A PCR assay for the amplification of small subunit ribosomal DNA (SSU rDNA) of Euryarchaea was developed and used to detect archaeal rDNA in 37 (77%) out of 48 pooled subgingival plaque samples from 48 patients suffering from periodontal disease. One major group of cloned periodontal sequences was identical to Methanobrevibacter oralis and a second minor group to Methanobrevibacter smithii. These two groups and a third novel group were found to be more than 98% similar to each other over an 0.65-kb segment of the 16S rRNA gene sequenced. M. oralis was found to be the predominant archaeon in the subgingival dental plaque. Phylogenetic analysis of partial SSU rDNA sequences revealed evidence for a distinct cluster for human and animal Methanobrevibacter sp. within the Methanobacteriaceae family. ß

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Plasmid pl5B and the genome of bacteriophage P1 are closely related, but their site-specific DNA inversion systems, Min and Cin, respectively, do not have strict structural homology. Rather, the complex Min system represents a substitution of a Cin-like system into an ancestral pl5B genome. The substituting sequences of both the min recombinase gene and the multiple invertible DNA segments of pl5B are, respectively, homologous to the pin recombinase gene and to part of the invertible DNA of the Pin system on the defective viral element e14 ofEscherichia coli K-12. To map the sites of this substitution, the DNA sequence of a segment adjacent to the invertible segment in the P1 genome was determined. This, together with already available sequence data, indicated that both P1 and pl5B had suffered various sequence acquisitions or deletions and sequence amplifications giving rise to mosaics of partially related repeated elements. Data base searches revealed segments of homology in the DNA inversion regions of pl5B, e14, and P1 and in tail fiber genes of phages Mu, T4, P2, and K. This result suggests that the evolution of phage tail fiber genes involves horizontal gene transfer and that the Min and Pin regions encode tail fiber genes. A functional test proved that the pl5B Min region carries a tail fiber operon and suggests that the alternative expression of six different gene variants by Min inversion offers extensive host range variation.Plasmid p15B is a P1 prophage-related resident of Escherichia coli 15T-(18). Electron microscopy heteroduplex studies revealed that approximately 82% of the 94-kb pl5B DNA hybridizes with the P1 genome (23). Since no phage particle with a P1-like morphology could be produced in E. coli 15T-(1, 18), pl5B DNA has been considered to be a defective prophage. One region of nonhomology between pl5B and P1 DNA covers the invertible segment of the P1 DNA (16, 23). Its corresponding region on pl5B carries a complex site-specific DNA inversion system called Min (17,34 Fig. 4).The complex Min system is closely related to simple DNA inversion systems which contain two crossover sites and constitute a two-state switch for gene expression: Cin of phage P1, Gin of phage Mu, Pin of the e14 element on E. coli K-12, and Hin of Salmonella typhimurium The crossover sites and recombinase genes of these systems are structurally homologous and can mutually substitute for their functions (for a review of these DNA inversion systems, called here the Din family, see reference 7; see also Fig. 4) The comparison of the DNA sequences of the pl5B Min region with previously determined sequences of the Cin region of phage P1 suggested that the invertible segment and part of the recombinase gene had been substituted by nonhomologous DNA on an ancestral pl5B genome (33). While one of the breakpoints from homologous to nonhomologous sequences had already been located within the recombinase genes, the second breakpoint remained undetermined because of incomplete P1 sequence data of this region.In this paper, the DNA ...

Plasmid pl5B of Escherichia coli 15T-carries a 3.5-kilobase segment that undergoes frequent DNA inversion mediated by the DNA inversion enzyme Min, a member of the Din family of site-specific recombinases. While the previously described Din inversion systems invert a DNA segment between two crossover sites in inverted orientation, the Min system produces more complex DNA rearrangements. These have been physically characterized by electron microscopy and by restriction cleavage analysis. The results can best be explained by a model that involves six crossover sites (called mix) and predicts 240 isomeric forms of the invertible region.The model was confirmed by sequencing the six mix sites in plasmids that contain the invertible DNA segments in a frozen configuration. All mix sites fit the dix consensus sequence, and they are all good substrates for DNA inversion when carried in inverted orientation. Recombination between two mix sites in direct orientation was rare, in line with the notion that Din inversion systems are topologically biased to the inversion reaction. Another recently described multiple inversion system, the shufflon of the E. coli plasmid R64, is neither functionally nor structurally related to the Min system of pl5B.gene also encodes a recombinational enhancer element (unpublished data). In contrast to all previously described Din systems, which invert a DNA segment between two crossover sites, the Min system (Min for multiple DNA inversion) causes more complex DNA rearrangements. The characterization of this mobile DNA region of p15B is the subject of this report.During our studies another example of clustered DNA inversion segments has been described on the R64 plasmid of E. coli (10). In this system, which has been named "shufflon," four DNA segments can invert independently or in groups resulting in complex DNA rearrangements. DNA inversion is brought about by site-specific recombination mediated by the Rci recombinase. Although the Min system of pl5B and the shufflon of R64 show a similar clustering of overlapping invertible DNA segments, they are not related because the DNA sequences of neither the recombinase genes (unpublished data) nor, as we show here, the crossover sites are homologous. In turn, the Rci enzyme shares significant sequence homologies with the recombinases of the integrase family (11).The prokaryotic site-specific DNA recombination systems that have been well characterized so far can be classified into two groups on the basis of their structural and functional homologies and of their mechanistic properties (1,2). One group of loosely related enzymes includes the integrases of phage A (3) and other lambdoid phages, the Cre recombinase of phage P1 (4), and the eukaryotic FLP enzyme of yeast (5).The other group (6) is subdivided into two families: one comprises the resolvases of the Tn3 family of transposons, while the other family consists of a number of DNA invertases (Din family). Site-specific recombinases for the latter are found in the genomes of phages P1 (Ci...

