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Journal of Bacteriology, February 2005, p. 1001-1013, Vol. 187, No. 3
0021-9193/05/$08.00+0     doi:10.1128/JB.187.3.1001-1013.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Combined Inactivation and Expression Strategy To Study Gene Function under Physiological Conditions: Application to Identification of New Escherichia coli Adhesins

Agnès Roux,1 Christophe Beloin,1 and Jean-Marc Ghigo1*

Groupe de Génétique des Biofilms-CNRS URA 2172, Institut Pasteur, Paris, France1

Received 9 August 2004/ Accepted 11 October 2004

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.

* Corresponding author. Mailing address: Groupe de Génétique des Biofilms-CNRS URA 2172, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France. Phone: (33) 01 40 61 39 17. Fax: (33) 01 40 61 39 70. E-mail: jmghigo{at}pasteur.fr.

Journal of Bacteriology, February 2005, p. 1001-1013, Vol. 187, No. 3
0021-9193/05/$08.00+0     doi:10.1128/JB.187.3.1001-1013.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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