Transcriptional analysis of the bglP gene from Streptococcus mutans
© Cote and Honeyman; licensee BioMed Central Ltd. 2006
Received: 23 February 2006
Accepted: 21 April 2006
Published: 21 April 2006
An open reading frame encoding a putative antiterminator protein, LicT, was identified in the genomic sequence of Streptococcus mutans. A potential ribonucleic antitermination (RAT) site to which the LicT protein would potentially bind has been identified immediately adjacent to this open reading frame. The licT gene and RAT site are both located 5' to a beta-glucoside PTS regulon previously described in S. mutans that is responsible for esculin utilization in the presence of glucose. It was hypothesized that antitermination is the regulatory mechanism that is responsible for the control of the bglP gene expression, which encodes an esculin-specific PTS enzyme II.
To localize the promoter activity associated with the bglP locus, a series of transcriptional lacZ gene fusions was formed on a reporter shuttle vector using various DNA fragments from the bglP promoter region. Subsequent beta-galactosidase assays in S. mutans localized the bglP promoter region and identified putative -35 and -10 promoter elements. Primer extension analysis identified the bglP transcriptional start site. In addition, a terminated bglP transcript formed by transcriptional termination was identified via transcript mapping experiments.
The physical location of these genetic elements, the RAT site and the promoter regions, and the identification of a short terminated mRNA support the hypothesis that antitermination regulates the bglP transcript.
Beta-glucosides are carbohydrates that contain a core glucose molecule that has an R-group attached to the C-l carbon. The R-group may be another carbohydrate or various other molecules such as amino acids or ringed compounds. Beta-glucosides are commonly found in nature in various sources such as tuberous vegetables. These compounds are generally broken down for the glucose molecule, while the R-group may or may not be metabolized. Beta-glucoside utilization systems have been described in several bacteria including Escherichia coli , Erwinia chrysanthemi , Clostridium longisporum , Lactobacillus plantarium , Bacillus subtilis [5, 6], and Streptococcus mutans . All of these organisms rely on the phosphoenolpyruvate-dependent phosphotransferase system (PTS)  for the transport of beta-glucosides into the bacterial cell.
It has been previously shown that the S. mutans bglP gene encodes a beta-glucoside-specific enzyme II of the PTS . An additional open reading frame adjacent to bglP has been isolated, sequenced, and examined for a possible regulatory function . The open reading frame encodes a putative regulatory protein of the BglG family of transcriptional antiterminators . Antiterminator proteins are believed to be involved in the transcriptional regulation of the beta-glucoside-specific genes present within E. coli , E. chrysanthemi , L. lactis , L. plantarium , B. subtilis [5, 6], and C. longisporum . This type of antiterminator protein has been shown to bind to a ribonucleic antitermination (RAT) site [12, 13], which is a palindromic sequence that may form multiple hairpin structures in the mRNA, some of which would act as a transcriptional terminator. Binding of one of these potential RNA structures by the antiterminator protein prevents the formation of the transcription terminator hairpin structure  and results in the elongation of the mRNA via transcriptional read through into the region encoding the beta-glucoside-specific gene product that is not normally transcribed. Thus, the mechanism of transcriptional regulation by antitermination allows for the expression of the beta-glucoside-specific loci only in the presence of a beta-glucoside .
The molecular mechanisms behind the process of antitermination have been studied most extensively in E. coli. and B. subtilis. The bglGFB operon in E. coli encodes an antiterminator protein (bglG), an EII component of the PTS that is specific for beta-glucosides (bglF), and a phospho-beta-glucosidase (bglB) . The BglG protein is responsible for binding two transcription terminators flanking the bglG gene [1, 15], which results in the production of a full-length transcript and full induction of the bgl operon . It has subsequently been shown that the EII protein (BglF) also plays a regulatory role in antitermination .
The molecular mechanisms responsible for the regulation of beta-glucoside-specific loci in B. subtilis by the LicR and LicT proteins are less documented. The licBCAH operon in B. subtilis has been shown to be responsible for the catabolism of cellobiose and lichenan . The licBCA genes encode the EII component of the PTS and a phospho-beta-glucosidase is encoded by the licH gene . This lic operon was determined to be regulated in part by the LicR protein . The LicR protein has been shown to contain several PTS regulation domains (PRDs) , and, presumably, the LicR protein is stimulated upon the phosphorylation of these PRDs . The activity of the LicR protein has also been shown to be negatively effected by phosphorylation of the EIIA domain encoded by the licBCA genes .
