University of South Florida College of Medicine, Department of Medical Microbiology and Immunology, Tampa, FL 33612, USA
Correspondence
Allen L. Honeyman
ahoneyman{at}tambcd.edu
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ABSTRACT |
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Present Address: Texas A&M Health Science Center, Baylor College of Dentistry, 3302 Gaston Avenue, Dallas, TX 75246, USA.
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INTRODUCTION |
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In this report, we determine that the LicT protein regulates the S. mutans -glucoside-specific PTS regulon in the presence of glucose via a putative antitermination mechanism. Additional evidence that LicT acts as an antiterminator is provided by the fact that this protein activates a cryptic Escherichia coli operon. Based upon protein similarities and potential RAT structures, we believe that the E. coli bglGFB operon is induced by the S. mutans LicT protein. LicT is shown to be essential for the efficient expression of bglP and aesculin hydrolysis by S. mutans in the presence of glucose. LicT also acts as a negative regulator of the bglA locus.
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METHODS |
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The licT gene was PCR-amplified from chromosomal DNA of S. mutans NG8 by primers designed using DNA sequence data obtained from the S. mutans genome database at the University of Oklahoma (http://www.genome.ou.edu/smutans.html). The primers were bglP1 (5'-CGCAAGCGAGTAACACAGTG-3') and licT (5'-GATTAGCAAAATGAAGGCAG-3'). The annealing temperature used for this primer pair was 45 °C. The polymerase used was KlenTaq-LA (Clontech) and was used according to the manufacturer's recommendations for elongation times and temperatures.
Aesculin transport and hydrolysis in S. mutans.
To measure the relative amount of aesculin transported and hydrolysed by the cell, the hydrolysis product of aesculin, 6,7-dihydroxycoumarin, was measured in the following manner. S. mutans cells were grown overnight as 10 ml cultures in CDM (van de Rijn & Kessler, 1980) supplemented with 0·5 % glucose and 0·5 % aesculin. The cells were collected by centrifugation and washed twice with TES buffer (10 mM Tris, 1 mM EDTA, 150 mM NaCl, pH 8·0). The cells were resuspended in 250 µl TES buffer and transferred to a 1·5 ml screw-cap microfuge tube (Sarstedt). A small amount of 0·1 mm zirconia/silica beads (Biospec Products) was added to each tube (approx. 1/6 of the total tube volume). The cells were lysed by shaking the tubes in a Mini-BeadBeater-8 (Biospec Products) for 80 s. Cleared cell lysates were formed by centrifugation in a microcentrifuge for 10 min at 13 000 r.p.m. The protein concentration of the lysate was determined using the Coomassie Plus Protein Assay reagent (Pierce). Thirty micrograms of protein from the lysate was diluted with TES to a final volume of 100 µl, and 200 µl of 0·002 M ferric citrate was added to each sample. The samples were mixed briefly and added to wells of a clear, flat-bottomed microtitre plate. The absorbance of the samples was read on a Molecular Devices
Max microplate reader set at 595 nm. TES buffer served as the blank, while lysates of S. mutans cells grown in the absence of aesculin served as the negative control. In this assay, the breakdown product of aesculin cleavage, 6,7-dihydroxycoumarin, reacts with the ferric citrate to form a black product. The relative amount of product formed was assayed by the absorbance at 595 nm. The obtained optical density (OD) values were divided by the OD value of the lysate from wild-type S. mutans NG8 cells. The ODs were plotted as the percentage of the wild-type and the data presented are a mean of at least three independent experiments.
Construction of lacZ fusions.
The lacZ transcriptional fusions with the bglP, bglC and bglA promoter regions were created using the gene reporter vector pALH122 (Honeyman et al., 2002). This plasmid is a derivative of the shuttle vector pVA838 (Macrina et al., 1982
) and contains the lacZ gene from E. coli for use as a reporter of transcriptional activity (Honeyman et al., 2002
).
Construction of the plasmids pALH187, pALH188 and pALH189 has been reported previously (Cote & Honeyman, 2002). Briefly, a 490 bp XbaIHindIII fragment, a 260 bp PCR fragment and a 233 bp PCR fragment were inserted into the unique SmaI site of pALH122, resulting in the reporter constructs pALH187, pALH188 and pALH189, respectively. These plasmids contained the promoter regions of the bglP, bglC and bglA genes, respectively. S. mutans strains NG8 and ALH201 containing these plasmids were used to monitor the transcriptional activity of the indicated genes.
