A unique nine-gene comY operon in Streptococcus mutans

Justin Merritt1, Fengxia Qi2 and Wenyuan Shi1,2

1 UCLA Molecular Biology Institute, 10833 Le Conte Avenue, Los Angeles, CA 90025, USA
2 UCLA School of Dentistry, 10833 Le Conte Avenue, Los Angeles, CA 90025, USA

Correspondence
Wenyuan Shi
wenyuan{at}ucla.edu


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Many Gram-positive and Gram-negative bacteria possess natural competence mechanisms for DNA capture and internalization. In Bacillus subtilis, natural competence is absolutely dependent upon the presence of a seven-gene operon known as the comG operon (comGAG). In species of Streptococcus, this function has been described for a four-gene operon (comYAD in Streptococcus gordonii and cglAD in Streptococcus pneumoniae). In this study, a nine-orf operon (named comYAI) required for natural competence in Streptococcus mutans was identified and characterized. Orf analysis of this operon indicates that the first four Orfs (ComYA–D) share strong homology with ComYA–D of S. gordonii and CglA–D of S. pneumoniae, the fifth to seventh Orfs (ComYE–G) match conserved hypothetical proteins from various species of Streptococcus with ComYF possessing a predicted ComGF domain, the eighth Orf (ComYH) shows a strong homology to numerous DNA methyltransferases from restriction/modification systems, and the ninth Orf (ComYI) is homologous to acetate kinase (AckA). RT-PCR analysis of the orf junctions confirmed that all nine orfs were present in a single transcript, while real-time RT-PCR analysis demonstrated that these orfs were expressed at a level very similar to that of the first orf in the operon. Mutations were constructed in all nine putative orfs. The first seven genes (comYAG) were found to be essential for natural competence, while comYH and comYI had reduced and normal natural competence ability, respectively. Analyses of S. mutans comY–luciferase reporter fusions indicated that comY expression is growth-phase dependent, with maximal expression at an OD600 of about 0·2, while mutations in ciaH, comC and luxS reduced the level of comY expression. In addition, comY operon expression appears to be correlated with natural competence ability.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Streptococcus mutans is widely considered to be the primary pathogen responsible for dental caries in humans (Quivey et al., 2001). These bacteria typically exist as biofilm-dwelling organisms and coexist with hundreds of other species of bacteria present in the dental biofilm (Tanzer et al., 2001). One feature that seems to be fairly ubiquitous among oral bacteria such as S. mutans is their ability to actively internalize DNA from the surrounding environment. This may be mediated through direct conjugation or natural competence. In addition, both types of genetic exchange mechanisms seem to be enhanced in a biofilm lifestyle (Hausner & Wuertz, 1999; Hendrickx et al., 2003; Li et al., 2001; Licht et al., 1999). In fact, in some instances, the uptake of DNA can even aid in the development of biofilm (Ghigo, 2001). Given that a biofilm creates an environment in which there are many intimate contacts among residential bacteria, there are presumably numerous opportunities to transfer genetic material between cells. In multispecies communities such as the dental biofilm, the possibilities for genetic exchange probably increase even more, as there is a constant supply of genetic material released from scores of Gram-positive and Gram-negative species of bacteria. Not surprisingly, many of the sequenced oral bacteria show a plethora of insertion sequences and other evidence of transposition (Califano et al., 2003; Kapatral et al., 2002). This is also true for S. mutans, which contains a variety of loci that appear to have resulted from horizontal gene transfer (Ajdic et al., 2002).

Typically, when a specific density of cells has accumulated within a population, a signalling cascade is initiated that results in a window of time in which the genes necessary for natural competence are induced. In species of Streptococcus, natural competence begins with the production of a peptide quorum-sensing signal called competence stimulating peptide (CSP), which is encoded by the constitutively expressed comC gene (Cheng et al., 1997; Li et al., 2002; Lunsford & London, 1996; Pestova et al., 1996). When a critical concentration of the peptide has accumulated, it is sensed by a two-component system encoded by comD and comE (Cheng et al., 1997; Havarstein et al., 1996; Pestova et al., 1996). The comE response regulator will then activate the expression of a natural competence-related minor sigma factor known as ComX (Lee & Morrison, 1999; Li et al., 2002; Lunsford & London, 1996). This sigma factor will then activate the expression of all of the late competence genes required to actually bind and internalize exogenous DNA (Lee & Morrison, 1999; Luo & Morrison, 2003).

