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1 UCLA School of Dentistry, Department of Oral Biology, Los Angeles, CA 90025, USA
2 China Medical University, Department of Microbiology and Parasitology, Shenyang, China
3 UCLA Molecular Biology Institute, Los Angeles, CA 90025, USA
Correspondence
Justin Merritt
justin-merritt{at}ouhsc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this work.
Present address: University of Oklahoma Health Sciences Center BRC364, 975 NE 10th St, Oklahoma City, OK 73104-5419, USA.
Present address: University of Oklahoma Health Sciences Center, College of Dentistry, Oklahoma City, OK 73104, USA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Therefore, these species commonly exist in an environment that can be hundreds to thousands of times denser than a typical laboratory broth culture. The cariogenic species Streptococcus mutans is a regular inhabitant of dental plaque, and thus routinely grows within an exceptionally high-cell-density environment. Interestingly, we have previously observed that the expression of at least two bacteriocins (mutacins) of S. mutans is largely dependent upon a cell density similar to that attained in the dental plaque or within a cell colony. While both bacteriocins normally exhibit little or no expression in broth cultures, we have demonstrated that the centrifugation and incubation of pelleted exponential-phase cultures can strongly induce the expression of both bacteriocins (Kreth et al., 2005; Merritt et al., 2005b). Thus we hypothesized that bacteriocin expression may be controlled by density-dependent regulators that are responsive to extremely high cell density. Using this approach, we identified a previously uncharacterized two-gene operon consisting of a putative transcription regulator and membrane protein. A transcription fusion to this operon confirmed its inducibility during high-density incubation, and phenotypic analysis suggested a role in the control of various density-dependent phenomena such as bacteriocin production, genetic competence and biofilm formation. These results suggest that this operon could be a mediator of the response of the cell to a high-cell-density environment. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. All S. mutans strains were grown in either brain heart infusion (BHI) medium or Todd–Hewitt (TH) medium (Difco). For the selection of antibiotic-resistant colonies, TH plates were supplemented with 15 µg erythromycin ml–1 (Sigma), 800 µg spectinomycin ml–1 (Sigma) or 800 µg kanamycin ml–1 (Sigma). All S. mutans strains were grown anaerobically (80 % N2, 10 % CO2, 10 % H2) at 37 °C. Escherichia coli cells were grown in LB medium (Fisher) with aeration at 37 °C. E. coli strains carrying plasmids were grown in LB medium containing 250 µg erythromycin ml–1 or 150 µg spectinomycin ml–1.
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). Primer sequences were designed using sequence data obtained from the Los Alamos National Laboratory Oral Pathogens Sequence Database. The PCR fragments were then cloned into the vector pCR2.1 (Invitrogen), digested with XbaI/SphI, and ligated to compatible sites on the suicide vector pJY4164 (Achen et al., 1986) to generate pLZ1689-i and pLZ1690-i. The resulting constructs were confirmed by restriction analysis and PCR before integration onto the chromosome of UA140 via single-crossover homologous recombination. Antibiotic-resistant isolates were tested by PCR to confirm the genotypes, and representative clones of both hdrR and hdrM mutants were frozen and renamed LZ89ins and LZ90, respectively.
Construction of the hdrR deletion mutant.
Two fragments corresponding to approximately 1 kb upstream and downstream of hdrR were generated by PCR using PfuUltra polymerase (Stratagene) and the primer pairs 1689upF/1689upR and 1689downF/1689downR (Table 2). The resulting fragments were cloned into the vector pCR2.1, cut with HindIII/BamHI (upstream fragment) and KpnI/SacI (downstream fragment), and ligated to compatible sites in pJY4164. The resulting plasmid was digested with BamHI, blunted with Klenow (NEB), and ligated with a spectinomycin resistance cassette (aad9) removed from pFW5 (Podbielski et al., 1996) using SmaI/HincII to generate the plasmid pLZ1689-d. After confirmation of the resulting plasmid by restriction analysis and PCR, the entire insert was PCR-amplified using PfuUltra and transformed into S. mutans (Li et al., 2001). Antibiotic-resistant isolates were tested with PCR using several combinations of primer sets to confirm the genotype. A confirmed clone was renamed LZ89del and frozen for future use.
Construction of the hdrR markerless in-frame deletion mutant.
