Characterization of Porphyromonas gingivalis insertion sequence-like element ISPg5.

Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobe, is found in periodontitis lesions, and its presence in subgingival plaque significantly increases the risk for periodontitis. In contrast to many bacterial pathogens, P. gingivalis strains display considerable variability, which is likely due to genetic exchange and intragenomic changes. To explore the latter possibility, we have studied the occurrence of insertion sequence (IS)-like elements in P. gingivalis W83 by utilizing a convenient and rapid method of capturing IS-like sequences and through analysis of the genome sequence of P. gingivalis strain W83. We adapted the method of Matsutani et al. (S. Matsutani, H. Ohtsubo, Y. Maeda, and E. Ohtsubo, J. Mol. Biol. 196:445-455, 1987) to isolate and clone rapidly annealing DNA sequences characteristic of repetitive regions within a genome. We show that in P. gingivalis strain W83, such sequences include (i) nucleotide sequence with homology to tRNA genes, (ii) a previously described IS element, and (iii) a novel IS-like element. Analysis of the P. gingivalis genome sequence for the distribution of the least used tetranucleotide, CTAG, identified regions in many of the initial 218 contigs which contained CTAG clusters. Examination of these CTAG clusters led to the discovery of 11 copies of the same novel IS-like element identified by the repeated sequence capture method of Matsutani et al. This new 1,512-bp IS-like element, designated ISPg5, has features of the IS3 family of IS elements. When a recombinant plasmid containing much of ISPg5 was used in Southern analysis of several P. gingivalis strains, including clinical isolates, diversity among strains was apparent. This suggests that ISPg5 and other IS elements may contribute to strain diversity and can be used for strain fingerprinting.

Genetic characterization of a Streptococcus mutans LraI family operon and role in virulence.

Proteins belonging to the LraI (for "lipoprotein receptor antigen") family function as adhesins in several streptococci, as a virulence factor for endocarditis in at least one of these species, and potentially as metal transporters in many bacteria. We have identified and characterized the chromosomal locus containing the LraI family gene (designated sloC) from Streptococcus mutans, an agent of dental caries and endocarditis in humans. Northern blot analysis indicated that sloC is cotranscribed with three other genes. As with other LraI operons, the sloA and sloB genes apparently encode components of an ATP-binding cassette transport system. The product of the fourth gene, sloR, has homology to the metal-dependent regulator from Corynebacterium diphtheriae, DtxR. A potential binding site for SloR was identified upstream from the sloABCR operon and was conserved upstream from LraI operons in several other streptococci. Potential SloR homologs were identified in the unfinished genomic sequences from two of these, S. pneumoniae and S. pyogenes. Mutagenesis of sloC in S. mutans resulted in apparent loss of expression of the entire operon as assessed by Northern blot analysis. The sloC mutant was indistinguishable from its wild-type parent in a gnotobiotic rat model of caries but was significantly less virulent in a rat model of endocarditis. Virulence for endocarditis was restored by correction of the sloC mutation but not by provision of the sloC gene in trans, suggesting that virulence requires the expression of other genes in the sloC operon.

Hemoglobinase activity of the lysine gingipain protease (Kgp) of Porphyromonas gingivalis W83.

Porphyromonas gingivalis, an important periodontal disease pathogen, forms black-pigmented colonies on blood agar. Pigmentation is believed to result from accumulation of iron protoporphyrin IX (FePPIX) derived from erythrocytic hemoglobin. The Lys-X (Lys-gingipain) and Arg-X (Arg-gingipain) cysteine proteases of P. gingivalis bind and degrade erythrocytes. We have observed that mutations abolishing activity of the Lys-X-specific cysteine protease, Kgp, resulted in loss of black pigmentation of P. gingivalis W83. Because the hemagglutinating and hemolytic potentials of mutant strains were reduced but not eliminated, we hypothesized that this protease played a role in acquisition of FePPIX from hemoglobin. In contrast to Arg-gingipain, Lys-gingipain was not inhibited by hemin, suggesting that this protease played a role near the cell surface where high concentrations of hemin confer the black pigmentation. Human hemoglobin contains 11 Lys residues in the alpha chain and 10 Lys residues in the beta chain. In contrast, there are only three Arg residues in each of the alpha and beta chains. These observations are consistent with human hemoglobin being a preferred substrate for Lys-gingipain but not Arg-gingipain. The ability of the Lys-gingipain to cleave human hemoglobin at Lys residues was confirmed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry of hemoglobin fragments resulting from digestion with the purified protease. We were able to detect several of the predicted hemoglobin fragments rendered by digestion with purified Lys-gingipain. Thus, we postulate that the Lys-gingipain of P. gingivalis is a hemoglobinase which plays a role in heme and iron uptake by effecting the accumulation of FePPIX on the bacterial cell surface.

