Cloning, characterization, and possible origin of the Prevotella loescheii dnaK homolog.

Heat shock proteins play an important role in bacterial survival and response to environmental stress. We cloned the Prevotella loescheii HSP70 homolog (dnaK) and characterized the coding sequence, regulatory regions, and evolutionary relationships to other bacteria. Predicted proteins encoded by the P. loescheii dnaK homolog (open reading frame ORF-1) and two downstream coding regions, ORF-2 and ORF-3, are highly homologous to the proteins encoded by ORF-4 (dnaK), ORF-5, and ORF-6 from the dnaK region of Porphyromonas gingivalis. The dnaK promoter resembles other HSP (heat shock protein) promoters. Alignment of the predicted protein encoded by ORF-2 showed significant homology to the Bacteroides fragilis tnpA gene from the transposon Tn4555, whereas the ORF-3 protein showed homology to B. fragilis transposase (Tn5220) and integrase (Tn4555) proteins. This suggests a transposition-like event may be responsible for transfer of these genes between Porphyromonas and Prevotella.

PCR monitoring for tetracycline resistance genes in subgingival plaque following site-specific periodontal therapy. A preliminary report.

BACKGROUND: The selection of antibiotic resistance genes during antibiotic therapy is a critical problem complicated by the transmission of resistance genes to previously sensitive strains via conjugative plasmids and transposons and by the transfer of resistance genes between gram-positive and gram-negative bacteria. The purpose of this investigation was to monitor the presence of selected tetracycline resistance genes in subgingival plaque during site specific tetracycline fiber therapy in 10 patients with adult periodontitis. METHOD: The polymerase chain reaction (PCR) was used in separate tests for the presence of 3 tetracycline resistance genes (tetM, tetO and tetQ) in DNA purified from subgingival plaque samples. Samples were collected at baseline, i.e., immediately prior to treatment, and at 2 weeks, and 1, 3, and 6 months post-fiber placement. The baseline and 6-month samples were also subjected to DNA hybridization tests for the presence of 8 putative periodontal pathogenic bacteria. RESULTS: PCR analysis for the tetM resistance gene showed little or no change in 5 patients and a decrease in detectability in the remaining 5 patients over the 6 months following tetracycline fiber placement. The results for tetO and tetQ were variable showing either no change in detectability from baseline through the 6-month sampling interval or a slight increase in detectability over time in 4 of the 10 patients. DNA hybridization analysis showed reductions to unmeasurable levels of the putative periodontal pathogenic bacteria in all but 2 of the 10 patients. CONCLUSIONS: These results complement earlier studies of tet resistance and demonstrate the efficacy of PCR monitoring for the appearance of specific resistance genes during and after antibiotic therapy.

Mutant analysis of Prevotella sp. plaA-lacZ fusion protein expression in Escherichia coli: support for an essential role of the stem-loop.

This study investigated the involvement of RNA folding in the synthesis of a fusion protein with beta-galactosidase activity. The coding gap region of the Prevotella loescheii adhesin gene plaA was fused in-frame with the Escherichia coli lacZ gene on plasmid pSK105. N-Terminal sequencing of the expressed plaA-lacZ protein indicated that it resulted from translational initiation at a fortuitous ribosomal-binding site within the plaA sequence at nt 570. Specific mutations were introduced in the stem-loop region that precedes the gap sequence. Analysis of stem-loop mutants, together with the introduction of compensatory mutations that restored activity, supports a requirement for stem-loop formation within the plaA sequence preceding the translational initiation site. A mutation reducing the predicted size of the loop, but preserving the stem structure, inactivated fusion protein synthesis. A suppressor mutation predicted to restore the size of the loop restored efficient fusion protein synthesis. In addition, the sequence preceding the translational start site of the plaA-lacZ fusion has several similarities to sequences that function as translational enhancers in prokaryotes. These include a stem-loop structure, an A-U rich region preceding the initiation codon, and a region of homology to 16S rRNA.

The translational hop junction and the 5' transcriptional start site for the Prevotella loescheii adhesin encoded by plaA.

The Prevotella loescheii adhesin gene, plaA, contains a coding gap between a small open reading frame (ORF-1) and a large open reading frame (ORF-2). Translation of the plaA mRNA requires bypassing this 29-nt coding gap on the plaA transcript. We have determined the N-terminal peptide sequence of the SO34 adhesin beyond the gap sequence. This sequence shows that the peptide junction between ORF-1 and ORF-2 is continuous in the adhesin and supports the conclusion that synthesis of the SO34 adhesin occurs by a ribosomal hop mechanism. To elucidate upstream signals, we used the 5' RACE (rapid amplification of cDNA ends) technique to map the start point of the plaA mRNA. DNA sequencing of plasmids with the 5' RACE products placed the 5' end of plaA mRNA 270 nt upstream from the plaA start codon. A region corresponding to a Bacteroides fragilis promoter consensus sequence precedes this start site.

Atypical palatal papillomatosis treated by excision and full-thickness grafting.

