Jürg Meyer

The plasmid cloning vector pBR325 contains a 482 base-pair-long inverted duplication

The plasmid pBR325 is a cloning vector constructed in vitro by addition of the chloramphenicol resistance (Cmr) gene of an IS1-flanked transposon to pBR322 (Bolivar, 1978). It is a 5 995 bp plasmid carrying no sequence originating from IS1. DNA-sequence data suggest that its Cmr segment was derived from a Cm transposon longer than Tn9. The plasmid pBR325 carries between the Cmr and Tcr genes a 482 bp sequence which duplicates, in the opposite orientation, a section pf pBR322 located at the end of the tcr gene. The same structure was found in pBR328, a deletion derivative of pBR325 (Soberon et al., 1980). The possible implications of this inverted duplication on cloning experiments are discussed.

Does the insertion element IS1 transpose preferentially into A+T-rich DNA segments?

SummaryIS1-mediated insertion and deletion formation occur preferentially into A+T-rich regions of DNA of bacteriophage P1 and of the r-determinant of the R plasmid NR1. The significance of this correlation is discussed in view of other published data.

The insertion element IS1 is a natural constituent of coliphage P1 DNA

Abstract The presence of one copy of the insertion element IS1 in P1 DNA at map unit 20 of the physical genome map is revealed by restriction enzyme cleavage patterns and electron microscopy. This IS1 element is cleaved once by the restriction endonuclease PstI and extends about 500 to 600 base pairs to the left and 200 to 300 base pairs to the right of the unique PstI cleavage site of P1 DNA. Two P1Cm derivatives, P1Cm246 and P1Cm89, carrying a chloramphenicol resistance determinant contain DNA insertions with two terminal directly repeated IS1 elements. Insertion of such IS1-mediated transposition elements may occur at the IS1 site in the P1 genome or at other sites. The significance of IS1 as a natural constitutent of P1 DNA is discussed.

Plasmid analysis and restriction fragment length polymorphisms of chromosomal DNA allow a distinction between Borrelia burgdorferi strains.

We examined the relationships of the genomes of five strains of Borrelia burgdorferi isolated from ticks, two from North America, including the type strain B31, and three from Switzerland. We determined restriction fragment length polymorphisms by using eight cloned DNA fragments as hybridization probes to genomic Southern blots. Two divergent patterns were observed, represented by B31 and one Swiss strain on the one hand and the two other Swiss strains on the other. The second American strain resembled B31. One of the DNA probes allowed distinction between the closely related strains within a group. The close resemblance of one Swiss strain to the North American strains suggests the possibility of their European origin. All five strains carried a circular plasmid of about 29 kb, and three contained an additional species of about 9 kb, both of which exhibited homology between the strains. The profiles of linear plasmids revealing species of 5.1 kb to 58 kb reflected the polymorphisms of chromosomal DNAs. Linear plasmids of similar size shared DNA sequence homology. Some of the smaller plasmids tended to become lost during cultivation.

Antimicrobial susceptibility of periodontopathogenic bacteria

OBJECTIVES The aim of this study was to evaluate the resistance profiles of Aggregatibacter (Actinobacillus) actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia/Prevotella nigrescens and to detect possible changes in antibiotic resistance over the time period of 1991-2005. METHODS A. actinomycetemcomitans (125 strains), P. gingivalis (152 strains) and P. intermedia/P. nigrescens (326 strains) isolated during the years 1991-2005 were tested for their susceptibility to amoxicillin/clavulanic acid, clindamycin, metronidazole, phenoxymethylpenicillin and tetracycline using the Etest. RESULTS No antibiotic resistance was detected in P. gingivalis, whereas a few isolates of P. intermedia were not susceptible to clindamycin (0.9%), phenoxymethylpenicillin (13.5%) or tetracycline (12.6%). Amoxicillin/clavulanic acid, tetracycline and metronidazole were the most effective antibiotics against A. actinomycetemcomitans with 0%, 0.8% and 20.8% non-susceptible isolates, respectively. However, 88% of the A. actinomycetemcomitans isolates were non-susceptible to phenoxymethylpenicillin and 88% to clindamycin. When strains isolated in the years 1991-94 were compared with those isolated in the years 2001-04, there was no statistically significant difference in the percentage of A. actinomycetemcomitans strains non-susceptible to clindamycin, metronidazole or phenoxymethylpenicillin, or in the percentage of P. intermedia strains non-susceptible to phenoxymethylpenicillin or tetracycline (P > 0.4 each). CONCLUSIONS Increasing antibiotic resistances in periodontopathogenic bacteria are not yet a problem in the Northern part of Switzerland.