B. subtilis has a second beta-glucoside specific locus, the bglPH operon, that is responsible for the breakdown of the beta-glucosides salicin and arbutin . The bglPH operon, encoding the EII component of the PTS (bglP) and a phospho-beta-glucosidase (bglH), is regulated by the LicT antiterminator protein . The LicT protein has been shown to be phosphorylated by HPr-phosphate , which allows the formation of an active LicT dimer .
This report provides a transcriptional analysis of the S. mutans bglP locus that is putatively regulated by the mechanism of transcriptional antitermination in the presence of esculin. The LicT protein is essential for efficient expression of the bglP gene and esculin hydrolysis by S. mutans in the presence of glucose. Physical examination of the bglP gene and its RNA transcripts has generated evidence that supports the hypothesis that the bglP locus is regulated by an antitermination mechanism mediated by the LicT protein. This report is the first demonstration of the antitermination process in S. mutans.
Results and discussion
Localization of the bglP promoter
The DNA sequence from the Xba I-Hind III fragment located 5' to the region contained in pALH211 (nucleotides 1–177 relative to the Xba I site) did not contribute significantly to the transcriptional activity associated with the bglP gene. This point is observed when examining the relative activity of pALH187 and pALH211 (Figure 5). However, further deletion of the DNA toward the putative -35 region in pALH217 does affect the activity of the promoter. This construct contains only 8 bp 5' to the putative -35 region and exhibits approximately 50% of the relative activity of pALH187 and pALH211. The additional 36 bases contained in pALH211 upstream of the -35 region appear to enhance transcription from the bglP promoter. It was also demonstrated that the region located 3' of the -10 promoter region, as contained in pALH214, is not associated with detectable levels of β-galactosidase activity (Figure 5).
A reporter plasmid that contained only the -35 and -10 region (relative nucleotides 178–260) was also constructed. In this construct, the entire RAT sequence (relative nucleotides 262–290) was absent. This construct would be predicted to have constitutive expression because both the sequence required for transcription termination in the absence of beta-glucosides and the sequence required for antitermination in the presence of beta-glucosides were removed. This plasmid was highly unstable in S. mutans and underwent spontaneous deletion events (data not shown). Presumably, unfettered expression of the E. coli beta-galactosidase enzyme from the consensus -35 and -10 promoter regions of the bglP gene was toxic to the S. mutans cells.
Determination of the transcriptional start site of the bglP gene
Northern blot analysis of the bglP transcript
Size determination of the terminated bglP transcript
In this report, we have demonstrated that the bglP gene of S. mutans produces a short, truncated mRNA transcript of 109 bases in the absence of the inducer, esculin. In the presence of esculin, a 2.1 kb transcript is produced. The start site of transcription for both of these mRNA molecules is located 7 bases 3' to the -10 promoter element and 4 bases 5' to a putative RAT site at which the antiterminator protein LicT would presumably bind to allow expression of the bglP gene. The locations of these genetic elements are shown in Figure 1. In addition, the LicT protein has been shown to be essential for the expression of the bglP gene by use of transcriptional gene fusions in a S. mutans licTi::Ω-Kan2 genetic background. These data and the fact that the LicT protein and the RAT site are highly conserved with other putative antitermination regulatory systems imply that the LicT protein interacts with the bglP RAT site to control expression of this PTS permease gene.
It has been previously illustrated that the licT gene product regulates the expression of the bglP gene . It was hypothesized that this regulation involved transcriptional antitermination of the bglP transcript. This report supports this hypothesis that the LicT antiterminator protein is regulating the bglP gene via binding of the RAT structure. The S. mutans bgl regulon has been previously shown to be unique from other beta-glucosidase operons because of several characteristics. This regulon has a novel arrangement of four adjacent genes with at least four separate transcriptional units . This includes the bglC gene, which is located in the middle of the regulon and is transcribed on the opposite DNA strand from the other members of the regulon. The BglC protein, a member of the XylS/AraC family of transcriptional regulators, has been shown to be a positive regulator of the bglA gene, which encodes the phospho-beta-glucosidase . This regulon has also been shown to display a glucose-resistant phenotype of catabolite repression . This report now adds transcriptional regulation by antitermination to the list of interesting characteristics of this beta-glucoside-specific PTS regulon from S. mutans.
