RESUMO
The transcriptional organization of the gene cluster encoding the F1845 fimbrial adhesin of a diarrhoea-associated Escherichia coli was investigated. Genes daaA to daaE were determined to constitute a single transcriptional unit under the control of the daaA promoter. The nucleotide sequence of daaA and that of an upstream open reading frame encoded on the opposite strand, designated daaF, were determined to share limited homology with the papB and papI genes of the P fimbrial adhesin, respectively. The 5' termini of the daaF and daaABCDE transcripts were mapped by primer extension and nuclease protection analyses. The promoters for these transcripts were associated with potential regulatory sequences including two consensus leucine-responsive regulatory protein (Lrp)-binding sites which contained differentially methylated GATC sequences, a cAMP-CRP-binding site, and an integration host factor (IHF)-binding site. Expression of the daa locus was determined to be dependent on Lrp, subject to catabolite repression, and dependent on IHF.
Assuntos
Antígenos de Bactérias , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Fímbrias , Fímbrias Bacterianas/metabolismo , Genes Bacterianos , Transcrição Gênica , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência de Bases , AMP Cíclico/fisiologia , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteína Reguladora de Resposta a Leucina , Dados de Sequência Molecular , Fases de Leitura Aberta , Óperon , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Alinhamento de Sequência , Fatores de Transcrição/metabolismoRESUMO
F1845, the fimbrial adhesin of a diarrhea-associated Escherichia coli, confers upon the bacteria the ability to adhere to cultured epithelial cells in a diffuse pattern. The fimbrial subunit gene, daaE, is encoded on a polycistronic mRNA which is processed endoribonucleolytically to produce a stable message encoding only daaE. The processing event occurs in bacterial strains with mutations in RNase III or RNase E, the only endoribonucleases which have been implicated in the processing of E. coli mRNA. Sequences encoding a stem-loop structure downstream of daaE play an essential role in determining the stability of the daaE mRNA. Rapid degradation of the sequences upstream of the cleavage site occurs upon processing, suggesting that processing of the F1845 polycistronic mRNA results in differential expression of genes involved in the biogenesis of fimbriae.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , RNA Mensageiro/genética , Adesinas de Escherichia coli , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ribonuclease III , Transcrição Gênica , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
F41, K88, and CS31A are fimbrial adhesins associated with enterotoxigenic Escherichia coli. These adhesins are distinct from one another in the composition of their structural subunits and the adherence characteristics associated with their expression. Despite these differences, extensive homology exists between the genetic determinants mediating the expression of these adhesins, extending throughout the region of each determinant encoding the accessory proteins involved in adhesin biogenesis. This suggests that the regulatory and assembly systems mediating expression of these adhesins may be functionally interchangeable. In the present study we demonstrated that the accessory systems of the F41, K88, and CS31A determinants are able to mediate the functional expression of heterologous fimbrial subunit proteins. Plasmid constructs containing the isolated fimbrial subunit gene of the F41 or CS31A determinant were prepared and introduced into E. coli harboring the F41, K88, and CS31A accessory genes contained on compatible plasmid vectors. The ability of each of the three accessory systems to mediate stable expression of the F41 or CS31A fimbrial subunit peptide was demonstrated by Western blot (immunoblot) analysis. Functional expression of the F41 or CS31A subunit on the bacterial cell surface was demonstrated by the ability of these proteins to confer mannose-resistant hemagglutination of human erythrocytes or in vitro adherence to epithelial cells, respectively. The accessory system of an unrelated adhesin determinant, F1845, did not mediate functional expression of F41 adherence. Taken together, these data indicate that the genetic determinants mediating expression of the F41, K88, and CS31A adhesins are members of a closely related family and suggest that a mechanism exists in the family for the more rapid divergence of genes encoding antigenic and adhesive determinants.