To generate the markerless in-frame deletion construct, a cloning strategy similar to that described above was used initially to clone the same 1 kb hdrR upstream and downstream fragments into pJY4164. This construct (pJY4164-upstream-downstream) was digested with HindIII/SacI to remove the entire insert, blunted with Klenow, and ligated to an SmaI-digested pIFD-Sm vector (Merritt et al., 2007). The resulting plasmid (pLZ1689-ifd) was confirmed by PCR and restriction analysis and transformed into the in-frame deletion recipient strain IFD140. The counter-selection procedure was performed as described previously (Merritt et al., 2007). Ten antibiotic-sensitive isolates were randomly selected and screened by PCR to confirm the presence of the expected markerless deletion. A confirmed clone was renamed LZ89ifd and frozen for later use.
Construction of the hdrR and hdrM double-deletion mutant.
The hdrR upstream fragment was amplified with PfuUltra and cloned into pCR2.1. This insert was removed from pCR2.1 with HindIII/BamHI, while the spectinomycin cassette was cut from pFW5 using BamHI/SmaI. These two fragments were ligated to pJY4164 digested with HindIII/SmaI. The resulting construct (pJY4164-upstream-aad9) was checked by restriction analysis and PCR for the proper configuration. Next, the hdrM downstream fragment was amplified by PCR using PfuUltra and the primer pair 1690downF/1690downR (Table 2) and cloned into the pGEM-T Easy vector (Promega. The downstream fragment was then released from pGEM-T Easy with EcoRI, blunted with Klenow, and ligated to pJY4164-upstream-aad9 cut with SmaI. Clones were analysed for the proper configuration by PCR and restriction analysis, and a confirmed isolate was renamed pLZ8990-d. This vector was subsequently linearized and transformed into UA140. Antibiotic-resistant clones were screened with PCR to confirm the expected mutation. A confirmed mutant was renamed LZ8990 and saved for future analysis.
Plate assay for mutacin production.
To assay for mutacin I production, the wild-type UA140 and various mutant strains were first grown overnight in liquid cultures under standard anaerobic conditions. A 5 µl volume of the overnight culture was spotted onto TH plates and incubated anaerobically at 37 °C overnight. The following day, the plates were overlaid with a soft agar suspension of the indicator strain (Streptococcus sobrinus strain OMZ176) and incubated anaerobically for an additional 16 h. Zones of inhibition were indicative of mutacin I production.
Construction of luciferase operon fusions.
To construct a luciferase transcriptional reporter fusion to the hdrRM operon, a fragment containing the promoter region was amplified using the primer pair 1689upF/1689upR (Table 2). This fragment consisted of 
1 kb of sequence upstream of the hdrR start codon as well as a small portion (50 bp) of coding sequence. The resulting fragment was cloned into pCR2.1, and later digested from pCR2.1 using SpeI/XhoI and ligated to the NheI/XhoI sites on the luciferase reporter plasmid pFW5-luc (Podbielski et al., 1999) to create pLZ1689-luc. This plasmid was transformed and integrated onto the chromosome of wild-type UA140 via single-crossover homologous recombination to generate the luciferase reporter strain LZ89-luc. The reporter plasmid was also transformed into the hdrR and hdrM mutant strains to create the mutant reporter strains KO1689-luc and KO1690-luc, respectively. Antibiotic-resistant isolates were screened with PCR for the expected insertions, and multiple clones were assayed for similar reporter activities.
Luciferase assays.