Bacteriophage P1 mutants with the 8.86-kb region between the invertible C-segment and the residential IS1 element deleted from their genome are still able to grow vegetatively and to lysogenize stably, but they show several phenotypic changes. These include the formation of minute plaques due to delayed cell lysis, the abundant production of small-headed particles, a lack of specific internal head proteins, sensitivity to type I host restriction systems, and altered properties to mediate generalized transduction. In the wild-type P1 genome, the accessory genes encoding the functions responsible for these characters are localized in the darA operon that is transcribed late during phage production. We determined the relevant DNA sequence that is located between the C-segment and the IS1 element and contains the cin gene for C-inversion and the accessory genes in the darA operon. The darA operon carries eight open reading frames that could encode polypeptides containing >100 amino acids. Genetic studies indicate that some of these open reading frames, in particular those residing in the 5' part of the darA operon, are responsible for the phenotypic traits identified. The study may contribute to a better comprehension of phage morphogenesis, of the mobilization of host DNA into phage particles mediating generalized transduction, of the defense against type I restriction systems, and of the control of host lysis.

Actinobacillus actinomycetemcomitans (Aa) strain ST1 carries the tetracycline (Tc) resistance transposon Tn916 and the Aa phi ST1 prophage, which is closely related to temperate bacteriophage Aa phi 23. High titre phage preparations were obtained from this strain by mitomycin C induction and were used to transduce the TcR determinant to the TcS recipient strains ZIB1001 and ZIB1015 (MIC 2 micrograms Tc/ml). TcR transductants (MIC > or = 32 micrograms Tc/ml) were detected at frequencies of 3 x 10(-6) to 5 x 10(-8) per pfu. All TcR transductants examined contained the entire Tn916 inserted at several different locations within the Aa genome. They appear to have resulted from generalized transduction. In addition both bacteriophages, Aa phi 23 and Aa phi ST1, were capable of transducing the chloramphenicol (Cm) resistance marker of plasmid pKT210 (transduction frequencies of 2 x 10(-5) to 3 x 10(-7) per pfu) to the recipient strain ZIB1001 (MIC 8 micrograms Cm/ml). Eleven CmR ZIB1001 transductants (MIC > or = 100 micrograms Cm/ml) studied carried a plasmid indistinguishable from pKT210 by restriction analyses. In view of the high prevalence of this phage family, and the increasing use of tetracycline in periodontitis therapy, these findings may have clinical importance.

Actinobacillus actinomycetemcomitans is a suspected etiologic agent in destructive periodontal diseases. The detection of bacteriophages in A. actinomycetemcomitans in the subgingival plaque of patients with rapidly destructive forms of periodontitis led to the hypothesis that bacteriophage infection might increase the virulence of this bacterium (19). A. actinomycetemcomitans was isolated from 68 subjects from the Netherlands and Switzerland with localized juvenile periodontitis, rapidly progressing periodontitis, or adult periodontitis, and was tested for the presence of temperate bacteriophage with the overlay plate technique. More than half of the A. actinomycetemcomitans strains were found to release bacteriophage which formed individual plaques on indicator strains. Electron microscopy of preparations from 7 strains revealed virions with an icosahedral head and a contractile tail typical for double-stranded DNA bacteriophages. The presence of A. actinomycetemcomitans carrying temperate bacteriophage was not correlated with the composition of the subgingival microflora nor with the clinical form of periodontal disease. Destructive periodontal disease of subjects with phage-carrying A. actinomycetemcomitans was not more severe than of subjects with phage-free A. actinomycetemcomitans as determined by several clinical parameters. In contrast, the pocket depth and the attachment loss were significantly lower for adult periodontitis subjects with phage-carrying A. actinomycetemcomitans. It seems unlikely that the frequently occurring temperate bacteriophages increase significantly the virulence of A. actinomycetemcomitans.

Plasmid p15B is a bacteriophage P1-related resident of Escherichia coli 15T-. Both genomes contain a segment in which DNA inversion occurs, although this part of their genomes is not identical. This DNA segment of p15B was cloned in a multicopy vector plasmid. Like its parent, the resulting plasmid, pAW800, undergoes complex multiple DNA inversions: this DNA inversion system is therefore called Min. The min gene, which codes for the p15B Min DNA invertase, can complement the P1 cin recombinase gene. The Min inversion system is thus a new member of the Din family of site-specific recombinases to which Cin belongs. The DNA sequence of the min gene revealed that Min is most closely related to the Pin recombinase of the e14 defective viral element on the E. coli K12 chromosome. Like other members of the Din family, the min gene contains a recombinational enhancer element which stimulates site-specific DNA inversion 300-fold.