RESUMO
Microcins are gene-encoded peptide antibiotics produced by enterobacteria that act on strains of gram-negative bacteria. In this work, we concentrated on higher-molecular-mass microcins, i.e., those possessing 60 or more amino acids. They can be subdivided into unmodified and posttranslationally modified peptides. In both cases, they exhibit conserved C-terminal sequences that appear to be characteristic of each subgroup. In the hypothesis that these sequences could correspond to domains, gene fusions between the activity genes for the unmodified microcin colicin V and the modified microcin H47 were constructed. These two microcins differ in their mode of synthesis, uptake, target, and specific immunity. Through this experimental approach, chimeric peptides with exchanged C-terminal sequences were encoded. Cells carrying the fusions in different genetic contexts were then assayed for antibiotic production. Many of them produced antibiotic activities with recombinant properties: the toxicity of one microcin and the mode of uptake of the other. The results led to the identification of a modular structure of colicin V and microcin H47, with the recognition of two domains in their peptide chains: a toxic N-terminal domain and an uptake C-terminal domain. This modular design would be shared by other microcins from each subgroup.
Assuntos
Antibacterianos/química , Bacteriocinas/química , Colicinas/química , Peptídeos/química , Sequência de Aminoácidos , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos , Bacteriocinas/biossíntese , Bacteriocinas/genética , Bacteriocinas/farmacologia , Colicinas/biossíntese , Colicinas/genética , Colicinas/imunologia , Colicinas/toxicidade , Sequência Conservada , Dissulfetos/química , Escherichia coli K12/genética , Fusão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Peso Molecular , Testes do Emplastro , Peptídeos/genética , Peptídeos/imunologia , Peptídeos/metabolismo , Peptídeos/toxicidade , Plasmídeos , Precursores de Proteínas/química , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Receptores de Catecolaminas/genética , Receptores de Catecolaminas/metabolismo , Recombinação Genética , Homologia de Sequência de AminoácidosRESUMO
Bacteriocins have been isolated both as simple proteins and as proteins in association with carbohydrates, lipids, etc. Colicins are commonly inducible and extracellular. Their molecular masses range from 30 to 90 kDa. Pure colicin S8 was obtained in three steps from supernatant of induced cells: (i) Ammonium sulfate precipitation; (ii) anion exchange chromatography; and (iii) phenyl-Sepharose hydrophobic chromatography, either by preparative or fast performance liquid chromatography (FPLC) analytical purification procedure. In our hands, purified colicin S8 was an aggregation of extremely related polypeptides. Composition of those active fractions was the same: five polypeptides of molecular weight around 55 kDa. Behavior on molecular filtration indicated a molecular weight higher than 200 kDa. Similar results were obtained when purification was carried out through FPLC. Producing strains contain a single plasmid that encodes colicin S8; in minicells, this plasmid was shown to specify a 60 kDa polypeptide. We conclude that more than one form of colicin S8 exists. The forms are structurally related and can be recognized by antibodies raised against one of the polypeptides. Consistent with this conclusion, comparison of peptides produced after hydrolysis with chlorosuccinamide indicated that the active proteins contained both shared and unique components.
Assuntos
Colicinas/química , Colicinas/isolamento & purificação , Escherichia coli/metabolismo , Amidas/química , Amidas/metabolismo , Proteínas de Bactérias/química , Cromatografia , Colicinas/genética , Colicinas/imunologia , Hidrólise , Immunoblotting , Substâncias Macromoleculares , Plasmídeos , SuccinatosRESUMO
Escherichia coli e Enterobacter foram isolados de grupos de indivíduos hospitalizados (H), recém-hospitalizados (P), sendo estudados quanto ao perfil de resistência às drogas ampicilina, cefalotina, cloranfenicol, estreptomicina, tetraciclina, gentamicina, canamicina e ao bicloreto de mercúrio, por meio da técnica de diluiçäo em meio sólido, e quanto à produçäo de colicinas. A resistência aos antibióticos betalactâmicos foi maior nas amostras isoladas dos grupos de portadores näo-hospitalizados (P) (modelo ampicilina-cefalosporina), em especial para o gênero Enterobacter. Por outro lado, a freqüencia das amostras colicinogênicas descresceu no grupo H em comparaçäo com amostras colicinogênicas decresceu no grupo H em comparaçäo com as amostras do grupo P. É provável que, em ambientes seletivos pela presença de elevadas concentraçöes de antibióticos, as linhagens portadoras de fenótipos sejam selecionadas e a colicinogênese, deslocada ou substituída, sendo mantida em ambientes näo-seletivos, devido à competitividade as linhagens de uma mesma espécie