Papillary lesions of the oral cavity are extremely common, and inflammatory palatal hyperplasia is well known to dental practitioners. Advanced sophistication in viral laboratory technologies makes it apparent that various forms of the human papilloma virus are often causative. However, this is not true for inflammatory palatal hyperplasia. This article describes a patient with anatomically well-demarcated, multiple squamous cell papillomas of the palate that could not be classified as inflammatory palatal hyperplasia, nor could a viral etiology be ascertained, despite exhaustive laboratory studies. The lesion recurred despite numerous surgical ablation attempts. Eradication was achieved only after applying free soft-tissue grafts over the areas of excision. The differential diagnosis of papillary lesions with an emphasis on viral etiology, laboratory studies associated with their identification, and a hypothesis that explains why grafting was the only successful means of treatment are also discussed.

In vitro effect of the Sensonic toothbrush on Treponema denticola.

The purpose of this in vitro study was to compare the effects of the Sensonic. Oral-B Braun mechanical and Oral-B manual toothbrushes upon the morphology and cellular integrity of Treponema denticola. This spirochete was chosen because of its frequent isolation from active lesions of inflammatory periodontal disease and its pathogenic potential. T. denticola, strain ATCC 33421, was grown in an anaerobic nitrogen rich atmosphere in enhanced 1186 mycoplasma broth. 160, 5-ml aliquots of cultured microbes were assigned to 1 of 3 brushing treatment groups and a control group. Samples were further divided into 4 groups of 10 samples each and assigned to one of 4 brushing exposure times: 15, 30, 45, and 60 seconds. After treatment, 0.2 ml of each sample was applied to a millipore filter and examined by SEM. Intact microbes were counted from 10 non-overlapping fields at 4500x. Remaining treated samples were pelleted and examined by TEM. A statistically significant reduction of intact microbes for the Sensonic treatment group at each exposure time was found when compared to Oral-B Braun, Oral-B manual, and non-treated controls. TEM examination of Sensonic treated samples revealed separation of the outer membrane at lower exposure times and only cellular debris after exposures of 45 and 60 s. These results suggest that exposure to the sonic frequency generated by the Sensonic toothbrush is capable of severely disrupting the structural integrity of T. denticola.

Expression of the Prevotella loescheii adhesin gene (plaA) is mediated by a programmed frameshifting hop.

The 2.4-kb plaA gene, which encodes a Prevotella loescheii galactoside-specific adhesin, contains a programmed frameshifting hop. The frameshift region consists of two UAA termination codons, two repeats of four identical bases between the terminators, and a stem-loop structure that has the potential to form a pseudoknot located downstream from the second UAA. The stem-loop and pseudoknot are features found in a number of retroviruses where frameshifting is a more common occurrence. The terminators, sequence repeats, and secondary structures were identified in both the P. loescheii plaA gene and the mRNA transcript. An in-frame fusion of the entire plaA frameshift region between codons 9 and 10 of the lacZ gene permitted relatively efficient expression (4 to 25% of that of the control) of beta-galactosidase in Escherichia coli.

The gene encoding a Prevotella loescheii lectin-like adhesin contains an interrupted sequence which causes a frameshift.

We cloned and sequenced the Prevotella loescheii gene plaA, which encodes a lectin-like adhesin that mediates the coaggregation of P. loescheii 1295 with Streptococcus oralis 34. A probe derived from the N-terminal amino acid sequence of the purified adhesin was used to identify the plaA gene from a P. loescheii genomic library constructed in lambda GEM-11. Sequence analysis of plaA indicates that the initial translation product contains a 22-amino-acid leader. The reading frame of the plaA gene is interrupted after amino acid 28 of the mature protein by a TAA termination codon. Amplification of the P. loescheii genomic DNA in the region surrounding this codon by the polymerase chain reaction followed by DNA sequencing of the cloned DNA fragment established that this stop codon was not an experimental artifact. A frameshift beginning 29 bp downstream of the ochre terminator was required to access the only large open reading frame in the gene. Amino acid sequences of six purified peptides derived by limited proteolysis of adhesin with endoproteinase Lys-C matched the downstream amino acid sequence derived by translation of the large open reading frame. The gene coding sequence of 2.4 kb contains sufficient information for the synthesis of an 89-kDa protein. A putative rho-independent terminator (delta G = -25.5 kcal/mol [ca. -107 kJ/mol]) was detected 38 bp downstream from the plaA stop codon.

RepC is rate limiting for pT181 plasmid replication.

The effect on pT181 plasmid replication of the concentration of the plasmid-coded initiator protein, RepC, has been analyzed. In one type of experiment, plasmid replication was found to stop immediately after the addition of an inhibitory concentration of chloramphenicol (Cm) to growing cultures. Chromosomal replication showed the slow turnoff that is usual for Cm inhibition. Because plasmid replication rate is determined autogenously, no host factor can be rate limiting, suggesting that the specific factor affected is Rep C. In another type of experiment, we constructed a translational fusion between the repC coding sequence and a translationally inducible Cm-acetylase gene, cat-86, using pUB110 as the carrier replicon. The fusion plasmid showed an eightfold amplification of its own copy number and a similar amplification of a co-resident pT181 plasmid upon Cm induction. The amplified plasmids did not show autocatalytic runaway replication but rather established stable elevated copy numbers, indicating the existence of a secondary level of regulation. These results suggest that RepC is rate limiting for pT181 replication and support the hypothesis that pT181 replication is regulated at the level of RepC synthesis. The nature of the secondary regulation is unknown.