A novel ß-glucoside-specific PTS locus from Streptococcus mutans that is not inhibited by glucose

Christopher K. Cote1, Dennis Cvitkovitcha,2, Arnold S. Bleiweis2 and Allen L. Honeyman1

Department of Medical Microbiology and Immunology, University of South Florida College of Medicine, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612, USA1
Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, FL 32611, USA2

Author for correspondence: Allen L. Honeyman. Tel: +1 813 974 2362. Fax: +1 813 974 4151. e-mail: ahoneyma{at}com1.med.usf.edu


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
A regulon from Streptococcus mutans that plays a role in the utilization of ß-glucosides has been isolated, sequenced and subjected to sequence analysis. This regulon encodes a ß-glucoside-specific Enzyme II (EII) component (bglP) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) and a phospho-ß-glucosidase (bglA) which is responsible for the breakdown of the phospho-ß-glucosides within the cell. Both the bglP and bglA gene products have significant similarity with proteins that have similar functions from Clostridium longisporum, Listeria monocytogenes, Erwinia chrysanthemi, Escherichia coli, Klebsellia oxytoca and Bacillus subtilis. The potential functions of the BglP and BglA proteins are supported by phenotypic data from both S. mutans and E. coli. A chromosomal deletion in S. mutans spanning the bglP and bglA genes resulted in a strain that was unable to hydrolyse the ß-glucoside aesculin in the presence of glucose. When glucose was removed from the medium, the deletion strain regained the ability to break down aesculin. These data suggest that S. mutans possesses an alternative mechanism from the one described in this report for breaking down ß-glucosides. This second mechanism was repressed by glucose while the regulon described here was not. Complementation studies in E. coli CC118 also suggest a potential role for this regulon in the utilization of other ß-glucosides. When a plasmid containing the 8 kb ß-glucoside-specific regulon was transformed into E. coli CC118, the transformed strain was able to break down the ß-glucoside arbutin.

Keywords: Streptococcus mutans, ß-glucosides, aesculin, phosphoenolpyruvate-dependent phosphotransferase system

Abbreviations: CTAB, cetyltrimethylammonium bromide; EII, Enzyme II; PTS, phosphoenolpyruvate-dependent phosphotransferase system

The GenBank accession number for the sequence reported in this paper is AF206272.

a Present address: Dental Research Institute, University of Toronto School of Dentistry, Toronto, Ontario, Canada.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The phosphoenolpyruvate-dependent phosphotransferase system (PTS) is important for carbohydrate acquisition in many bacteria (Postma et al., 1993 ). The PTS consists of several proteins that transport PTS-dependent carbohydrates in both Gram-negative and Gram-positive organisms. The PTS components include a non-specific Enzyme I (EI), a heat-stable histidine protein (HPr) and a sugar-specific Enzyme II (EII) (Postma et al., 1993 ). The functional domains of the EII protein (EIIA, EIIB and EIIC) may be found on one or more separate proteins (Saier et al., 1988 ). It is this multi-domain complex that participates in the carbohydrate-specific translocation across the membrane. This translocation ultimately leads to the utilization of the PTS-dependent carbohydrate by the bacterium. Streptococcus mutans relies upon the PTS for the uptake of many carbon sources, including glucose, lactose and sucrose (Hamada & Slade, 1980 ). The PTS has been clearly associated with the utilization of common dietary carbohydrates, but its role in ß-glucoside utilization is less substantiated. ß-Glucosides, such as cellobiose, arbutin, salicin and aesculin, can be found in foods containing plant extracts. The general structure of these compounds is a glucose moiety with various groups attached at the C-1 hydroxyl of the glucose core. The ability of S. mutans to metabolize ß-glucosides can be viewed as a mechanism to ensure survival under starvation conditions (Kruger & Hecker, 1995 ). S. mutans will preferentially utilize more readily metabolized carbon sources, such as glucose, before turning to ß-glucosides as a carbon source. Other bacteria, including Escherichia coli, Bacillus subtilis, Clostridium longisporum and Erwinia chrysanthemi, have been shown to use the PTS for the translocation of ß-glucosides (Brown & Thomson, 1998 ; el Hassouni et al., 1990 ; Fox & Wilson, 1968 ; Le Coq et al., 1995 ).