In Bacillus subtilis, initial DNA binding to the cell surface seems to be critically dependent on an apparent seven-gene operon known as the comG operon. Each of these late competence genes is predicted to encode membrane-associated proteins and have been shown to be essential for both the binding of DNA to the cell surface as well as for natural transformability in general (Chung et al., 1998; Chung & Dubnau, 1998; Dubnau, 1999). In published reports of various Streptococcus species, this function has been shown to be encoded in an apparent four-gene operon (cglAD in Streptococcus pneumoniae and comYAD in Streptococcus gordonii) (Lunsford & Roble, 1997; Pestova & Morrison, 1998). Current data suggests that these genes are required for the assembly of a multi-protein complex that provides the structural framework for a DNA binding protein (comEA in B. subtilis) involved with exogenous DNA capture (Chung & Dubnau, 1998). Presumably, loss of any of these genes disrupts the complex and prevents comEA from performing its DNA binding function.

Among the oral streptococci, there are several reports that suggest a connection between the process of competence development and other cellular functions. For example, it has been shown in both S. gordonii and S. mutans that the quorum-sensing systems responsible for regulating competence are also involved in regulating the process of biofilm formation (Li et al., 2002; Loo et al., 2000; Yoshida & Kuramitsu, 2002). This suggests that some signalling overlap may exist between these processes. Therefore, to complement our studies on the function of quorum sensing in biofilm formation, we began an investigation of genes involved with competence development. To this end, we have identified a nine-gene operon in S. mutans that is absolutely required for natural transformation and regulated by the same signal transduction systems reported to be involved in biofilm formation.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and culture conditions.
Bacterial strains used in this study and their relevant characteristics are listed in Table 1. Construction of comY mutants and comYluc fusion strains will be described below. The ciaH (34), comC and mutR (unpublished strains by F. Qi, J. Merritt and W. Shi) mutants were constructed in our laboratory during other studies. All S. mutans strains were grown in Todd–Hewitt (TH, Difco) medium or on brain heart infusion (BHI, Difco) agar plates. For selection of antibiotic resistant colonies, BHI plates were supplemented with either 800 µg kanamycin ml–1 (Fisher), 15 µg tetracycline ml–1 (Fisher) or 800 µg spectinomycin ml–1 (Sigma). All S. mutans strains were grown anaerobically (80 % N2, 10 % CO2 and 10 % H2) at 37 °C. Escherichia coli cells were grown in Luria–Bertani (LB, Fisher) medium with aeration at 37 °C. E. coli strains carrying plasmids were grown in LB medium containing 150 µg kanamycin ml–1 or 250 µg spectinomycin ml–1.


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Table 1. Bacterial strains used in this study

 
Identification and analysis of the comY locus in S. mutans.
Orthologues of the S. gordonii comY genes were identified in S. mutans using the University of Oklahoma Streptococcus mutans Genome BLAST server (http://www.genome.ou.edu/smutans.html) and the TBLASTP program. The query sequence (S. gordonii ComYA) was obtained from the NCBI database, accession no. AAC45310 The sequences of the surrounding Orfs were then determined using DNA Strider software.

