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Infection and Immunity, June 2005, p. 3568-3576, Vol. 73, No. 6
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.6.3568-3576.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Inducible Antisense RNA Expression in the Characterization of Gene Functions in Streptococcus mutans

Bing Wang and Howard K. Kuramitsu*

Department of Oral Biology, State University of New York, Buffalo, New York 14214

Received 12 November 2004/ Returned for modification 6 January 2005/ Accepted 15 February 2005


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ABSTRACT
 
In order to examine gene function in Streptococcus mutans, we have recently initiated an antisense RNA strategy. Toward this end, we have now constructed and evaluated three Escherichia coli-S. mutans shuttle expression vectors with the fruA and scrB promoters from S. mutans, as well as the tetR-controlled tetO promoter from Staphylococcus aureus. Among these, the tetO/tetR system proved to be the most tightly controlled promoter. By using this shuttle plasmid system, modulation of gene function by inducible antisense RNA expression was demonstrated for comC antisense fragments of different sizes as well as for distinct gtfB antisense fragments. It was demonstrated that the size, but not the relative position, of an antisense DNA fragment is important in mediating the antisense phenomenon. Furthermore, by constructing and screening random DNA libraries with the tet expression shuttle system, 78 growth-retarded transformants harboring antisense DNA fragments were also identified. Almost all of them corresponded to homologous essential genes in other bacteria. In addition, a novel essential gene, the coaE gene, encoding dephospho-coenzyme A kinase, which is involved in the final step of coenzyme A catabolism in S. mutans, was identified and characterized. These results suggest that the antisense RNA strategy can be useful for identifying novel essential genes in S. mutans bacteria as well as further characterizing the physiology (including potential virulence factors) of these organisms.


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INTRODUCTION
 
Streptococcus mutans is a primary etiologic agent of tooth decay in humans. One of the most important virulence traits of this pathogen is sucrose-dependent colonization of tooth surfaces (biofilm formation). This property is dependent upon the expression of the glucosyltransferase (GTF) genes (gtf) of this organism (22). One of these gene products, GtfB, is important for insoluble glucan synthesis (1) and was shown to be inducible in biofilms (26, 45). S. mutans is a naturally transformable bacterium. The comC gene product, a competence-stimulating peptide (CSP), appears to play an important role in facilitating the natural transformation of S. mutans bacteria as well as biofilm formation and the aciduricity of the organisms (23). Bacteria adapt to environmental changes by modifying these biological processes via adjusting the expression levels of the genes involved. Since there are numerous gene products associated with these biological processes, evaluating these biological processes via modulation of specific target gene expression is important in evaluating the role of the target genes in the physiology of the organism. By using a new tetO/tetR-regulated S. mutans shuttle vector, we constructed a series of antisense strains modulating the expression of the comC genes as well as that of the gtfB genes. Natural transformation of the comC antisense strains and biofilm formation by the gtfB antisense strains were evaluated by induction of the expression of these antisense DNAs.

Bacterial pathogens often regulate the expression of virulence genes in a coordinated manner in response to changes in the environment. The specific modulation of target gene expression is a very powerful strategy to study the contribution of a given gene product to bacterial growth and virulence. Compared to traditional gene inactivation strategies which result in the total loss of gene function, regulated gene expression can provide quantitative information about the functional importance of a gene product. It also allows the creation of conditional lethal phenotypes in essential genes. In addition, manipulating the expression of genes is an important tool for understanding regulatory cascade mechanisms and the importance of gene regulation in pathogenesis.

Interference of mRNA expression with antisense RNA has been used effectively in eukaryotic systems to inhibit gene expression (5). Recently, this approach has also been used with Staphylococcus aureus (19), mycobacteria (32) and Candida albicans (6) to identify essential genes and virulence factors as well as to study gene functions of microorganisms (17, 46). For these purposes, a regulated promoter system is essential. Although a variety of promoters and vectors have been developed to allow quantitative modulation of gene expression in gram-negative bacteria (3, 7, 10, 14, 43), many of these systems do not function well in gram-positive organisms. This likely results from the more-stringent control of promoter usage in gram-positive species relative to that in gram-negative species (28, 29). Although several useful promoter systems were reported for S. aureus, Streptococcus pneumoniae, and Bacillus subtilis (8, 20, 12, 21, 34, 36, 44), few strong regulatory systems are available in S. mutans. Among these gram-positive bacterial promoter systems, Ptet is induced by tetracycline and its derivative doxycycline, which are not common components in bacterial media and which allow for the induction of promoter activity without interfering with bacterial metabolism. Recently, several promoters which are induced by sugars in S. mutans were identified: the scrB promoter is induced nearly threefold by sucrose (16) and the fruA promoter is induced approximately fourfold by inulin relative to fructose (4). In this study, we adapted the tetO/tetR promoter from an S. aureus expression system, as well as the fruA and scrB promoters from S. mutans, to construct shuttle expression vectors and evaluate the relative strengths and induction properties of these promoters.