Luciferase assays were performed similarly to ones described previously (Merritt et al., 2005a). A 50 µl volume of 1 mM D-luciferin (Sigma) suspended in 0.1 M pH 6 citrate buffer was added to 200 µl cell culture. All samples were measured using a TD 20/20 luminometer (Turner Biosystems). For all luciferase assays, luminescence values were normalized relative to sample optical densities, and luciferase values were determined from the mean of three independent samples taken for each time point. To determine the influence of high cell density upon hdrRM expression, overnight cultures of LZ89-luc were diluted 1 : 30 into fresh TH broth and incubated anaerobically as batch cultures for 2.5 h (OD600 0.08–0.09). The cultures were then divided into 1 ml aliquots in microcentrifuge tubes and further incubated anaerobically as planktonic cells or as cell pellets. Pelleted cells were centrifuged at 16 000 g for 1 min before incubation. After incubation, both planktonic and pelleted cells were resuspended by vortexing before the luciferase activities and optical densities were measured. Luciferase data were expressed as a percentage of the activity measured at the start of the assay. The initial luciferase activity was arbitrarily defined as 100 %. To determine the effect of high cell density upon the hdrR and hdrM mutants, the reporter strains LZ89-luc, KO1689-luc and KO1690-luc were all assayed as described above with the following modifications. After the initial incubation for 2.5 h (OD600 0.08–0.09), cells from each reporter strain were divided into planktonic cells and cell pellets, and incubated for an additional 2.5 h. After the incubation period, luciferase and optical density measurements were made for each sample. The luciferase activity data were expressed as a percentage of the activity relative to the wild-type planktonic sample. The activity of the wild-type planktonic sample was arbitrarily defined as 100 %.
Transformation assays.
Determination of natural transformation efficiency was performed using a method similar to that described previously (Li et al., 2001; Merritt et al., 2005c). UA140 and the various hdrR and hdrM mutants were grown anaerobically to OD600 0.2–0.3 in TH broth supplemented with 0.4 %, w/v, BSA. S. mutans genomic DNA containing a kanamycin marker was then added to each culture at a final concentration of 1.2 µg ml–1. The cultures were then incubated for an additional 3 h before plating. Transformation efficiency was determined by calculating the ratio of c.f.u. on selective plates to that on non-selective plates.
Biofilm imaging.
Fluorescent images obtained from confocal laser scanning microscopy (CLSM) were produced as described previously (Kreth et al., 2004). Biofilms were grown as static cultures in modified Lab-Tek II chamber slides (Nalge Nunc International) containing a total volume of 700 µl of chemically defined medium (CDM) supplemented with 1 %, w/v, sucrose. In some cases, 1 %, w/v, glucose was substituted for sucrose to measure sucrose-independent surface attachment and growth. Biofilm inoculation was performed by first growing overnight cultures of wild-type UA140 and the hdrR and hdrM mutants in CDM. The following day, stationary-phase cultures were gently sonicated to disperse the cells using a water sonicator (Misonix), and optical density adjustments were made to equalize the overnight cultures before diluting each sample 1 : 100 in the chamber wells. The chambers were then incubated anaerobically for 24 h at 37 °C to allow biofilm development to reach maturity in each sample. After the incubation period, the spent medium was removed and replaced with 500 µl fresh CDM plus 1 µl CellTracker Orange CMTMR (Molecular Probes), followed by further incubation at 37 °C for 1 h. After incubation, the wells were washed three times with PBS before imaging. CLSM was performed using an LSM 5 PASCAL confocal laser scanning microscope with LSM 5 PASCAL software (Carl Zeiss).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Of these, the most strongly affected genes were found to be the stress regulator hrcA (9x induction) and a conserved hypothetical ORF (11x induction) annotated as SMU.1854 (NCBI database)/SMu1689 (Los Alamos database) (Table 3). Sequence analysis of this ORF yielded strong homologies to the DNA-binding domains of putative LytTR family regulators from various Gram-positive species. This ORF also appeared to be the first gene of a two-gene operon, since its stop codon overlaps with a putative start codon of a downstream ORF. The downstream ORF also appeared to be the end of the operon, since the next three putative ORFs are transcribed from the opposite DNA strand. Bioinformatic analysis using the Los Alamos National Laboratoy Oral Pathogen Sequence Database predicted that the second gene of the putative operon encodes a membrane protein with two transmembrane segments (SMU.1855, NCBI/SMu1690, Los Alamos). The operon structure was also reminiscent of the membrane sensor/response regulator arrangement typical of two-component system operons. However, there was no identifiable kinase domain homology within the predicted amino acid sequence of the putative membrane protein.
