RESUMO
The AmpC beta-lactamase gene and a small portion of the regulatory ampR sequence of Enterobacter aerogenes 97B were cloned and sequenced. The beta-lactamase had an isoelectric point of 8 and conferred cephalosporin and cephamycin resistance on the host. The sequence of the cloned gene is most closely related to those of the ampC genes of E. cloacae and C. freundii.
Assuntos
Proteínas de Bactérias , Enterobacter aerogenes/genética , beta-Lactamases/genética , Sequência de Aminoácidos , Sequência de Bases , Cefalosporinas/farmacologia , Cefamicinas/farmacologia , Cromossomos Bacterianos , DNA Bacteriano/análise , Resistência Microbiana a Medicamentos , Enterobacter aerogenes/efeitos dos fármacos , Enterobacter aerogenes/enzimologia , Focalização Isoelétrica , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , beta-Lactamases/química , beta-Lactamases/classificaçãoRESUMO
A 7-kb fragment of pACM1 (fragment 90¿91) containing one or more kor (kill-override) loci was sequenced, and 28 open reading frames (ORFs; >/=50 codons) were identified. The nucleotide sequence has no significant homologs in the GenBank database except for a 1.3-kb region 98.6% identical to the iml (insensitivity to phage PhiM-mediated lysis) determinant fragment of IncM plasmid R446. Deduced amino acid sequences for several ORFs are homologous to those of known proteins, including the Sog DNA primases of IncI1 plasmids R64 and ColIb-P9 and the TraL, TraM, and TraN products of ColIb-P9. Two protein products of the putative primase ORF (ORF 1, 1100 amino acids) were detected by SDS-PAGE. The 158- and 107-kDa proteins were designated PriL and PriS, respectively. PriS is apparently produced by an in-frame reinitiation of the ORF 1 transcript at a second start codon located between a Sau96I site and a PstI site. The motif EGYATA, conserved among primases and associated with primase function, occurs in the first one-third of the deduced amino acid sequence of PriL and is not included in PriS. Partial suppression of the temperature-sensitive dnaG3 mutation in BW86 was demonstrated by recombinants that overexpressed both PriL and PriS, but not by constructs overexpressing only PriS. Therefore, primase function can be assigned to PriL. Fragment 90/91 represents a portion of the IncM tra region, which has not previously been examined in detail.
Assuntos
Conjugação Genética , DNA Primase/genética , DNA Bacteriano/análise , Klebsiella/genética , Plasmídeos/análise , Sequência de Aminoácidos , Aminoácidos/análise , Proteínas de Bactérias/genética , Escherichia coli/genética , Expressão Gênica , Klebsiella/enzimologia , Dados de Sequência Molecular , Mutagênese , Nucleotídeos , Fases de Leitura Aberta , Análise de Sequência/métodosRESUMO
pACM1 is a conjugative multiresistance plasmid from Klebsiella oxytoca that encodes SHV-5 extended-spectrum beta-lactamase (ESBL) and has two integrons. The first is a type I (sul type); the second, detected by hybridization with an intI gene probe, has been putatively identified as a defective type I integron. The cassette region of the first integron has now been fully sequenced and contains three aminoglycoside resistance determinants (aac(6')-Ib, aac(3)-Ia, and ant(3")-Ia) and two open reading frames of unknown function. In addition, sequencing of a region downstream from the qacEDelta1-sulI-ORF 5 gene cluster of the first integron revealed a copy of insertion sequence IS6100 flanked by inverted copies of sequence from the 11.2-kb insert (In2) of Tn21. This arrangement is similar to that found in In4 of Tn1696. The coincidence of an ESBL gene and mobile elements on a conjugative plasmid has potential implications for the spread of ESBL-mediated drug resistance, though evidence of bla((SHV-5)) movement mediated by these elements has not been found.