RNA extraction and transcriptional analysis.
An overnight culture of S. mutans was diluted 1 : 30 in competence media (TH+0·4 % BSA) in a total volume of 300 µl. Cells were allowed to grow to an OD600 of ~0·2, after which they were pelleted by centrifugation and the supernatant was aspirated. Pellets were resuspended in 700 µl Tris/EDTA buffer plus 200 µl 10 % SDS plus 1·5 ml hot acidic phenol (65 °C, pH 4·3) (Fisher). The solution was then incubated at 65 °C for 15 min with frequent mixing. After incubation, the solution was centrifuged and the supernatant saved for extraction. The supernatant was extracted twice with acidic phenol, followed by two extractions with a 25 : 24 : 1 mixture of phenol/chloroform : isoamyl alcohol. RNA was precipitated with a 1/10 volume of 3M sodium acetate plus 2·5 volumes of 100 % ethanol. After drying, the RNA pellet was resuspended in 89 µl RNase-free water plus 10 µl 10x DNase buffer plus 1 µl RNase-free DNase (Promega). The mixture was incubated at 37 °C for 2–3 h. After incubation, samples were further purified with an RNeasy spin column (Qiagen) and eluted in 50 µl RNase-free water. Three micrograms of total RNA was used for cDNA synthesis using Stratascript RT (Stratagene) according to the manufacturer's protocol. RT-PCR and real-time PCR primer combinations of the various orfs and junctions are all listed in Table 2. All of the respective orfs in Table 2 are listed by their GeneID, as specified by the Los Alamos National Laboratory Oral Pathogen Sequence Database (http://www.oralgen.lanl.gov/oralgen/bacteria/smut/index.html). For real-time RT-PCR, SYBR green (Bio-Rad) was used for fluorescence detection with the iCycler (Bio-Rad) real-time PCR system according to the manufacturer's protocol.


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Table 2. Primers used in this study

Primers denoted ‘KO’ were used for gene mutations; ‘RT’ primers were used for RT-PCR; ‘Real-Time’ primers were used for real-time RT-PCR.

 
Construction of mutants.
Mutants in each of the putative comY genes were created via double crossover homologous recombination. For each construct, two fragments were generated by PCR which corresponded to approximately 1 kb of upstream and downstream sequence relative to the target gene. Primers were designed using genome data obtained from the University of Oklahoma S. mutans Genome Sequence Database (http://www.genome.ou.edu/smutans.html) and from the Los Alamos National Laboratory Oral Pathogen Sequence Database (http://www.oralgen.lanl.gov/oralgen/bacteria/smut/index.html) and all are listed in Table 2. All fragments were generated using Pfu polymerase (Stratagene) and subsequently treated with Taq polymerase to generate 5' overhangs. These fragments were subsequently cloned into the pCR2·1 vector from Invitrogen. Individual upstream and downstream fragments were then cut from the pCR2·1 vector and gel purified (Qiagen). Quick ligase (NEB) was used to anneal purified upstream and downstream fragments to compatible restriction sites of a kanamycin resistance cassette cut from plasmid pBsK (J. Merritt and W. Shi, unpublished results) and a linearized pUC18 vector. E. coli was subsequently transformed with the ligation reaction using standard protocols, and the resulting plasmids carrying knockout constructs were confirmed by restriction analysis and PCR. For the transformation of knockout constructs into S. mutans, the plasmids were linearized using a unique AatII site on the vector and later transformed using typical protocols (Perry & Kuramitsu, 1981; Shah & Caufield, 1993). Confirmation of DNA integration was performed by extracting genomic DNA from antibiotic-resistant clones and PCR amplifying various combinations of fragments. Multiple isolates of each confirmed mutant strain were saved for later phenotypic analysis. RT-PCR analysis confirmed the expression of downstream genes following integration of the deletion construct (see Results).

Construction of luciferase reporter strains.
To construct a luciferase transcriptional reporter fusion to the putative comY operon, a fragment containing the comY promoter region was ligated into the vector pFW5-luc (Podbielski et al., 1999) to generate the plasmid pComYluc. This fragment consisted of 1 kb of sequence upstream of the ComYA start codon as well as a small portion (~50 bp) of coding sequence. The resulting construct was confirmed by restriction analysis and PCR, and integrated into the chromosome of S. mutans via single-crossover homologous recombination. The expected integration was confirmed in antibiotic-resistant clones by both PCR and the analysis of multiple clones for similar reporter activity.