The availability of complete bacterial genome sequences offers the possibility of uncovering the physiological functions of many unknown genes of microorganisms. Recently, we reported the utilization of the antisense RNA strategy to identify common essential genes in heterogeneous bacteria by employing an E. coli expression system to clone DNA fragments of S. mutans into Escherichia coli (41). In the present study, we constructed DNA libraries with a tetO/tetR regulatory expression shuttle vector to identify essential genes in this microorganism by screening for antisense transformants with retarded growth. One of these previously uncharacterized genes codes for dephospho-coenzyme A kinase activity which is involved in the final step of coenzyme A catabolism and appears to be essential for the growth of S. mutans.


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MATERIALS AND METHODS
 
Bacterial strains and plasmids. The S. mutans strains used in this study were LT11 (37), a spontaneous mutant of UA159 with high transformation efficiency; GS5 (13); S. mutans NG8{Delta}comC (24); and S. mutans LN62, a gtfB mutant (33). E. coli DH5{alpha} (supE44 hsdR17 recA1 endA1 gyrA96 thi-1 relA1) was utilized as a general cloning host. S. mutans strains were grown in Todd-Hewitt broth (THB; Invitrogen, Carlsbad, CA) and on tryptic soy broth agar (TSA) plates (Difco, Detroit, Mich.). E. coli strains were cultured in L-broth (Invitrogen), and transformants were selected on L-agar plates supplemented with the indicated antibiotics.

Plasmids used in this study were pUC19 (43), pMM223 (40), pACL2073 (2), pTS749 (35), its derivative pSCR749 (unpublished data), pKS::fruA (4), and pCR2.1 TOPO (Invitrogen).

DNA manipulations and sequence similarity analysis. DNA isolation, restriction endonuclease digestion, PCR, sequencing, Southern blotting, ligation, transformation, and other DNA manipulations were carried out as described previously (42). Restriction endonucleases and other DNA-modifying enzymes were obtained from Invitrogen, New England Biolabs Inc. (Beverly, MA), and Promega Corp. (Madison, WI) and used according to the specifications of the manufacturers. DNA fragments were isolated from agarose gels by using a QIAEXII kit (QIAGEN, Valencia, CA). Double-stranded PCR template sequencing was performed by using the Sequenase 7-deaza-dGTP sequencing kit (USB, Cleveland, OH). Sequence similarity searches were performed with BLAST against the S. mutans strain UA159 genome sequence (found at the University of Oklahoma website [http://www.genome.ou.edu/smutans.html]) and Swiss-Prot databases.

The general strategy for gene inactivation in S. mutans involved designing two pairs of PCR primers corresponding to the upstream and downstream fragments (average size, 500 bp) of the target gene. The PCR products were then ligated with an erythromycin resistance gene, erm (35). PCR amplification of the recombinant DNA fragment containing the upstream fragment-erm-downstream fragment was performed using a pair of primers corresponding to the 5' end of the upstream DNA fragment and 3' end of the downstream fragment. The resultant recombinant DNA fragment for which part of or the entire target gene was replaced by the erm gene was cloned into the TOPO PCR cloning plasmid (Invitrogen) screening for erythromycin-resistant (200-µg/ml) transformants. The resultant plasmid was next digested with EcoRI and transformed into S. mutans LT11 (37). Following selection for erythromycin-resistant (5-µg/ml) transformants, the target gene was inactivated by a double-crossover recombination mechanism.

