Department of Oral Biology, University of Florida, Gainesville, FL 32610, USA1
Center for Oral Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA2
Author for correspondence: Robert A. Burne. Tel: +1 352 392 4370. Fax: +1 352 392 7357. e-mail: rburne{at}dental.ufl.edu
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ABSTRACT |
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Keywords: cis-elements, pHregulation, urease gene expression, oral streptococci
Abbreviations: cat and CAT, chloramphenicol acetyltransferase; PureI, promoter of the ure operon
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INTRODUCTION |
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Urea is probably the most abundant nitrogen source in the oral cavity. Urea is secreted in saliva and crevicular fluids at concentrations of 310 mM (Golub et al., 1971 ; Kopstein & Wrong, 1977
) and is rapidly hydrolysed by microbial ureases to yield ammonia and CO2. Ammonia may inhibit the initiation and progression of dental decay by neutralizing acids generated from bacterial glycolysis (Clancy et al., 2000
). In addition, the less acidic conditions resulting from ureolysis provide a more favourable growth environment for those bacterial species that do not compete well at low pH with cariogenic organisms. Thus, ureolysis may have a major impact on oral biofilm ecology (Li et al., 2000
; Clancy et al., 2000
). Consequently, recent studies have begun to explore the molecular architecture and regulation of the ureases of oral bacteria. For example, the urease gene cluster of Streptococcus salivarius, one of the most abundant and highly ureolytic oral micro-organisms, is regulated differently than other known bacterial urease gene clusters. In particular, the S. salivarius urease genes are induced by acidic pHs and expression of these genes can be markedly enhanced by growing the organism in acidic conditions and in an excess amount of carbohydrate (Chen & Burne, 1996
; Weaver et al., 2000
). It is known that oral bacteria commonly deal with large and rapid fluctuations in their environment, especially in pH and in the amount and type of carbohydrate available to them (Burne, 1998
). It is reasonable to predict that oral organisms that could not appropriately respond to these environmental changes would be at a competitive disadvantage and would not persist in the biofilms colonizing the teeth and soft tissues. Thus, enhanced expression of S. salivarius urease in acidic conditions and with excess amounts of carbohydrate is consistent with the role of urease in protection of the organism from acid killing and the ability of the organism to use urea as a primary source of nitrogen (Chen et al., 2000
) both of these factors could provide a strong advantage for this organism to persist in its natural environment.
The S. salivarius urease gene cluster (ure) is arranged as an operon, beginning with ureI, followed by the genes that encode the structural subunits, ureABC, and the genes that encode the proteins required for assembly of the active enzyme, ureEFGD (Chen et al., 1998b ). A
70-like promoter that is responsible for differential expression of these genes in response to pH changes and carbohydrate concentrations has been localized immediately 5' to ureI (PureI). Using promoter fusions of PureI and a deletion derivative lacking 100 bp 5' to the -35 element to a chloramphenicol acetyltransferase (cat) gene, it was found that the expression of PureI is negatively regulated (Chen et al., 1998b
), with nearly complete repression of expression at neutral pH. However, no ORFs encoding proteins with characteristics of transcriptional regulators could be identified in the regions flanking the ure gene cluster, raising the possibility that the expression of the ure operon may be regulated by factor(s) that are not specific to urease. The purpose of this study was to identify the cis-acting elements that are responsible for the differential expression of urease and to explore the possibility that the S. salivarius urease may be part of a global circuit that controls gene expression in streptococci in response to pH.
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METHODS |
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Construction of integration vectors for S. salivarius 57.I and S. gordonii DL1.
The integration vector pMC195 was constructed as follows (Fig. 1). The S. salivarius 57.I lacZ gene was first amplified by PCR using Taq DNA polymerase with primer pairs homologous to lacZ of Streptococcus thermophilus A054 (accession no. M63636), as detailed previously (Chen et al., 2002
). The PCR product was initially cloned onto pCRII and the identity of the product was confirmed by sequence analysis. The internal 2·45 kbp PstIXbaI fragment of the PCR product was subsequently purified, blunt-ended by T4 polymerase and cloned into pGEM-3Zf(+) that had been digested with HindIII and EcoRI followed by Klenow treatment to remove the multiple-cloning sequences (MCSs) to generate pMC194. A DNA fragment containing a kanamycin-resistance gene flanked by T4 transcription/translation terminator signals (
kan) (Perez-Casal et al., 1991
) and MCSs from pGEM-7Zf(+) was subsequently cloned onto a unique ClaI site within the PCR product. The resulting plasmid, pMC195, allowed the integration of foreign genes into the S. salivarius 57.I chromosome at the lacZ locus with the acquisition of a kanamycin-resistant phenotype.
