RESUMO
The recombinant plasmid pIP1713 was constructed to analyse the transpositional activity of the insertion sequence IS1181 in Staphylococcus aureus RN4220, Staphylococcus carnosus TM300 and Listeria monocytogenes EGD. This 11.3-kb plasmid contains two genetically different elements: (i) a pE194ts-derived replicon, the ermC gene of which confers resistance to erythromycin in Gram-positive bacteria of several species, and (ii) a copy of IS1181, cloned from S. aureus BM3121, in which the tetracycline resistance gene, tet(T), has been inserted between the transposase-encoded gene and the downstream inverted repeat. When introduced by electroporation into the three bacterial hosts, pIP1713 delivered IS1181 omega tet(T) to various chromosomal sites. Cointegrate structures between pIP1713 and the host chromosome were occasionally detected. Transposition was associated with 8-bp repeats at the insertion sites. IS1181 omega tet(T) could be used for random mutagenesis in Gram-positive bacteria.
Assuntos
Elementos de DNA Transponíveis , Genoma Bacteriano , Listeria monocytogenes/genética , Mutagênese Insercional/métodos , Staphylococcus aureus/genética , Staphylococcus/genética , Sequência de Bases , Clonagem Molecular/métodos , DNA Bacteriano/química , DNA Bacteriano/genética , Vetores Genéticos , Plasmídeos , Mapeamento por RestriçãoRESUMO
Tn5405 is a 12 kb staphylococcal composite transposon delimited by two inverted copies of the insertion sequence IS1182. This transposon carries two aminoglycosides resistance genes, aphA-3 and aadE, an altered gene similar to sat4 from Campylobacter coli BE/G4, and three open reading frames of unknown functions.
Assuntos
Antibacterianos/farmacologia , Elementos de DNA Transponíveis , Resistência Microbiana a Medicamentos/genética , Staphylococcus aureus/genética , Sequência de Aminoácidos , Aminoglicosídeos , Sequência de Bases , Dados de Sequência MolecularRESUMO
Tn5405 (12 kb) is a staphylococcal composite transposon delimited by two inverted copies of IS1182, one of which contains IS1181. The internal part of this transposon carries three antibiotic resistance genes, aphA-3, aadE, and sat4, and three open reading frames (ORFs), orfx, orfy, and orfz, of unknown function. The dispersion of Tn5405 and the genes and ORFs included in this transposon were investigated in 50 epidemiologically unrelated staphylococci carrying aphA-3. Twenty-three maps, distinguishable by the presence or absence of the investigated genes or ORFs and/or by the sizes of the restriction fragments carrying them, were identified. Four isolates carried Tn5405, and 15 other isolates contained a Tn5405-related element. IS1182 was not detected in the aphA-3 regions mapped in 31 isolates which carried the following combinations: orfx, orfy, aadE, sat4, and aphA-3 +/- orfz; orfy, aadE, sat4, and aphA-3 +/- orfz; and aadE, sat4, aphA-3, and orfz. In all isolates, the genes and ORFs investigated were in relative positions similar to those in Tn5405. Thus, the internal part of Tn5405 appeared to be partially conserved with the maintenance, in all of the isolates, of at least the three antibiotic resistance genes.
Assuntos
Genoma Bacteriano , Staphylococcus/genética , DNA Bacteriano/isolamento & purificação , Resistência Microbiana a Medicamentos/genética , Mapeamento de Nucleotídeos , Reação em Cadeia da Polimerase , Staphylococcus/efeitos dos fármacosRESUMO
The repeated nucleotide sequence isolated from a methicillin-resistant Staphylococcus aureus isolate displays the characteristic features of an insertion sequence and was named IS1181. It has a size of 1512 bp and consists of a 1359-bp open reading frame that encodes a 439-amino-acid protein which is predicted to be highly basic and 23-bp terminal inverted complementary repeated sequences exhibiting six mismatches. The three copies of IS1181 isolated from distinct parts of the chromosome of S. aureus, BM3121, are flanked at their ends by 8-bp direct repeats, suggesting a duplication of the target sequence. IS1181 exhibits similarities with IS1165 from Leuconostoc mesenteroides and IS1001 from Bordetella parapertusis. IS1181 was detected in at least two to eight copies in 41 of the 52 S. aureus isolates tested, whereas none of the 26 coagulase-negative staphylococci, 24 streptococci, or 11 enterococci analyzed carried nucleotide sequences hybridizing with IS1181.
