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
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ABSTRACT |
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INTRODUCTION |
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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.
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METHODS |
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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 23 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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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 ml1 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.
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RESULTS |
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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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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 comYluciferase 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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Analysis of comY expression in different mutation backgrounds
The successful construction of the comYluciferase 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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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Received 12 August 2004;
revised 21 September 2004;
accepted 22 September 2004.
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