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Construction of PureIcat recombinant fusion strains.
The wild-type PureI and a set of deletions of the 5' flanking regions of PureI were generated by PCR using the primers listed in Table 2. Briefly, the 5' portions of PureI
21cat, PureI
40cat, PureI
60cat and PureI
80cat were amplified from the S. salivarius 57.I chromosome by PCR using primer pairs pMC43S-4+PureI-70HincII, pMC43S-4+PureI-100HincII, pMC43S-4+PureI-120HincII and pMC43S-4+PureI-140HincII, respectively. To be noted, primer pMC43S-4 contains a SpeI recognition site, and a HincII recognition site is present in primers PureI-70HincII, PureI-100HincII, PureI-120HincII and PureI-140HincII (Table 2
). Subsequently, the PCR products were digested with SpeI and HincII and then cloned into XbaI- and HincII-digested pGEM-3Zf(+) to generate plasmids pMC162, pMC159, pMC160 and pMC161, respectively. The 3' portion of all of the deletion derivatives was generated from HincII and SphI double-digested pMC63 (Chen et al., 1998b
), which resulted in a fragment containing the 60 bp region located immediately 5' to the ureI start codon (ATG) that had been fused with a promoterless cat (Promega). This HincIISphI fragment was subsequently cloned into pMC162, pMC159, pMC160 and pMC161 at unique HincII and SphI sites. The sequence of each construct was confirmed by sequence analysis. The correct promotercat fusions were subsequently cloned into pMC195 and pMJB8, and the resulting chimeric plasmids were introduced into S. salivarius 57.I by electroporation (Chen et al., 1998b
) or into S. gordonii DL1 by transformation (LeBlanc & Hassell, 1976
), respectively. The correct integration of each construct via allelic exchange into S. salivarius and into S. gordonii was confirmed by Southern-blot analysis using a lacZ- and a gtfG-specific probe, respectively.
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Site-directed mutagenesis.
Site-directed mutagenesis of the AT-rich regions 5' to PureI was carried out by using the Chameleon double-stranded, site-directed mutagenesis kit (strain M1; Stratagene) or by PCR (strains M2 and M3). Mutagenic oligonucleotides for constructing strains M1 (5'-GCAAAATTTCTGTCTAGACGTTGACATGTG-3') and M2 (5'-CACATGTCAACGAATTTTCAGATCTCTAGAGCAACATTTAC-3') were designed to create an XbaI site (shown in bold) that could be used to screen for clones containing the correct mutations. Oligomer 5'-CACATGTCAACGAATTTTCAGAAGATCTGCAACATTTAC-3' was used to generate strain M3. The sequences of all of the constructs with the desired mutations were confirmed and promotercat constructs carrying the correct mutations were introduced into S. salivarius 57.I or S. gordonii DL1, as described above.
CAT assay.
Recombinant S. salivarius and S. gordonii strains were grown in BHI containing 50 mM KPB (pH 7·5) or in BHI/HCl (pH 5·5) to an OD600 value of 0·5. Cells were harvested and washed once with an equal volume of 10 mM Tris (pH 7·8), and then resuspended in 1/40 of the original culture volume in the same buffer. Concentrated cell suspensions were subjected to mechanical disruption in the presence of an equal volume of glass beads (0·1 mm diameter) by homogenization in a Bead Beater (Biospec Products) for a total of 40 s at 4 °C. The concentration of each protein lysate was measured by using the Bio-Rad Protein Assay based on the method of Bradford (1976)
. BSA served as the standard. CAT activities were calculated based on the rates of chloramphenicol acetylation, as detailed by Shaw (1979)
.
