dependent glucose transport in oral streptococci. J Dental Res 52, 1209-1215.[Medline]
Southern, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98, 503-517.[Medline]
Tabor, S. & Richardson, C. C. (1987). DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A 84, 4767-4771.[Abstract]
Takami, H., Nakasone, K., Ogasawara, N. & 7 other authors (1999). Sequencing of three lambda clones from the genome of alkaliphilic Bacillus sp. strain C-125. Extremophiles 3, 2934.[Medline]
Vellanoweth, R. L. & Rabinowitz, J. C. (1992). The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. Mol Microbiol 6, 1105-1114.[Medline]
Yamane, K., Kumano, M. & Kurita, K. (1996). The 25°36° region of the Bacillus subtilis chromosome: determination of the sequence of a 146 kb segment and identification of 113 genes.Microbiology 142, 3047-3056.[Abstract]
Received 10 December 1999;
revised 10 March 2000;
accepted 20 March 2000.