ß-Glucoside utilization, facilitated by the PTS, has been best characterized in Escherichia coli (Fox & Wilson, 1968 ; Schaefler, 1967 ). The E. coli PTS ß-glucoside operon (the bglGFB operon) contains all of the genetic elements for the uptake and utilization of ß-glucosides, but is cryptic in nature (Schnetz et al., 1987 ). A genetic mutation is required for E. coli to utilize ß-glucosides (Schnetz & Rak, 1988 ). The majority of the mutations in E. coli leading to the activation of the bgl operon are the result of the transposition of an insertion sequence into the region of the operon that is upstream of bglG (Schnetz & Rak, 1988 ). Once activated, the bglGFB operon is subject to regulation via the antiterminator protein BglG. The bglF gene encodes the EII of the PTS, while bglB encodes a phospho-ß-glucosidase (Amster-Choder et al., 1989 ; Chen et al., 1997 ). Both the BglF and the BglG proteins are components of a sensory system that induces the bgl genes in the presence of ß-glucosides (Chen & Amster-Choder, 1998 ).

Erwinia chrysanthemi has a system for the metabolism of ß-glucosides very similar to that encoded by the bglGFB operon of Escherichia coli. The arb genes in Er. chrysanthemi are similar in both structure and function to the bglGFB operon of E. coli, but are not cryptic (el Hassouni et al., 1990 ). In Er. chrysanthemi, the arbF gene encodes a ß-glucoside-specific EII permease and the arbB gene encodes a phospho-ß-glucosidase. In both Er. chrysanthemi and E. coli, the PTS EIIs that are encoded by the arbF and bglF genes, respectively, are specific for the aromatic ß-glucosides arbutin and salicin. While neither of these organisms can utilize aesculin as a sole carbon source, the proteins encoded by the E. coli bglGFB genes and the arbGFB genes in Er. chrysanthemi allow for the hydrolysis of this glucoside (el Hassouni et al., 1990 ).

Gram-positive bacteria have been shown to contain ß-glucoside systems similar to those of the Gram-negative bacteria. In Bacillus subtilis, the bglPH operon is responsible for the production of a PTS EII (bglP) and a phospho-ß-glucosidase (bglH) (Kruger et al., 1996 ). In this organism, the synthesis of the BglP protein is induced by the presence of either arbutin or salicin. A second ß-glucoside system has also been identified and characterized in B. subtilis (Tobisch et al., 1997 ). This system is responsible for the transport and utilization of cellobiose and lichenan. A ß-glucoside-specific EII is encoded by the licABC operon, and a 6-phospho-ß-glucosidase is encoded by licH (Tobisch et al., 1997 ).

Clostridium longisporum, a ruminal Gram-positive bacterium, also relies on the PTS for the uptake of ß-glucosides. C. longisporum utilizes an aryl-ß-glucoside uptake and utilization system that is composed of several abg genes (Brown & Thomson, 1998 ). This includes a PTS EII that is specific for salicin and arbutin, which is encoded by the abgF gene. The phospho-ß-glucosidase in C. longisporum is encoded by the abgA gene.

In both Gram-negative and Gram-positive organisms, the operons associated with ß-glucoside utilization are repressed in the presence of a more readily metabolized carbon source, such as glucose. It has been noted that the regulation of ß-glucoside systems is markedly similar in both Gram-negative and Gram-positive bacteria (Rutberg, 1997 ). One common feature of these PTS-mediated systems is the unusual regulation that is associated with the catabolite repression of these genes. Throughout the ß-glucosidase operons discussed, antitermination is the main regulatory mechanism (Bardowski et al., 1994 ; Kruger & Hecker, 1995 ; Schnetz & Rak, 1988 ; Schnetz et al., 1996 ). Thus, the study of the ß-glucoside-specific PTS operon from S. mutans is of importance because of the potential regulatory mechanism for these genes.

The work presented here describes a ß-glucoside-specific PTS regulon isolated from S. mutans. This regulon contains genes encoding a ß-glucoside-specific permease (bglP) and a phospho-ß-glucosidase (bglA). The region of DNA between these genes contains three additional ORFs, one of which is transcribed in the opposite direction. Two of these ORFs are hypothesized to encode functional proteins.