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RESULTS |
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All of the promoter fusions were cloned into pMC195 (Fig. 1) with the
kan element 5' to the PureIcat fusions and integrated into the S. salivarius lacZ locus. In addition, the PureIcat fusions were positioned such that transcription from PureI was in either orientation with respect to the lacZ gene. Although repression of expression from PureI is not as complete in batch culture at neutral pH values as it is in a chemostat, the use of chemostats was not practical for obtaining data from the large number of constructs examined in this study. Thus, we elected to measure cat expression in each recombinant strain grown batch-wise in media buffered at pH 7·5 or adjusted to pH 5·5. Importantly, the use of buffered media and batch cultivation in this study allowed for observation of significant differences in the expression from the PureI promoter at the two pH values tested. The results indicated that the orientation of the promoter fusions with respect to the lacZ gene did not significantly affect the levels of CAT activity; hence, only the results from fusion strains with PureI transcribed in the same orientation as the interrupted lacZ gene are presented here (Table 3
). In agreement with previous observations, wild-type PureI was sensitive to environmental pH, with higher levels of expression observed in cells grown at pH 5·5, as compared to cells grown at pH 7·5. The recombinant strain containing a 21 bp deletion immediately 5' to the -35 element (strain PureI
21cat) expressed more than 10-fold and fourfold higher levels of CAT than the wild-type promoter at pH 7·5 and 5·5, respectively, suggesting the presence of target site(s) within this region for repressor(s) binding. When an additional 19 bp (strain PureI
40cat) or 39 bp (strain PureI
60cat) immediately 5' to the 21 bp region were deleted, the levels of CAT expression in the resulting recombinant strains were reduced compared to those observed in strain PureI
21cat at both pH values, suggesting the presence of sequences required for optimal expression within this 39 bp region. However, when 80 bp were deleted immediately 5' to the -35 element (PureI
80cat), the CAT activities of the resulting recombinant strain were greater than the activities of the wild-type PureIcat strain and strains PureI
40cat and PureI
60cat, but they were less than the activities of strain PureI
21cat at both pH values, raising the possibility that the region located between positions -96 and -115 could also contribute to repression of the urease promoter. Even though marked derepression of expression occurred in all of the deletion derivatives at neutral pH, some sensitivity to pH was consistently retained in the deletion derivatives, albeit at variable levels and at levels below that seen for the intact PureI promoter region.
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Functional analysis of the cis-elements of PureI in an urease-negative host
To determine whether a global circuit might be involved in the regulation of PureI expression in response to environmental signals, and principally to pH, the expression of the various PureI derivatives was evaluated in a non-ureolytic Streptococcus sp., S. gordonii DL1. The urease-negative phenotype is an established phenotype for S. gordonii (Whiley & Beighton, 1998 ). To establish the PureIcat fusions in S. gordonii, we constructed a vector (pMJB8; Fig. 1
) that allowed the integration of foreign DNA into the gtfG locus by allelic exchange. All promotercat fusions were cloned into pMJB8 with the
kan element 5' to PureIcat and with the PureI fusions transcribed either in the same or the opposite orientation with respect to gtfG. The CAT activities of all of the recombinant PureIcat strains were measured in batch-grown cells at pH 7·5 and 5·5. The orientation of PureI fusions in relation to the gtfG gene did not influence the results significantly, so only data from constructs carrying fusions in the same orientation as gtfG are presented. Four primary findings arose from the use of S. gordonii as the urease-negative host. First, the pH sensitivity of expression of PureIcat was conserved in an organism lacking urease genes, with a fivefold increase in CAT activity in cells grown at pH 5·5 versus those grown at pH 7·5 (Table 3
). Second, the patterns of regulation in recombinant S. gordonii strains harbouring the various deletion derivatives of PureIcat essentially paralleled those observed in recombinant S. salivarius strains, although lower levels of activity were consistently observed in S. gordonii with each of the deletion constructs (Table 3
). Third, S. gordonii M1 expressed higher levels of CAT activities than S. gordonii carrying the wild-type PureIcat at pH 7·5 and 5·5, although the levels were not as high as those observed in S. gordonii PureI
21cat (Table 4
). Additionally, mutations in the AT-rich region further upstream of the -35 element (strains M2 and M3) did not significantly alter the expression of PureI at either pH value tested, when compared to S. gordonii PureIcat. These results indicated that although the primary target for the negative regulation of PureI was recognized by the regulatory machinery of S. gordonii, there appear to be differences in the binding specificity of the repressor-binding site between S. salivarius and S. gordonii. Finally, when the functions of the potential positive cis-element(s) located 5' to the primary repressor-binding site were also evaluated in the S. gordonii M1 background (Table 4
), it was found that all three deletion derivatives expressed levels of CAT activities comparable to the parent M1 strain at pH 7·5 and 5·5. Therefore, the sequences 5' to the primary repressor-binding site did not appear to enhance transcription from PureI as was observed in S. salivarius.
