RESUMEN
mTn 10 transposon mutagenesis of Escherichia coli producing K88 fimbria was carried out in order to identify host factors involved in the regulation of the fae (K88) operon and the production of K88 fimbriae. Five independent chromosomal insertion mutants were obtained which showed an increased expression of K88 fimbriae. Inverse PCR and nucleotide sequencing were carried out to characterize the mutations. One insertion affected the Ipp gene, encoding the major outer membrane lipoprotein. Another mutation was found to be located in the Irp gene, encoding the 'global' regulatory protein Lrp (leucine responsive regulatory protein). A third mutant was found to affect the expression of rfaF, encoding heptosyltransferase II, which resulted in a partially wild-type and partially Re-Rd1 type of LPS. A fourth mutation affected sseB, a gene involved in serine-sensitivity of E. coli cells. Another mutant contained an insertion in an unknown region of the E. coli genome. The mutants were further characterized with respect to K88 as well as K99 fimbriae production.
Asunto(s)
Antígenos Bacterianos , Toxinas Bacterianas , Elementos Transponibles de ADN , Proteínas de Escherichia coli , Escherichia coli/química , Proteínas Fimbrias , Fimbrias Bacterianas/genética , Factores de Transcripción , Secuencia de Aminoácidos , Antígenos de Superficie/biosíntesis , Proteínas Bacterianas/genética , Secuencia de Bases , Mapeo Cromosómico , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Proteína Reguladora de Respuesta a la Leucina , Lipopolisacáridos , Datos de Secuencia Molecular , Mutagénesis InsercionalRESUMEN
Expression of the K88 (fae) operon is negatively controlled by the co-operative binding of Lrp and FaeA to the fae regulatory region and is dependent on the methylation status of three GATC sites present in this region. In this paper, we describe the binding of Lrp to a T-rich DNA helix between GATC site I and site II. FaeA stabilized and modified the Lrp binding, thereby extending the Lrp footprint over GATC site I and site III. Methylation of GATC site I prevented the binding of Lrp/FaeA at this site and appeared to be essential for the cells, since mutation of this site into GTTC resulted in a lethal overproduction of K88 fimbriae. Methylation of GATC site II and site III reduced the stability of Lrp/FaeA binding. Moreover, methylation of GATC site III stimulated faeB promoter activity. The plasmid population in cells harbouring multiple copies of a K88 plasmid consisted of two differentially methylated forms. Form A plasmids with a methylated GATC site I and site III and a nonmethylated site II (+,-,+) represented 20% of the population and were responsible for high-level expression. Form B plasmids with a methylated GATC site I and a non-methylated site II and site III (+,-,-) represented 80% of the population and were responsible for low-level expression. Apparently, K88 fimbriae expression in vivo is balanced at its maximal possible level by modulation of the methylation status of GATC site III. The ratio (1:4) between these populations is stabilized by a constitutive synthesis of FaeA resulting from the presence of an IS1 insertion upstream of faeA. This IS1 insertion separates the faeA promoter from the FaeB-binding sites, thereby neutralizing the control by FaeB activity on expression of FaeA. Instead, faeA transcription is stimulated by binding of FaeA to the faeA promoter region.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli , Regulación Bacteriana de la Expresión Génica , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Elementos Transponibles de ADN , ADN Bacteriano/química , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Desoxirribonucleasa I , Genes Bacterianos , Proteína Reguladora de Respuesta a la Leucina , Metilación , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Plásmidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Mapeo RestrictivoRESUMEN
Tn10 transposon mutagenesis of Escherichia coli producing K99 fimbriae was carried out to identify host factors involved in regulation of biosynthesis of fimbriae. Two chromosomal mutants were obtained that showed a strongly reduced cell surface expression of K99 fimbriae upon colony blotting and ELISA. Analysis by inversed PCR and nucleotide sequencing showed that one mutant (EP14) contained the Tn10 transposon in rfaQ, affecting the expression of the rfaQGP gene cluster, whereas the other mutant (EP35) was affected in a, to date, unknown region of the genome. Immunoblotting analysis confirmed a Rd1 type of LPS of mutant strain EP14. These findings for the first time indicated an effect of LPS core biosynthesis on the biogenesis of fimbriae at the cell surface. Preliminary experiments indicated that K99 major subunits, in contrast to K88 subunits, strongly bind LPS molecules.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/genética , Proteínas Fimbrias , Lipopolisacáridos/biosíntesis , Anticuerpos Monoclonales , Proteínas Bacterianas/biosíntesis , Secuencia de Bases , Sondas de ADN , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Immunoblotting , Lipopolisacáridos/inmunología , Datos de Secuencia Molecular , Mutagénesis Insercional , Reacción en Cadena de la PolimerasaRESUMEN
Nucleotide sequence analysis of the fae operon encoding the biosynthesis of K88 fimbriae revealed the presence of two divergently transcribed regulatory genes, faeA and faeB, separated by two inverted IS1 insertions. The amino acid sequences of the regulatory proteins FaeA and FaeB show similarity to the primary structure of corresponding regulatory proteins involved in the biosynthesis of Pap and S fimbriae. Expression of faeA is positively controlled by the FaeA protein, whereas K88 fimbriae production is negatively controlled by the co-operative activity of FaeA and the leucine-responsive regulatory protein (Lrp). Exchange of FaeA for Papl, a positive regulator of Pap fimbriae expression, also represses K88 production indicating that the combination Papl/Lrp has opposite effects on fae and pap expression. Mutations in faeB had no effect on the biosynthesis of K88 fimbriae. The presence of the two IS1 insertions is hypothesized to neutralize part of the repression of K88 biosynthesis by FaeA/Lrp. Like pap, the fae operon does not respond to exogenous leucine.
Asunto(s)
Antígenos Bacterianos , Antígenos de Superficie/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Elementos Transponibles de ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas Fimbrias , Regulación Bacteriana de la Expresión Génica , Operón , Proteínas Represoras , Factores de Transcripción , Secuencia de Aminoácidos , Proteínas Bacterianas/biosíntesis , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Genes Reguladores , Proteína Reguladora de Respuesta a la Leucina , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
The nucleotide sequences of the genes faeF, faeH, faeI, and faeJ encoding K88 minor fimbrial subunits were determined. Analysis of the primary structure of the gene products revealed that all four proteins are synthesized with an amino-terminal signal sequence. The molecular masses of the mature FaeF, FaeH, FaeI, and FaeJ proteins were calculated to be 15,161, 25,461, 24,804, and 25,093 Da, respectively. FaeH, FaeI, and FaeJ showed significant homology with FaeG, the major fimbrial subunit of K88 fimbriae. Mutations in the respective genes were constructed. Analysis of the mutants showed that the minor fimbrial subunits FaeF and FaeH play an essential role in the biogenesis but not in the adhesive properties of the K88 fimbriae. Mutations in faeI or faeJ had no significant effect on K88 production or adhesive capacity. Specific antisera against FaeF and FaeH were raised by immunization with hybrid Cro-LacZ-FaeF and Cro-LacZ-FaeH proteins. Immunoblotting and immunoelectron microscopy revealed that FaeF and FaeH are located in or along the K88 fimbrial structure.