Construction of regulatory systems in shuttle plasmids pMM223 and pTS749 and lacZ reporter plasmid pTetELac. The tetO/tetR promoter DNA fragment was amplified by PCR using a pair of primers corresponding to the flanking sequences of the tetO/tetR region of pACL2073 with additional EcoRI and BglII sites at the 5' and 3' ends, respectively (Ptet-5, 5'-GGAATTCAAGCTTGCATCCCTGCAG-3'; Ptet-6, 5'-GAAGATCTATTCGAGCTCGGTACCC-3' [with the EcoRI and BglII sites, respectively, underlined). The EcoRI-BglII-digested tetO/tetR fragment was then cloned into the EcoRI-BglII-digested vector pMM223 and transformed into E. coli DH5{alpha}. The resultant plasmid pTetE contained tetO controlling the transcription of downstream reporter genes, aad and lacZ. Plasmid pTetR with the tetR gene located upstream of the same reporter genes was constructed by the cloning of the EcoRI-PstI fragment containing tetO/tetR of pACL2073 into the EcoRI-PstI sites of pMM223. Plasmids pTetE and pTetR were a pair of shuttle plasmids with tetO/tetR in the opposite orientation (Fig. 1). For the construction of a S. mutans transformable shuttle vector, plasmid pSCR749(B), a derivative with a BglII site deleted and a multicloning site (MCS) from pTS749, was digested by NcoI and EcoRI. The HpaI-digested fragment of pMM223 was next cloned into the blunted fragment of NcoI-EcoRI digested vector, pSCR749(B), yielding plasmid pSM. The EcoRI-BglII-digested tetO/tetR fragment was finally cloned into EcoRI-BglII-digested vector pSM to yield shuttle plasmid pSCR.



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  		FIG. 1. Construction of inducible expression shuttle vectors.

To clone the fruA promoter into the pMM223 MCS, a BamHI-PstI-digested fragment from pKS::fruA containing the fruA promoter fragment was introduced into the BamHI-PstI sites of pMM223. The resultant plasmid, pMfru, contains the fruA promoter upstream of the aad gene (Fig. 1). To clone the scrB promoter into the pMM223 MCS, the PCR product of the scrB promoter region digested by BglII was introduced into the EcoRI/BglII sites of pMM223. The resultant plasmid pMscrB contains the scrB promoter upstream of the aad gene (Fig. 1).

To construct the pTet::lacZ-expressing plasmid, a lacZ reporter gene from pSBL20 (16) was amplified by PCR using a pair of primers (scrB-9, 5'-GAAGATCTATTATTAGGAGTAGATAAACG-3', with the BglII site underlined; scrB-10, 5'-CCAACAATAGGATAAAATTGTCC-3') and was introduced into the BglII-SmaI sites of pTetE. The recombinant plasmid pTetELac was then transformed into S. mutans LT11.

Preparation and screening of genomic DNA libraries. S. mutans LT11 chromosomal DNA fragments (200 to 400 bp) partially digested by Sau3AI were cloned into the BglII site of pTetR, transformed into E. coli DH5{alpha}, and grown on L-agar containing 150 µg/ml erythromycin at 30°C. The transformants were pooled and inoculated into LB containing 150 µg/ml of erythromycin at 30°C. The plasmid pool was then extracted from the cells and transformed into S. mutans LT11. The transformants were plated on TSA-5 µg/ml erythromycin and grown at 37°C anaerobically. The colonies were then replica plated onto TSA-5 µg/ml erythromycin plates in the presence/absence of 50 ng/ml doxycycline. The transformants which showed smaller colonies or no colonies on the doxycycline plate compared to the plate without antibiotic were then selected for further analysis. The growth-retarded phenotype was confirmed by examining growth in broth cultures in the presence/absence of doxycycline at an optical density at 600 nm (OD600).

Construction of scrA::yacE in S. mutans LT11. By use of a pair of primers (Sc-1, 5'-CATTTGCAAAAAACTCC-3'; Sc-2, 5'-CTTGCCAATTCTTAATATC), an 800-bp DNA fragment containing the start codon and promoter of scrA and part of the scrB gene was produced by PCR. The yacE structural gene from the 5' terminus without the ATG start codon to the stop codon was amplified by PCR from E. coli, using the following pair of primers: yac-10 (5'-GGAAGATCTCATTACGGTTTTTCCTG-3', with the BglII site underlined) and yac-11 (5'-AGGTATATAGTTGCCTTAAC-3'). The scrAB PCR fragment treated by XbaI-T4 kinase was ligated with the BglII-T4 kinase-treated yacE PCR fragment. The PCR product of scrA::yacE was amplified from the ligation reaction described above using primers yac-10 and Sc-3 (5'-TATCTAGACTTTGTGCTCCAACAATAG-3, with the XbaII site underlined). The BglI-XbaI-digested scrA::yacE fragment was next cloned into the BamHI-XbaI sites of pUCSp, which was derived from pUC18 by insertion of the aad gene from pSCSP-1 (42) into the FspI sites to inactivate the ß-lactamase gene. The recombinant plasmid pSY was transformed into S. mutans LT11 to select for spectinomycin-resistant transformants containing a chromosomal copy of LT11 scrA::yacE (Fig. 2).