Construction and immunologic evaluation of a Porphyromonas gingivalis subsequence peptide fused to hepatitis B virus core antigen.

Several proteins of Porphyromonas gingivalis contain multiple copies of a 47 amino acid conserved repeated sequence. A fusion protein was constructed in which the P. gingivalis peptide was fused to the carboxy terminus of the hepatitis B core protein. This fusion protein was expressed in Escherichia coli, purified, and used to vaccinate mice that were later challenged with P. gingivalis W83 using the mouse abscess model. Although the mice were not protected against bacterial challenge, Western blot analysis showed that sera from the mice and from rabbits immunized with the fusion protein reacted with a number of vesicle proteins from P. gingivalis W83. These data suggested that this peptide is recognized by the host's immune system but that the antibodies are not protective.

Localization of HArep-containing genes on the chromosome of Porphyromonas gingivalis W83.

We have mapped a group of virulence genes of Porphyromonas gingivalis to a single large fragment of the genome. These genes (rgpA, kgp, and hagA) all contain a consensus repeat sequence (HArep). rgpA and kgp encode cysteine proteases with Arg-X and Lys-X specificity, respectively, and hagA encodes a hemagglutinin. Genomic DNA fragments separated by pulse-field gel electrophoresis were blotted and probed in order to localize the genes to a 0.25-Mb NheI fragment of the P. gingivalis W83 genome. Further hybridization analyses with single- and double-restriction digestion allowed us to generate a physical map of the fragment and determine the precise locations of the protease and hemagglutinin genes. In addition, we found an insertion-like sequence, IS195, near the ends of the 0. 25-Mb NheI fragment. A similarly sized fragment carrying HArep sequences was also demonstrated in the P. gingivalis W12 and W50 genomes.

Molecular genetics and nomenclature of proteases of Porphyromonas gingivalis.

The strategies used by bacterial pathogens to establish and maintain themselves in the host represent one of the fundamental aspects of microbial pathogenesis. Characterization of these strategies and the underlying molecular machinery offers new opportunities both to our understanding of how organisms cause disease in susceptible individuals and to the development of novel therapeutic measures designed to undermine or interfere with these determinants of successful survival. With respect to the microbial aetiology of the periodontal diseases, a growing body of evidence suggests that the proteolytic enzymes of Porphyromonas gingivalis represent key survival and, by extrapolation, virulence determinants of this periodontal bacterium. This in turn has led to international efforts to characterize these enzymes at the gene and protein level. Approximately 20 protease genes of P. gingivalis with different names and accession numbers have been deposited in the gene databases and a correspondingly heterogeneous nomenclature system is employed for the products of these genes in the literature. However, it is evident, through comparison of these gene sequences and through gene inactivation studies, that the genetic structure of the proteases of this organism, particularly those with specificity for arginyl and lysyl peptide bonds, is less complicated than originally thought. The major extracellular and surface associated arginine specific protease activity is encoded by 2 genes which we recommend be designated rgpA and rgpB (arg-gingipains A & B). Similarly we recommend that the gene encoding the major lysine specific protease activity is designated kgp (lys-gingipain). These three genes, which account for all the extracellular/surface arginine and lysine protease activity in P. gingivalis, belong to a family of sequence-related proteases and haemagglutinins.

