RESUMO
We have cloned and sequenced the DNA needed for production of CS2 pili in Escherichia coli K-12. The four open reading frames, cotB, cotA, cotC, and cotD, show homology with the genes needed for production of CS1 and CFA/I pili, which are also found on enterotoxigenic E. coli associated with human diarrheal disease. We also report that CotA plus CotB interact with the CS1 gene products CooC and CooD to form pili that can be visualized by electron microscopy and, conversely, that the CS1 gene products CooA and CooB interact with CotC and CotD to form pili.
Assuntos
Antígenos de Bactérias/genética , Escherichia coli/genética , Fímbrias Bacterianas/genética , Genes Bacterianos , Sequência de Aminoácidos , Antígenos de Superfície/genética , DNA Bacteriano , Escherichia coli/imunologia , Fímbrias Bacterianas/ultraestrutura , Teste de Complementação Genética , Humanos , Dados de Sequência Molecular , Família Multigênica , Fases de Leitura AbertaRESUMO
Many strains of enterotoxigenic Escherichia coli (ETEC) isolated from patients with diarrhoeal disease exhibit CS1 pili on their surfaces. These appendages, which are thought to be important for colonization of the upper intestine, are composed largely of multiple identical protein subunits encoded by cooA. We have sequenced the DNA directly downstream of cooA and identified two open reading frames, cooC and cooD, transcribed in the same direction as cooB and cooA. Following cooD is DNA homologous to an insertion sequence, so cooB, A, C and D appear to encode all the information needed for E. coli K-12 to synthesize CS1 pili. Complementation analysis of mutants cloned in E. coli K-12 and constructed in an ETEC-derived strain indicates that cooC and cooD are not required for stability of the major CS1 pilin protein or for its transport to the periplasm, but, like cooB, both are needed for assembly of cooA into pili.
Assuntos
Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Fímbrias Bacterianas/metabolismo , Genes Bacterianos/genética , Proteínas de Membrana , Sequência de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sequência de Bases , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/ultraestrutura , Proteínas de Fímbrias , Fímbrias Bacterianas/ultraestrutura , Dados de Sequência Molecular , Mapeamento por Restrição , Análise de Sequência de DNA , Deleção de Sequência , Homologia de Sequência de AminoácidosRESUMO
CS1 pili are filamentous proteinaceous appendages found on many enterotoxigenic Escherichia coli (ETEC) strains isolated from human diarrhoeal disease. They are thought to effect colonization of the upper intestine by facilitating binding to human ileal epithelial cells. We have identified a gene, cooB, which lies directly upstream of cooA, the gene that encodes the major structural CS1 protein. When translated in vitro, the protein product of cooB migrates in sodium dodecyl sulphate/polyacrylamide gel with an apparent molecular mass of 26 kDa, which is consistent with that predicted from its DNA sequence. We constructed a mutant allele (cooB-1) by insertion of the omega fragment, which inhibits transcription and translation, into the cooB gene in vitro. In a derivative of an ETEC strain with the cooB-1 mutation (JEF100) and a plasmid that encodes Rns (pEU2030), the positive regulator required for CS1 expression, no cooB and a greatly reduced level of cooA product was detectable in total cell extracts. The reduction of cooA in this strain appears to result from polarity of the cooB mutation because introduction of the wild-type cooA gene in trans causes production of CooA protein, which is found in cell pellet extracts, in extracts containing only surface proteins and in the culture supernatant. Therefore, in the absence of CooB, CooA is stable and it is transported through both inner and outer membranes. However, the cooB-1 strain with cooA in trans does not cause haemagglutination of bovine erythrocytes (the model system used to assay adherence mediated by coli surface antigen 1 (CS1) pili).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/genética , Fímbrias Bacterianas/metabolismo , Genes Bacterianos , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Western Blotting , DNA Bacteriano , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Proteínas de Fímbrias , Hemaglutinação , Microscopia Eletrônica , Biossíntese de Proteínas , Transcrição GênicaRESUMO
We have constructed and tested a single-copy-plasmid vector (pEU720) based on the IncFII-group plasmid, R100, that is useful for cloning promoters in front of lacZ. The vector is 15 kb long and contains a unique XhoI site in front of lacZ.