Construction of the licT : : -Kan2 mutant strain.
The PCR fragment containing the licT gene was isolated following amplification by agarose gel electrophoresis. A PCR fragment of approximately 2·15 kb was isolated from the agarose, digested with the restriction endonuclease HindIII and cloned into the HindIII site of the vector pACKS30, a derivative of pBluescript II (Alting-Mees & Short, 1989) that has a pACYC184 (Chang & Cohen, 1978
) origin of replication (A. L. Honeyman, unpublished data). This results in the construction of pALH185. A licT gene interruption was created by inserting the
-Kan2 cassette (Perez-Casal et al., 1991
) into the unique StyI site located 293 bp 3' to the LicT initiation codon within the cloned licT gene contained on pALH185. This resulted in the creation of pALH186. Plasmid pALH186 was then linearized by restriction endonuclease digestion with the enzyme XhoI and the linear restriction fragment transformed into S. mutans. Integration of the gene interruption into the S. mutans NG8 chromosome was selected for by kanamycin resistance. This resulted in the S. mutans strain ALH201. The interruption of the licT gene in this strain was confirmed by Southern blot analysis of chromosomal DNA isolated from the kanamycin-resistant transformant (data not shown). The described lacZ transcriptional fusions containing the various promoter regions from the bgl regulon were then transformed into this licT : :
-Kan2 strain, ALH201, to generate the strains listed in Table 1
.
Construction of the bglP : : -Kan2 mutant strain.
To create a S. mutans strain that has an insertional mutation in the bglP gene, plasmid pALH163 (Cote et al., 2000) was altered. Plasmid pALH163, which contains the S. mutans bglPBCA genes, was digested with the restriction endonucleases ClaI and EcoRI to remove a HindIII site located in the multiple cloning region of the vector. The linearized plasmid was treated with the Klenow fragment to generate blunt ends and then re-ligated to itself. The resulting plasmid contains a unique HindIII site located within the cloned bglP gene. This plasmid was digested with HindIII and the 5' overhanging ends were converted to blunt ends using the Klenow fragment. The
-Kan2 cassette (a SmaI restriction fragment) was ligated to the linearized plasmid (Perez-Casal et al., 1991
). The insertion of the
-Kan2 cassette into the unique HindIII site located within the bglP gene resulted in plasmid pALH198. This plasmid was then linearized with the restriction endonuclease XbaI and the resulting DNA fragment was transformed into S. mutans NG8. Growth on kanamycin selected for the integration of the bglP : :
-Kan2 gene interruption into the chromosome of S. mutans NG8 to generate strain ALH376.
Fluorescent -galactosidase assays.
S. mutans strains containing the lacZ transcriptional fusions were grown in CDM media (van de Rijn & Kessler, 1980) supplemented with glucose, aesculin or a combination of glucose and aesculin. Each carbohydrate was at a concentration of 0·5 %. The cells were physically lysed with a Mini-BeadBeater-8 and fluorescent
-galactosidase assays were performed as described previously (Cote & Honeyman, 2002
). All data presented are the result of at least five independent assays and the standard deviations are indicated.
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RESULTS AND DISCUSSION |
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Transport of aesculin into specific S. mutans strains
The phenotype observed when S. mutans hydrolyses aesculin on an agar plate, a blackening of the agar, is the result of the interaction of the aesculin hydrolysis product, 6,7-dihydroxycoumarin, and the ferric citrate present in the agar medium. Experiments were performed to further characterize the transport of aesculin into the S. mutans cell. Cell lysates from S. mutans cultures grown in the presence of glucose and aesculin were able to change a ferric citrate solution from colourless or pale-orange to black, indicating the presence of hydrolysed aesculin within the cell lysate. The bglP : : -Kan2 mutant strain ALH376 and the licT : :
-Kan2 mutant strain ALH201 both exhibited significantly lower levels of intracellular hydrolysed aesculin (approx. 2050 % of wild-type) when compared to the levels observed in the wild-type S. mutans NG8 (data not shown). These results support the hypothesis that the licT and bglP gene products are involved with the transport of aesculin into the S. mutans cell in the presence of glucose. The existence of a second transport system for
-glucosides in S. mutans is also supported by the fact that aesculin transport is not totally eliminated in the mutant strains. However, it is apparent that the licT and bglP gene products are necessary for optimal levels of aesculin translocation into S. mutans.