IS195, an insertion sequence-like element associated with protease genes in Porphyromonas gingivalis.

Porphyromonas gingivalis is recognized as an important etiologic agent in adult and early-onset periodontal disease. Proteases produced by this organism contribute to its virulence in mice. Protease-encoding genes have been shown to contain multiple copies of repeated nucleotide sequences. These conserved sequences have also been found in hemagglutinin genes. In the process of studying the genetic loci containing the conserved repeated sequences, we have characterized a prtP gene homolog from P. gingivalis W83 encoding a cysteine protease with Lys-X specificity. However, this prtP gene was interrupted by an insertion sequence-like element which we designated IS195. Furthermore, IS195 and another element, IS1126, were present downstream of prtP gene homologs (kgp) found in P. gingivalis H66 and 381. IS195, a 1,068-bp insertion sequence-like element, contained 11-bp inverted repeats at its termini and was bordered by 9-bp direct repeats presumed to be a transposition-mediated target site duplication. Its central region contained one large open reading frame encoding a predicted 300-amino-acid protein which appeared to be a transposase. We isolated two naturally occurring variants of P. gingivalis W83, one carrying IS195 within the coding region of the prtP gene and another containing an intact prtP gene. Biochemical characterization revealed a lack of trypsin-like Lys-X specific proteolytic activity in the P. gingivalis W83 variant carrying the disrupted prtP gene. Studies using a mouse model revealed a reduction of virulence resulting from insertion of IS195 into the coding region of the prtP gene. An allelic-exchange mutant defective in the prtP gene also was constructed and tested in vivo. It displayed intermediate virulence compared to that of the wild-type and prtP::IS195 mutant strains. We conclude that the Lys-X cysteine protease contributes to virulence in soft tissue infections.

Nucleotide sequence of the Porphyromonas gingivalis W83 recA homolog and construction of a recA-deficient mutant.

Degenerate oligonucleotide primers were used in PCR to amplify a region of the recA homolog from Porphyromonas gingivalis W83. The resulting PCR fragment was used as a probe to identify a recombinant lambda DASH phage (L10) carrying the P. gingivalis recA homolog. The recA homolog was localized to a 2.1-kb BamHI fragment. The nucleotide sequence of this 2.1-kb fragment was determined, and a 1.02-kb open reading frame (341 amino acids) was detected. The predicted amino acid sequence was strikingly similar (90% identical residues) to the RecA protein from Bacteroides fragilis. No SOS box, characteristic of LexA-regulated promoters, was found in the 5' upstream region of the P. gingivalis recA homolog. In both methyl methanesulfonate and UV survival experiments the recA homolog from P. gingivalis complemented the recA mutation of Escherichia coli HB101. The cloned P. gingivalis recA gene was insertionally inactivated with the ermF-ermAM antibiotic resistance cassette to create a recA-deficient mutant (FLL33) by allelic exchange. The recA-deficient mutant was significantly more sensitive to UV irradiation than the wild-type strain, W83. W83 and FLL33 showed the same level of virulence in in vivo experiments using a mouse model. These results suggest that the recA gene in P. gingivalis W83 plays the expected role of repairing DNA damage caused by UV irradiation. However, inactivation of this gene did not alter the virulence of P. gingivalis in the mouse model.

Immunization with FimA protects against Streptococcus parasanguis endocarditis in rats.