Bacterial strains and media
S. mutans NG8 was used as the source of chromosomal DNA for PCR reactions as well as the host strain for all S. mutans transformations. E. coli CC118 [Δ(ara-leu) 7697 araD 139 ΔlacX 74 galE galK ΔphoA 20 thi-1 rpsE rpoB argE(Am) recA 1]  was used for recombinant DNA procedures. S. mutans strains were grown on Brain-Heart Infusion (BHI) agar plates or in BHI broth (Difco, Detroit, MI). S. mutans strains were grown under antibiotic selection with kanamycin (500 μg/ml), erythromycin (20 μg/ml), or a combination of both antibiotics, as necessary. All E. coli strains were grown on Lennox Broth (LB) agar plates or in LB broth (Gibco-BRL, Rockville, MD). E. coli was grown under antibiotic selection with ampicillin (100 μg/ml) or chloramphenicol (20 μg/ml), as necessary. S. mutans cultures were grown statically at 37°C, while E. coli cultures were grown at 37°C with shaking aeration. Unless otherwise noted, all chemicals and reagents were purchased from Sigma (St. Louis, MO).
DNA isolation, manipulation, and PCR amplification
Streptococcal DNA was isolated as previously described . Mini-plasmid preparations were performed using the QIAprep 8 Miniprep kits or the QIAprep Spin Miniprep Kits (Qiagen, Valencia, CA). Recombinant plasmid constructs were electroporated as previously described  and S. mutans was transformed as previously described [25, 26].
The licT gene was amplified from the chromosome of S. mutans NG8 by using primers designed from the sequence data obtained from the S. mutans genome database at the Oral Pathogen Sequence Database maintained by Los Alamos National Laboratory . The primers were bglP-1 (5'-CGCAAGCGAGTAACACAGTG-3') and licT (5'-GATTAGCAAAATGAAGGCAG-3'). The annealing temperature used for this primer pair was 45°C. The KlenTaq-LA polymerase (Clontech, Palo Alto, CA) was used according to the manufacturer's recommendations. All DNA manipulations and enzyme procedures were done according to the manufacturer's instructions and under NIH recombinant DNA guidelines.
Construction of lacZ fusions
The lacZ transcriptional fusions were created using the parent plasmid, pALH122 . Briefly, this plasmid is a derivative of the shuttle vector pVA838  and contains the lacZ gene from E. coli for use as a reporter of transcriptional activity. This vector can replicate in both Gram-negative and Gram-positive bacteria. It contains an erythromycin resistance marker for selection in S. mutans and a chloramphenicol resistance marker for selection in E. coli.
The construction of pALH187, which displays regulated promoter activity, has been previously reported , Briefly, a 490 bp Xba I-Hind III fragment, containing the entire bglP promoter region, was inserted into the Sma I site of pALH122. In order to further localize the promoter activity of the bglP gene, five additional reporter constructs were made. These constructs contain various segments of the putative promoter region. A diagram of the regions cloned is shown in Figure 2. In this series, various PCR fragments were cloned into the Sma I site of pALH122 to create different lacZ reporter plasmids.
All of the reporter constructs were analyzed by restriction endonuclease digestion, DNA sequencing, and/or PCR analysis. These analyses were performed to verify that a single copy of the amplified PCR fragment was cloned into pALH122 and to ensure that the desired fragment was cloned in the correct orientation to express the lacZ reporter gene. The reporter constructs were then transformed into S. mutans NG8 as described to determine their relative promoter activity in the appropriate S. mutans host background.
