Department of Oral Surgery and Hospital Dentistry, School of Dentistry1,2 and Department of Microbiology and Immunology,1 School of Medicine, Indiana University, Indianapolis, IN 46202, USA
Author for correspondence: M. M. Vickerman. Tel: +1 317 278 3250. Fax: +1 317 278 6244. e-mail: mvickerm{at}iupui.edu
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ABSTRACT |
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Keywords: oral streptococci, glucans, rgg
Abbreviations: GTF, glucosyltransferase; IPCR, inverse PCR; Spp, sucrose-promoted phenotype
The nucleotide sequence of the S. gordonii chromosomal region reported in this paper has been appended to the gtfG entry in GenBank with accession number U12643.
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INTRODUCTION |
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At the time rgg was first identified, there were no similar genes in the genetic databases (Sulavik et al., 1992 ). Since then, determinants with significant similarity to rgg have been identified in a number of related bacterial species. Nucleotide sequence and Southern blot hybridization analyses have identified rgg-like determinants in Streptococcus oralis (Fujiwara et al., 2000
) and Streptococcus sanguis (Vickerman et al., 1995
), early colonizers of dental plaque (Frandsen et al., 1991
) which, like S. gordonii, appear to have only one GTF enzyme; these rgg-like determinants appear to be located near their gtf determinants, suggesting that GTF expression in these species may be regulated in a manner similar to that of S. gordonii (Vickerman et al., 1995
). rgg-like determinants have also been characterized in other streptococcal (Lyon et al., 1998
; Chaussee et al., 1999
; Qi et al., 1999
) and lactococcal (Sanders et al., 1998
) species, and have been found to regulate a variety of proteins with different functions. These data suggest that rgg-like genes are members of a family of important streptococcal regulatory determinants.
Genetic data suggest that the regulation of S. gordonii GTF activity is complex and may involve genes in addition to rgg and gtfG. Both rgg and gtfG are preceded by DNA inverted repeats, suggestive of possible regulatory factor binding sites (Sulavik et al., 1992 ). Two distinct genetic loci that influence GTF activity have been identified by chemical mutagenesis (Haisman & Jenkinson, 1991
). Nucleotide sequence analysis has shown that S. gordonii strains with only 2030% of the parental level of GTF activity have no differences in the 5·95 kb genome region encoding rgg, gtfG and their immediate flanking regions (Vickerman et al., 1997a
). Because functionally linked genes are often located in close proximity on bacterial chromosomes, it was hypothesized that genes flanking the rgg/gtfG locus could be involved in GTF regulation. However, insertional inactivation of the lemA and htpX genes immediately upstream of rgg did not affect the level of GTF expression (Vickerman et al., 2001). Therefore, the present study was undertaken to examine the S. gordonii chromosomal region downstream of gtfG and to determine potential influences of any genes identified in this region on GTF activity.
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METHODS |
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Southern blot hybridization analyses.
S. gordonii chromosomal DNA was digested with appropriate restriction enzymes, electrophoresed on 0·7% agarose gels and transferred to a Hybond-N membrane via capillary action under neutral conditions (Ausubel et al., 1987 ). Probe DNA was labelled with digoxigenin-dUTP, hybridized to membranes and washed under stringent conditions. Hybridized probe was detected by chemiluminescence with the Genius System (Roche Molecular Biochemicals), according to the manufacturers directions.
Recovery of S. gordonii chromosomal DNA downstream of gtfG.
Nucleotide sequencing of the downstream region was originally done using the E. coli plasmid pAMS21, which carries the downstream 3·4 kb HindIII fragment of pAMS40 subcloned in pBluescript (Vickerman et al., 1997b ; Fig. 1a
). However, difficulty identifying ORFs raised the possibility that there were difficulties cloning this region. Therefore, using primers derived from the pAMS21 sequence, three independent identical PCR products from this region were sequenced directly. The results indicated that deletions had occurred in the ORF immediately downstream of gtfG in pAMS21. Subsequent attempts to clone these PCR products confirmed that deletions occurred in E. coli cloning vectors. Consequently, the nucleotide sequence of the region downstream of gtfG was determined directly from PCR products. At least three overlapping independent products were sequenced for each region.
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Nucleotide sequence determination and analysis.
