1 Department of Oral Surgery and Hospital Dentistry, School of Dentistry, School of Medicine, Indiana University, Indianapolis, IN, USA
2 Department of Microbiology and Immunology, School of Medicine, Indiana University, Indianapolis, IN, USA
3 Department of Biochemistry and Molecular Biology, School of Medicine, Indiana University, Indianapolis, IN, USA
Correspondence
M. M. Vickerman
mvickerm{at}iupui.edu
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ABSTRACT |
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INTRODUCTION |
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Parental levels of GTF allow S. gordonii cells growing in the presence of sucrose to synthesize 1,3-- and 1,6-
-glucans that confer a sucrose-promoted phenotype (Spp+) to colonies on sucrose agar plates (Tardif et al., 1989
). These glucans facilitate bacterial accumulation on surfaces and have been implicated as colonization determinants (Vickerman et al., 1991
). Cells with decreased levels of GTF activity (Spp-) are less able to grow in these bacterial biofilms in the presence of sucrose. The nucleotide sequence of one spontaneous Spp- strain was examined and found to contain a point mutation near the 3' end of rgg (Vickerman et al., 1997
). This strain had only approximately 25 % of the parental level of GTF activity (Tardif et al., 1989
) and significantly decreased levels of gtfG transcript (Vickerman et al., 1995
). To confirm that the rgg point mutation was responsible for the decreased GTF activity, strain CHC102 was constructed by allelic exchange (Vickerman et al., 1997
). As expected, strain CHC102 had only approximately 25 % of the parental level of GTF activity. The rgg allele of strain CHC102, designated rggC1, has a cytosine replacing the parental guanine at nucleotide number 811 in the 891 bp rgg ORF. This results in a codon change from a negatively charged aspartate to a positively charged histidine at amino acid number 271 of the encoded protein RggC1 (Vickerman et al., 1997
). The computer-predicted pI of RggC1 is 6·12 compared with the parental Rgg pI of 5·78. The present genetic and biochemical studies were undertaken to determine if the decreased GTF activity in strain CHC102 was due to the change in the encoded RggC1 protein or due to the change in the chromosomal region near the 3' end of rgg, which may function as a DNA-binding site for Rgg. The results provide insights into the binding interaction of Rgg with the chromosomal region upstream of gtfG, and have implications for understanding the functional domains and specificity of the Rgg protein.
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METHODS |
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Construction of S. gordonii strain CH51C1 by allelic exchange.
To construct S. gordonii strain CH51C1, which had an engineered translational stop to encode a truncated Rgg, as in strain DS512, and also contained the point mutation at nucleotide number 811 in rgg, as in strain CHC102, PCR amplification was performed using strain CHC102 chromosomal DNA as template. The forward primer, 5'-TCCTCGAGGGTAGGATTATTAAGATGCTACTTAATG-3', had an engineered 5' XhoI site (underlined) compatible with SalI, an in-frame engineered translational stop (bold), and was designed to anneal immediately downstream of the SalI site of rgg (Fig. 1). The reverse primer, 5'-TTGAGCTCAACTGCAAAGTT-3', was designed to anneal to the SstI site at nucleotide number 750 in the gtfG ORF. The resulting approximately 1·1 kb PCR product was digested with XhoI and SstI and cloned into SalISstI-digested pMI2201 (Vickerman & Minick, 2002
), a pGEM7 (Stratagene) derivative that carries an approximately 2·25 kb ApaISstI fragment encoding the 3' end of the upstream determinant, htpX, rgg and the 5' end of gtfG of S. gordonii strain CH1. After confirmation of the correct nucleotide sequence, the resulting 2·25 kb fragment containing the inserted mutations was transformed into strain CH1 to allow homologous recombination between similar chromosomal and linear DNA on both sides of rgg; this resulted in integration of the desired sequence into the S. gordonii chromosome. Spp- colonies, presumably arising from mutations in rgg, were selected on 3 % sucrose agar plates.