Fluorescent beta-galactosidase assays
S. mutans strains containing the various lacZ transcriptional fusions were grown in Chemically Defined Medium (CDM)  supplemented with glucose, esculin, or a combination of glucose and esculin. All carbohydrates were at a final concentration of 0.5%. After being harvested by centrifugation, the cells were physically lysed with a Mini-BeadBeater (Biospec, Inc., Bartlesville, OK) and fluorescent beta-galactosidase assays were then performed as previously described . Briefly, the cleared cell lysates were allowed to incubate in a reaction well containing 40 μl of AB buffer (60 mM K2HPO4, 40 mM KH2PO4, 100 mM NaCl)  and 10 μl of resorufin-β-D-galactopyranoside (0.4 mg/ml in DMSO) (Molecular Probes, Eugene, OR). After 30 min, the reaction was slowed by the addition of excess AB buffer. Sodium bicarbonate was added to adjust the pH of the reaction mixture to increase the sensitivity of the assay by approaching the optimal extinction coefficient of resorufin salt. Two hundred microliters of the reaction mixture was then added to a black opaque microtiter plate and read in a fluorescent plate reader (Molecular Devices, Sunnyvale, CA). The data were obtained as relative fluorescent units (RFUs). The protein concentrations of the bacterial cell lysates were determined using the Coomassie Plus Protein Assay Reagent (Pierce, Rockford, IL). The data were standardized as RFUs/μg of cell lysate protein prior to conversion to specific activities using a resorufin salt standard. The specific activity was expressed as ng resorufin salt formed/μg protein. Specific activities were divided by the activity from the S. mutans strain containing pALH187 grown in glucose. This served as an internal control for daily growth variation and yielded relative transcriptional activities, which were then plotted (Figure 3).
One ml of an overnight S. mutans NG8 starter culture was used to inoculate a 15 ml screw cap tube containing 14 ml of prewarmed CDM supplemented with various carbohydrates at 0.5% final concentration. The cultures were allowed to grow at 37°C for 6–8 hr; at this time, the cultures were approximately at the mid-log phase of growth. Cells were harvested by centrifugation and transferred to a 1.5 ml microfuge tube. The cells were washed in 1 ml of TE buffer and then resuspended in 1 ml of Trizol reagent (Gibco BRL). The microfuge tubes containing the cell/Trizol suspensions were filled approximately 1/2 full with zirconia/silica beads and physically lysed in a Mini-BeadBeater for 80 sec. The samples were placed on ice, 200 μl of chloroform added to the lysate, and the mixture agitated again for 20 sec. The samples were then microcentrifuged for 10 min at 4°C. The aqueous phase was removed and precipitated with isopropanol overnight at -20°C. The RNA pellet was washed with 100 μl of 100% ethanol and resuspended in 100 μl of DEPC-treated water. The sample was treated with RQI RNase-Free DNase (Promega, Madison, WI) as recommended by the manufacturer. The resulting RNA was purified further and concentrated using an RNeasy Total RNA kit (Qiagen).
Primer extension reactions
Primer extension experiments were performed using the Primer Extension-AMV Reverse Transcriptase kit (Promega, Madison, WI) and S. mutans RNA isolated from cells grown in CDM supplemented with 0.5% esculin. A DNA primer (called Primer Extension-2) (5'-CCAAATAAATACAACAAAACACTCG-3') was end-labeled with gamma-32P-ATP and was used in both the primer extension and the DNA sequencing reactions using the fmol sequencing system (Promega). For reverse transcriptase mapping, the primer was hybridized with approximately 30 μg of RNA at 42°C and the primer extension reaction was carried out as described by the manufacturer. Di-deoxynucleotide sequencing reactions, using the same primer and a DNA template, were electrophoresed with the primer extension product on a denaturing polyacrylamide sequencing gel in order to determine the endpoints of the primer extension product.