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  		FIG. 2. Construction of the LT11 scrA::yacE strain, which expresses YacE of E. coli from the chromosome of S. mutans.

Construction, overexpression, and purification of SMU.1613c-histidine tag fusion protein (SMU.1613c-H). A pair of primers complementary to the SMU.1613c open reading frame without the start codon (coaE-1, 5'-CCAAGCTTCATTCATTTTGCAACCTC-3'; coaE-2, 5'-CGGGATCCACAAAAATTATTGGGATTACAGGC-3') was designed to amplify the SMU.1613c gene. The PCR product was digested by BamHI-HindIII and cloned into the BamHI-HindIII sites of pQE80L-1 (QIAGEN), which contains the six-His tag upstream of the MCS. The recombinant plasmid pQC contained the six-His SMU.1613c under the control of the T5 promoter and the lac operator. The DNA sequence of SMU.1613c-H was verified by sequencing.

The E. coli transformant harboring pQC was cultured in 1.0 liter of LB-100 µg/ml ampicillin at 30°C to an optical density of 0.5 to 0.6 at 600 nm and then induced by 0.1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG). After induction at 30°C for 3 h with aeration, the bacteria were harvested by centrifugation at 4,000 x g for 20 min and resuspended in 20 ml of lysis buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0). A soluble fraction obtained by sonication and centrifugation was applied to the Ni-nitrilotriacetic acid spin column (QIAGEN), and SMU.1613c-H was eluted according to the instructions of the supplier, with slight modifications. Protein purity was evaluated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with RGS.His antibody (QIAGEN).

Measurement of MICs. Each S. mutans strain grown overnight at 37°C anaerobically was inoculated at 105 CFU into duplicate THB media containing serially diluted concentrations of spectinomycin and growth measured in the presence and absence of doxycycline inducer. After the strains were grown overnight at 37°C anaerobically, the cell density was measured by determining the OD600. The MIC of spectinomycin for each tested strain was determined as the lowest concentration inhibiting bacterial growth.

Determination of the transformation efficiency of S. mutans. Bacteria were cultured in THB-10% horse serum overnight at 37°C and then inoculated (1/10) into fresh THB-10% horse serum in the presence/absence of 50 ng/ml doxycycline. Bacteria were incubated for 2 h and 10 µg/100 µl of chromosomal DNA from the GS5::{Delta}gtfD mutant (Tetr) (15) was added. The cells were incubated at 37°C for another 2 h and serially diluted on TSA-10 µg/ml tetracycline agar plates. After incubation at 37°C anaerobically for 48 h, the transformation efficiency was determined as the number of transformants/total cells transformed.

Dephospho-coenzyme A kinase activity assay. The enzymatic determination with myokinase (MK) (ATP:AMP phosphotransferase), pyruvate kinase (PK), and lactate dehydrogenase (LDH) quantitates the decrease of NADH, as measured by the change in extinction at 340 nm, as follows:{zii00605488800s1} Dephospho-coenzyme A (CoA) kinase activity was measured as described previously (25) with the following modifications. Each 1.0-ml assay mixture contained KCl (20 mM), MgCl2 (10 mM), ATP (10 mM), NADH (0.3 mM), phosphoenolpyruvate (PEP) (0.4 mM), pyruvate kinase (10 U), and lactic dehydrogenase (4 U) in 50 mM Tris buffer (pH 8.5). After the addition of the dephospho-coenzyme A kinase sample, the reaction was initiated by the addition of dephospho-coenzyme A (0.4 mM). The dephospho-coenzyme A kinase activity was determined by the measurement of NADH oxidation at OD340.