Functional characterization of the prokaryotic mobile genetic element IS26

SummaryIS26L and IS26R are the 820 bp long elements found as direct repeats at both ends of the kanamycin resistance transposon Tn2680. They can mediate cointegration in E. coli K12 which contains no IS26 in its chromosome. Cointegration occurs in rec+ or recA- strains with similar frequency. Upon cointegration mediated by either IS26R or IS26L, the element is duplicated and integrated into one of many different sites. Both IS26L and IS26R carry 14 bp perfect terminal inverted repeats and generate 8 bp direct repeats at their target sequences. Deletion formation mediated by IS26R was also observed. These functional and structural features of IS26 are characteristic of a prokaryotic mobile genetic element.

Amplification of chloramphenicol resistance transposons carried by phage P1Cm in Escherichia coli.

SummaryWe have characterized a number of P1Cm phages which contain the resistance genes to chloramphenicol and fusidic acid as IS1-flanked Cm transposons. Restriction cleavage and electron microscopic analysis showed that these Cm transposons were carried as monomers (M) or tandem dimers (D). Lysogens of P1Cm (D) are more resistant to chloramphenicol than those of its P1Cm (M) presumably as a result of an increased gene dosage. Amplification of the Cm transposons to tandem multimers was frequently observed in P1Cm (D) lysogens grown in the presence of high concentrations of chloramphenicol or fusidic acid and was also detected in P1Cm (M) lysogens. The degree of amplification varied in different clones which suggests that cells containing spontaneously amplified Cm transposons were selected by high doses of the antibiotics. The dimeric as well as the amplified Cm transposons carried in P1Cm lysogens grown in the absence of chloramphenicol displayed considerable stability. Mechanisms for the amplification of the IS1-flanked transposons are discussed.

The kanamycin resistance transposon Tn2680 derived from the R plasmid Rts1 and carried by phage P1Km has flanking 0.8-kb-long direct repeats.

Abstract Phage P1Km carries within the invertible DNA segment a 5-kb insertion with 0.8-kb terminal direct repeats flanking the kanamycin resistance determinant. The same structure was also found on the R plasmid Rts1, from which the Km resistance segment of P1Km was derived. Obviously, this Km resistance segment translocated as a unit to the P1 genome and it is therefore called Tn 2680 . Loss of the Km resistance determinant due to recombination between the flanking direct repeats occurs during vegetative growth of P1Km. Amplification of Tn 2680 to tandem oligomers is documented and is thought to result from recombination between the flanking direct repeats. The flanking 0.8-kb repeats are different from known IS elements.

Complete Genomic Nucleotide Sequence of the Temperate Bacteriophage AaΦ23 of Actinobacillus actinomycetemcomitans

The entire double-stranded DNA genome of the Actinobacillus actinomycetemcomitans bacteriophage Aa Phi 23 was sequenced. Linear DNA contained in the phage particles is circularly permuted and terminally redundant. Therefore, the physical map of the phage genome is circular. Its size is 43,033 bp with an overall molar G+C content of 42.5 mol%. Sixty-six potential open reading frames (ORFs) were identified, including an ORF resulting from a translational frameshift. A putative function could be assigned to 23 of them. Twenty-three other ORFs share homologies only with hypothetical proteins present in several bacteria or bacteriophages, and 20 ORFs seem to be specific for phage Aa Phi 23. The organization of the phage genome and several genetic functions share extensive similarities to that of the lambdoid phages. However, Aa Phi 23 encodes a DNA adenine methylase, and the DNA packaging strategy is more closely related to the P22 system. The attachment sites of Aa Phi 23 (attP) and several A. actinomycetemcomitans hosts (attB) are 49 bp long.

Transduction of antibiotic resistance markers among Actinobacillus actinomycetemcomitans strains by temperate bacteriophages Aaφ23

Abstract.Actinobacillus actinomycetemcomitans (Aa) strain ST1 carries the tetracycline (Tc) resistance transposon Tn916 and the Aaφ ST1 prophage, which is closely related to temperate bacteriophage Aaφ23. 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 μg Tc/ml). TcR transductants (MIC ≥ 32 μg Tc/ml) were detected at frequencies of 3 × 10−6 to 5 × 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, Aaφ23 and AaφST1, were capable of transducing the chloramphenicol (Cm) resistance marker of plasmid pKT210 (transduction frequencies of 2 × 10−5 to 3 × 10−7 per pfu) to the recipient strain ZIB1001 (MIC 8 μg Cm/ml). Eleven CmR ZIB1001 transductants (MIC ≥ 100 μg 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.