Both strands of the DNA template were sequenced using a PRISM-Ready Reaction Dye Deoxy Terminator Sequencing kit (Applied Biosystems) and an automated DNA sequencer (model 373, Applied Biosystems), using customized oligonucleotide primers. Searches for homologues to S. gordonii nucleotide and amino acid sequences were done using the BLAST algorithm (Altschul et al., 1990 ) on the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov), streptococcal genome databases at the University of Oklahoma (http://genome.ou.edu) and The Institute for Genomic Research database (http://www.tigr.org). Comparisons of entire genes and their encoded proteins were done using MacVector (version 7.0, Oxford Molecular Group) and the GCG package (Wisconsin Package version 10) for nucleotide and protein similarity. Encoded signal peptide and transmembrane helices were predicted with the Signal P (Nielsen et al., 1997
) and TmPred (Hofmann & Stoffel, 1993
) programs. Potential protein structural motifs were identified via the pfam database (Bateman et al., 2000
).
Insertional inactivation of ORFs.
Oligonucleotide primers with engineered flanking restriction sites and translational stops were used with pAMS40 template to produce internal gene fragments by PCR. The resulting fragments were digested and directionally cloned into the BamHI and HindIII sites of pVA891 (Macrina et al., 1983 ). After construction and verification in E. coli DH5
, purified plasmid DNA was transformed into strain CH1. Putative S. gordonii transformants with the expected plasmid insertion were verified by two methods. Southern blots of digested chromosomal DNA were probed with both pVA891 and the cloned internal gene fragment. Transformant strains that had the expected hybridization patterns were confirmed by direct sequence analysis of PCR products. Primers designed to anneal adjacent to the BamHI and HindIII sites of the vector (5'-ACGATGCGTCCGGCGTAGAG-3' and 5'-AGGTGCTGACTTTCAACTGC-3', respectively) were used with primers designed to anneal to S. gordonii chromosomal sequences flanking the region of each expected plasmid insertion. Resulting PCR products with each transformant template were sequenced to confirm the correct integration of each plasmid for gene disruption.
Determination of GTF activity.
Relative amounts of GTF activity for each strain were measured via glucan production in acrylamide gels, as described by Tardif et al. (1989) . Briefly, strains to be compared were grown to the same mid- to late-exponential stage (OD520
1·6). Cell pellets were extracted with 1% (w/v) SDS (Vickerman & Clewell, 1997
) and equal volumes of cell extracts and cell-free culture supernatants were run on an 8·75% acrylamide SDS-PAGE. After electrophoresis, gels were incubated overnight at 37 °C in a solution of 3% sucrose and 0·5% Triton X-100 in 10 mM sodium phosphate (pH 6·8). The resulting glucan bands were stained with pararosaniline, as described by Tardif et al. (1989)
. Band intensities reflect the relative amount of GTF protein and activity (Vickerman et al., 1996
). Relative GTF activity for each strain was determined via laser densitometry (LKB Ultrascan XL) in at least four independent gels. GTF activity was determined as a percentage of the parental strain activity (set at 100% for the parental strain on each gel).
Northern hybridization analyses.
S. gordonii cells were grown in 0·5% (w/v) glycine medium to the same density. Total RNA was prepared using a Purescript RNA isolation kit (Gentra Systems) essentially to the manufacturers directions, but with the following modifications: Cell pellets were resuspended in 16·1 mg lysozyme ml-1 and 100 units of mutanolysin in GET buffer (50 mM glucose, 10 mM EDTA, 25 mM Tris/HCl, pH 8·0) and incubated at 37 °C for 10 min. After addition of the SDS solution, cells were incubated on ice for 5 min, followed by 30 s at 65 °C for lysis. Prepared RNA was further purified with two phenol/chloroform/isoamyl alcohol extractions and an ethanol precipitation. Equal amounts of RNA were electrophoresed on a 2% formaldehyde/1% agarose gel, transferred to Hybond-N membranes, probed with digoxigenin-labelled DNA fragments and washed under stringent conditions, according to the instructions for the Genius system (Roche Biochemicals). All solutions were prepared using sterilized dimethyl pyrocarbonate-treated water.
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RESULTS |
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Disruption of dsg influences GTF activity
Colonies of strain CH8942 had the hard, cohesive Spp+ colony phenotype on sucrose agar plates, indicating that they synthesized glucan polymers. However, the colonies were macroscopically more glassy and translucent than parental strain CH1 Spp+ colonies, suggesting some difference in the GTF activities of these two strains. Scanning of GTF activity gels indicated that strain CH8942 only had 57·4±8·2% SD of the extracellular GTF activity of strain CH1 (Fig. 3a). Similar results were seen for cell-associated GTF activity: strain CH8942 had only 53·0±8·4% SD the parental level of cell-associated GTF activity. However, Northern blots (Fig. 4a
) indicated that the amount of the 5·4 kb gtfG-specific and 6·4 kb polycistronic rgg/gtfG transcripts were similar in strains CH1 and CH8942, suggesting that dsg may be involved in post-transcriptional modifications that result in decreased levels of GTF protein.