Luciferase assays.
Luciferase assays were performed as previously described (Loimaranta et al., 1998). Reactions were measured in 250 µl total volume [200 µl cell culture plus 50 µl 1 mM D-luciferin (Sigma) solution suspended in 0·1 M citrate buffer, pH 6·0]. All samples were measured using a TD 20/20 luminometer (Turner Biosystems). For analysis of comY expression over the growth curve, S. mutans cells were grown overnight in TH broth containing 0·4 % BSA to stimulate natural competence (Shah & Caufield, 1993). The cells were then diluted 1 : 30 into fresh medium and allowed to grow in the absence of O2 to various OD600 values. These samples were then assayed for luciferase activity in the presence of O2. To compare maximum comY induction between wild-type strains and mutants, 1 : 30 diluted overnight cultures were grown in the absence of O2 in competence media to an OD600 of about 0·2. In addition, a portion of this culture was saved for the determination of transformation efficiency. For all luciferase assays, luminescence values were normalized based on sample optical densities. All luciferase values were determined from the mean of three independent samples taken for every time point.

Transformation assays.
Cells were grown as mentioned above to an OD600 of about 0·2. Half of each culture was used for luciferase measurements, and the other half was used for the transformation assay. The transformation assay was performed by adding transforming genomic DNA to a final concentration of 10 µg ml–1 for each reaction, and the cultures were subsequently allowed to grow for an additional 1·5 h. After the incubation period, cultures were briefly sonicated to disperse cell chains and plated on antibiotic-containing BHI agar plates as well as on non-selective BHI plates. Successful transformation was scored based on acquired tetracycline resistance following transformation, and the calculation of total viable cells was determined by counting the number of colonies growing on non-selective plates. Transformation efficiency was determined by calculating the ratio of transformants to total viable cells.

Analysis of comY expression in different mutation backgrounds.
Mutations in comC, ciaH and mutR had all been previously generated in other strains of S. mutans, and the respective mutations were transferred to the UA140 comY luciferase reporter strain (140Y-luc) by transformation of genomic DNA. The resulting strains were tested for luciferase activities and transformation efficiencies (as described above) under the various conditions specified in Results.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification and analysis of the putative comY locus in S. mutans
In an effort to find a suitable candidate for the analysis of late competence in S. mutans, we chose to search for orthologues of the S. gordonii comY genes (comYAYD), which were previously shown to be essential for natural transformation of S. gordonii (Lunsford & Roble, 1997). The sequence of the translated product of the S. gordonii comYA gene was used as query to search the completed Streptococcus mutans Genome Database (http://www.genome.ou.edu/smutans.html). Using the program TBLASTP, we found a match with 68 % identity and 81 % similarity which corresponded to an Orf encoded by bases 1 856 166–1 855 207. A large region (>10 kb) surrounding this sequence was then analysed for the presence of Orfs using DNA Strider software. Based on this analysis, we found at least seven Orfs whose arrangement was highly suggestive of a polycistronic operon (Fig. 1a). In addition, there were two additional downstream Orfs which were candidate members of this putative operon (Fig. 1a). A protein alignment of the first four (SMu1804–1801) Orfs exhibited strong homology to the published sequence of the S. gordonii comY operon gene products ComYA (68 % identity/81 % similarity), ComYB (60 %/78 %), ComYC (71 %/87 %) and ComYD (44 %/69 %) (Fig. 1a). A BLASTP search was conducted for each of the remaining Orfs. Orf 5 (SMu1800), Orf 6 (SMu1799) and Orf 7 (SMu1798) matched strongly to conserved hypothetical proteins from other species of Streptococcus. Interestingly, Orf 6 (SMu1799) was predicted to contain a ComGF-like domain. ComGF is the sixth gene product in the comG operon of B. subtilis (Fig. 1a) as well as Listeria monocytogenes. Orf 8 (SMu1797) exhibited a strong homology to numerous DNA methyltransferases from restriction/modification systems, which suggested a potential role in competence. Orf 9 (SMu1796) was predicted to be an acetate kinase, AckA, which did not have any known connection to natural competence.