FimA, a surface-associated protein of Streptococcus parasanguis, is associated with initial colonization of damaged heart tissue in an endocarditis model (D. Burnette-Curley, V. Wells, H. Viscount, C. Munro, J. Fenno, P. Fives-Taylor, and F. Macrina, Infect. Immun. 63:4669-4674, 1995). We have evaluated the efficacy of recombinant FimA as a vaccine in the rat model of endocarditis and investigated in vitro the mechanism for the protective role of immunization. FimA-immunized and nonimmunized control animals were catheterized to induce heart valve damage and infected intravenously with 10(7) CFU of wild-type S. parasanguis FW213 bacteria. The presence of bacteria associated with platelet-fibrin vegetations 24 h postchallenge was evaluated. Immunized rats were significantly less susceptible to endocarditis (2 cases among 34 animals) than the control group (21 cases among 33 animals) (P < 0.001). Incubation of S. parasanguis FW213 with rabbit anti-FimA immune serum decreased the mean percent adherence (0.34% of added cells) to platelet-fibrin matrix in vitro compared with that of preimmune normal serum (5.04% of added cells; P < 0.001). Adsorption of immune serum with FimA-positive S. parasanguis FW213 yielded antiserum that failed to block adherence to the platelet-fibrin matrix. We assessed the vaccine potential of FimA as a common immunogen able to provide cross-protection in streptococcal endocarditis by determining the occurrence and expression of fimA in the viridans group streptococci and enterococci. We detected the presence of fimA homologs by Southern hybridization and PCR amplification analyses and determined by immunoblotting the expression of FimA-like proteins among a variety of streptococci and enterococci that frequently cause endocarditis. Eighty-one percent (26 of 32) of streptococcal and enterococcal strains isolated from bacteremic patients expressed proteins that comigrated with FimA and were reactive with polyclonal anti-FimA serum. Streptococcal DNA from strains that were positive by Western blot (immunoblot) analysis hybridized to the full-length fimA probe. Our studies suggest that FimA immunization results in antibody-mediated inhibition of bacterial adherence, a critical early event in the pathogenesis of endocarditis. Our data demonstrate that a majority of streptococcal strains associated with endocarditis have genes that encode FimA-like proteins. Taken together, these results suggest that FimA is a promising candidate for an endocarditis vaccine.

Characterization of IS199 from Streptococcus mutans V403.

The complete nucleotide sequence of the insertion sequence IS199 has been determined. This novel element was found in Streptococcus mutans V403 where DNA hybridization studies suggested that it was present in multiple copies on the genome. IS199 is 1220 bp and appears unique to the mutans streptococci. A limited epidemiological survey revealed this element to occur infrequently in this group (4/50 strains examined). IS199 belongs to the IS3 family of procaryotic insertion sequence elements. It contained tandem open reading frames (ORFs) reminiscent of the structure of the IS3 class. Additionally, the larger of the two ORF products (ORFB) exhibited amino acid sequence similarities to comparable proteins from IS3 and other elements of this family. Transposition of IS199 gave rise to a 3-bp target site duplication. We were able to insert an antibiotic resistance gene into IS199 enabling us to follow the transposition of this element in S. mutans V403.

Immunologic characteristics of a Streptococcus mutans glucosyltransferase B sucrose-binding site peptide-cholera toxin B-subunit chimeric protein.

Glucosyltranferases (Gtfs) produced by the mutans streptococci are recognized as virulence factors in dental caries, and the inhibition of Gtfs by secretory immunoglobulin A is predicted to provide protection against this disease. The basis of such mucosal immunity is linked to the ability to reliably stimulate production of secretory immunoglobulin A against Gtfs. In this regard, we are exploring the immunogenicities of various Gtf peptides genetically fused to the B subunit of cholera toxin (CTB), a known mucosal adjuvant. In this work, we have created a gene fusion linking the GtfB active-site (AS) peptide DANFDSIRVDAVDNVDADLLQIA to the amino terminus of CTB. This sequence, deduced from the nucleotide sequence of gtfB from Streptococcus mutans GS5, has been found to be strongly conserved in Gtfs from several mutans streptococci. We have purified this recombinant protein (AS:CTB) from Escherichia coli carrying the fusion gene under the control of the lactose operon promoter. This protein was immunogenic in rabbits and produced specific serum antibodies against both the Gtf peptide and the CTB moiety. The antiserum was tested for its ability to inhibit GtfB activity obtained from a mutant of S. mutans able to make only this enzyme and none of the other usual Gtfs or fructosyltransferase. Approximately 50% of the GtfB activity was inhibited in such assays. These results suggest that the AS of this enzyme is accessible to antibody binding and that this region of the protein may be considered a vulnerable target for vaccine design and development. The AS:CTB was able to bind GM1, ganglioside in enzyme-linked immunosorbent assays, indicating that the recombinant protein retained this property, which is though to be critical to the mucosal immunoadjuvant properties of CTB. Thus, this protein may be promising as a candidate anticaries vaccinogen alone or in combination with other Gtf peptides or conjugates.

