Molecular cloning and characterisation of the genes for a non-fimbrial adhesin from Escherichia coli

Barbara A. Hales; Helen Beverley-Clarke; Nicky J. High; Klaus Jann; Rachel Perry; Janina Goldhar; Graham J. Boulnois

doi:10.1016/0882-4010(88)90076-9

Molecular cloning and characterisation of the genes for a non-fimbrial adhesin from Escherichia coli

Barbara A. Hales, Helen Beverley-Clarke, Nicky J. High, Klaus Jann, Rachel Perry, Janina Goldhar, Graham J. Boulnois^*

^*Corresponding author for this work

Clinical Microbiology and Immunology

Research output: Contribution to journal › Article › peer-review

Abstract

A non-fimbrial adhesin (NFA-1) from the uropathogenic Escherichia coli strain 827 responsible for agglutination of human erythrocytes was cloned using the cos 4 cosmid vector. A clone was isolated which promoted haemagglutination and showed the same biological properties as the adhesin produced by the wild type strain. Both express adhesin at 37 °C, but not 18 °C nor in the presence of 1% glucose. Adhesin purified from the clone formed high molecular weight aggregates which were resolved to the 21 K dalton subunit protein seen in the wild type strain on denaturation. Binding to human kidney cells by the clone and the wild type E. coli, from which the genes were cloned, were compared in an ELISA assay and shown to be the same. The genes for the adhesin were isolated on a 15.5 kilobase BamHI-EcoRI fragment which was subjected to γδ mutagenesis. The NFA-1 operon was localised to a 6.5kb region of this fragment.

Original language	English
Pages (from-to)	9-17
Number of pages	9
Journal	Microbial Pathogenesis
Volume	5
Issue number	1
DOIs	https://doi.org/10.1016/0882-4010(88)90076-9
State	Published - Jul 1988

Keywords

adhesin
cloning
haemagglutination
mutagenesis
non-fimbrial
protein purification

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title = "Molecular cloning and characterisation of the genes for a non-fimbrial adhesin from Escherichia coli",

abstract = "A non-fimbrial adhesin (NFA-1) from the uropathogenic Escherichia coli strain 827 responsible for agglutination of human erythrocytes was cloned using the cos 4 cosmid vector. A clone was isolated which promoted haemagglutination and showed the same biological properties as the adhesin produced by the wild type strain. Both express adhesin at 37 °C, but not 18 °C nor in the presence of 1% glucose. Adhesin purified from the clone formed high molecular weight aggregates which were resolved to the 21 K dalton subunit protein seen in the wild type strain on denaturation. Binding to human kidney cells by the clone and the wild type E. coli, from which the genes were cloned, were compared in an ELISA assay and shown to be the same. The genes for the adhesin were isolated on a 15.5 kilobase BamHI-EcoRI fragment which was subjected to γδ mutagenesis. The NFA-1 operon was localised to a 6.5kb region of this fragment.",

keywords = "adhesin, cloning, haemagglutination, mutagenesis, non-fimbrial, protein purification",

author = "Hales, {Barbara A.} and Helen Beverley-Clarke and High, {Nicky J.} and Klaus Jann and Rachel Perry and Janina Goldhar and Boulnois, {Graham J.}",

note = "Funding Information: This work was supported by grants from the Medical Research Council and the Wellcome Trust to GJB and the Bundes Ministerium fur Forschung and Technologie (BMFT) to KJ . NJH acknowledges an MRC studentship . GJB is a Lister-Jenner Research Fellow .",

year = "1988",

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AU - Hales, Barbara A.

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AU - High, Nicky J.

AU - Jann, Klaus

AU - Perry, Rachel

AU - Goldhar, Janina

AU - Boulnois, Graham J.

N1 - Funding Information: This work was supported by grants from the Medical Research Council and the Wellcome Trust to GJB and the Bundes Ministerium fur Forschung and Technologie (BMFT) to KJ . NJH acknowledges an MRC studentship . GJB is a Lister-Jenner Research Fellow .

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N2 - A non-fimbrial adhesin (NFA-1) from the uropathogenic Escherichia coli strain 827 responsible for agglutination of human erythrocytes was cloned using the cos 4 cosmid vector. A clone was isolated which promoted haemagglutination and showed the same biological properties as the adhesin produced by the wild type strain. Both express adhesin at 37 °C, but not 18 °C nor in the presence of 1% glucose. Adhesin purified from the clone formed high molecular weight aggregates which were resolved to the 21 K dalton subunit protein seen in the wild type strain on denaturation. Binding to human kidney cells by the clone and the wild type E. coli, from which the genes were cloned, were compared in an ELISA assay and shown to be the same. The genes for the adhesin were isolated on a 15.5 kilobase BamHI-EcoRI fragment which was subjected to γδ mutagenesis. The NFA-1 operon was localised to a 6.5kb region of this fragment.

AB - A non-fimbrial adhesin (NFA-1) from the uropathogenic Escherichia coli strain 827 responsible for agglutination of human erythrocytes was cloned using the cos 4 cosmid vector. A clone was isolated which promoted haemagglutination and showed the same biological properties as the adhesin produced by the wild type strain. Both express adhesin at 37 °C, but not 18 °C nor in the presence of 1% glucose. Adhesin purified from the clone formed high molecular weight aggregates which were resolved to the 21 K dalton subunit protein seen in the wild type strain on denaturation. Binding to human kidney cells by the clone and the wild type E. coli, from which the genes were cloned, were compared in an ELISA assay and shown to be the same. The genes for the adhesin were isolated on a 15.5 kilobase BamHI-EcoRI fragment which was subjected to γδ mutagenesis. The NFA-1 operon was localised to a 6.5kb region of this fragment.

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KW - cloning

KW - haemagglutination

KW - mutagenesis

KW - non-fimbrial

KW - protein purification

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Molecular cloning and characterisation of the genes for a non-fimbrial adhesin from Escherichia coli

Abstract

Keywords

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