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Fig. 1. Genetic analysis of the putative comY operon. (a) The comY operon of S. mutans labelled by orf number, Los Alamos Gene ID and sequence homology with known genes. Solid black spacers indicate predicted intergenic regions. (b) RT-PCR of the junctions between the various orfs in this region. Beginning with orf1 (comYA), each probed Orf junction is listed by its order within the putative operon (1–2, 2–4, 4–6, 5–7, 7–8 and 8–9). Each sample was run in three reactions with three different templates: genomic DNA (+), cDNA (C) and no reverse transcriptase control (–). (c) Real-time RT-PCR analysis of transcripts of orf1 (comYA), orf8 (comYH) and orf9 (comYI), and the junctions between orfs 7 and 8, and between orfs 8 and 9. The relative abundance of each indicated transcript was normalized to that of the first orf (comYA). The results presented are the average of two independent cDNA synthesis and real-time RT-PCR reactions.

 
Since this operon appeared to be quite different from the comY operon of S. gordonii, an obvious question was whether these nine orfs were indeed co-transcribed as a single operon during competence. To address this issue, we isolated RNA from cells grown in competence medium and performed RT-PCR on all of the predicted junctions between the Orfs, beginning upstream of orf1 and continuing downstream of orf9. As shown in Fig. 1(b), no amplification of the cDNA was observed in the junction before orf1 (SMu1804) and after orf9 (SMu1796). However, we were able to amplify cDNA from all of the junctions between each of the nine Orfs. This result suggested that orf1 was likely the first gene of the operon, and that all nine orfs were potentially co-transcribed (Fig. 1a).

Since there were small intergenic regions located after the first seven genes and between the last two genes, it was unclear whether the nine-gene transcript was the primary unit or if additional promoters were possible in the junctions between orf7 and orf8 or between orf8 and orf9 (Fig. 1a). Therefore, it was necessary to determine whether the expression of these genes was primarily due to the comY promoter or was independent of comY transcription. To address this concern, we performed real-time RT-PCR on the intergenic regions between orf7 and 8 and between orf8 and 9, as well as within the coding portions of orf8 and 9. The relative abundance of each of these amplified cDNAs was then compared to that of comYA. As shown in Fig. 1(c), the amplified cDNA from each intergenic region showed a very similar abundance to that obtained from the coding region of each orf including comYA. These results suggested that the last two genes were likely co-transcribed with the first seven genes without any significant contribution from additional promoters functioning in the intergenic regions during competence. Therefore, we referred to orf19 as comYAI, respectively.

Construction and phenotypic characterization of comY mutants
Due to the unexpected structure of the S. mutans comY operon, we were next interested in determining whether all nine genes of the operon would be required for natural competence. We constructed deletion mutations in various combinations of all nine genes via allelic replacement, as described in Methods and illustrated in Fig. 2. To minimize the possibility of polar effects created by inserting an antibiotic resistance cassette, the kanamycin resistance gene was modified by deletion of the transcription terminator. In previous publications, this modification was shown to permit efficient read-through to downstream genes (Qi et al., 1999b), which was also confirmed with RT-PCR in this study (data not shown). After transformation of the constructs into the wild-type strain UA159, multiple isolates of each single gene mutant were confirmed and tested for competence ability. As shown in Table 3, deletions in each of the first seven genes (comYAG) completely eliminated the natural transformability of S. mutans. A deletion in comYH, the putative DNA methyltransferase, reduced competence to about 40 % of the wild-type level, while a mutation within comYI showed no noticeable defect in natural competence ability.



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Fig. 2. Strategy used to delete the comY genes by double cross-over recombination. All mutants were created by the same allelic replacement strategy with a terminatorless kanamycin cassette. See Methods for details.

 

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Table 3. Effect of comY gene mutations on transformation efficiency

Transformants were scored as colonies with acquired tetracycline resistance to medium containing 15 µg tetracycline ml–1 following transformation with gDNA, while the total number of colony forming units (c.f.u.) was determined from the number of colonies counted after dilution and plating on a non-selective medium. Transformation efficiency was calculated as the ratio of transformants to total c.f.u. Transformation efficiency in UA159 was calculated to be 9·63x10–7. Percentage transformation efficiency was calculated as a ratio of the mutant transformation efficiency versus the weight. The wild-type sample was arbitrarily assigned the value 100 %. All experiments are expressed as the mean of three independent experiments.

