RESUMO
Analysis of the 50-kb R-plasmid pTP10 from the clinical isolate Corynebacterium xerosis M82B revealed that the erythromycin resistance gene, ermCX, is located on a 4524-bp composite transposable element, Tn5432. The ends of Tn5432 are identical, direct repeats of an insertion sequence, designated IS1249, encoding a putative transposase of the IS256 family. IS1249 consists of 1385 bp with 45/42 imperfect terminal inverted repeats. The nucleotide sequence of the 1754-bp Tn5432 central region is 99% identical to the previously sequenced erythromycin resistance region of the Corynebacterium diphtheriae plasmid pNG2. It encodes the erythromycin resistance gene, ermCX, and an ORF homologous to the amino-terminal end of the transposase of IS31831 from Corynebacterium glutamicum. Transposons with regions flanking the insertion sites were recovered from the C. glutamicum chromosome by a plasmid rescue technique. Insertion of Tn5432 created 8-bp target site duplications. A Tn5432-induced isoleucine/valine-auxotrophic mutant was found to carry the transposon in the 5' region of the ilvBNC cluster; in pTP10 the transposon is inserted in a region similar to replication and partitioning functions of the Enterococcus faecalis plasmid pAD1 and the Agrobacterium tumefaciens plasmid pTAR.
Assuntos
Corynebacterium/genética , Elementos de DNA Transponíveis , Genes Bacterianos , Sequência de Aminoácidos , Antibacterianos/farmacologia , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Corynebacterium/efeitos dos fármacos , Corynebacterium diphtheriae/efeitos dos fármacos , Corynebacterium diphtheriae/genética , DNA Nucleotidiltransferases/genética , Replicação do DNA/genética , DNA Bacteriano/genética , Resistência Microbiana a Medicamentos/genética , Eritromicina/farmacologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Sequências Repetitivas de Ácido Nucleico , TransposasesRESUMO
The clinical isolate Corynebacterium xerosis M82B carries the 50-kb R-plasmid pTP10 that confers resistance to the antibiotics chloramphenicol, kanamycin, erythromycin, and tetracycline. A detailed restriction map of pTP10 was constructed by cloning and analyzing restriction fragments of pTP10 in Escherichia coli. The resistance determinants of pTP10 were located by studying the phenotype of the recombinant plasmids in E. coli and Corynebacterium glutamicum. Restriction patterns of fragments encoding the kanamycin and erythromycin resistances revealed striking similarity to the kanamycin resistance of transposon Tn903 and the erythromycin resistance on plasmid pNG2 from Corynebacterium diphtheriae, respectively. Expression of the resistance determinants in E. coli and C. glutamicum ATCC 13032 led to high resistance levels in both strains, with the exception of the tetracycline resistance gene, which could be expressed only in C. glutamicum. Furthermore, the erythromycin resistance gene was found to be located on a transposable element which is functional in C. glutamicum strains.