Size determination of the terminated bglP transcript
Transcript mapping was performed on S. mutans RNA isolated from cells grown in 0.5% lactose using mung bean nuclease (New England Biolabs, Beverly, MA). Lactose was used because it represses the bglP gene more than glucose does and yields more of the terminated transcript (data not shown). A radiolabeled DNA probe was generated by amplifying a 277 bp region of S. mutans DNA using the primers bglP-1 and LicT-3 (5'-TCTGCTATAATATCTTTGTAAGG-3'). This PCR reaction was carried out as previously described, but with the addition of 5 μl each of alpha-32P-dGTP and alpha-32P-dCTP (NEN-Perkin Elmer Life Sciences, Boston, MA). The PCR product was denatured in boiling water for 10 min and immediately chilled on ice for 10 min. This radiolabeled probe was added to 40 μg of total RNA that was isolated from S. mutans as previously described. The DNA probe/RNA mixture was ethanol precipitated and washed with 70% ethanol. The nucleic acid pellets were air dried for 30 min prior to resuspension in 20 μl of hybridization buffer (80% formamide, 40 mM PIPES (pH 6.4), 400 mM NaCl, and 1 mM EDTA (pH 8)) , The samples were denatured at 65°C for 10 min and then placed in a 40°C heat block and allowed to hybridize over night. Following hybridization, 300 μl of mung bean nuclease reaction mix (1X enzyme buffer and 500 Units/ml of mung bean nuclease) was added to each sample (New England Biolabs). The reaction tubes were incubated at 37°C for 2 hr. The mung bean nuclease was inactivated by the addition of EDTA to a final concentration of 1 mM. The mixture was ethanol precipitated and washed with 70% ethanol. The final pellet was dried with a Speed-Vac SC100 (Savant, Waltham, MA) for 5 min. The signal intensity was estimated and the products were diluted accordingly with TE buffer and formamide loading dye (Promega). The transcript mapping products were denatured in boiling water for 10 min and then chilled on ice for 10 min prior to visualization on an 8% polyacrylamide sequencing gel. The DNA ladder was produced by a sequencing reaction using the LicT-3 primer. Thus, the size of the transcript could be determined by comparing it to the ladder of the known DNA sequence.
Total RNA was subjected to Northern blot analysis. Approximately 10 μg of total S. mutans RNA (final volume of 11 μl) was combined with 9 μl of 12.3 M formaldehyde, 5 μl of 10X MOPS running buffer (0.4 M MOPS (pH 7.0), 0.1 M sodium acetate, and 0.01 M EDTA), and 25 μl formamide. The samples were then incubated at 55°C for 15 min before being loaded onto a denaturing formaldehyde gel. The formaldehyde gel was composed of 1% of agarose, IX MOPS running buffer, and 6.6% formaldehyde. The total RNA was run on the denaturing formaldehyde gel at approximately 100 volts until the bromphenol blue dye had migrated approximately 2/3 the length of the gel. The gel was then blotted onto a positively charged nylon membrane (PALL Corporation, Ann Arbor, MI) using the Genie Electrophoretic Blotter (Idea Scientific, Minneapolis, MN). As suggested by the manufacturer, the blotting was performed at 12 volts for two hrs in Northern transfer buffer (20 mM MOPS, 5 mM sodium acetate, and 0.5 mM EDTA). The membrane was then washed with 0.2X SSC for 5 min, gently dried, and the RNA crosslinked to the membrane using a Stratalinker UV generator (120,000 microjoules) (Stratagene, La Jolla, CA).
The membrane was stained with 0.03% (w/v) methylene blue in 0.3 M sodium acetate for 45 sec. The membrane was then destained with de-ionized H2O. Membrane staining was performed to identify the ribosomal RNA bands. The image of the stained membrane was electronically scanned for future reference. The membrane was then permitted to prehybridize for at least 1 hr at 55°C in a Hybaid Hybridization oven (Labnet Systems, Edison, NJ). The North2South Northern Blot kit (Pierce) was used as described by the manufacturer to identify the transcripts of interest. The DNA probe was generated using the same PCR primers that were used for probe generation in the transcript mapping experiments. However, the probe was generated using biotinylated nucleotides instead of being radiolabeled. This allowed for non-radioactive, chemiluminescent detection of the transcripts on the Northern blot. The biotinylated probe was detected with the Neutravidan antibody and developed with luminol enhancer and stable peroxide solution as described in the methodology for Southern blots (Pierce).
This work was supported by a Public Health Service grant from the NIH National Institute of Dental and Craniofacial Research (DE10890) to ALH.