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DISCUSSION |
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A practical by-product of this study was the generation of two integration vectors that were designed specifically for use in the study of gene regulation in two of the most abundant commensal oral streptococci, S. salivarius and S. gordonii. The organization of the integration vectors permits simple cloning and stable integration of gene fusions or heterologous DNA into these oral streptococci, and the placement of strong transcription/translation terminator signals (the element of the
kan cassette) between the cloning site and the upstream gene of the host assures that transcriptional read-through will not affect results obtained in gene-fusion studies or adversely influence the expression of heterologous genes. The high degree of homology between the lacZ locus of S. salivarius and that of S. thermophilus (93% identity) may allow one to use pMC195 as an integration vector in S. thermophilus as well, although we have not yet determined whether this is possible. A recent genome-sequencing effort has been undertaken for S. gordonii (http://tigrblast.tigr.org/ufmg/), and the integration plasmid detailed in this study should prove highly valuable for future regulation and virulence studies involving this organism.
The results of deletion analyses provided consistent evidence that transcription from PureI is repressed at neutral pH through cis-acting elements located immediately 5' to the -35 element of the promoter. However, there is always some concern with deletion studies that the observed effects on regulation do not result directly from the removal of sequences, but rather from the juxtapositioning of non-native sequences closer to the promoter element(s). The use of site-directed mutagenesis obviated this concern, providing strong evidence that the two AT-rich direct-repeats within the 21 bp region 5' to the -35 element were target sites for repression of the ure operon. Although this 21 bp region is clearly the primary target site for repression, we cannot exclude the possibility that a secondary repressor-binding site may exist for the ure operon. Specifically, the potential presence of such an element became apparent in the PureI80cat deletion derivative, which showed a rebound in cat expression over the PureI
40cat and PureI
60cat constructs. Thus, at this stage, we propose that the primary control of urease expression occurs through repressor binding in the 21 bp region 5' to the -35 element, and possibly at nucleotides -96 to -115 as well. It is also noteworthy that there is no obvious sequence conservation between the 21 bp region and the -96 to -115 region.
Another consistent observation in this study was that we were never able to completely eliminate the pH sensitivity of PureI expression with any of the constructs used in this study. The simplest interpretation of this observation is that the repressor-binding site may actually overlap with the -35 sequence and, regardless of what occurs upstream of the promoter, there will always be some residual repressor binding. Isolation of the repressor and subsequent footprint analysis should help to resolve whether this idea is valid. Alternatively, additional sequences located 3' to the -35 element may also be involved in the regulation of urease expression in a pH-dependent fashion. Since we utilized the cognate urease ribosome-binding site in all of our fusions, this could also reflect some level of translational control in response to pH. Translational control of gene expression is not atypical for growth-rate-dependent expression (Carter-Meunchau & Wolfe, 1989 ), but we are not aware of any cases where pH-dependent gene expression is modulated translationally. Notably, the urease mRNA of Helicobacter pylori is more stable at low pH (Akada et al., 2000
). However, our previous analyses of the urease mRNA levels in S. salivarius grown under various conditions would indicate that the expression of the ure operon is mainly controlled by transcriptional initiation. A definitive demonstration of the pathways involved in expression of the ure operon, other than that involved in the control of transcriptional initiation of the urease genes, will be performed when the urease repressor gene is isolated.