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1 kb upstream fragment of SMU.1854/SMu1689 was cloned into the luciferase reporter vector pFW5-luc to create the transcription reporter strain LZ89-luc. To test the response of LZ89-luc to high cell density, the reporter was first grown to a very low optical density (OD600<0.1) and divided into dispersed and pelleted samples. Optical density and luciferase measurements were made every 30 min until the dispersed samples were well into stationary phase about 5 h later. As shown in Fig. 1(a), the dispersed samples exhibited increasing reporter activity at the beginning of the assay and plateaued about 90 min later (OD6000.2). The pelleted cells also exhibited a similar increase in luciferase activity in the early time points of the assay (Fig. 1a). However, these samples continued to increase in activity until reaching a much higher maximal activity 210 min (t test, P=0.0006) after the start of the assay (Fig. 1a). Based on the response of this operon to high cell density, we subsequently renamed SMU.1854/SMu1689 as ‘high density responsive regulator’ (hdrR) and SMU.1855/SMu1690 as ‘high density responsive membrane protein’ (hdrM).
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). As shown in Fig. 1(b), the mutant reporter strains showed virtually no response to high cell density, whereas the wild-type reporter exhibited the expected increase in activity after centrifugation. In addition, the weak activity of the mutant reporter strains suggested that hdrR and hdrM are both required for induction of the hdrRM operon under both low- and high-cell-density conditions.
The hdrRM operon regulates the production of mutacin I
Previously, we reported that the expression of the bacteriocin mutacin I could be induced by growth at a high cell density (Merritt et al., 2005b). Therefore, we were interested to determine whether the hdrRM operon was required for the production of mutacin I. Initially, we tested the insertion-duplication mutants of both hdrR and hdrM, LZ89ins and LZ90, respectively. With these mutants, we consistently found that only LZ90 exhibited mutacin I deficiency (Fig. 2). This was surprising, given that LZ89ins was likely to have had a polar effect upon hdrM. To rule out any unexpected artefacts that may have occurred with the hdrR insertion, we also tested mutacin I production in both a markerless in-frame deletion mutant (LZ89ifd) and a polar allelic replacement of hdrR (LZ89del). As shown in Fig. 2, these hdrR mutant strains were also able to produce mutacin I. Thus, the two polar mutations and the non-polar mutation of hdrR all had no effect upon mutacin I. Finally, we deleted the entire operon and found that this strain (LZ8990) was similarly proficient at mutacin I production (Fig. 2). Consequently, it appeared that only the hdrM mutation could abolish mutacin I production and this phenotype could be rescued by mutation of hdrR.
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), we were next interested to determine whether the hdrRM operon had any influence on competence development. Based on the results with the previous mutacin I assay, we decided to measure the natural competence ability of the hdrR and hdrM mutants, as well as the double-deletion strain. Surprisingly, we found that LZ90 exhibited a transformation frequency that was 80-fold higher than those of the wild-type UA140 and other mutant strains (data not shown). As for the previous mutacin I results, only a mutation in hdrM could produce the phenotype, whereas deletion of both genes in the operon restored competence to normal levels. This suggested that the hdrRM operon acts as a negative regulator of natural competence.
The hdrM mutant has a reduced growth rate
While performing the transformation assays, we noticed that LZ90 consistently took longer to reach the optimal optical density for natural competence induction. Based on this observation, we decided to compare the growth curves of LZ89ifd, LZ90 and LZ8990 with that of the wild-type UA140. As shown in Fig. 3, UA140, LZ89ifd, and LZ8990 all had growth profiles that were largely indistinguishable, whereas LZ90 had a noticeably longer doubling time and a lower terminal optical density than all the other strains. Once again, the double-deletion strain seemed to compensate for the hdrM phenotype.
