Sequence requirements for target activity in site-specific recombination mediated by the Int protein of transposon Tn 1545.

Excision and integration of Tn1545 occur by reciprocal site-specific recombination between 6 (or 7) bp variable sequences present in the recombining attachment (att) sites and designated overlap regions. We devised an assay for Tn1545 transposition in which derivatives containing the cis-acting transposition sequences (attTn 1545) integrate into a target replicon when complemented in trans by the transposon-encoded integrase Int-Tn. This assay was used to determine the characteristics of the DNA sequence that influence target site selection.

[Multifactorial analysis of the phenotypes for beta-lactams of 1044 Escherichia coli strains].

1,044 E. coli strains were randomly collected by the beginning of 1992. Their susceptibility for seven beta-lactam antibiotics: amoxycillin, augmentin, ticarcillin, claventin, cephalothin, cefoxitin and cefotaxime, was studied routinely by the agar diffusion method.

Genetic basis of tetracycline resistance in clinical isolates of Listeria monocytogenes.

The genetic basis of tetracycline resistance was studied in 25 clinical isolates of Listeria monocytogenes. Resistance to tetracycline was associated with resistance to minocycline and due to the presence of the tet(M) gene in 24 strains. Association of tet(M) with int-Tn, the gene encoding the protein required for the movements of Tn1545-like conjugative transposons, was found in all strains.

An integrative vector exploiting the transposition properties of Tn1545 for insertional mutagenesis and cloning of genes from gram-positive bacteria.

We have constructed and used an integrative vector, pAT112, that takes advantage of the transposition properties (integration and excision) of transposon Tn1545. This 4.9-kb plasmid is composed of: (i) the replication origin of pACYC184; (ii) the attachment site (att) of Tn1545; (iii) erythromycin-and kanamycin-resistance-encoding genes for selection in Gram- and Gram+ bacteria; and (iv) the transfer origin of IncP plasmid RK2, which allows mobilization of the vector from Escherichia coli to various Gram+ recipients.

Nucleotide sequences specific for Tn1545-like conjugative transposons in pneumococci and staphylococci resistant to tetracycline.

The distributions of tet(M) and conjugative transposons related to Tn1545 were studied by hybridization in 47 clinical isolates of Streptococcus pneumoniae resistant to tetracycline. Resistance to tetracycline was always associated with resistance to minocycline and was due to the presence of the tet(M) gene. Association of tet(M) with int-Tn, the gene encoding the protein required for the movements of Tn1545-like transposons, was found in all but one strain of S.

Conjugative transposition of Tn916 requires the excisive and integrative activities of the transposon-encoded integrase.

Transposon Tn916 is a 16.4-kb broad-host-range conjugative transposon originally detected in the chromosome of Enterococcus faecalis DS16. Transposition of Tn916 and related transposons involves excision of a free, nonreplicative, covalently closed circular intermediate that is substrate for integration.

Shuttle vectors containing a multiple cloning site and a lacZ alpha gene for conjugal transfer of DNA from Escherichia coli to gram-positive bacteria.

The mobilizable shuttle cloning vectors, pAT18 and pAT19, are composed of: (i) the replication origins of pUC and of the broad-host-range enterococcal plasmid pAM beta 1; (ii) an erythromycin-resistance-encoding gene expressed in Gram- and Gram+ bacteria; (iii) the transfer origin of the IncP plasmid RK2; and (iv) the multiple cloning site and the lacZ alpha reporter gene of pUC18 (pAT18) and pUC19 (pAT19). These 6.6-kb plasmids contain ten unique cloning sites that allow screening of derivatives containing DNA inserts by alpha-complementation in Escherichia coli carrying the lacZ delta M15 deletion, and can be efficiently mobilized by self-transferable IncP plasmids co-resident in the E.

The integration-excision system of the conjugative transposon Tn 1545 is structurally and functionally related to those of lambdoid phages.

Excision of Tn1545 and related conjugative transposons of Gram-positive bacteria occurs by reciprocal site-specific recombination between non-homologous regions of the transposon-target junctions. Excisive recombination requires two transposon-encoded proteins designated Xis-Tn and Int-Tn. We have shown that, following excision, Tn1545 is a circular structure with ends separated by either of the two hexanucleotides that were present at the transposon-target junctions.

Transferable plasmid-mediated antibiotic resistance in Listeria monocytogenes.

A strain of Listeria monocytogenes, isolated from a patient with meningoencephalitis, was resistant to chloramphenicol, erythromycin, streptomycin, and tetracycline. The genes conferring resistance to these antibiotics were carried by a 37-kb plasmid, pIP811, that was self-transferable to other L monocytogenes cells, to enterococci-streptococci, and to Staphylococcus aureus. The efficacy of transfer and the stability of pIP811 were higher in enterococci-streptococci than in the other gram-positive bacteria.

Antibacterial and plasmid curing activity of lomefloxacin in vitro.

The in vitro activity of lomefloxacin in comparison to other recently developed quinolones was evaluated by determination of MICs for 89 test strains. Organisms tested included clinical isolates of gram-positive and gram-negative bacteria susceptible or resistant to quinolones, resistant mutants of Enterobacteriaceae, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis obtained in vitro, and the wild type strain Escherichia coli K12 with its mutants na1A, nalB, nalC and nalB. The activity of lomefloxacin was similar to that of pefloxacin against gram-positive cocci and similar to that of norfloxacin against gram-negative bacteria.

Molecular characterization of two proteins involved in the excision of the conjugative transposon Tn1545: homologies with other site-specific recombinases.

Excision is probably the initial and rate-limiting step of the movements of conjugative transposons of Gram-positive bacteria such as Tn916 and Tn1545. We have shown, by molecular cloning and DNA sequencing, that a 2058 bp Sau3A right-junction fragment of transposon Tn1545 specifies two gene products that are involved in the excision of the element. The DNA sequence of these genes, designated orf1 and orf2, has been determined and the corresponding proteins, ORF1 and ORF2, have been identified in a bacterial cell-free coupled transcription-translation system.

[Effect of various antibiotics on granulopoiesis, in mice treated with cyclophosphamide, in an in vivo model of experimental C. albicans infection].

The effect of piperacillin (PIP), cefotaxime (CTX), cefoxitin (CXT) on the natural resistance to C. albicans infection has been evaluated in vivo, in normal or neutropenic mice, in correlation with the PMN count in the peripheral blood. In neutropenic mice treated with PIP or CTX, the number of PNN increased more rapidly and higher than in CXT treated or control mice.