   METHODS
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INTRODUCTION
METHODS
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Bacterial strains and media.
S. mutans NG8 (ALH76) was used as the source of chromosomal DNA and served as the recipient strain for transformations. E. coli strains TB1 (F- ara {Delta}(lac–proAB) rpsL [{phi}80dlacZ{Delta}M15] thi hsdR) (Anonymous, 1984 ; Gibco-BRL) and CC118 [{Delta}(ara–leu)7697 araD139 {Delta}lacX74 galE galK {Delta}phoA20 thi-1 rpsE rpoB argE(Am) recA1] (Manoil & Beckwith, 1985 ) were used for recombinant DNA manipulations. All S. mutans strains were grown on Brain-Heart Infusion (BHI) plates or in BHI broth (Difco). E. coli strains were grown on Lennox Broth (LB) plates or in LB broth (Gibco-BRL). Antibiotics used for selection of E. coli strains were either ampicillin (100 µg ml-1) or chloramphenicol (20 µg ml-1). Selection for S. mutans strains was with either erythromycin (10 µg ml-1) or kanamycin (500 µg ml-1). Purple Broth Base (Difco) was used to make agar (1·5%) plates containing 1% carbohydrate for ß-glucoside specificity experiments. Aesculin plates were composed of 0·5% neopeptone, 0·5% heart infusion broth, 0·1% aesculin, 0·1% dextrose and 0·05% ferric citrate. Aesculin plates were also made as stated above, but without the addition of dextrose. Minimal medium plates with 1% of the desired ß-glucoside as a carbon source were used to check the phenotype of E. coli strains harbouring various plasmids carrying the S. mutans bgl genes (Curtiss, 1965 ). Strains and plasmids generated in these study are listed in Table 1.


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Table 1. Strains and plasmids

 
DNA isolation and manipulation.
Streptococcal chromosomal DNA was isolated as follows. A 50 ml tube of BHI broth was inoculated with a 5 ml starter culture and grown overnight at 37 °C in a screw-cap centrifuge tube. The cells were harvested by centrifugation, resuspended in 4·1 ml TE buffer (10 mM Tris, 1 mM EDTA, pH 8·0) and incubated at 65 °C for 30 min. The cell suspension was cooled on ice and 0·4 g lysozyme, 200 µl mutanolysin (5000 U ml-1), and 200 µl lyticase (final concn 500 U ml-1) were added. The mixture was incubated at 37 °C for 1 h. The cells were lysed by the addition of 450 µl 20% SDS (final concn 1·5%). Upon lysis, 14 µl Proteinase K (20 mg ml-1) was added to the lysate, mixed, and incubated at 65 °C for 1 h. After incubation, 0·9 ml 5 M NaCl was added and the lysate was mixed gently. This was followed by the addition of 710 µl 10% CTAB/0·7 M NaCl (final CTAB concn 1%). The mixture was incubated again at 65 °C for 10 min. The solution was cooled and was extracted with an equal volume of chloroform/isoamyl alcohol (24:1, v/v). The DNA was precipitated from the aqueous phase by the addition of 0·6 vol. 2-propanol and was collected by spooling onto a glass rod. The spooled DNA was washed with 70% ethanol and resuspended in 1 ml TE buffer.

Large-scale plasmid DNA preparations were done as described by Sambrook et al. (1989) , but with omission of the PEG precipitation step. Mini-plasmid preparations were performed using QIAprep Spin Miniprep Kits and QIAprep 8 Miniprep Kits (Qiagen). All DNA manipulations and enzyme procedures were done according to the manufacturer’s instructions and under NIH recombinant DNA guidelines.

Recombinant plasmid constructs were electroporated into E. coli (Dower, 1990 ). Prior to electroporation, ligation mixtures were butanol precipitated. Briefly, 500 µl butanol was added to a 50 µl ligation reaction. The mixture was vortexed briefly and microcentrifuged for 15 min. The butanol was removed and the pellet was washed with 500 µl 100% ethanol. The pellet was air-dried and resuspended in 12 µl water. Three microlitres of the resuspended ligation was used for electroporation. S. mutans was transformed using the method described by Murchison et al. (1986) . All chemicals and reagents were purchased from Sigma unless otherwise noted.