Determination of ß-galactosidase activity. S. mutans LT11 strains harboring pTetO and pSMT were grown in THB containing 5 µg/ml erythromycin to mid-log phase. Doxycycline was then added to the broth and the cells induced for 2 h. The cells were then collected by centrifugation and assayed for ß-galactosidase activity as described previously (16).

Sucrose-mediated biofilm formation assay. Biofilm formation was quantified as previously described (45). Flat-bottom polystyrene microtiter plates (96-well Easy Wash enzyme immunoassay-radioimmunoassay plates; Corning Inc., Corning, N.Y.) containing 100 µl of THB-0.5% sucrose per well were inoculated with S. mutans (1.7 x 105 CFU per well) from a 24-h growth in THB. After 24 h of incubation at 37°C, 25 µl of 1% (wt/vol) crystal violet solution was added to each well. After 15 min, the wells were rinsed three times with 200 µl of distilled water and air dried. The crystal violet on the abiotic surfaces was solubilized in 95% ethanol, and the optical density at 570 nm was measured. Growth was determined by measuring the turbidities (optical densities at 570 nm) of parallel wells following resuspension of the sessile organisms together with the planktonic cells.

Determination of GTF activity. Relative amounts of GTF protein were determined by measuring the enzyme activity in polyacrylamide gels as previously described (38, 39). Briefly, strains were grown to the same mid- to late-log stage as determined by cell density (OD600 in THB medium) in the presence or absence of 50 ng/ml doxycycline. The cells were then collected and washed with phosphate-buffered saline buffer (pH 7.4). The cells were next resuspended in phosphate-buffered saline buffer and adjusted to the same OD600 of 0.85 to 0.875. Twenty microliters of each cell suspension was run on 8.75% SDS-PAGE gels. After electrophoresis, the gels were incubated overnight at 37°C in 3% sucrose, 0.5% Triton X-100, and 10 µg/ml dextran T10 in 10 mM sodium phosphate, pH 6.8, at 37°C, and the resulting glucan bands were treated with periodic acid and pararosanilin. The intensities of the stained bands reflect the relative amounts and activities of the GTF proteins.


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RESULTS
 
Evaluation of inducible expression systems in S. mutans. In order to maximize the effects of antisense RNA expression on the physiology of S. mutans, it was first necessary to construct for this organism a shuttle plasmid system which would allow for high-level induction of cloned antisense fragments. A comparison of the activities of the promoters of the scrB and fruA genes of S. mutans as well as that of a tetracycline-inducible promoter expression system in S. mutans was performed using vector pMM223. This was accomplished by initially measuring spectinomycin MICs of the individual strains harboring the respective plasmids in the presence and absence of inducer. pTetE expressed the highest level of aad gene product of the three expression systems (the MIC for this strain was 64 mg/ml in the absence of doxycycline and 128 mg/ml in the presence of doxycycline), 8 times higher than pFru-controlled aad gene expression (the MIC for this strain was 16 mg/ml in the absence/presence of 0.5% inulin) and 32 times higher than pScr controlled aad gene expression (the MIC for this strain was 4 mg/ml in the absence/presence of 0.5% sucrose), compared to the strain harboring only the vector pMM223 (MIC, 500 µg/ml). Therefore, we chose pTetEB to further examine promoter activities in the presence/absence of inducer. Since MIC measurement is a semiquantitative evaluation, a pTet::lacZ reporter plasmid, pTetElac, was constructed and the resulting strain was examined for its ability to express ß-galactosidase activity when induced. Experiments examining the kinetics of induction and inducer concentrations indicated that steady-state lacZ gene expression reached its maximum within 3 h after induction by 100 ng/ml doxycycline and that 50 to 100 ng/ml doxycycline promoted maximum induction without significant inhibition of growth. When fully induced, the pTetE system expressed the highest level of ß-galactosidase activity, 120 units, which is 12 times the basal expression (11 units) (Fig. 3). Thus, the pTetE system was selected to construct the inducible antisense RNA strains.



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  		FIG. 3. LacZ activities of LT11::pTetEBLac. LacZ activities (ß-galactosidase activity, in Miller units) were measured in wild-type LT11 grown in the presence of 100 ng/ml of doxycycline and LT11::pTetElac grown in the absence/presence of doxycycline. (A) Time course after adding doxycycline. Triangle line, LT11::pTetElac in the presence of 100 ng/ml doxycycline; square line, LT11::pTetElac in the absence of doxycycline; diamond line, LT11 in the presence of 100 ng/ml doxycycline. (B) Dosage of doxycycline and the growth of LT11::pTetElac. The data are the averages of three samples, and standard errors are shown.

