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DEVELOPMENT OF GENETIC METHODS IN GRAM-NEGATIVE BACTERIA

A new donor strain to perform RP4 conjugation-based transposon mutagenesis

We showed that SM10 lpir and S17-1 lpir, two popular donor strains widely used to perform random and targeted mutagenesis in many different bacteria also transfer a bacteriophage Mu into recipient strains. We demonstrated that this silent, marker-less contamination finds its origin in the original strain construction and leads to systematic transposon/Mu double mutagenesis events. We carried on a detailed genetic analysis of bacteriophage Mu insertions in the contaminated strains and we re-constructed a new Mu-free donor strain. Since the publication of these results (December 2010, we received and responed to 27 requests for the MFDpir strain.

Our findings demonstrate that bacteriophage Mu contaminated almost all mutant screens performed over the past 30 years in Mu-sensitive species, leading to potential misinterpretation of the transposon mutant phenotype. The constructed Mu-free donor strain MFDpir will significantly simplify the analysis of mutant hunts in Escherichia coli and other Mu-sensitive RP4 host bacteria.

Ferrieres, L., Hemery, G., Nham, T., Guerout, A. M., Mazel, D., Beloin, C. and Ghigo, J. M. (2010) Silent mischief: bacteriophage Mu insertions contaminate products of Escherichia coli random mutagenesis performed using suicidal transposon delivery plasmids mobilized by broad-host-range RP4 conjugative machinery J Bacteriol.192:6418-27.


Expression strategy to study gene function in physiological conditions: application to the identification of new adhesins in E. coli.

In bacteria, plasmid complementation strategies are still subjected to limitations such as cloning difficulties, non-physiological level of gene expression or need for antibiotics as plasmid selection pressure. In particular the use of plasmids can lead to biases when studying biological process such as adhesion or biofilm formation, due to pleiotropic modifications of the cell physiology.
We developed a plasmid-free approach that combines the lambda-red linear DNA recombination method with the site-directed insertion of repression/expression (RExBAD, RexTeT) or constitutive (PcL) cassettes, which places functional promoters upstream of a target gene. We showed that this method permits both the inactivation and modulation of most Escherichia coli gene expression, including toxin and essential genes. We applied this strategy to study putative adhesion and bacterial biofilm functions of previously uncharacterized genes encoding putative adhesins. Beyond the intrinsic interest of the elucidation of the environmental conditions that lead to the expression of these adhesins, the REx or PcL approach can be used in several enterobacteria to study the function of cryptic or uncharacterized genes in large-scale post-genomic functional analyses.

• Roux, A, Beloin, C. and Ghigo, J.M.(2004 / 2005) A combined inactivation/expression strategy to study gene function in physiological conditions: application to the identification of new adhesins in E. coli. J. Bacteriol. 187:1001-13

• Da Re, S., Le Quere, Ghigo, J.M. and C. Beloin .(2007). Tight modulation of Escherichia coli bacterial biofilm formation through controlled expression of adhesion factors. Appl. Environl Microbiol. 73:3391-403

Development of gene inactivating methods in Gram- bacteria.

We developed, in parallel with other laboratories a rapid PCR-based method to inactivate target genes in E. coli ,based on an initial report by Ken Murphy (see (Murphy, 1998; Chaveroche et al., 2000; Datsenko and Wanner, 2000; Yu et al., 2000) In collaboration with E. Carniel’s group, (Yersiniae Unit, I. Pasteur)), this method was further adapted to allow the rapid and cloning-free disruption of genes in many enterobacteriacea (see ).

• Chaveroche, M. K., Ghigo, J. M. and d'Enfert, C. (2000). “A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans.” Nucleic Acids Res 28(22): E97.

• Derbise, A ; B. Lesic, D. Dacheux, J.M. Ghigo and E. Carniel . (2003)« A rapid and simple method for inactivating chromosomal genes in Yersinia”. FEMS I. Med Microbiol. Sep 22;38(2):113-6