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To compare trans effects of rggD to those of rgg in S. gordonii, the rggD, its putative promoter and its transcriptional termination regions were amplified by PCR using primers 5'-taGATATCCTTCAGCCAAGTCTAGCTTC-3' and 5'-taGATATCCGTAAGGCAAGTTTCATA-3'. The resulting 1397 bp EcoRV-flanked fragment was cloned into the compatible HaeIII site of the streptococcal plasmid pVA749. Nucleotide sequence analysis confirmed that the orientation of the rggD fragment in the resulting plasmid, pMI226, was the same as that of rgg in the plasmid pAMS57 previously characterized by Sulavik et al. (1992) .
rggD does not affect GTF activity
Although their nucleotide and deduced amino acid sequences were similar, the specificities of rgg and rggD appeared to differ. Northern blots indicated that disruption of rggD in strain CH891 did not affect levels of rgg/gtfG or gtfG transcript (Fig. 4a). The GTF activities of strains CH1 and CH8961 were similar (Fig. 3a
), indicating that disruption of rggD did not affect potential post-transcriptional modifications that would result in altered levels of GTF activity. Effects of plasmid-borne rgg and rggD were also specific. Plasmid-borne rgg in pAMS57 has been shown to increase GTF activity approximately sixfold in the parental strain CH1; pAMS57 has also been shown to increase GTF activity to the same level in strain DS512 (Sulavik et al., 1992
). Due to a frameshift mutation resulting in a premature translation stop in rgg, strain DS512 has only
3% of the parental level of GTF activity (Sulavik & Clewell, 1996
). However, plasmid-borne rggD did not increase GTF activity in strains CH1 or DS512 to a level above that of the plasmid-free strain (Fig. 3b
). Thus, rggD in trans did not affect GTF activity, even in the absence of a functional chromosomal rgg. These results suggest that rggD does not influence gtfG expression and may provide important insights into the specificity of rgg-like genes.
Effects of rggD on transcription of flanking genes
To determine if rggD and dsg were co-transcribed, strain CH1 RNA was probed with internal fragments of rggD and dsg in Northern blots (Fig. 4b, c
). A
1·0 kb rggD-specific transcript and a
1·3 kb dsg-specific transcript were detected. No polycistronic transcript was noted.
Previously characterized rgg-like genes positively regulate transcription of adjacent genes (Sulavik & Clewell, 1996 ; Lyon et al., 1998
; Sanders et al., 1998
; Qi et al., 1999
; Chaussee et al., 1999
). Furthermore, rgg-like genes are often separated from the gene they regulate by DNA secondary structures, such as inverted repeats. Accordingly, the inverted repeats between rggD and dsg suggested the possibility that rggD might affect dsg transcription. However, in Streptococcus pyogenes, the rgg-like gene that regulates the gene for streptococcal erythrogenic toxin B, speB, is transcribed in the divergent reading direction (Lyon et al., 1998
) Therefore, the possibility that rggD regulates the gene further downstream was also investigated. Nucleotide sequencing identified a 531 bp ORF downstream of rggD, in the divergent reading direction (Fig. 1a
). This gene encodes a predicted 19·9 kDa cytoplasmic protein with a pI of 3·89. Examination of genome databases indicated that this gene is most similar to the ylbN-like gene of unknown function of Lactococcus lactis subsp. cremoris. The S. gordonii ylbN-like gene is associated with a
2·4 kb transcript (Fig. 4d
), suggesting that it is co-transcribed with determinants further downstream. Indeed, partial sequence of an ORF, designated orf8 in Fig. 1(a)
, showed a putative ribosome-binding site for translation of the encoded protein, but no apparent transcriptional termination sequences were noted in the region between the ylbN-like gene and orf8.