 
As a further test to show that this phenomenon was not strain specific, the same mutations were transferred into another wild-type strain, UA140, and found to exhibit similar phenotypes (data not shown). In addition, similar transformation phenotypes were obtained when plasmid DNA was used instead of genomic DNA (data not shown). Furthermore, the effects of these comY mutations on other cellular functions, such as growth rate and biofilm formation in the presence and absence of sucrose, were tested, and no obvious defects were detected (data not shown).

Analysis of comY operon expression through a luciferase reporter system
To see whether expression of the comY operon correlated with the period of peak competence, we constructed comY–luciferase reporter gene fusions in two wild-type S. mutans strains, UA159 and UA140 (Fig. 3a). Luciferase activities of the reporter strains were measured throughout exponential phase, and luminescence values were normalized based on optical density. For both reporter strains, luciferase activity peaked early in exponential phase at an OD600 of approximately 0·2 (Fig. 3b). This result agreed well with the established transformation procedure for S. mutans, in which transforming DNA is typically added when the culture achieves an OD600 of approximately 0·2 (Perry & Kuramitsu, 1981; Shah & Caufield, 1993). However, there was a difference in the level of comYluc activity exhibited between these two wild-type strains, with UA140 displaying a much higher luciferase activity than UA159 (Fig. 3b).



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Fig. 3. Analysis of comY expression by a luciferase reporter gene fusion. (a) Construction of a comY luciferase reporter. The plasmid pComY-luc was generated by cloning a 1 kb upstream region of comYA into the promoterless luciferase vector pFW5-luc. This plasmid was integrated into the chromosome in a single copy and resulted in a duplication of the upstream promoter region. (b) comYluc gene expression during exponential phase. Both strains UA140 : : comYluc ({blacklozenge}) and UA159 : : comYluc ({blacksquare}) were grown in competence medium, and samples were taken at designated time points. Luciferase activity and cell density were measured and plotted as a function of time. Solid line, luciferase activity expressed in relative light units (RLU); dashed line, cell density expressed as OD600. All data were determined from the mean of three independent samples.

 
Apparent correlation of comYluc expression with competence
Given the apparent difference in maximal expression of the comY operon in UA140 compared with UA159, we were interested in determining if there was a correlation between the level of comY expression and the transformation efficiency of these strains. Both UA140 and UA159 reporter strains were grown in competence media to OD600 ~0·2 (peak comY induction) and simultaneously assayed for luciferase activity as well as transformation efficiency. As expected, UA140 had a fivefold higher comY expression than UA159, which correlated well with a substantial increase in competency (data not shown). UA140 had a calculated mean transformation efficiency of 2·17x10–5, while UA159 had an efficiency of 9·63x10–7. In UA159, this corresponded to a value approximately 4 % of that in UA140.

Analysis of comY expression in different mutation backgrounds
The successful construction of the comY–luciferase reporter gene fusions also allowed us to study genetic factors responsible for regulating comY expression. comC and ciaH are two regulatory genes known to affect competence (Lee & Morrison, 1999; Magnuson et al., 1994; Mascher et al., 2003; Qi et al., 2004). comC, the structural gene for Streptococcus quorum-sensing signalling peptides, has been shown to be required for the activation of transcription of a competence-specific sigma factor (comX), which subsequently activates the expression of late competence genes (Lee & Morrison, 1999; Magnuson et al., 1994), while ciaH, the histidine kinase gene of a two-component signal transduction system, was recently shown to affect the transcription of comC (Mascher et al., 2003). Although the luxS mutation has not been previously reported to affect competence, we have consistently encountered a considerable natural competence defect in this strain. Therefore, we decided to transfer the comC, ciaH and luxS mutations into the comYluc reporter strain and test their effect on comY expression. As a control, we also introduced a mutation into mutR in this same reporter strain. mutR is a specific transcriptional activator for the lantibiotic bacteriocin mutacin I in UA140 (Chen et al., 1999; Qi et al., 1999a) and was not expected to be involved in the regulation of competence. Luciferase activity and transformation efficiency assays were performed simultaneously, as described in previous experiments. As shown in Fig. 4(a), both comC and luxS mutations reduced comYluc gene expression approximately sevenfold, while the ciaH mutation reduced expression approximately twofold. In contrast, mutR had a negligible effect on comY expression. Concurrently, all three mutations reduced transformation efficiency in a manner similar to their effect on comY expression, with the comC and luxS mutations causing about a 35-fold reduction in competence and the ciaH mutation causing a fivefold reduction (Fig. 4b). As expected from the luciferase data, the mutR mutation did not exhibit any inhibitory effect on competence (Fig. 4b).