Effect of the LicT protein on the bgl regulon
The S. mutans licT : : -Kan2 strain, ALH201, was transformed with the reporter constructs containing the lacZ transcriptional fusions with the promoters from the bglP, bglC and bglA genes. The specific activity associated with each reporter construct, pALH187, pALH188 and pALH189, was determined by
-galactosidase assays following growth in various media. The specific activity of the bglC promoter region was not significantly influenced by the absence of LicT (Fig. 4
) when compared to its expression level in the wild-type host. However, the transcriptional activity of the bglP promoter is significantly lowered in the absence of LicT regardless of the carbohydrate examined (Fig. 4
). When glucose is present as the sole carbon source, a fourfold decrease in activity of the bglP promoter occurs. In the presence of both glucose and aesculin, the bglP promoter is approximately eight times less active in the absence of LicT. When aesculin is the only carbohydrate available, the bglP promoter is approximately fourfold less active in the absence of LicT. In all cases, LicT appears to act as a positive regulator of the bglP locus. It is our assumption that LicT acts as an antiterminator, which is a positive regulator of the bglP transcription. While this locus is somewhat insensitive to glucose repression, it is interesting to note that the largest decrease in bglP transcriptional activity is in the licT-negative strain in the presence of both glucose and aesculin.
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Effect of the BglP protein on the bglA gene
It was unclear if the increased transcriptional activity of the bglA promoter observed in the absence of LicT was due directly to the lack of LicT or due to a decrease in the level of BglP mediated by the licT : : -Kan2 mutation. The bglA : : lacZ reporter construct (pALH189) was transformed into the bglP : :
-Kan2 strain ALH376 to generate strain ALH378. The data generated from
-galactosidase assays performed on strain ALH378 following growth on various carbohydrate sources suggested that the absence of BglP does not induce the expression of the bglA promoter (Fig. 5
) to above the wild-type expression level in a manner similar to the induction seen in the licT mutant strain. Thus, LicT must act as a negative regulator of the bglA locus. In the absence of BglP, the bglA promoter is expressed less than in the wild-type host strain. However, it is uncertain if this slight reduction in transcriptional activity of the bglA promoter is due to the absence of BglP or due to the decreased levels of intracellular aesculin that are mediated by the bglP : :
-Kan2 mutation. Regardless of the effect of BglP on bglA, these data suggest that LicT negatively regulates the expression of the bglA promoter while positively affecting the expression of the bglP promoter. It is unclear at this time if the effect of LicT on the bglA locus is direct or is mediated by another protein.
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These observations can potentially be explained by examining the products obtained from the hydrolysis of -glucosides, specifically aesculin. Hydrolysis of aesculin results in the formation of a glucose 6-phosphate molecule and a 6,7-dihydroxycoumarin molecule. It is hypothesized that if this glucose 6-phosphate, either intra- or extracellularly, or extracellular glucose, which is transported to form glucose 6-phosphate, tightly repressed or regulated the bglP gene, the subsequent transport of additional aesculin would be inhibited. This would effectively block further utilization of an available substrate that is present in the environment. Both extra- and intracellular glucose 6-phosphate has been shown in E. coli to induce catabolite repression (Hogema et al., 1998a
, b
). We propose that a similar phenomenon occurs in S. mutans. Therefore, bglP must be somewhat insensitive to catabolite repression by the hydrolysis product of the
-glucoside substrate, glucose 6-phosphate, and must also be insensitive to repression by glucose, which generates glucose 6-phosphate following transport by the PTS.
This -glucoside-specific region of the S. mutans chromosome has been shown to contain several interesting features. These characteristics include a unique regulon structure and the fact that this bgl regulon is not totally repressed to non-induced levels by the presence of glucose. This region also contains an additional transcriptional regulator encoded by the bglC gene that partially controls the expression level of the bglA gene (Cote & Honeyman, 2002
). This type of regulatory element is not found in any other
-glucoside PTS described previously (Cote et al., 2000
).
This report adds transcriptional antitermination to the list of regulatory mechanisms that may govern -glucoside utilization by S. mutans. We have shown that LicT is essential for optimal expression of the
-glucoside-specific enzyme II of the PTS encoded by the bglP gene. Based upon analogy with other
-glucoside PTSs, and our data, we believe that LicT positively regulates this locus at the level of transcription. It is proposed that LicT acts on the bglP locus via an antitermination mechanism of transcriptional regulation which occurs at the RAT site located upstream of the bglP gene. The binding of LicT to the RAT site would result in transcription through the putative terminator structure and allow the expression of the BglP protein, which translocates
-glucosides such as aesculin into the cell. The proposal of an antitermination mechanism to control the expression of bglP is supported by the activation of a cryptic operon in E. coli by the presence of the S. mutans licT gene. Based upon the amino acid sequence similarity between the LicT protein of S. mutans and the E. coli BglG protein, and their corresponding RAT sites, we believe that the bglGFB operon is induced in E. coli by the expression of the S. mutans gene.