It was also of interest that deletion of the DNA sequences located within 39 bp 5' of the 21 bp element resulted in decreased expression from PureI. Again, the possibility that these results were obtained because of a change in spacing or moving of non-native sequences closer to the promoter was diminished, but not eliminated, by the findings that the M1 deletion derivatives also showed diminished expression from the constructs lacking the -52 to -96 sequences. Based on computer predictions, we could locate a potential stemloop structure (G°=-19·0 kcal mol-1) from -62 to -84 (Fig. 2
), but the role of this secondary structure in regulation is not clear at this point. Furthermore, the use of computer algorithms did not reveal the presence of any sequences within this region that were bound by known transcriptional activators.
Previous studies using continuous chemostat cultures have shown that carbohydrate availability and the sugar:phosphoenolpyruvate phosphotransferase system are involved in the regulation of urease expression (Chen & Burne, 1996 ; Chen et al., 1998a
; Weaver et al., 2000
); these studies linked a system capable of sensing carbohydrate source and availability with induction of the urease genes, although pH was still the dominant influence. To help clarify whether the influences of pH and carbohydrate are exerted through a single regulator or through two separate regulatory proteins in S. salivarius, we also measured CAT activity in chemostat-grown S. salivarius PureIcat, PureI
21cat and M1 at pH 7·0 and 5·5, with limiting or excess amounts of carbohydrate (data not shown). The idea, in this case, was that if the effects of carbohydrate were exerted through a different cis-acting site than that to which the repressor bound, pH sensitivity would be lost but carbohydrate sensitivity would be retained. We found that, unlike with the intact PureI promoter, carbohydrate availability did not influence the expression of PureI in the strains that lacked the primary repressor-binding site, regardless of the growth pH, suggesting that pH and carbohydrate regulation of urease expression do not operate independently. In addition, we also found that the pattern of cat expression in each strain when grown under both pH values paralleled that observed with batch-grown cultures, indicating that the batch-culture system was adequate for this study.
Based on sequence analysis we could not identify any ORF that could potentially encode a DNA-binding protein in the flanking sequences of the ure operon, nor could we identify any known regulatory sequences around the PureI region based on BLAST searches against the GenBank database. The lack of a linked regulatory gene, coupled with the fact that urease transcription is rapidly induced at low pH (Burne & Chen, 1998 ) and, thus, could be part of an acid-shock or acid-tolerance response, led us to postulate that perhaps urease was controlled as part of a global regulon for the low-pH induction of genes. To test this idea, we introduced the PureIcat fusions into the urease-negative oral Streptococcus sp. S. gordonii. The most notable findings from this experiment included that the PureIcat fusions were pH-sensitive in S. gordonii and that the target sequences for repression that were recognized by S. salivarius were also recognized by S. gordonii. However, there were two notable differences in the regulation of PureI in S. gordonii compared to its regulation in S. salivarius. The first was the lack of involvement of the upstream sequences in positive regulation of the urease promoter in the M1 background. This may simply reflect an absence of, or an absence of recognition by, accessory factors in S. gordonii that fine-tune urease expression in S. salivarius. There was also some slight variability in the behaviour of the M2 and M3 mutants in S. gordonii when compared with the behaviour of these targeted mutations in S. salivarius, possibly due to slight variations in the target-site specificity of the trans-acting factor that governs expression in response to pH. Notwithstanding these facts, in all cases the behaviour of the derivatives with a mutated primary repressor-binding site in an organism that lacks a urease gene cluster (i.e. S. gordonii) paralleled the behaviour of the same mutants in the parent S. salivarius. Interestingly, we have also noted that the urease promoter is sensitive to pH in Streptococcus mutans, but the magnitude of induction at low pH is only about twofold (Y.-H. Li & R. A. Burne, unpublished data). Collectively, these data support that urease may be the target for global regulators that control gene expression in response to environmental pH and possibly carbohydrate availability and growth rate as well. Thus, the study of the low-pH-inducible urease operon is highly relevant to the development of a thorough understanding of how abundant oral streptococci regulate gene expression in response to those environmental factors that have been shown to be crucial for the development of caries and other common oral health maladies.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 20 March 2002;
revised 28 May 2002;
accepted 15 July 2002.