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Fig. 4. Effect of different genetic mutations on comY expression. UA140 : : comYluc derivative strains containing ciaH, comC, luxS and mutR mutations were grown in competence media to an OD600 of 0·2, and used for both luciferase activity (a) and transformation efficiency assays (b). Values are expressed as a percentage of the wild-type value. All data were determined from the mean of three independent samples.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this paper, we describe the identification and characterization of an S. mutans operon required for competence. This operon contains orthologues to the comY operon of S. gordonii, the cgl operon of S. pnemoniae, and the comG operon of B. subtilis. Through a combination of RT-PCR and real-time RT-PCR, we demonstrated that this operon contains nine genes that are primarily co-transcribed in one polycistronic message following competence induction. Further genetic analyses show that eight out of these nine genes are directly involved in competence, with the first seven genes being absolutely required for competence. The regulation of this operon was also partially characterized through the use of a luciferase reporter that was integrated into the chromosome in a single copy. In both S. mutans strains UA140 and UA159, induction of this operon correlated well with induction of natural competence. Differences in the expression of this operon, either as a result of strain variation or from mutation, seemed to have a similar effect on transformation efficiency as well. The ciaH and comC genes, which were both previously reported to regulate competence and biofilm formation (Cheng et al., 1997; Cvitkovitch, 2001; Havarstein et al., 1996; Li et al., 2001, 2002; Mascher et al., 2003; Qi et al., 2004; Yoshida & Kuramitsu, 2002) in several species of Streptococcus, also reduced the expression of the comY luciferase reporter. In addition, it was also found that the luxS mutation had an effect on competence similar to that of the comC mutation in both the luciferase and transformation assays. These findings suggest that the comYluc reporter strain may be used as a marker for late competence, and may also be useful to aid in screening for additional regulators of competence.

Within the past few years, a great deal of data has been generated regarding the process of natural competence induction and the gene responses that follow (Claverys & Martin, 2003). It is interesting to note that the first seven genes of the comY operon in S. mutans have a similar arrangement to that of B. subtilis; however, only the first four genes have been reported in S. pneumoniae and S. gordonii (Lunsford & Roble, 1997; Pestova & Morrison, 1998). This result was initially quite surprising, given that these two organisms share extensive sequence homology with S. mutans. However, closer examination of sequence data from the NCBI database shows that the downstream region of the cgl operon in S. pneumoniae contains additional Orfs homologous to the comYEI genes of S. mutans. Therefore, a likely explanation for the discrepancy is simply a failure to detect these Orfs. This is not surprising because, in addition to being quite small, some of these Orfs, such as the comYE orthologue, contain an atypical start codon: GTG in S. mutans as well as in S. pneumoniae. In both L. monocytogenes and Listeria innocua, this gene also contains an unusual start codon: TTG. At the time of this report, the complete genome sequence of S. gordonii was not available, but it is expected that this organism also has additional Orfs downstream of its comYD gene. Indeed, Lunsford & Roble (1997) suggested in their analysis of the comY operon of S. gordonii that there were potential Orfs downstream of the four comY genes which they characterized. Given the evolutionary distance between organisms such as B. subtilis and S. mutans, it seems likely that the seven-gene operon is a highly conserved feature of many naturally competent Gram-positive organisms. In addition, it would not be surprising if the unique nine-gene operon structure of S. mutans was, in fact, a common feature among the streptococci.