Assuntos
Elementos de DNA Transponíveis/genética , DNA Bacteriano/química , Nucleotidiltransferases/genética , Staphylococcus aureus/genética , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , DNA Bacteriano/isolamento & purificação , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Dados de Sequência Molecular , Nucleotidiltransferases/biossíntese , Fases de Leitura Aberta , Plasmídeos , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Staphylococcus aureus/enzimologia , TransposasesRESUMO
A new staphylococcal composite transposon, designated Tn5405, carrying the genes aphA-3 and aadE, which encode resistance to aminoglycosides, was partially characterized. The transposon is 12 kb long and is flanked by inverted repeated sequences displaying the characteristic features of an insertion sequence, named IS1182. This insertion sequence is 1864 bp long and has 23/33-bp imperfect inverted repeats at its ends. One of the IS1182 copies delimiting Tn5405 contains a copy of IS1181 flanked by 8-bp direct repeats. Tn5405 was found in the chromosome of MRSA clinical isolate BM3121, within a Tn552-related transposon, Tn5404. Tn5404 was previously characterized following its transposition onto a beta-lactamase plasmid harbored by BM3121. Two forms of the recombinant beta-lactamase-encoding plasmid generated by the inversion of Tn5405 within Tn5404 were detected. IS1182 was not detected in the DNA of 4 of the 17 tested MRSA isolates containing aphA-3 and resistant to streptomycin. Thus, aphA-3 and aadE genes are not disseminated only by Tn5405 or related transposons delimited by IS1182.
Assuntos
Aminoglicosídeos/farmacologia , Elementos de DNA Transponíveis , DNA Bacteriano , Resistência Microbiana a Medicamentos/genética , Staphylococcus aureus/genética , Sequência de Aminoácidos , Sequência de Bases , Inversão Cromossômica , Cromossomos Bacterianos , Dados de Sequência Molecular , Plasmídeos , Mapeamento por Restrição , Análise de Sequência de DNARESUMO
The plasmid plP1066, harboured by by a methicillin-resistant Staphylococcus aureus strain isolated in France, carries genes specifying beta-lactamase. This plasmid undergoes numerous rearrangements. One of these was insertion, between the genes binR and sin encoding resolvases, of a 16 kb element which displayed the characteristic features of a transposon. This putative transposon, named Tn5404, carried genes encoding proteins involved in its transposition, as well as a resolution system, which were indistinguishable from those of the S. aureus transposon Tn552. These were: p480 encoding a probable transposase, p271 encoding a putative ATP-binding protein, binL encoding a resolvase, and a resolution site, resL. In addition, Tn5404 carried aminoglycoside-resistance genes (aphA, str) and the insertion sequence IS1181. Tn5404 contained at its termini 116 bp imperfect inverted repeats, similar to those of Tn552, and was flanked by 6 bp direct repeats. Insertion of Tn5404 close to resR and to the structural and regulatory beta-lactamase genes (blaZ, blal, blaR1) of pIP1066, generated a 3.5 kb invertible segment flanked by inversely repeated resolution sites (resR, resL). This invertible segment, which carried p480, p271 and binL, generated in Tn552 or Tn5404, depending on its orientation. Thus, these two transposons share their transposition and resolution systems.
Assuntos
Inversão Cromossômica , Elementos de DNA Transponíveis/genética , Resistência a Meticilina/genética , Plasmídeos/genética , Staphylococcus aureus/genética , beta-Lactamases/genética , Sequência de Bases , Clonagem Molecular , DNA Nucleotidiltransferases/genética , Escherichia coli/genética , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Análise de Sequência de DNA , Staphylococcus aureus/enzimologia , TransposasesRESUMO
Yersinia pestis, the causative agent of plague, is a highly uniform clone that diverged recently from the enteric pathogen Yersinia pseudotuberculosis. Despite their close genetic relationship, they differ radically in their pathogenicity and transmission. Here, we report the complete genomic sequence of Y. pseudotuberculosis IP32953 and its use for detailed genome comparisons with available Y. pestis sequences. Analyses of identified differences across a panel of Yersinia isolates from around the world reveal 32 Y. pestis chromosomal genes that, together with the two Y. pestis-specific plasmids, to our knowledge, represent the only new genetic material in Y. pestis acquired since the the divergence from Y. pseudotuberculosis. In contrast, 149 other pseudogenes (doubling the previous estimate) and 317 genes absent from Y. pestis were detected, indicating that as many as 13% of Y. pseudotuberculosis genes no longer function in Y. pestis. Extensive insertion sequence-mediated genome rearrangements and reductive evolution through massive gene loss, resulting in elimination and modification of preexisting gene expression pathways, appear to be more important than acquisition of genes in the evolution of Y. pestis. These results provide a sobering example of how a highly virulent epidemic clone can suddenly emerge from a less virulent, closely related progenitor.