- Schnetz K, Toloczyki C, Rak B: Beta-glucoside (bgl) operon of Escherichia coli K-12: nucleotide sequence, genetic organization, and possible evolutionary relationship to regulatory components of two Bacillus subtilis genes. J Bacteriol. 1987, 169 (6): 2579-2590.PubMed CentralPubMedGoogle Scholar
- el Hassouni M, Chippaux M, Barras F: Analysis of the Erwinia chrysanthemi arb genes, which mediate metabolism of aromatic beta-glucosides. J Bacteriol. 1990, 172 (11): 6261-6267.PubMed CentralPubMedGoogle Scholar
- Brown GD, Thomson JA: Isolation and characterisation of an aryl-beta-D-glucoside uptake and utilisation system (abg) from the gram-positive ruminal Clostridium species C. longisporum. Mol Gen Genet. 1998, 257 (2): 213-218. 10.1007/s004380050641.View ArticlePubMedGoogle Scholar
- Marasco R, Salatiello I, De Felice M, Sacco M: A physical and functional analysis of the newly-identified bglGPT operon of Lactobacillus plantarum. FEMS Microbiol Lett. 2000, 186 (2): 269-273. 10.1111/j.1574-6968.2000.tb09116.x.View ArticlePubMedGoogle Scholar
- Le Coq D, Lindner C, Kruger S, Steinmetz M, Stulke J: New beta-glucoside (bgl) genes in Bacillus subtilis: the bglP gene product has both transport and regulatory functions similar to those of BglF, its Escherichia coli homolog. J Bacteriol. 1995, 177 (6): 1527-1535.PubMed CentralPubMedGoogle Scholar
- Tobisch S, Glaser P, Kruger S, Hecker M: Identification and characterization of a new beta-glucoside utilization system in Bacillus subtilis. J Bacteriol. 1997, 179 (2): 496-506.PubMed CentralPubMedGoogle Scholar
- Cote CK, Cvitkovitch D, Bleiweis AS, Honeyman AL: A novel β-glucoside-specific PTS locus from Streptococcus mutans that is not inhibited by glucose. Microbiology. 2000, 146 (7): 1555-1563.View ArticlePubMedGoogle Scholar
- Postma PW, Lengeler JW: Phosphoenolpyruvate:carbohydrate phosphotransferase system of bacteria. Microbiol Rev. 1985, 49 (3): 232-269.PubMed CentralPubMedGoogle Scholar
- Cote CK, Honeyman AL: The LicT protein acts as both a positive and a negative regulator of loci within the bgl regulon of Streptococcus mutans. Microbiol. 2003, 149 (5): 1333-1340. 10.1099/mic.0.26067-0.View ArticleGoogle Scholar
- Stulke J, Arnaud M, Rapoport G, Martin-Verstraete I: PRD – a protein domain involved in PTS-dependent induction and carbon catabolite repression of catabolic operons in bacteria. Mol Microbiol. 1998, 28 (5): 865-874. 10.1046/j.1365-2958.1998.00839.x.View ArticlePubMedGoogle Scholar
- Bardowski J, Ehrlich SD, Chopin A: BglR protein, which belongs to the BglG family of transcriptional antiterminators, is involved in beta-glucoside utilization in Lactococcus lactis . J Bacteriol. 1994, 176 (18): 5681-5685.PubMed CentralPubMedGoogle Scholar
- Declerck N, Vincent F, Hoh F, Aymerich S, van Tilbeurgh H: RNA recognition by transcriptional antiterminators of the BglG/SacY family: functional and structural comparison of the CAT domain from SacY and LicT. J Mol Biol. 1999, 294 (2): 389-402. 10.1006/jmbi.1999.3256.View ArticlePubMedGoogle Scholar
- Houman F, Diaz-Torres MR, Wright A: Transcriptional antitermination in the bgl operon of E. coli, is modulated by a specific RNA binding protein. Cell. 1990, 62 (6): 1153-1163. 10.1016/0092-8674(90)90392-R.View ArticlePubMedGoogle Scholar
- Rutberg B: Antitermination of transcription of catabolic operons. Mol Microbiol. 1997, 23 (3): 413-421. 10.1046/j.1365-2958.1997.d01-1867.x.View ArticlePubMedGoogle Scholar
- Mahadevan S, Wright A: A bacterial gene involved in transcription antitermination: regulation at a rho-independent terminator in the bgl operon of E. coli. Cell. 1987, 50 (3): 485-494. 10.1016/0092-8674(87)90502-2.View ArticlePubMedGoogle Scholar