Isolation of the S. mutans ß-glucoside-specific regulon.
A 780 bp PCR fragment was obtained from the chromosome of S. mutans NG8 using degenerate primers designed for the glutamine synthetase gene. The PCR fragment was cloned and its nucleotide sequence determined. Sequence analysis revealed that this PCR fragment would encode a protein that had similarity with the ß-glucoside-specific PTS EIIs from various bacteria rather than the glutamine synthetase protein. The cloned PCR fragment was then moved into the E. coli–S. mutans shuttle vector pVA891 (Macrina et al., 1983 ), resulting in the construction of pALH103. This non-replicating shuttle vector was transformed into S. mutans NG8 and transformants were selected for by erythromycin resistance. One such transformant, ALH96, which contained pALH103 integrated into the chromosome, was used as the source of DNA for the isolation of the bgl regulon.

To isolate the bgl genes surrounding the integration site of pALH103, chromosomal DNA was isolated from S. mutans ALH96 and was digested with various restriction endonucleases that cut outside the origin of replication and the antibiotic-resistance marker of the integrated vector. The restriction digests were phenol/chloroform extracted and the DNA was self-ligated overnight at low DNA concentrations. This resulted in the self-ligation of part of the integrated shuttle vector to the adjacent chromosomal DNA that flanked the original integration site. The ligation mixtures were butanol precipitated prior to electroporation into E. coli. Chloramphenicol resistance was used to select for cells containing plasmids carrying the chromosomal DNA that flanks the integration site. This procedure resulted in a 13·9 kb plasmid when the restriction endonuclease BamHI was used. This construct, pALH104, contained approximately 8 kb of chromosomal DNA located 3' from the original integration site. Repeating the described procedure with the chromosomal DNA isolated from ALH96 using the restriction enzyme XbaI resulted in a 7·5 kb plasmid. This construct, pALH105, contained approximately 2 kb of chromosomal DNA located 5' from the original integration site. The original integration into the chromosome and the subsequent clones that contained flanking DNA are shown in Fig. 1.



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Fig. 1. Diagram illustrating the integration of vector pALH103 into the chromosome of S. mutans NG8. The plasmids that were rescued from the chromosome of strain ALH96 that contained the bgl genes are shown below the integration. Also included are the restriction fragments used to generate a chromosomal deletion of the bgl genes to create strain ALH167.

 
DNA sequence analysis of regions from pALH104 and pALH105 determined that the bgl utilization system covered the entire area isolated. To determine the nucleotide sequence of this region, the DNA flanking the integration site of pALH103 was required in its native state. Therefore, the region flanking the integration site was isolated by PCR amplification using S. mutans NG8 chromosomal DNA as the template. PCR primers were synthesized using DNA sequence data previously obtained. The primers were located just outside the HindIII sites shown in Fig. 2. The amplified product was digested with HindIII prior to cloning into pACKS30 (A. L. Honeyman, unpublished) to generate pALH112. This construct is shown in Fig. 2 and enabled the native chromosomal DNA sequence to be completed flanking the integration site of pALH103 (Fig. 1).



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Fig. 2. Organization of the characterized ß-glucoside-specific regulon of S. mutans. The figure indicates the chromosomal DNA fragments contained in the listed plasmids or the DNA present in the chromosome of the various deletion or insertion mutants.

 
DNA sequence determination and analysis.
The nucleotide sequence of the ß-glucoside-specific PTS region was determined on both strands. Manual sequencing was performed on selected subcloned DNA fragments using the fmol DNA sequencing system (Promega). To facilitate the sequencing of the regulon, a deletion series containing progressive, unidirectional deletions of the cloned DNA was created using the Erase-a-Base system (Promega). DNA sequencing was performed on the deletion series at the Moffit Cancer Center at the University of South Florida and the Core Molecular Biology facility at the University of Florida. Primers were subsequently designed from the sequences obtained from the deletion series to complete the nucleotide sequence. Nucleotide sequence analysis was performed with GCG (Devereux et al., 1984 ) and DNA Strider (Marck, 1988 ) software.

PCR amplification.
S. mutans chromosomal DNA was used as the template in PCR reactions. The polymerase KlenTaq-LA (Clonetech) was used, according to the manufacturer’s recommendations. The primers used to isolate the native chromosome surrounding the integration site of pALH103 into the bgl genes were Bgl-5' (GTCTCCCATGTTGGTGGC) and Bgl-3' (GGAGCAATTTGCCAACCCC).