Modulation of the expression of S. mutans genes by inducible antisense RNA production depends on the size, but not the relative position, of the antisense DNA fragment. To study the regulation of the physiological functions of individual genes via inducible antisense RNA production and the impact of the position and size of antisense DNA fragments on such changes, initially, a series of comC DNA fragments were PCR amplified and cloned into the KpnI-SmaI sites in the antisense orientation downstream of the tetO promoter in vector pTetE (Fig. 4). The resultant plasmids were introduced into LT11, yielding comC antisense RNA strains. Transformation in the strains was evaluated by the introduction of chromosomal DNA of an LT11::{Delta}gtfD mutant. The results indicated that the location of antisense DNA fragments from either the 3' or 5' terminus of comC resulted in similar inhibitions of transformation. The minimal size of the antisense fragment to achieve optimal inhibition was estimated at >20 bp (Table 1). The supplementation of CSP restored transformation efficiency in the antisense inhibited strains (data not shown). This result confirmed that the inhibition of transformation efficiency in the comC antisense strains was mediated by the reduced expression of CSP in the presence of doxycycline.



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  		FIG. 4. Genetic arrangement of the comC gene in wild-type (wt) S. mutans LT11 and antisense strains. Ptet, promoter of tetO; solid arrows, antisense comC fragments in the recombinant plasmids; open arrow, comC gene. The sizes of the antisense DNA fragments cloned into the vectors are indicated to the right of the strain name.


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  		TABLE 1. Transformation efficencies of LT11 comC antisense strains

Biofilm formation is an important physiological adaptation of oral bacteria. The gtfB gene, of approximately 4,500 bp, was reported previously to be important in sucrose-mediated biofilm formation of strain GS5 (1). We therefore constructed a series of gtfB antisense RNA strains by the same strategy used for the comC antisense strains by use of pSCR. A series of gtfB DNA fragments were PCR amplified and cloned into the KpnI-SmaI sites in pSM. Next, a tetO/tetR fragment was cloned into the EcoRI-BglII sites of the respective plasmids, resulting in shuttle plasmids with gtfB fragments in the antisense orientation downstream of the tetO promoter (Fig. 5C). The resultant plasmids were transformed into strain GS5, yielding a series of gtfB antisense RNA strains. Sucrose-dependent biofilm formation of the antisense strains was compared with that of GS5 harboring pSCR only, as well as that of the gtfB null mutant LN62 (33) in the presence of 50 ng/ml doxycycline (Fig. 5A). After induction, the gtfB antisense strains showed reduced biofilm formation compared to GS5 and GS5::pSCR but elevated activity relative to LN62. These results are also similar to the comC antisense strains, since the antisense fragments from the 3' and 5' termini of gtfB produced similar inhibitions of biofilm formation (Fig. 5A and B). The minimum size of the antisense fragment necessary for significant inhibition was >100 bp. This size represents about 4% of the intact gtfB gene. Glucosyltransferase activity assays confirmed that GTF activities in the antisense strains were lower than those in GS5 harboring only pSMT (Fig. 5D). GS5::gtf-3 with a 60-bp antisense gtfB fragment had little impact on GTF expression (Fig. 5D), as well as minimal effects on biofilm formation.



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  		FIG. 5. Modulation of expression of the gtfB gene by gtfB antisense strains. wt, wild type. (A) Bacterial growth and biofilm formation of S. mutans wild-type GS5, gtfB mutant LN62, GS5 harboring shuttle vector pSCR (pSCR), GS5 gtfB antisense strains (gtf-1, gtf-2, gtf-3, and gtf-4) in 1/4 strength THB-mucin-0.5% sucrose with doxycycline (50 ng/ml). Growth (white bars) and biofilm formation (black bars) were measured under anaerobic conditions. The data are the averages of three samples, and standard errors are shown. (B) Data from the same experiment with the absence of doxycycline. (C) Genetic arrangement of the gtfB gene in wild-type S. mutans GS5 and antisense strains. Ptet, promoter of tetO; solid arrows, antisense gtfB fragments in the recombinant plasmids; open arrow, gtfB gene. The sizes of the antisense DNA fragments cloned into the vectors are indicated to the right of the strain name. (D) GTF activity detected following SDS-PAGE analysis and periodic acid-Schiff staining of the S. mutans strains mentioned above grown in the presence (top panel) and absence (bottom panel) of 50 ng/ml doxycycline.