When comparing sequence data between S. pneumoniae, S. gordonii and S. mutans, it is apparent that the comY operon contains a high degree of sequence conservation. According to annotations in the NCBI database, all three of these organisms share a fairly large gap between the first and second Orfs (~100 bp), but, based on Northern blot analysis in S. gordonii, it seems that these genes should all be co-transcribed (Lunsford & Roble, 1997). In S. mutans, we also found the comYB orthologue to be co-transcribed with comYA. However, we suspect that, at least in S. mutans, comYB may actually begin translation much closer to the end of comYA than predicted. There is a potential alternative start site (TTG) just downstream of the stop codon of comYA. This site also contains a feasible ribosome-binding site (RBS) in the region just upstream of the TTG (AGAAGAAATTG), while the annotated comYB Orf has no obvious RBS in the vicinity of its predicted ATG start codon.

Based on data presented in studies of the comG operon in B. subtilis, there is reason to believe that this operon in S. mutans shares a similar function. However, it is peculiar that S. mutans should have two additional genes in this operon that are dispensable for natural competence. It seems plausible that if the eighth gene of this operon is truly a DNA methyltransferase, it could be useful during competence. Presumably, such a gene could be involved with the restriction/modification system, which is likely to be employed during DNA uptake and recombination. The function of comYI (an orthologue of ackA) during competence seems a bit more enigmatic. Traditionally, the AckA enzyme functions in the conversion of acetyl-CoA to acetate. It may be that, during competence, there is a metabolic shift towards acetate production from pyruvate. In this case, it would make sense for this gene to be upregulated during competence induction. Alternatively, DNA could be metabolized for energy during competence, which may yield an alternative explanation for upregulating this gene. The ackA gene of B. subtilis has been previously shown to be transcriptionally regulated based on carbon-source utilization, and has also been shown to be important in ribose catabolism in Lactobacillus sakei (Grundy et al., 1993; Stentz & Zagorec, 1999). It is also interesting to note that, in the B. subtilis genome, the ackA gene is located adjacent to the DNA methytransferase ytxK. When the protein sequence of ytxK is used as a query to search the S. mutans genome database, the only significant match is to the comYH gene product from the S. mutans comY operon (35 % identical, 59 % similar). The fact that these two genes (comYHcomYI and ytxKackA) are located next to each other in both S. mutans and B. subtilis suggests that they may function in related pathways.

The data presented in this paper may provide a potential mechanism for the observed differences in the level of competence among different strains of S. mutans. For example, in working with UA159 and UA140, we have consistently observed a difference in transformability between the two strains (unpublished observations). Data presented in this study (Fig. 3) suggest that this difference may be partially due to variability in the level of transcription of the comY operon. This is also true for comY gene expression and competence in the ciaH, comC and luxS backgrounds (Fig. 4). In both cases, the level of comY gene expression correlated well with natural competence ability. Therefore, it would be feasible to use this operon as an indicator for predicting and/or comparing competency between various wild-type or mutant strains of S. mutans.


   ACKNOWLEDGEMENTS
 
We would like to thank Dr A. Podbielski for plasmid pFW5-luc and Dr D. Cvitkovitch for helpful discussions. This work was supported in part by NIH grant R01 DE 014757 to F. Qi, an NIH MPTG Training Grant T32-AI07323 and NIDCR T32 DE007296 Training grant to J. Merritt, and a BioStar/C3 Scientific Corporation grant and a Washington Dental Service grant to W. Shi. We greatly appreciate the public release of the S. mutans sequence data from the Streptococcus mutans Genome Sequencing Project funded by USPHS/NIH grant from the Dental Institute and the help of B. A. Roe, R. Y. Tian, H. G. Jia, Y. D. Qian, S. P. Lin, S. Li, S. Kenton, H. Lai, J. D. White, R. E. McLaughlin, M. McShan, D. Ajdic and J. Ferretti from the University of Oklahoma. We also would like to acknowledge the Los Alamos National Laboratories Oralgen website for useful annotation of the S. mutans genome.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 12 August 2004; revised 21 September 2004; accepted 22 September 2004.



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