Construction of deletion strains and plasmids.
Table 1 and Figs 1 and 2 show the various strains and plasmids used in this report. To create a S. mutans strain containing an insertional mutation of the bglP gene, the E. coli–S. mutans shuttle construct pALH103 was integrated into the chromosome. This was accomplished via homologous recombination between the cloned PCR fragment and the chromosome of S. mutans, resulting in strain ALH96. Strain ALH168, containing an insertional mutation of the bglA gene, was created by the insertion of an {Omega}-Kan cassette (Perez-Casal et al., 1991 ) into an EcoRV site within the cloned bglA gene. The construct containing the desired mutation of bglA was linearized with XbaI and integrated into the chromosome of S. mutans NG8 via a double homologous recombination.

Strain ALH167, containing a nearly complete deletion of the entire regulon was created by cloning the 2·0 kb XbaI–BamHI fragment from pALH105 (Fig. 1) and the 1·2 kb PstI–EcoRI fragment from pALH104 into the same plasmid vector. This construct, pALH168, has these two restriction fragments in the same orientation as they are found in the native S. mutans chromosome. An {Omega}-Kan cassette was inserted into a unique SmaI site located between these two subcloned restriction fragments to create pALH169. This plasmid was linearized within the vector sequence and the deletion integrated into the chromosome by selection for the {Omega}-Kan marker. This produced the strain ALH167.

Subcloned fragments from this ß-glucoside-specific regulon were used to create plasmids for expression of these S. mutans genes in E. coli. The bglP deletion construct pALH164 was created by cloning a 5·3 kb EcoRI fragment from the isolated regulon into pACKS30. This fragment was located downstream from the bglP gene, but contains the bglC and bglA genes. The bglA deletion construct pALH165 contains the XhoI–EcoRV fragment from the 5' end of the regulon. This region contains the bglP and the bglB genes and deletes all of the bglA gene. The construct pALH163, which contains the entire regulon described here, was generated from several smaller subclones (Fig. 2).


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Nucleotide sequence analysis
The nucleotide sequence of the entire bgl regulon has been determined on both strands from the XbaI site to the EcoRI site in pALH163 (Fig. 2; GenBank accession no. AF206272). This regulon is composed of 8006 bp and has a G+C content of 34 mol%. Sequence analysis determined that this region contains five ORFs (Fig. 2). Four of these ORFs are thought to encode functional proteins. Based upon analysis of the deduced amino acid sequences, two of these proteins have similarity with proteins associated with the utilization of ß-glucosides as carbon sources in other bacteria. The nucleotide sequence 3' to the EcoRI site located at the end of the genes reported here has been reported previously by Ferretti et al. (1989) .

bglP.
The first ORF spans 1935 nucleotides (base-pairs 420–2354 in the submitted sequence) and would encode a deduced protein of 645 amino acids. A putative ribosome-binding site, with the sequence AAGGAG, is located 6 bases upstream from the start of this ORF. This sequence resembles binding sites in other Gram-positive bacteria (De Vos, 1987 ; Moran et al., 1982 ). The predicted protein has extensive homology with the ß-glucoside-specific PTS EIIs from other organisms (Table 2a). This protein is likely responsible for the translocation of the carbohydrate into the bacterial cell. The PTS EIIs can have three domains that are present on one or more proteins. The ß-glucoside-specific PTS EII encoded by this ORF is predicted to contain all three domains (EIIB, EIIC and EIIA) on a single protein (EIIBCA). The similarity observed between this deduced protein and the PTS EIIs is significant throughout the full length of the deduced protein. Due to the similarity between the EII gene products, which act as permeases, and the deduced protein, the first ORF has been named bglP.


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Table 2. Identities and similarities of deduced proteins from the bgl regulon of S. mutans

 
bglB.
The second ORF is 1512 bp long (base-pairs 2509–4020) and the deduced protein would be 504 amino acids long. The start of this ORF is 155 bp downstream from the end of bglP. There is also a putative ribosome-binding site, AAGGAGG, located 8 bases upstream from the start of this ORF. The deduced protein from this ORF displayed similarity to several GenBank entries. However, this similarity is limited to an internal 70 amino acid sequence in the deduced protein. This region exhibits similarity to several polyhydroxybutyrate depolymerases from Pseudomonas lemoignei, Ralstonia pickettii and Alcaligenes faecalis (data not shown). Based upon amino acid similarity comparisons, a potential function of this protein cannot be determined. Due to its central location within the protein, the homologous region may serve as a potential binding site. However, there are no apparent structural similarities between polyhydroxybutyrate and ß-glucosides. Although the function of this putative protein is in question, the gene encoding this protein has been named bglB because of its close proximity to bglP and the fact that it is situated between bglP and bglA.