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, the DIC images demonstrated that both LZ89ifd and LZ8990 had an overall biofilm structure that was very similar to that of UA140. The biofilms were very smooth, confluent layers that covered the entire surface with a few large aggregates present in each. In contrast, LZ90 appeared sparsely populated on the surface and failed to achieve noticeable confluence, and a sizeable portion of the biofilm appeared to be localized within extremely large, densely packed aggregates (Fig. 4e). At high magnification, the differences in the LZ90 biofilm became even more apparent. The horizontal section of the LZ90 fluorescent image was taken very near to the bottom of the biofilm, because much of the surface contained only a very thin layer of biofilm that could not be seen in the higher layers of the image (Fig. 4f). However, the vertical sections of the LZ90 biofilm were in stark contrast. As shown in Fig. 4(f), the aggregates formed in the LZ90 biofilm were extremely tall: about 55 versus 30 µm for each of the other biofilms. Thus, the hdrM mutant formed a very weak biofilm over much of the surface, while in some localized areas there were clusters of biofilm that were almost double the height attained by the other strains. Interestingly, this phenotype was reproducible for both sucrose-dependent and sucrose-independent biofilms (data not shown). In addition, the fluorescent images of LZ89ifd suggested that it formed a more densely packed homogeneous biofilm than did UA140, whereas LZ8990 had a biofilm architecture somewhere between those of UA140 and LZ89ifd (Fig. 4b, d, h). Since both LZ90ifd and LZ8990 formed biofilms of approximately the same height and overall external structure, these mutations did not seem to have much effect upon basic biofilm-forming ability, but they did seem to produce some minor differences in biofilm architecture.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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An interesting feature of this operon is the consistency of phenotypes associated with the hdrM mutation and not the hdrR or hdrRM mutations. The CiaRH two-component system of S. mutans also exhibits similar behaviour. Inactivation of the CiaH membrane sensor has been shown to create numerous phenotypes related to acid tolerance, growth rate, natural competence, bacteriocin production and biofilm formation. However, inactivation of either the CiaR response regulator or the whole CiaRH operon causes no observable phenotype (Ahn et al., 2006; Qi et al., 2004). Our genetic data also suggest a couple of features of the hdrRM operon. First, both hdrR and hdrM are likely to function in the same pathway(s), since the polar hdrR mutations and the double mutation of hdrRM all exhibited wild-type behaviour for each of the phenotypes observed in the hdrM mutant. For example, bacteriocin production, natural competence, growth rate and biofilm formation were all affected in the hdrM mutant, while each of the mutations that affected both hdrR and hdrM behaved similarly to the wild-type. Secondly, in addition to the hdrRM whole-operon mutants, wild-type behaviour was also observed with the hdrR in-frame deletion mutant. Therefore, it can be hypothesized that the activity of HdrR is regulated, possibly by HdrM or via some intermediate. In this scenario, the phenotypes caused by the hdrM mutation could be attributed to the production of an unregulated HdrR. However, it is currently unclear whether this regulation is positive or negative. Since no identifiable kinase domain homology was observable in the HdrM sequence, the mechanism of signal transduction may be distinct from the typical phosphorelay scheme used in two-component systems.
Another interesting feature of this operon is the connection with natural competence. While there are numerous examples of mutations that lower the natural competence ability of S. mutans (Abranches et al., 2006; Ahn et al., 2005; Ahn & Burne, 2006; Hussain et al., 2006; Merritt et al., 2005c; Qi et al., 2004; Rolerson et al., 2006; Senadheera et al., 2005, 2007; Wang & Kuramitsu, 2006; Wen et al., 2005), to our knowledge, there is only one other report of a mutation that increases natural competence (Tao et al., 1993). This mutation was generated by chemical mutagenesis and its identity has, so far, not been published. Therefore, the hdrM competence phenotype is very unusual within the S. mutans literature. Since the genetic pathway leading to natural competence has been well characterized in S. mutans and other streptococci (Martin et al., 2006), it will be very interesting to determine whether the hdrM mutation affected the expression of the known competence regulators or had an alternative mechanism to affect the expression of the DNA-uptake complex. Also, as shown in Fig. 3, the growth defect of the hdrM mutant was first noticeable as the cells achieved OD600 0.2. This is the period of the growth curve at which S. mutans initiates its natural competence programme (Perry et al., 1983; Shah & Caufield, 1993). Furthermore, as shown in Fig. 1(a), dispersed cells of the hdrRM luciferase reporter achieved maximum expression about 90 min after the start of the assay. At this time point, the OD600 was also 0.2. Therefore, it is conceivable that there is a regulatory connection between the hdrM phenotypes associated with growth and natural competence. This result would not be surprising, given that competence escape in Bacillus subtilis is linked to cell growth (Hahn et al., 1995; Haijema et al., 2001). In addition, it has been demonstrated that natural competence in S. mutans is greatly enhanced within the biofilm (Li et al., 2001). So the biofilm phenotype may also be connected to the growth and natural-competence phenotypes. Experiments are currently in progress to elucidate the pathway(s) leading to these phenotypes. More extensive high-cell-density microarray studies are also currently in progress.
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ACKNOWLEDGEMENTS |
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Edited by: R. J. Lamont
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 23 February 2007;
revised 11 April 2007;
accepted 12 April 2007.
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