Putative transformants were examined by Southern hybridization analysis (Ausubel et al., 1987) to confirm the presence of the upstream rgg mutation resulting from religation of compatible SalI- and XhoI-digested DNA. SalI-digested chromosomal DNA was electrophoresed on 0·7 % agarose gels, transferred to Hybond-N membranes (Amersham) and probed with a double-stranded digoxigenin-dUTP-labelled DNA probe for rgg, using the Genius System (Roche Molecular Biochemicals), according to the manufacturer's directions. Strains that showed loss of the rgg SalI site were chosen for nucleotide sequence analysis to confirm that in addition to the presence of the engineered translational stop codon, the strains also had the desired rgg point mutation.
Construction of RecA-deficient strains.
To minimize the potential of recombination between plasmid-borne and chromosomal copies of rgg in complementation studies, RecA-deficient derivatives of strains CHC102 and CH51C1, designated CHC102R and CH51C1R, respectively, were constructed. The method used was that described previously to construct RecA-deficient derivatives of strains CH1 [parental RecA-deficient strain CHR3 (Vickerman et al., 1993)], DS512 and CH
R1 [RecA-deficient strains DS512R and CH
R1R, respectively (Vickerman & Minick, 2002
)]. Briefly, strains were transformed with pAM6200, an integrative plasmid carrying a spectinomycin-resistance determinant and an approximately 300 bp internal fragment of the S. gordonii recA gene. Expected integration of pAM6200 into the streptococcal chromosome, resulting in disruption of the recA gene, was confirmed by Southern hybridization analysis using the internal 300 bp recA fragment as a probe (Vickerman et al., 1993
).
Plasmids for determining trans effects of rgg.
The rgg of strain CHC102, designated rggC1, was cloned into the streptococcal vector pVA749 (Macrina et al., 1981) to compare trans effects of rgg to those of rggC1 in S. gordonii. The oligonucleotide primers 5'-GTGGATCCTGCAAAAACTTTTCTACACTCAC-3' and 5'-AGGGATCCAGAGAGGTAACAGCAATCGCTAC-3' were used in PCR with strain CHC102 chromosomal template to amplify rggC1 and its flanking region. The BamHI-flanked approximately 1·49 kb PCR fragment was cloned into a BamHI site which had been created in the HaeIII site (GGCC) of the streptococcal plasmid pVA749 by insertion of two nucleotides (AT). Plasmid DNA was sequenced to confirm PCR fidelity and orientation. The plasmids chosen for these studies, pMIC1, which carried rggC1, and pAMS57 (Sulavik et al., 1992
), which carries the parental rgg in the HaeIII site of pVA749, differed only by the expected point mutation in rgg and the two nucleotides (AT) at the engineered cloning site.
Determination of GTF activity.
Relative amounts of GTF activity for each strain were determined by measuring glucan synthesized in acrylamide gels, as described previously (Tardif et al., 1989). Briefly, strains were grown to the same mid-to-late exponential stage in FMC (OD520 value of
1·6) and equal volumes of cell-free culture supernatants were run on SDS-8·75 % PAGE. After electrophoresis, gels were incubated overnight in 3 % sucrose, 0·5 % Triton X-100 in 10 mM sodium phosphate, pH 6·8, at 37 °C. Resulting glucan bands were stained with pararosanaline and densitometrically scanned to determine Intensity Units (Alpha Imager TM2200; Alpha Innotech). Band intensities reflected the relative levels of GTF activity. Results shown are representative of at least four independent experiments.
Determination of effects of plasmid-borne rgg on expression of gtfG promoter : : lacZ fusions in E. coli.