LicT also regulates the transcriptional activity of the bglA promoter in a negative manner. This is also a unique characteristic of this regulon. At this time it remains unclear why LicT would negatively regulate the expression of a gene that encodes the catabolic protein responsible for the breakdown of the translocated carbohydrate while positively regulating the gene encoding the protein responsible for transporting the carbohydrate. A possible explanation for this is that the second -glucosidase enzyme present in S. mutans also acts upon the substrates transported by BglP and that LicT does not regulate this locus. This dual regulatory role of LicT is very interesting and is currently under further study.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Alting-Mees, M. A. & Short, J. M. (1989). pBluescript II: gene mapping vectors. Nucleic Acids Res 17, 9494.[Medline]
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (2001). Current Protocols in Molecular Biology. Edited by V. B. Chanda. New York: Wiley.
Bardowski, J., Ehrlich, S. D. & Chopin, A. (1994). BglR protein, which belongs to the BglG family of transcriptional antiterminators, is involved in -glucoside utilization in Lactococcus lactis. J Bacteriol 176, 56815685.[Abstract]
Brehm, K., Ripio, M. T., Kreft, J. & Vasquez-Boland, J. A. (1999). The bvr locus of Listeria monocytogenes mediates virulence gene repression by -glucosides. J Bacteriol 181, 50245032.
Brown, G. D. & Thomson, J. A. (1998). Isolation and characterisation of an aryl--D-glucoside uptake and utilisation system (abg) from the gram-positive ruminal Clostridium species C. longisporum. Mol Gen Genet 257, 213218.[CrossRef][Medline]
Chang, A. C. & Cohen, S. N. (1978). Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134, 11411156.[Medline]
Cote, C. K. & Honeyman, A. L. (2002). The transcriptional regulation of the Streptococcus mutans bgl regulon. Oral Microbiol Immunol 17, 18.[CrossRef][Medline]
Cote, C. K., Cvitkovitch, D., Bleiweis, A. S. & Honeyman, A. L. (2000). A novel -glucoside-specific PTS locus from Streptococcus mutans that is not inhibited by glucose. Microbiology 146, 15551563.
Cvitkovich, D. G., Gutierrez, J. A., Crowley, P. J., Wojciechowski, L., Hillman, J. D. & Bleiweis, A. S. (1998). Tn917 transposon mutagenesis and marker rescue of interrupted genes of Streptococcus mutans. Methods Cell Sci 20, 112.[CrossRef]
De Vos, W. M. (1987). Gene cloning and expression in lactic streptococci. FEMS Microbiol Rev 46, 281295.[CrossRef]
Debarbouille, M., Arnaud, M., Fouet, A., Klier, A. & Rapoport, G. (1990). The sacT gene regulating the sacPA operon in Bacillus subtilis shares strong homology with transcriptional antiterminators. J Bacteriol 172, 39663973.[Medline]
Dower, W. J. (1990). Electroporation of bacteria: a general approach to genetic transformation. In Genetic Engineering Principles and Methods, pp. 275296. New York: Plenum.
el Hassouni, M., Chippaux, M. & Barras, F. (1990). Analysis of the Erwinia chrysanthemi arb genes, which mediate metabolism of aromatic -glucosides. J Bacteriol 172, 62616267.[Medline]
el Hassouni, M., Henrissat, B., Chippaux, M. & Barras, F. (1992). Nucleotide sequences of the arb genes, which control -glucoside utilization in Erwinia chrysanthemi: comparison with the Escherichia coli bgl operon and evidence for a new
-glycohydrolase family including enzymes from eubacteria, archaebacteria, and humans. J Bacteriol 174, 765777.[Abstract]
Franz, C. M., Worobo, R. W., Quadri, L. E., Schillinger, U., Holzapfel, W. H., Vederas, J. C. & Stiles, M. E. (1999). Atypical genetic locus associated with constitutive production of enterocin B by Enterococcus faecium BFE 900. Appl Environ Microbiol 65, 21702178.
