RESUMO
The proteolytic cleavage of BlaI was shown to correlate with beta-lactamase synthesis in Staphylococcus aureus. BlaI was found to be autoregulatory when expressed from the blaZ promoter. Insertion of a 10-bp linker into the SnaBI site of blaRI resulted in constitutive synthesis of beta-lactamase.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Repressoras/metabolismo , Staphylococcus aureus/metabolismo , beta-Lactamases/biossíntese , Proteínas de Bactérias/metabolismo , Western Blotting , Indução Enzimática , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/genética , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genéticaRESUMO
Purified BlaI, the putative repressor of the beta-lactamase operon in Staphylococcus aureus, binds specifically to two regions of dyad symmetry (operators) located in the blaZ-blaR1 intergenic region. BlaI binds with similar affinity to the two regions and to the related sequence upstream of the mec gene found in methicillin-resistant strains of S. aureus, providing physical evidence for the cross-talk previously observed between these systems. A change from a lysine in the N-terminus of BlaI to an alanine or deletion of the C-terminal 23 amino acids severely reduces its DNA-binding ability, demonstrating the functional importance of both the N- and C-termini. An operator DNA-protein complex observed with crude cell lysates from repressed cells, indistinguishable from that observed with purified BlaI, was eliminated by induction of the beta-lactamase operon. Furthermore, BlaI is proteolytically cleaved in response to the addition of inducer in a blaR1-dependent manner, providing primary evidence for the molecular basis of induction. Thus, BlaI is shown to be the repressor of the beta-lactamase system.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Proteínas Repressoras/biossíntese , Proteínas Repressoras/metabolismo , Staphylococcus aureus/metabolismo , beta-Lactamases/biossíntese , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , DNA Bacteriano , Proteínas de Ligação a DNA/genética , Endopeptidases/metabolismo , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Staphylococcus aureus/genética , Inibidores de beta-LactamasesRESUMO
The nucleotide sequence of the ileS gene conferring high-level resistance to mupirocin in Staphylococcus aureus J2870 has been determined. The gene sequence is substantially different from that of the native ileS gene of S. aureus, indicating that high-level resistance to mupirocin results from the acquisition of a novel ileS gene.
Assuntos
Mupirocina/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Sequência de Bases , Clonagem Molecular , Códon , DNA Bacteriano/análise , Resistência Microbiana a Medicamentos , Dados de Sequência Molecular , Fases de Leitura Aberta , RNA de Transferência de Isoleucina/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
A plasmid known to be associated with mupirocin resistance of Staphylococcus aureus has been isolated and a restriction enzyme map constructed. An EcoRI fragment of 4.05 kb from this plasmid has been cloned into an Escherichia coli-Staphylococcus aureus shuttle vector and shown to carry the gene for resistance to mupirocin. The DNA sequence of a small section of the gene has been determined and the derived amino acid sequence compared with a data bank. The amino acid sequence is identical for eight amino acids with the sequence of isoleucyl tRNA synthetase of E. coli. This finding adds to the evidence that mupirocin resistance is the result of a modified isoleucyl tRNA synthetase.
Assuntos
Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos/genética , Genes Bacterianos , Fatores R , Staphylococcus aureus/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Escherichia coli/enzimologia , Escherichia coli/genética , Ácidos Graxos/farmacologia , Isoleucina-tRNA Ligase/genética , Dados de Sequência Molecular , Mupirocina , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Staphylococcus aureus/efeitos dos fármacosRESUMO
A plasmid encoded beta-lactamase gene from Staphylococcus aureus (strain 3804) was cloned and sequenced. The nucleotide sequence is very similar to those obtained for other such beta-lactamase genes. The beta-lactamase has an N-terminal region characteristic of an exported protein and assay of activity shows that the enzyme is found extracellularly in S. aureus. A residue in the N-terminal region near to the postulated cleavage site was changed by site-directed mutagenesis from a serine into a proline. Comparison of beta-lactamase activity outside the cell in strains containing the cloned wild-type and mutagenised genes shows that this single amino acid completely prevents the appearance of the enzyme in the medium.
Assuntos
Sinais Direcionadores de Proteínas/genética , Staphylococcus aureus/genética , beta-Lactamases/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/genética , Genes Bacterianos , Dados de Sequência Molecular , Mutação , Sinais Direcionadores de Proteínas/metabolismo , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Staphylococcus aureus/enzimologia , beta-Lactamases/metabolismoRESUMO
The nucleotide sequence of a small (1273 bp) plasmid (pOX1000) of Staphylococcus aureus has been determined and compared with similar plasmids. The sequence includes a single open reading frame; two large palindromes and a 22 bp palindrome that is contiguously repeated three times upstream of the open reading frame. Composite plasmids of pE194ts and pOX1000 were constructed with pUC18 separately inserted into five different sites on pOX1000 and used to analyse the replication functions of the cryptic plasmid.