Identification of essential genes by construction and screening of inducible antisense RNA libraries of S. mutans LT11. To construct stable antisense RNA libraries in E. coli, we introduced the strain LT11 chromosomal DNA (200 to 400 bp) partially digested by Sau3AI into the BglII site of pTetR, downstream of the tetO promoter. The random genomic library was amplified by passage through E. coli to provide sufficient amounts of DNA from the plasmid pool and was then transformed into S. mutans LT11. By screening for growth-retarded transformants following replica plating on TSA with/without 50 ng/ml doxycycline (confirmed by determination of OD600 in broth cultures in the presence or absence of inducer, as well as sequence analysis), a total of 78 growth-retarded transformants harboring the antisense DNA fragments of putative essential genes was identified (Table 2) out of approximately 3,000 transformants.


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  		TABLE 2. Essential genes of S. mutans detected by the antisense RNA strategy

Identification and characterization of the essential gene encoding dephospho-coenzyme A kinase (coaE) of S. mutans. Since one of the growth-retarded transformants, W213, apparently harbored a novel gene of unknown function at the time of this analysis downstream of the tetO promoter in the antisense RNA orientation, it was subjected to further analysis. Following the sequencing of the insert fragment and a homology search of the S. mutans UA159 database, the antisense DNA fragment corresponded to a conservative hypothetical protein, SMU.1613c, with an optimal Blastp hit with the ytaG gene in Bacillus subtilis. It also shares 33% identity with the yacE gene of E. coli. The gene product of yacE of E. coli has been recently characterized as dephospho-coenzyme A kinase, an enzyme involved in the last critical step of coenzyme A biosynthesis (27). Homologues of the yacE gene exist in a broad spectrum of microorganisms, as well as eukaryotic cells, indicating that this enzyme is important in cell metabolism. However, a gene encoding dephospho-coenzyme A kinase in gram-positive bacteria has not yet been characterized. Therefore, the gene essentiality of SMU.1613c was initially confirmed by attempts at gene inactivation. However, no erythromycin-resistant transformants were recovered following transformation experiments designed to inactivate this gene by using a transcriptional terminator-negative erythromycin resistance gene replacing part of the SMU.1613c gene. This result is compatible with the essential nature of the gene in S. mutans.

In order to further characterize the essential nature of the SMU.1613c gene, a gene complementation strategy was carried out by introducing the E. coli yacE gene into the chromosome of strain LT11 downstream of the scrA promoter of this S. mutans strain. The resultant transformant, the LT11 scrA::yacE strain, was then subjected to gene inactivation of the SMU.1613c gene. Erythromycin-spectinomycin-resistant transformants were then recovered and compared with the control LT11 wild-type strain. Truncation of SMU.1613c by double crossover in the transformants was confirmed by PCR (data not shown). The successful inactivation of the SMU.1613c gene indicated that expression of the yacE gene of E. coli in S. mutans complemented the function of the SMU.1613c product. Therefore, the SMU.1613c gene product has a function similar to that of the yacE product, i.e., the former gene also expresses dephospho-coenzyme A kinase (CoaE) activity.

To further confirm the function of the SMU.1613c gene product, a histidine-tagged SMU.1613c fusion protein plasmid was constructed by using vector pQE80L-1. The resultant plasmid, pQC, was transformed into E. coli. The SMU.1613c-H was expressed and purified from the transformant. The E. coli CoaE-His tag fusion protein (CoaE-H) was expressed and purified from the E. coli strain harboring pESC124, a histidine-tagged yacE fusion plasmid (31). Both proteins have the same molecular mass, about 22 kDa (data not shown). The CoaE activity assay showed that both fusion proteins expressed similar CoaE activities (Fig. 6A) compared to a negative control. In addition, ADP formation was dose dependent on the SMU.1613c-H concentration (Fig. 6B). Thus, these results confirm that SMU.1613c encodes dephospho-coenzyme A kinase activity in S. mutans. This is the first report of the identification and characterization of CoaE and its corresponding gene, coaE, in gram-positive bacteria.