FimA, a major virulence factor associated with Streptococcus parasanguis endocarditis.

Adherence of microorganisms to damaged heart tissue is a crucial event in the pathogenesis of infective endocarditis. In the present study, we investigated the role of the FimA protein as a potential virulence factor associated with Streptococcus parasanguis endocarditis. FimA is a 36-kDa surface protein that is a recognized adhesin in the oral cavity where it mediates adherence to the salivary pellicle. An insertion mutant and a deletion mutant of S. parasanguis were employed in the rat model of endocarditis to determine the relevance of FimA in endocarditis pathogenesis. Catheterized rats were infected with either the fimA deletion mutant VT929, the fimA insertion mutant VT930, or the isogenic, wild-type S. parasanguis FW213. Rats inoculated with FW213 developed endocarditis more frequently (50.9%) than animals inoculated with either the deletion mutant (2.7%) or the insertion mutant (7.6%) (P < 0.001). A series of in vitro assays were performed to explore the mechanism(s) by which FimA enhanced the infectivity of S. parasanguis. FimA did not inhibit the uptake or the subsequent killing of S. parasanguis by phagocytic granulocytes. Similarly, FimA did not play a role in the adherence to or the aggregation of platelets. Significant differences were noted between FW213 and VT929 (P < 0.05) and FW213 and VT930 (P < 0.001) in their abilities to bind to fibrin monolayers. The mean percent adherence of FW213 to fibrin monolayers (2.1%) was greater than those of VT929 (0.5%) and VT930 (0.12%). Taken together, these results indicate that FimA is a major virulence determinant associated with S. parasanguis endocarditis and further suggest that its role is associated with initial colonization of damaged heart tissue.

Characterization of six linked open reading frames necessary for pIP501-mediated conjugation.

pIP501 is a broad-host-range gram-positive conjugative plasmid. Previous transposon mutagenesis of a pIP501 derivative, pVA1702, identified two regions, A and B, that were involved in conjugation proficiency. Recently, we reported that the A region contained a functional oriT site similar to those found on plasmids of gram-negative origin (Wang and Macrina, 1995). We report here the nucleotide sequence of the complete A region. Analysis of this DNA sequence (8121 bp) revealed six contiguous open reading frames (orf1-6) immediately downstream from the oriT site. ORF1 was similar to gram-negative plasmid nickase proteins known to play a role in the initiation of conjugal transfer. The remaining ORF proteins (2-6) were unique. Complementation studies suggested that the gene products of ORFs 3-6 were able to function in trans. A single available insertion mutant in orf2 could not be complemented in trans. Thus, although the conjugative apparatus that initiates transfer (oriT and nickase) appears to be conserved between this class of gram-positive plasmid and gram-negative plasmids, most of the A region genes of pIP501 involved in transfer appear unique and their cellular and molecular roles are undefined.

Streptococcal plasmid pIP501 has a functional oriT site.

DNA sequence analysis suggested the presence of a plasmid transfer origin-like site (oriT) in the gram-positive conjugative plasmid pIP501. To test the hypothesis that the putative oriT site in pIP501 played a role in conjugal transfer, we conducted plasmid mobilization studies in Enterococcus faecalis. Two fragments, 49 and 309 bp, which encompassed the oriT region of pIP501, were cloned into pDL277, a nonconjugative plasmid of gram-positive origin. These recombinant plasmids were mobilized by pVA1702, a derivative of pIP501, at a frequency of 10(-4) to 10(-5) transconjugants per donor cell, while pDL277 was mobilized at a frequency of 10(-8) transconjugants per donor cell. These results indicated that the oriT-like site was needed for conjugal mobilization. To demonstrate precise nicking at the oriT site, alkaline gel and DNA-sequencing analyses were performed. Alkaline gel electrophoresis results indicated a single-stranded DNA break in the predicted oriT site. The oriT site was found upstream of six open reading frames (orf1 to orf6), each of which plays a role in conjugal transfer. Taken together, our conjugal mobilization data and the in vivo oriT nicking seen in Escherichia coli argue compellingly for the role of specific, single-stranded cleavage in plasmid mobilization. Thus, plasmid mobilization promoted by pVA1702 (pIP501) works in a fashion similar to that known to occur widely in gram-negative bacteria.

