Combined inactivation and expression strategy to study gene function under physiological conditions: application to identification of new Escherichia coli adhesins
| Autor: | Jean-Marc Ghigo, Agnès Roux, Christophe Beloin |
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| Přispěvatelé: | Génétique des Biofilms, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), J.-M.G. and C.B. were supported by the Institut Pasteur, Paris, France, and by CNRS URA2172 grants. A.R. was supported by a Ministère Français de l'Éducation Nationale, de l'Enseignement Supérieur et de la Recherche fellowship., Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: | angličtina |
| Rok vydání: | 2005 |
| Předmět: |
Operon
[SDV]Life Sciences [q-bio] Restriction Mapping Genetics and Molecular Biology MESH: Escherichia coli/genetics Biology medicine.disease_cause Microbiology 03 medical and health sciences MESH: Plasmids/genetics Plasmid MESH: Escherichia coli Proteins/genetics Gene expression MESH: Promoter Regions Genetic medicine Escherichia coli Promoter Regions Genetic Molecular Biology Gene MESH: Adhesins Escherichia coli/genetics MESH: Restriction Mapping 030304 developmental biology Genetics 0303 health sciences Adhesins Escherichia coli 030306 microbiology MESH: Glucose/metabolism Escherichia coli Proteins PBAD promoter Arabinose [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology Bacterial adhesin Complementation Mutagenesis Insertional Glucose MESH: Mutagenesis Insertional MESH: Escherichia coli/cytology MESH: Escherichia coli/metabolism MESH: Arabinose/metabolism MESH: Cell Division Cell Division Plasmids |
| Zdroj: | Journal of Bacteriology Journal of Bacteriology, American Society for Microbiology, 2005, 187 (3), pp.1001-1013. ⟨10.1128/JB.187.3.1001-1013.2005⟩ Journal of Bacteriology, 2005, 187 (3), pp.1001-1013. ⟨10.1128/JB.187.3.1001-1013.2005⟩ |
| ISSN: | 0021-9193 1098-5530 |
| DOI: | 10.1128/JB.187.3.1001-1013.2005⟩ |
| Popis: | In bacteria, whereas disruption methods have been improved recently, the use of plasmid complementation strategies are still subject to limitations, such as cloning difficulties, nonphysiological levels of gene expression, or a requirement for antibiotics as plasmid selection pressure. Moreover, because of the pleiotropic modifications of cell physiology often induced by plasmid-based complementation, these strategies may introduce biases when biological process such as adhesion or biofilm formation are studied. We developed a plasmid-free approach that combines the lambda-red linear DNA recombination method with site-directed insertion of a repression and expression (RExBAD) cassette which places a functional pBAD promoter upstream of a target gene. We showed that this method permits both inactivation and modulation of most Escherichia coli gene expression, including expression of toxin and essential genes. We used this strategy to study adhesion and bacterial biofilms by placing the RExBAD cassette in front of the tra operon, encoding the DNA transfer and pilus genes on the F conjugative plasmid, and in front of flu , the antigen 43 (Ag43) autotransporter adhesin-encoding gene. In silico analysis revealed the existence of 10 genes with homology to the Ag43 gene that were good candidates for genes that encode putative new adhesins in E. coli . We used the RExBAD strategy to study these genes and demonstrated that induction of expression of four of them is associated with adhesion of E. coli to abiotic surfaces. The potential use of the RExBAD approach to study the function of cryptic or uncharacterized genes in large-scale postgenomic functional analyses is discussed. |
| Databáze: | OpenAIRE |
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