- Reynolds AE, Mahadevan S, LeGrice SF, Wright A: Enhancement of bacterial gene expression by insertion elements or by mutation in a CAP-cAMP binding site. J Mol Biol. 1986, 191 (l): 85-95. 10.1016/0022-2836(86)90424-9.View ArticlePubMedGoogle Scholar
- Schnetz K, Rak B: Regulation of the bgl operon of Escherichia coli by transcriptional antitermination. EMBO J. 1988, 7 (10): 3271-3277.PubMed CentralPubMedGoogle Scholar
- Tobisch S, Stulke J, Hecker M: Regulation of the lic operon of Bacillus subtilis and characterization of potential phosphorylation sites of the LicR regulator protein by site-directed mutagenesis. J Bacteriol. 1999, 181 (16): 4995-5003.PubMed CentralPubMedGoogle Scholar
- Kruger S, Hecker M: Regulation of the putative bglPH operon for aryl-beta-glucoside utilization in Bacillus subtilis. J Bacteriol. 1995, 177 (19): 5590-5597.PubMed CentralPubMedGoogle Scholar
- Lindner C, Galinier A, Hecker M, Deutscher J: Regulation of the activity of the Bacillus subtilis antiterminator LicT by multiple PEP-dependent, enzyme I- and HPr-catalysed phosphorylation. Mol Microbiol. 1999, 31 (3): 995-1006. 10.1046/j.1365-2958.1999.01262.x.View ArticlePubMedGoogle Scholar
- Declerck N, Dutartre H, Receveur V, Dubois V, Royer C, Aymerich S, van Tilbeurgh H: Dimer stabilization upon activation of the transcriptional antiterminator LicT. J Mol Biol. 2001, 314 (4): 671-681. 10.1006/jmbi.2001.5185.View ArticlePubMedGoogle Scholar
- Cote CK, Honeyman AL: The transcriptional regulation of the Streptococcus mutans bgl regulon. Oral Microbiol Immunol. 2002, 17: 1-8. 10.1046/j.0902-0055.2001.00087.x.View ArticlePubMedGoogle Scholar
- Manoil C, Beckwith J: TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci USA. 1985, 82 (23): 8129-8133. 10.1073/pnas.82.23.8129.PubMed CentralView ArticlePubMedGoogle Scholar
- Dower WJ: Electroporation of bacteria: a general approach to genetic transformation. Genetic Engineering-Principles and Methods. 1990, New York: Plenum Publishing, 12: 275-296.Google Scholar
- Murchison HH, Barrett JF, Cardineau GA, Curtiss Rd: Transformation of Streptococcus mutans with chromosomal and shuttle plasmid (pYA629) DNAs. Infect Immun. 1986, 54 (2): 273-282.PubMed CentralPubMedGoogle Scholar
- Cvitkovich DG, Gutierrez JA, Crowley PJ, Wojciechowski L, Hillman JD, Bleiweis AS: Tn917 transposon mutagenesis and marker rescue of interrupted genes of Streptococcus mutans. Methods Cell Sci. 1998, 20: 1-12. 10.1023/A:1009711609646.View ArticleGoogle Scholar
- Oral Pathogen Sequence Databases.http://www.oralgen.lanl.gov/
- Honeyman AL, Cote CK, Curtiss R: Construction of transcriptional and translational lacZ gene reporter plasmids for use in Streptococcus mutans. J Microbiol Methods. 2002, 49: 163-171. 10.1016/S0167-7012(01)00368-2.View ArticlePubMedGoogle Scholar
- Macrina FL, Tobian JA, Jones KR, Evans RP, Clewell DB: A cloning vector able to replicate in Escherichia coli and Streptococcus sanguls. Gene. 1982, 19 (3): 345-353. 10.1016/0378-1119(82)90025-7.View ArticlePubMedGoogle Scholar
- van de Rijn I, Kessler RE: Growth characteristics of group A streptococci in a new chemically defined medium. Infect Immun. 1980, 27 (2): 444-448.PubMed CentralPubMedGoogle Scholar
- Youngman P: Use of transposon and integrational vectors for mutagenesis and construction of gene fusions in Bacillus species. Molecular Biological Methods for Bacillus. Edited by: Harwood CR, Cutting SM. 1990, New York: John Wiley & Sons, 221-259.Google Scholar
- Ausubel FM, Brent R, Kinston RE, Moore DM, Seidman JG, Smith JA, Struhl K, (eds.): Current protocols in molecular biology. 2001, Brooklyn, N. Y. Greene Publishing Associates
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