Derivatives of E. coli strain MC4100 with a promoterless lacZ (strain VA203) or gtfG promoter : : lacZ fusion (strain VA204) in single copy in the att site were used to examine trans effects of rgg on gtfG promoter expression, as described previously (Sulavik & Clewell, 1996
). To compare effects of rgg and rggC1, these genes were subcloned from pAMS57 and pMIC1 into pBCSK+ (Stratagene) to create pBCRgg and pBCRggC1, respectively. DNA ligation mixes were transformed into DH5
and selected on chloramphenicol agar plates. After restriction digest mapping to confirm that rgg and rggC1 were in the same orientation, the plasmids were transformed into the kanamycin-resistant strains VA203 and VA204. After growth to mid-exponential phase in LB medium,
-galactosidase activity in chloroform-permeabilized cells was measured spectrophotometrically (Pharmacia Ultraspec 2000) using ONPG as the enzyme substrate (Miller, 1972
). Total protein concentration was measured by the method of Markwell et al. (1978)
and activity was expressed in Miller units (Miller, 1972
). Comparisons between strains were done using a two-tailed Student's t-test. Experiments were repeated a minimum of three times.
Expression and purification of recombinant Rgg proteins.
Oligonucleotide primers 5'-AAGGGATCCATGCTTATCGTAAAGTCG-3' and 5'-GAGCTCGAGCTATTCTATTTGCAAATC-3' (start and stop codons are italicized) were used in PCR with pAMS57 or pMIC1 templates to amplify the rgg and rggC1 genes, respectively. The resulting PCR products were cloned into the in-frame, compatible BamHI and XhoI sites of pET28a (Novagen) to produce NH2-terminal hexahistidine-tagged recombinant proteins. E. coli B834(DE3) cells (Novagen) transformed with pET28a carrying either rgg or rggC1 were grown at 37 °C in LB medium containing 50 µg kanamycin ml-1 until an OD600 value of approximately 0·6 was reached. Cultures were then induced with 1 mM IPTG for 18 h at 16 °C. Cells were harvested by centrifugation and resuspended in a lysis buffer [50 mM HEPES, 500 mM NaCl, 1 % (v/v) Triton X-100, 1 mM benzamidine, 5 mM imidazole, pH 7·5] with 1 mM PMSF and 5 mM MgCl2. After the addition of 2 mg RNase A ml-1 and 500 U DNase, bacteria were lysed in a French pressure cell. The lysate was centrifuged (15 000 g for 20 min at 4 °C) and the supernatant was applied to an approximately 57 ml nickel-chelating Sepharose column (Amersham Pharmacia) pre-equilibrated with lysis buffer. The column was washed with 50 ml lysis buffer, then 100 ml of 500 mM NaCl/100 mM imidazole, pH 7·5. Histidine-tagged protein was eluted with a solution of 500 mM NaCl/500 mM imidazole, pH 7·5. Protein-containing fractions were pooled and dialysed against 500 mM NaCl/10 mM imidazole/5 mM EDTA/0·1 % (v/v) -mercaptoethanol with a sample : dialysis buffer volume ratio of 1 : 100, with two changes at approximately 8 h intervals. Protein recovered in this manner could attain concentrations as high as 9 mg ml-1, with a purity in excess of 99 % as estimated by SDS-PAGE.
Surface plasmon resonance (SPR) studies.
Binding interactions of Rgg and RggC1 recombinant proteins with DNA were monitored using the SPR method (Wang et al., 2000), which measured changes in refractive index that occurred when injected protein bound to DNA coupled to a biosensor surface. Double-stranded DNA fragments, representing the 107 bp region from the EcoRI site in rgg (Fig. 1
) through the ATG start codon of gtfG, were made using a 5' biotinylated reading strand primer (Integrated DNA Technologies) hybridized to the complementary strand in a 1 : 5 ratio to ensure that all biotinylated oligonucleotides were in duplex form. The resulting dsDNA was purified by electrophoresis in a native 8 % polyacrylamide gel (Ausubel et al., 1987
).