Northern blot analyses were used to examine the transcription of dsg and ylbN-like genes in strain CH8961 (in which rggD was insertionally inactivated) and in a parental strain carrying additional copies of rggD in pMI226 (Fig. 4b, c
). Unexpectedly, the results did not indicate any differences in transcription of either dsg or ylbN in these strains compared with the parental strain CH1. Recent studies in S. pyogenes have indicated that an rgg-like determinant influences transcription in stationary-phase cells (Chaussee et al., 2001
); hence the S. gordonii strains were also examined in late-exponential to stationary growth phase. However, as seen in the mid-exponential-phase cells, no differences were evident (data not shown). Similar results were also seen when the S. gordonii strains were grown in defined FMC medium and in complex ToddHewitt broth, indicating that components of these different media did not influence potential regulation of either gene by rggD. These results do not preclude the possibility that rggD affects transcription of the dsg or ylbN-like genes under conditions other than those in the present study.
Comparison of streptococcal chromosomal regions
The finding that rggD did not influence transcription of the adjacent genes raised the possibility that rggD regulates distally located genes on the S. gordonii chromosome. This hypothesis is supported by the comparison of similar chromosomal regions using available genome data for other streptococcal species (Fig. 1b). S. pyogenes has htpX- and ylbN-like genes adjacent to each other. However, S. gordonii has four genes between the htpX and ylbN-like genes, i.e. two convergent genes, gtfG and dsg, which are each preceded by similar rgg-like genes. Both Streptococcus mutans and Streptococcus pneumoniae have hypothetical ATP-binding proteins with no rgg-like determinants on the opposite reading strand between their htpX- and ylbN-like genes. These hypothetical genes, encoding ATP-binding proteins, do not have significant similarities to the S. gordonii rgg-flanked gtfG and dsg genes. However, there is a dsg-like gene (37% identity at the nucleotide level; 57% similarity of the encoded putative proteins) distally located on the S. pneumoniae chromosome between putative histidine tRNA ligase and dihydroxy-acid dehydratase genes. These findings suggest that the S. gordonii determinants between the htpX and ylbN-like genes may have resulted from recombinations between similar DNA regions within the S. gordonii chromosome, or via horizontal gene transfer. It is possible that such a recombination or rearrangement resulted in distancing rggD from the S. gordonii gene which it regulates.
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DISCUSSION |
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Directly 5' to dsg, an rgg-like determinant, rggD, was identified: this is the third rgg-like determinant reported in S. gordonii. In addition to the originally identified positive gtfG regulator, rgg (Sulavik et al., 1992 ), an rgg-like determinant, iviB, was recovered from a rabbit endocarditis model using in vivo expression technology (Kilic et al., 1999
); this suggests that iviB, or possibly a gene(s) that it regulates, is required for survival in vivo. Multiple rgg-like genes are present in S. pyogenes (Ferretti et al., 2001
), S. mutans (http://www.genome.ou.edu) and S. pneumoniae (http://www.tigr.org), suggesting that rgg-like genes make up a relatively widely occurring family of streptococcal regulatory genes. Under the conditions in the present studies, the S. gordonii gene that rggD regulates was not identified. Based upon the findings that most rgg-like determinants regulate adjacent genes (Sulavik & Clewell, 1996
; Lyon et al., 1998
; Sanders et al., 1998
; Qi et al., 1999
; Chaussee et al., 1999
) it was hypothesized that rggD would regulate the S. gordonii dsg or ylbN-like genes. Northern blots did not confirm this hypothesis. However, it is possible that changes in the levels of the dsg and ylbN transcript in strains with disruption or additional copies of rggD were too subtle to be detected by Northern blot hybridization analyses. Nevertheless, the dramatic sixfold differences in gtfG transcription due to rgg (Sulavik & Clewell, 1996
), along with the effects of rgg (ropB) on transcription of speB (Lyon et al., 1998
), were readily apparent on Northern blots, suggesting that the magnitude of positive regulation by rgg-like genes should be clearly evident by this assay. It is possible that rggD regulates either of the adjacent genes under conditions other than those in these studies. Although transcripts were examined from mid-exponential through to stationary growth phase in defined as well as complex media, there are innumerable conditions that could affect genetic regulation. Comparison of the chromosomal organizations of related streptococci, which show that the genes present in the regions between htpX- and ylbN-like genes differ among these species, suggest that DNA recombinations and rearrangements may have occurred in these intergenic regions. This finding gives support to additional speculation that rggD may regulate distally located S. gordonii gene(s) from which it was distanced by recombinational events. Additional S. gordonii genome sequence data may provide insights into this possibility.
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ACKNOWLEDGEMENTS |
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Received 6 June 2001;
revised 6 July 2001;
accepted 24 July 2001.