Hall, B. G. & Xu, L. (1992). Nucleotide sequence, function, activation, and evolution of the cryptic asc operon of Escherichia coli K12. Mol Biol Evol 9, 688706.[Abstract]
Hogema, B. M., Arents, J. C., Bader, R., Eijkemans, K., Inada, T., Aiba, H. & Postma, P. W. (1998a). Inducer exclusion by glucose 6-phosphate in Escherichia coli. Mol Microbiol 28, 755765.[CrossRef][Medline]
Hogema, B. M., Arents, J. C., Bader, R., Eijkemans, K., Yoshida, H., Takahashi, H., Aiba, H. & Postma, P. W. (1998b). Inducer exclusion in Escherichia coli by non-PTS substrates: the role of the PEP to pyruvate ratio in determining the phosphorylation state of enzyme IIAGlc. Mol Microbiol 30, 487498.[CrossRef][Medline]
Honeyman, A. L., Cote, C. K., & Curtiss, R., III (2002). Construction of transcriptional and translational lacZ gene reporter plasmids for use in Streptococcus mutans. J Microbiol Methods 49, 163171.[CrossRef][Medline]
Le Coq, D., Lindner, C., Kruger, S., Steinmetz, M. & Stulke, J. (1995). New -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 177, 15271535.[Abstract]
Li, Y. & Ferenci, T. (1996). The Bacillus stearothermophilus NUB36 surA gene encodes a thermophilic sucrase related to Bacillus subtilis SacA. Microbiology 142, 16511657.[Abstract]
Macrina, F. L., Tobian, J. A., Jones, K. R., Evans, R. P. & Clewell, D. B. (1982). A cloning vector able to replicate in Escherichia coli and Streptococcus sanguis. Gene 19, 345353.[CrossRef][Medline]
Manoil, C. & Beckwith, J. (1985). TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A 82, 81298133.[Abstract]
Marasco, R., Salatiello, I., De Felice, M. & Sacco, M. (2000). A physical and functional analysis of the newly-identified bglGPT operon of Lactobacillus plantarum. FEMS Microbiol Lett 186, 269273.[CrossRef][Medline]
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, 339346.[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, 273282.[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, 26172624.[Medline]
Postma, P. W. & Lengeler, J. W. (1985). Phosphoenolpyruvate : carbohydrate phosphotransferase system of bacteria. Microbiol Rev 49, 232269.[Medline]
Reynolds, A. E., Felton, J. & Wright, A. (1981). Insertion of DNA activates the cryptic bgl operon in E. coli K12. Nature 293, 625629.[Medline]
Rutberg, B. (1997). Antitermination of transcription of catabolic operons. Mol Microbiol 23, 413421.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schnetz, K., Toloczyki, C. & Rak, B. (1987). -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 169, 25792590.[Medline]
Schnetz, K., Stulke, J., Gertz, S., Kruger, S., Krieg, M., Hecker, M. & Rak, B. (1996). LicT, a Bacillus subtilis transcriptional antiterminator protein of the BglG family. J Bacteriol 178, 19711979.[Abstract]
Tangney, M. & Mitchell, W. J. (2000). Analysis of a catabolic operon for sucrose transport and metabolism in Clostridium acetobutylicum. J Mol Microbiol Biotechnol 2, 7180.[Medline]
Tobisch, S., Glaser, P., Kruger, S. & Hecker, M. (1997). Identification and characterization of a new -glucoside utilization system in Bacillus subtilis. J Bacteriol 179, 496506.[Abstract]
van de Rijn, I. & Kessler, R. E. (1980). Growth characteristics of group A streptococci in a new chemically defined medium. Infect Immun 27, 444448.[Medline]
Yoshida, K., Shindo, K., Sano, H., Seki, S., Fujimura, M., Yanai, N., Miwa, Y. & Fujita, Y. (1996). Sequencing of a 65 kb region of the Bacillus subtilis genome containing the lic and cel loci, and creation of a 177 kb contig covering the gntsacXY region. Microbiology 142, 31133123.[Abstract]
Zukowski, M. M., Miller, L., Cosgwell, P., Chen, K., Aymerich, S. & Steinmetz, M. (1990). Nucleotide sequence of the sacS locus of Bacillus subtilis reveals the presence of two regulatory genes. Gene 90, 153155.[CrossRef][Medline]
Received 22 October 2002;
revised 20 January 2003;
accepted 24 January 2003.