For preparation of biosensor surfaces with DNA, the biotinylated dsDNA was diluted to 1·5 nM in a buffer containing 10 mM HEPES, pH 7·4, 0·005 % polysorbate-20 and 1·0 M NaCl, and manually injected onto an immobilized Neutravidin surface of a BIACORE sensor chip B1 to 450 Response Units using a BIACORE 3000 (Biacore). Neutravidin surfaces were prepared using an amine coupling reaction according to the manufacturer's directions (BIAtechnology handbook). One flow cell was left underivatized to allow bulk refractive index change correction. Proteins were diluted in the running buffer containing 10 mM HEPES, pH 7·4, 150 mM NaCl and 0·005 % polysorbate-20. Following protein binding to DNA, regeneration was performed with a 30 s quick-injection of 0·25 % SDS in running buffer. For reproducibility, each experiment was repeated at least twice.
SPR analysis.
Bia-evaluation software supplied by the vendor was used to analyse binding data using a simple Langmuir 1 : 1 model (Myszka, 2000). In the present experiments, the analyte is the recombinant protein, ligand is the DNA and Rmax is the total surface-binding capacity. For the model [A]+[B]
[AB], where [A]=concentration of analyte, total ligand on the surface [B0]=Rmax, and initial response [AB0]=0, local numerical integration for each curve was performed using the differential equation d[AB]/dt=ka[A][B]-kd[AB]. The fit of the data was reported as a mean sum of differences (
2) between predicted and observed data and as standard errors. KD values for the proteinDNA complexes were calculated as the kd (dissociation rate constant) divided by the ka (association rate constant).
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RESULTS |
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Plasmid-borne rggC1 is unable to increase transcription of gtfG' : : lacZ fusions to the same extent as parental rgg
Since expression of rgg has both cis and trans effects on gtfG expression in S. gordonii (Vickerman & Minick, 2002), independent measurements to clarify the ability of trans-acting rgg and rggC1 gene products to increase activity of the gtfG promoter were performed in E. coli. Plasmids carrying these genes were transformed into strains VA203 and VA204. These strains had a promoterless lacZ or a gtfG promoter : : lacZ fusion, respectively, chromosomally integrated in single copy into the
att site.
-Galactosidase activities observed in strains VA203 and VA204 carrying pBCRgg were consistent with previously published results (Sulavik & Clewell, 1996
). The presence of pBCRgg in the gtfG promoter fusion strain VA204 resulted in an approximately 6·38-fold increase in
-galactosidase activity compared with activity seen with the pBCSK vector alone (Table 1
). Strain VA204(pBCRggC1) showed only an approximately 5·36-fold increase in levels of
-galactosidase activity compared to levels seen in VA204(pBCSK) (Table 1
). These results suggested that although the product of rggC1 increased the ability of the gtfG promoter to express lacZ, it was not able to achieve the same level as plasmid-borne parental rgg.
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DISCUSSION |
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Although previous studies in S. gordonii had demonstrated that deletion of a 264 bp DNA region flanking the rggC1 point mutation did not affect the ability of plasmid-borne rgg to increase GTF activity (Vickerman & Minick, 2002), the possibility existed that the deletion in strain CH
R1 allowed an upstream DNA region to act as an artificial binding site for Rgg. However, the present genetic studies suggest that parental rgg in pAMS57 can complement strain CH51C1, which had the chromosomal point mutation. This supports previous studies that had implicated only the involvement of the chromosomal region downstream of the rgg EcoRI site with Rgg (Vickerman & Minick, 2002
). Accordingly, a 107 bp dsDNA fragment, representing the rgg EcoRI site through the start codon of gtfG, was used in RggDNA-binding studies. Because the recombinant Rgg protein was relatively insoluble in the low salt conditions necessary for classical electrophoretic mobility shift assays (Ausubel et al., 1987
), SPR was used to measure the proteinDNA-binding interaction. The very dilute solutions used in this method minimized protein solubility and aggregation problems (Green et al., 2000
), so that in addition to monitoring binding in real time, kinetic studies were possible. The recombinant Rgg specifically bound to the 107 bp duplex at levels above the binding to the random poly dA/dT control dsDNA, supporting the RggDNA-binding model for gtfG regulation.