Assuntos
Escherichia coli/genética , Vetores Genéticos/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Clonagem Molecular , Expressão Gênica/genética , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Miniplasmids with the P1 copN22 mutation have a copy number about seven times that of the wild type. Selection for reduced copy number from this plasmid led to the isolation of second-site pseudorevertants, called poc mutants. DNA sequence analysis showed that all six independent poc mutants have a single base change in the same codon of the repA gene. This implicates the amino acid at this location, either directly or indirectly, in interactions important for copy number control.
Assuntos
Escherichia coli/genética , Mutação , Plasmídeos , Sequência de Bases , Códon/genética , DNA Bacteriano/genética , Dados de Sequência Molecular , Mapeamento por RestriçãoRESUMO
P1 and P7 are closely related plasmid prophages which are members of the same incompatibility group. We report the complete DNA sequence of the replication region of P7 and compare it to that of P1. The sequence predicts a single amino acid difference between the RepA proteins of these two plasmids, no differences in methylation sites or regions where dnaA protein is expected to bind, and no difference in the spacing of the major features of the two replicons. A P1 replicon with a mutation in repA, the gene that encodes an essential replication protein, is complemented for replication by providing either the P1 RepA protein (RepA1) or the P7 RepA protein (RepA7) in trans. Furthermore, when either of these proteins is supplied in trans, the plasmid copy number of P1 cop mutants drops to that of P1 cop+. However, when RepA7 is supplied, the copy number of P1 cop and P1 cop+ is higher than that when RepA1 is supplied. This indicates that the single amino acid difference between the two versions of the RepA protein plays an important role in determining the plasmid copy number.
Assuntos
Aminoácidos/genética , Proteínas de Bactérias , Bacteriófagos/genética , DNA Helicases , Replicação do DNA , Proteínas de Ligação a DNA , Plasmídeos , Proteínas , Transativadores , Sequência de Bases , Escherichia coli/genética , Teste de Complementação Genética , Dados de Sequência MolecularRESUMO
Mutant P1 Ap Cm lysogens were isolated in which the drug resistance genes resident on the plasmid prophage P1 Ap Cm are amplified by a novel mechanism. The first step required for amplification is IS1-mediated rearrangement of the P1 Ap Cm prophage. The drug resistance genes are amplified from the rearranged P1 Ap Cm prophage by the formation of a plasmid (P1dR) which contains the two resistance genes. The P1dR plasmid is an independent replicon about one-half the size of P1 Ap Cm that can be maintained at a copy number eightfold higher than that at which P1 Ap Cm can be maintained. It contains no previously identified replication origin and is dependent on the Rec+ function of the host.
Assuntos
Proteínas de Bactérias , Bacteriófagos/genética , Desoxirribonucleases de Sítio Específico do Tipo II , Amplificação de Genes , Replicon , Enzimas de Restrição do DNA/metabolismo , Desoxirribonuclease BamHI , Desoxirribonuclease EcoRI , Resistência Microbiana a Medicamentos , Fenótipo , Plasmídeos , Recombinação GenéticaRESUMO
Like low-copy-number plasmids including P1 wild type, multicopy P1 mutants (P1 cop, maintained at five to eight copies per chromosome) can suppress the thermosensitive phenotype of an Escherichia coli dnaA host by forming a cointegrate. At 40 degrees C in a dnaA host suppressed by P1 cop, the only copy of P1 is the one in the host chromosome. Trivial explanations of the lack of extrachromosomal copies of P1 cop have been eliminated: (i) during integrative suppression, the P1 cop plasmid does not revert to cop+; (ii) the dnaA+ function of the host is not required to maintain P1 cop at a high copy number; and (iii) integrative recombination does not occur within the region of the plasmid involved in regulation of copy number. Since there are no more copies of the chromosomal origin (now located within the integrated P1 plasmid) than in a P1 cop+-suppressed strain, the extra initiation potential of the P1 cop is not used to provide multiple initiations of the chromosome. When a P1 cop-suppressed dnaA strain was grown at 30 degrees C so that replication could initiate from the chromosomal origin as well as from the P1 origin, multicopy supercoiled P1 DNA was found in the cells. This plasmid DNA was lost again when the temperature was shifted back to 40 degrees C.