Increased opsonization of a prtH-defective mutant of Porphyromonas gingivalis W83 is caused by reduced degradation of complement-derived opsonins.

Periodontitis is a disease of the supporting structures of the teeth that is caused by bacteria whose common ecologic niche is the gingival crevice or the periodontal pocket. Tissue destruction occurs in spite of both local and systemic immune responses against such bacteria. Porphyromonas gingivalis is considered to be an important pathogen in some forms of human periodontitis and is particularly interesting because of its multiplicity of virulence factors. We have previously observed that phagocytosis-resistant invasive strains of P. gingivalis proteolytically degrade C3 and IgG and accumulate less C3-derived opsonins during complement activation. We recently have cloned the prtH gene from P. gingivalis W83 that encodes a 97-kDa active protease, which has the capacity to degrade purified C3 protein. By using this cloned gene we created an allelic exchange mutant of P. gingivalis W83, designated V2296, in which the prtH gene was inactivated. This mutant was previously shown to be less virulent than its parent strain W83 in a mouse model of bacterial invasiveness. In the present study we have assessed the relative capacity of V2296 and W83 to be opsonized by complement and to be taken up by PMNs. The data demonstrate that V2296, in comparison with its parent strain W83, is less able to degrade C3 and that it accumulates significantly greater numbers of molecules of C3-derived opsonins on the bacterial surface in the form of C3b and iC3b during complement activation. Furthermore, opsonized V2296 is taken up in much higher numbers by human PMNs than W83, suggesting that the prtH gene product may be important in evasion of host defense mechanisms.

Sucrose-derived exopolymers have site-dependent roles in Streptococcus mutans-promoted dental decay.

We have constructed a panel of mutants of S. mutans V403 which are defective in one or more of the glucosyltransferase genes (gtfB, C or D) or the fructosyltransferase gene (ftf). These strains have been tested for virulence in a gnotobiotic rat caries model with reference to both buccal (smooth surface) and sulcal (pit and fissure) carious lesions. Our data suggest differing roles for degradable and non-degradable polymers at buccal and sulcal sites. Non-degradable polymers (made by products of the gtfB and C genes) contributed significantly to the severity of smooth surface lesions. However, our studies suggested their role in pit and fissure lesions was not as important as the role of degradable polymers. Specifically, a mutant deficient in the major insoluble glucan synthesizing activity (product of the gtfB gene) was 25% more cariogenic on sulcal surfaces than was the wild-type V403 strain. We propose that extracellular glucosyltransferases and fructosyltransferase compete for sucrose and that this competition influences pathogenicity at differing tooth sites.

Virulence of a Porphyromonas gingivalis W83 mutant defective in the prtH gene.

In a previous study we cloned and determined the nucleotide sequence of the prtH gene from Porphyromonas gingivalis W83. This gene specifies a 97-kDa protease which is normally found in the membrane vesicles produced by P. gingivalis and which cleaves the C3 complement protein under defined conditions. We developed a novel ermF-ermAM antibiotic resistance gene cassette, which was used with the cloned prtH gene to prepare an insertionally inactivated allele of this gene. This genetic construct was introduced by electroporation into P. gingivalis W83 in order to create a protease-deficient mutant by recombinational allelic exchange. The mutant strain, designated V2296, was compared with the parent strain W83 for proteolytic activity and virulence. Extracellular protein preparations from V2296 showed decreased proteolytic activity compared with preparations from W83. Casein substrate zymography revealed that the 97-kDa proteolytic component as well as a 45-kDa protease was missing in the mutant. In in vivo experiments using a mouse model, V2296 was dramatically reduced in virulence compared with the wild-type W83 strain. A molecular survey of several clinical isolates of P. gingivalis using the prtH gene as a probe suggested that prtH gene sequences were conserved and that they may have been present in multiple copies. Two of 10 isolates did not hybridize with the prtH gene probe. These strains, like the V2296 mutant, also displayed decreased virulence in the mouse model. Taken together, these results suggest an important role for P. gingivalis proteases in soft tissue infections and specifically indicate that the prtH gene product is a virulence factor.