The KD values for Rgg and RggC1 suggest that the proteinDNA complexes are less stable than those seen with some transcriptional activators or repressors, although dissociation rate constants are similar (Poon et al., 2001; Stockley et al., 1998
). However, because of different functions of bacterial regulatory proteins, it is not necessarily valid to compare KD values among proteinDNA complexes. As in all in vitro systems, there are inherent limitations in interpretation of results. The KD ranges seen in the present SPR studies with purified recombinant Rgg may be influenced by several factors. In vivo, Rgg may form complexes with intracellular components such as sigma factors, RNA polymerase, or additional, as-yet-unidentified proteins. Indeed, previous genetic studies have indicated that distally located genes, which could potentially encode regulatory cofactors, influence rgg or gtfG expression (Vickerman et al., 1995
, 1997
). It is possible that such factors may influence the stability of the RggDNA binding. In addition, it is probable that only a small number of nucleotides directly interact with the helixturnhelix motif of Rgg (Wintgens & Rooman, 1996
). In the semi-solid phase technology used in the SPR studies, the relatively large number of nucleotides in the 107 bp dsDNA fragment may sterically hinder the binding and stability of the proteinDNA complex. Additional studies to delineate the DNA region within the 107 bp sequence that interacts with Rgg are in progress. Identification of specific nucleotides involved in the RggDNA interaction may demonstrate as-yet-unidentified general patterns in DNA regions regulated by Rgg-like proteins.
The basis for the decreased gtfG transcription and resulting decreased GTF activity (Vickerman et al., 1995) in strain CHC102 appears to be due to a change in the encoded Rgg protein. Comparison of Rgg-like sequences indicates that the amino acid change in RggC1 is located in a fairly conserved region near the carboxyl terminus (Vickerman et al., 2001
); no function has yet been assigned to this domain. Although RggC1 has the parental sequence in the helixturnhelix putative DNA-binding region (amino acids 1164; Bateman et al., 2000
), RggC1 was more efficient at binding DNA than the parental Rgg in the SPR studies. Although the molecular mechanism has not yet been determined, the increased DNA binding ability of RggC1 may be due to a conformational change in the protein that influences the DNA binding interaction at biochemical (e.g. due to local charge effects) or mechanical (e.g. torsional effects) levels (Travers & Muskhelishvili, 1998
). Point mutations that result in bacterial proteins that are defective in transcriptional initiation or activation, yet do not show decreased DNA-binding abilities, have been previously described in other bacteria (Gerber & Hinton, 1996
; Kelly & Hoover, 1999
). Accordingly, despite its increased DNA binding ability in SPR studies, RggC1 appears to be defective in transcriptional activation. The plasmid pMIC1, carrying rggC1, was unable to increase GTF activity in S. gordonii strains to the same extent as pAMS57, carrying rgg. Furthermore, plasmids carrying these rgg alleles differed in their ability to increase expression of gtfG promoter fusions in E. coli. Although these latter studies must be interpreted with care due to differences in promoter recognition in E. coli and streptococcal species (Dillard & Yother, 1991
), they nevertheless independently support the relative activities of the Rgg and RggC1 proteins for transcriptional activation of gtfG (Sulavik & Clewell, 1996
). It is not clear if the point mutation in rggC1 causes a direct change in an RggC1 domain involved in transcriptional activation, or if the changes observed in DNA binding and transcriptional activation are due to indirect conformational effects on the RggC1 protein. Nevertheless, identification of an amino acid essential for maintaining transcriptional activating activity in S. gordonii Rgg not only provides insights into the control of gtfG expression, but also may provide general insights into the specificity and functional domains among the family of Rgg-like proteins.
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ACKNOWLEDGEMENTS |
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Received 9 September 2002;
revised 18 November 2002;
accepted 19 November 2002.