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  		FIG. 6. Dephospho-coenzyme A kinase activity assays measured by ADP formation. (A) ADP formation catalyzed by purified YacE-His tag fusion protein from E. coli and purified fusion protein of S. mutans CoaE-H compared to the negative control without adding CoaE. (B) Dosage effect of S. mutans CoaE-H. The data are the averages of three samples, and standard errors are shown.


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DISCUSSION
 
The current availability of gram-positive bacterium-inducible expression systems is somewhat limited, especially in regard to regulated systems. There are a few systems for use with gram-positive bacteria: Pspac (44), xylA (20), nisA (8, 21), MalR-regulated promoter (30), C1-regulated promoter (34), and Ptet (36). Unfortunately, different species have very different responses to these systems (9). Most of them are regulated by carbohydrate sources which are essential for growth and may also be subject to carbon catabolite repression. There is no comparable inducible expression system for oral streptococci. We introduced the tetO/tetR, scrB, and fruA promoters into the streptococcal shuttle vector pMM223. The induction experiments showed that PtetO/tetR is a relatively strong inducible promoter compared to the other two promoters in S. mutans. One useful feature of this system is that the inducer is not known to interfere at the concentrations utilized with other aspects of S. mutans metabolism.

One of the recent approaches to studying gene function, especially those genes involved in essential cell functions, is the utilization of antisense RNA to manipulate the expression levels of target genes (2, 18). It was noticed that not all of the antisense fragments produced uniform inhibitory effects (18). In addition, it was unclear what effects the relative positions or the sizes of the antisense RNA fragments had on target gene expression. The comC and gtfB antisense strains constructed in this study produced different degrees of inhibition of cell function. The antisense DNA fragments from the 5' end of the target genes cloned downstream of the inducible promoter had the same inhibitory effects as those from the 3' fragments. Compared to the location of the antisense DNA fragments, the size of the fragments appears to be important to achieve maximal antisense inhibition. A minimum of >20 bp was required to produce inhibition of the expression of the comC gene, which is >5% of the length of the mRNA of the comC gene. For gtfB, since it is relatively large, a minimum size of 200 bp, which is about 4% of the whole length, was required to achieve significant antisense inhibition. Therefore, these results suggest that a DNA fragment larger than approximately 5% of the entire gene length is likely required to produce optimal inhibition of gene expression. Such size limitations on antisense RNA effects have not been reported before for bacteria. The resulting antisense RNA likely inhibits the translation of the target genes.

By a screening of the antisense library constructed in the tetO/tetR expression shuttle vector, several categories of essential genes in S. mutans were identified. These essential genes detected in this system are involved in the essential biological processes of DNA replication, recombination and repair, translation, ribosomal structure and biogenesis, transcription, and cell envelope biogenesis, as well as metabolism. Most of them appear to be involved in the maintenance of ribosomal structure or transcriptional processes. The relatively low frequency of essential gene detection in the S. mutans system compared to that reported for S. aureus (11, 19) may be due to plasmid instability in E. coli when some S. mutans antisense fragments are introduced into the gram-negative host. For example, this was observed when cloning the S. mutans scrB promoter into E. coli plasmids (42). In addition, the stronger induction of promoter activity in the other studies (11, 19) would also allow for more convenient identification of growth-inhibited transformants under inducing conditions. Nevertheless, the system utilized in the present study did allow for the identification of a number of essential genes in S. mutans and the characterization of a novel essential gene, coaE, in this organism. Furthermore, the coaE gene, encoding dephospho-coenzyme A kinase, was identified and characterized as an essential gene in S. mutans. Undoubtedly, the screening of additional antisense DNA clone banks by this strategy might uncover additional novel essential genes some of which may prove to be specific targets for novel anticaries approaches.


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ACKNOWLEDGMENTS
 
We thank B. Zaat, A. L. Cheung, and R. A. Burne for providing some of the plasmids used in this study.

This study was supported in part by National Institutes of Health grant DE03258.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Oral Biology, State University of New York at Buffalo, 3435 Main Street, Buffalo, NY 14214. Phone: (512) 249-5901. Fax: (716) 829-3942. E-mail: Kuramitsu{at}earthlink.net. Back

Editor: V. J. DiRita


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Infection and Immunity, June 2005, p. 3568-3576, Vol. 73, No. 6
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.6.3568-3576.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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