RESUMO
A DNA fragment that can functionally substitute for cfaD, the positive regulatory gene involved in expression of CFA/I fimbriae, has recently been cloned from an Escherichia coli strain of serotype O167:H5 that produces CS5 fimbriae. Nucleotide sequence determination showed that the fragment contained a gene, csvR (Coli Surface Virulence factor Regulator) homologous to the cfaD gene, which encoded for a protein of 301 amino acid residues. The csvR gene was found to be located between two different insertion sequences. Comparison of the amino acid sequence of the CsvR and CfaD proteins showed that CsvR is 34 amino acid residues longer at the C-terminus and, in the sequence, it also contains an insertion of two amino acid residues. The similarity between CfaD and Rns, the positive regulator of CS1 and CS2 expression, is much higher (97%) than between CsvR and CfaD (87%). This is reflected by the fact that the level of expression of CFA/I fimbriae induced by CsvR is not as high as when expression is induced by CfaD or Rns.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Fímbrias , Genes Reguladores , Plasmídeos , Transativadores , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Fímbrias Bacterianas , Genes Bacterianos , Dados de Sequência Molecular , Mapeamento por Restrição , Alinhamento de SequênciaRESUMO
The gene encoding suilysin was cloned from Streptococcus suis serotype 2 strain P1/7. Analysis of the nucleotide and translated amino acid sequence confirmed suilysin to be a member of the thiol activated cytolysin group (TACY). The pneumolysin from Streptococcus pneumoniae is the most closely related orthologous gene known. Suilysin was overexpressed in E. coli in an active haemolytic form. A strong correlation between the presence of the sly gene and haemolytic activity in the supernatant of S. suis field strains was found. Of 158 strains tested, 63% contained the gene. Within the (most prevalent) serotype 2, the sly gene was demonstrated in 95% of the strains isolated in Eurasia, but only in 7% of the strains from North America.
Assuntos
Genes Bacterianos , Proteínas Hemolisinas/genética , Streptococcus suis/genética , Animais , Anticorpos Monoclonais , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/análise , Escherichia coli/genética , Proteínas Hemolisinas/metabolismo , Hemólise , Dados de Sequência Molecular , Compostos Orgânicos , Filogenia , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes/biossíntese , Análise de Sequência de DNA , Sorotipagem , Streptococcus suis/classificaçãoRESUMO
The genes encoding the CS6 colonization factor were cloned from two human enterotoxigenic Escherichia coli strains of different serotypes. The DNA sequences from both clones were nearly identical and contained four open reading frames. Two of them have homology to genes encoding molecular chaperones and ushers found in many other operons encoding colonization factors. The two remaining open reading frames encode two heterologous major subunit proteins which makes CS6 unique because other colonization factors have only one major subunit. Upstream and downstream of the CS6 operon the DNA sequences of the clones diverged abruptly.
Assuntos
Antígenos de Superfície/genética , Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Antígenos de Bactérias/genética , Clonagem Molecular , DNA Bacteriano/análise , Escherichia coli/classificação , Regulação Bacteriana da Expressão Gênica/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Óperon/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , SorotipagemRESUMO
CFA/I fimbriae of enterotoxigenic Escherichia coli are expressed at 37 degrees C and not at 20 degrees C. Expression of CFA/I fimbriae requires two DNA regions (regions 1 and 2) which are separated by 40 kb on the wild type plasmid. Region 2 encodes a protein (CfaD) which activates the promoter in region 1. We investigated whether the histone-like protein H-NS (H1) of E.coli is involved in the temperature regulated expression of CFA/I fimbriae. As demonstrated recently with other temperature regulated genes, a mutation in the gene coding for this nucleoid-associated H-NS (H1) protein resulted in derepression of CFA/I expression. CFA/I fimbriae were now expressed both at 20 degrees C and 37 degrees C. More strinkingly, the positive regulator CfaD was not needed for CFA/I expression in an H-NS- strain. This indicates that CfaD diminishes an inhibitory effect of the H-NS nucleoid-associated protein. We also showed that in the H-NS- strain the CfaD protein still has a positive effect on the transcription of CFA/I.
Assuntos
Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Fímbrias , Fímbrias Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica , Antígenos de Bactérias/biossíntese , Autorradiografia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Northern Blotting , Western Blotting , Proteínas de Ligação a DNA/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , Óperon , Plasmídeos , Reação em Cadeia da Polimerase , RNA Bacteriano/metabolismo , Temperatura , Transcrição Gênica , beta-Galactosidase/metabolismoRESUMO
CFA/I fimbriae on human enterotoxigenic Escherichia coli are composed of the CfaB protein, the product of the second gene of the CFA/I operon. We show here that CfaB is expressed at a higher level than other proteins of the CFA/I operon. mRNA encoding the CfaB protein is much more abundant than mRNA encoding CfaA, the first protein, together with CfaB or mRNA encoding CfaA only. Only one promoter, upstream of cfaA, is present. These data indicate that a primary transcript containing cfaA and cfaB is processed into a cfaA-specific mRNA and a cfaB-specific mRNA. The cfaA mRNA is unstable, while the cfaB mRNA is stable and therefore accumulates in CFA/I-producing E. coli. The cfaB mRNA is probably stabilized by a stem-loop structure downstream of the cfaB gene. No distinct mRNA fragments could be detected encoding the other two proteins, CfaC and CfaE, of the CFA/I operon. These results indicate that cfaC- and cfaE-specific mRNAs degrade very rapidly and/or are produced in small amounts.