bglC.
The third ORF of this region is transcribed from the opposite strand of DNA. It consists of 906 nucleotides (base-pairs 4254–5159) and would encode a protein of 302 amino acids. There is no apparent ribosome-binding site located immediately 5' to this ORF. This deduced protein has similarity with several proteins in the XylS/AraC family of transcriptional regulators, which are involved with the regulation of various operons in Listeria monocytogenes, B. subtilis, E. coli and Haemophilus influenzae (Table 2b). These proteins contain helix–turn–helix DNA-binding domains. Therefore, this ORF would encode a putative transcriptional regulator and has been named bglC. No function has been assigned to this ORF at this time.

ORF 4.
The fourth ORF is 423 nucleotides in length (base-pairs 5344 to 5766) and the predicted protein has very low similarity with the TraG and TrsG regulatory proteins from Staphylococcus aureus. There is no apparent ribosome-binding site adjacent to this ORF and it is not known if the ORF encodes a functional protein. Therefore, this ORF has not been named at this time.

bglA.
The fifth ORF is 1440 nucleotides long (base-pairs 5974–7413) and would encode a protein of 480 amino acids. A putative ribosome-binding site, AAGGA, is positioned 6 bases from the start of this gene. This deduced protein would have high similarity to the phospho-ß-glucosidases from other organisms, including C. longisporum, B. subtilis, E. coli, Klebsiella oxytoca and Er. chrysanthemi (Table 2c). As with the S. mutans bglP gene product, this similarity is apparent throughout the entire deduced protein. This ORF has been named bglA because of the similarities between the deduced BglA protein and the phospho-ß-glucosidase from C. longisporum. It is the bglA gene product that is responsible for the hydrolysis of ß-glucosides.

Phenotypes of various bgl strains
Various deletion and interposon insertion strains were constructed in an attempt to assign phenotypes to the genes located in this region of DNA. Strain ALH167 has a partial deletion of both the bglP and bglA genes and the complete deletion of the bglB, bglC and ORF4 genes (see Methods). This deletion strain was tested on media containing various ß-glucosides as the carbon source in order to detect a phenotype and any potential functions of the proteins encoded by this locus. Strain ALH167 retained the ability to breakdown arbutin, salicin and cellobiose when assayed on Purple Broth plates containing these ß-glucosides as carbon sources (data not shown). The phenotype of this strain was also assayed on aesculin plates, which only detect the breakdown of aesculin and are not indicative of glucose fermentation. This strain was able to break down aesculin when grown on aesculin plates without glucose, but not when glucose was added to the aesculin plates (not shown). The wild-type S. mutans NG8 consistently hydrolysed aesculin in either the presence or the absence of glucose. These results, summarized in Table 3, indicate that this region is responsible, at least in part, for the utilization of the ß-glucoside aesculin, supporting the assignment of the bgl gene nomenclature.


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Table 3. Phenotypes of S. mutans strains when grown on aesculin plates

 
The aesculin hydrolysis data show that the ß-glucoside-specific regulon that we have isolated is not repressed in the presence of glucose, and also that another mechanism for ß-glucoside utilization exists in S. mutans that is significantly repressed by glucose. Multiple mechanisms of ß-glucoside utilization exist in several organisms, including E. coli and B. subtilis. It is possible that the glucose-specific PTS of S. mutans is able to transport ß-glucosides as an alternative substrate due to the glucose moiety in these compounds.

Additional knockout strains were constructed with a specific insertion into either the bglP or the bglA gene in order to determine the function of the individual gene products. The bglP knockout strain, ALH96, which would not produce the putative PTS EII responsible for ß-glucosidase translocation, was unable to break down aesculin in the presence of glucose; in the absence of glucose, it was able to break down the aesculin nearly as efficiently as the wild-type strain (Table 3). These phenotypic data indicate that bglP is necessary for the translocation of aesculin in the presence of glucose.

A knockout strain was also constructed that contained an insertional mutation in the bglA gene. This strain, ALH168, would not produce the putative phospho-ß-glucosidase. The bglA knockout strain and the wild-type strain were able to hydrolyse aesculin in both the presence and absence of glucose. However, aesculin hydrolysis was less pronounced in the presence of glucose. This indicates that one of the mechanisms for breaking down ß-glucosides is repressed in the presence of glucose, while another is not repressed by glucose. These data also indicate that the S. mutans bglA gene knockout is complemented by another ß-glucosidase encoded elsewhere on the chromosome.

