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Isolation and characterization of chromosomal mTn 10 insertion mutations affecting K88 fimbriae production in
T T Huisman1, E Pilipcinec, F Remkes
1Department of Molecular Microbiology, Vrije Universiteit Amsterdam, Netherlands.
Abstract:
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.
Insights
Transposon mutagenesis identified host factors regulating K88 fimbriae production in Escherichia coli. Mutations in Ipp, Lrp, rfaF, and sseB genes altered fimbrial expression, revealing new regulatory insights.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Escherichia coli uses fimbriae for adhesion, crucial for pathogenesis.
- Understanding K88 fimbriae regulation is key to controlling bacterial infections.
- The fae (K88) operon governs K88 fimbriae production.
Purpose of the Study:
- To identify host factors regulating the fae (K88) operon and K88 fimbriae production in Escherichia coli.
- To characterize mutations affecting K88 fimbriae expression using transposon mutagenesis.
Main Methods:
- mTn 10 transposon mutagenesis was employed to generate mutants.
- Inverse PCR and nucleotide sequencing were used to identify insertion sites.
- Mutants were analyzed for K88 and K99 fimbriae production.
Main Results:
- Five mutants with increased K88 fimbriae expression were identified.
- Mutations were found in Ipp (outer membrane lipoprotein), Lrp (global regulator), rfaF (heptosyltransferase II), and sseB (serine-sensitivity).
- One insertion occurred in an uncharacterized genomic region.
Conclusions:
- Host factors including Ipp, Lrp, rfaF, and sseB play roles in K88 fimbriae regulation.
- This study provides novel insights into the genetic control of fimbrial expression in E. coli.
- The identified genes offer potential targets for therapeutic intervention.
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