The case study approach to teaching scientific integrity in nursing and the biomedical sciences.

Scientific integrity courses for graduate trainees are emerging at many institutions. The content of such courses varies and is evolving as faculty strive to meet the needs of their specific student audiences. Typical course subject matters include responsible authorship, conflict of interest, handling of scientific misconduct, data management, and the use of humans and animals in biomedical experimentation. These and other topic areas are amenable to teaching methods that use the case study approach. In this article we discuss our experiences in teaching scientific integrity using the case study approach. Issues covered include underlying philosophy of the approach, preparation and use of cases, desired inclass preparation and teaching. Examples of short cases are presented and, where appropriate, discussed in the context of these issues.

Cloning and characterization of a new protease gene (prtH) from Porphyromonas gingivalis.

Porphyromonas gingivalis has been implicated as a contributing etiological agent of adult periodontitis and generalized forms of early-onset periodontitis. Proteases of P. gingivalis may contribute to its pathogenicity by destroying connective tissue as well as inactivating key plasma proteins that might mediate protective host functions. In order to explore this problem, antiserum raised against membrane vesicles of P. gingivalis W83 was used to screen a genomic library of strain W83 constructed by using the lambda DASH vector system. A recombinant phage (lambda 34) expressing a P. gingivalis protease from the library was identified and characterized. Casein substrate zymography of lambda 34 lysates revealed a protease with an apparent molecular mass of 97 kDa. The gene encoding this protease was designated prtH. It was localized to a 3.7-kb HindIII-BamHI fragment and specified an enzyme which hydrolyzed the human C3 complement protein under defined conditions. The nucleotide sequence of this 3.7-kb fragment was determined, and one 2.9-kb open reading frame (992 amino acids) corresponding to a 110-kDa protein was detected, suggesting it might be a precursor of the 97-kDa active protease. prtH is not similar to any previously cloned protease gene from P. gingivalis.

Genetic analysis of fructan-hyperproducing strains of Streptococcus mutans.

Fructan polymer, synthesized from sucrose by the extracellular fructosyltransferase of Streptococcus mutans, is thought to contribute to the progression of dental caries. It may serve as an extracellular storage polysaccharide facilitating survival and acid production. It may also have a role in adherence or accumulation of bacterial cells on the tooth surface. A number of clinical isolates of S. mutans which produce large, mucoid colonies on sucrose-containing agar as a result of increased production of fructan have been discovered. By using eight independent isolates, we sought to determine if such fructan-hyperproducing strains represented a genetically homogeneous group of organisms. Restriction fragment patterns of total cellular DNA were examined by using pulsed-field and conventional gel electrophoresis. Four genetic types which appeared to correlate with the serotype of the organism and the geographic site of isolation were evident. Southern blot analysis of several genetic loci for extracellular enzymes revealed some minor differences between the strains, but the basic genomic organizations of these loci were similar. To evaluate whether the excess fructan produced by these strains enhanced the virulence of these organisms in the oral cavity, it was of interest to create mutants deficient in fructosidase (FruA), the extracellular enzyme which degrades this polymer. The fruA gene was inactivated by allelic exchange in two fructan-hyperproducing strains as well as in S. mutans GS5, a strain which does not hyperproduce fructan. All of the fruA mutant strains were devoid of fructan hydrolase activity when levan was used as a substrate. However, the fructan-hyperproducing strains retained the ability to hydrolyze inulin, suggesting the presence of a second fructosidase with specificity for inulin in these strains.