The phenotypic data collected from various strains of S. mutans (summarized in Table 3) allow for several interesting inferences into the mechanisms that are used to utilize ß-glucosides in S. mutans. The bglP gene, isolated and discussed in this report, is not repressed in the presence of glucose. A second permease, which is strongly repressed in the presence of glucose, must exist that can also translocate aesculin into the bacterial cell. These data also indicate the presence of at least one additional mechanism that can breakdown ß-glucosides in S. mutans. Another gene, encoding a second catabolic enzyme, that is responsible for breaking down aesculin in the absence of the BglA protein, is also repressed by glucose. This second ß-glucosidase is not repressed by glucose to the extent that the second permease is. The bglA gene product is not necessary for aesculin utilization but does play a role in ß-glucoside breakdown in the presence of glucose (Table 3).

Other bacteria have been shown to have multiple operons or regulons in order to utilize various ß-glucosides (el Hassouni et al., 1990 ; Kruger & Hecker, 1995 ; Tobisch et al., 1997 ). A recurring characteristic of these organisms is the ablility to utilize different proteins which are specific for different groups of ß-glucosides. Some species have genes that are involved in the utilization of the aromatic ß-glucosides arbutin and salicin. These same organisms will also have a completely different set of genes involved in the metabolism of the ß-glucoside cellobiose. This phenomenon is exemplified by the bglPH and lic operons in B. subtilis (Kruger et al., 1996 ; Tobisch et al., 1997 ). In view of this carbohydrate specificity of the ß-glucoside systems, it is likely that S. mutans has other PTS systems involved in the utilization of other ß-glucosides as carbon sources.

ß-Glucoside-specific activity was assayed in E. coli CC118 containing the plasmid pALH163, which harbours the entire bgl region. E. coli CC118(pALH163) was able to break down arbutin, while the host strain CC118 was not. E. coli CC118 was also transformed with pALH164, to generate a strain containing the ß-glucoside regulon with a deletion of the bglP gene. Strain CC118(pALH164) was assayed for arbutin hydrolysis on minimal medium plates containing this ß-glucoside as the sole carbon source. It was unable to break down the arbutin. While not definitively due to the lack of the bglP gene, this result tends to indicate that the bglP gene product is necessary for arbutin utilization in E. coli. A third E. coli strain was created that lacked the bglA gene from S. mutans. This strain, E. coli CC118(pALH165), retained the ability to breakdown arbutin (Table 4). From these E. coli complementation studies, it appears that the EII encoded by bglP is necessary for arbutin hydrolysis in E. coli CC118, but that the putative phospho-ß-glucosidase encoded by the bglA gene is not.

While all of the elements required for ß-glucoside utilization are present in the bgl regulon of S. mutans, the arrangement of the genes is different from that in previously characterized operons. Generally, ß-glucoside PTS operons consist of an EII gene that is directly followed by the phospho-ß-glucosidase gene. However, in S. mutans, there are three ORFs between these genes (bglP and bglA) (Fig. 2). The idea that the bgl regulon of S. mutans is structurally different from those previously described is exemplified by the fact that the bglC gene is transcribed in the opposite direction from bglP, bglB and bglA.

In this report, we have described the isolation and characterization of a ß-glucoside-specific PTS from S. mutans. The bglP gene encodes a PTS EII component that is not repressed by glucose. The protein encoded by the S. mutans bglA gene is similar to the phospho-ß-glucosidases isolated from other bacterial PTSs. The bglC gene is thought to encode a transcriptional regulator and the potential functions of the proteins encoded by the bglB gene and ORF4 are still unknown. Further studies are being initiated to examine the possible functions of these proteins, as well as to understand the regulation of this region. This regulon will be used to further examine the unusual regulation normally associated with ß-glucoside utilization, as well as to further characterize the mechanisms responsible for catabolite repression in S. mutans.


   ACKNOWLEDGEMENTS
 
This work was supported by Public Health Service grants DE08007 to A.S.B. and DE10890 to A.L.H.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
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Received 3 December 1999; revised 17 March 2000; accepted 27 March 2000.