title: Rapid 96-well plates DNA extraction and sequencing procedures to identify genome-wide transposon insertion sites in a difficult to lyse bacterium: Lactobacillus casei.
abstract: Random transposon mutagenesis followed by adequate screening methods is an unavoidable procedure to characterize genetics of bacterial adaptation to environmental changes. We have recently constructed a mutant library of Lactobacillus casei and we aimed to fully annotate it. However, we have observed that, for L. casei which is a difficult to lyse bacterium, methods used to identify the transposon insertion site in a few mutants (transposon rescue by restriction and recircularization or PCR-based methods) were not transposable for a larger number because they are too time-consuming and sometimes not reliable. Here, we describe a method for large-scale and reliable identification of transposon insertion sites in a L. casei mutant library of 9250 mutants. DNA extraction procedure based on silica membranes in 96-column format was optimized to obtain genomic DNA from a large number of mutants. Then reliable direct genomic sequencing was improved to fit the obtained genomic DNA extracts. Using this procedure, readable and identifiable sequences were obtained for 87% of the L. casei mutants. This method extends the applications of a library of this type, reduces the number of insertions needed to be screened, and allows selection of specific mutants from an arrayed and stored mutant library. This method is applicable to any already existing mutant library (obtained by transposon or insertional mutagenesis) and could be useful for other bacterial species, especially for highly lysis-resistant bacteria species such as lactic acid bacteria.
authors.affiliation: UMR A PAM, AgroSup Dijon/Université de Bourgogne, 1 Esplanade Erasme, 21000, Dijon, France.
authors.forename: Hélène
authors.initials: H
authors.lastname: Scornec
authors.affiliation: Plate-forme Génomique, Département Génomes et Génétique, Institut Pasteur, 28 Rue du Docteur Roux, 75724, Paris cedex 15, France.
authors.forename: Magali
authors.initials: M
authors.lastname: Tichit
authors.affiliation: Plate-forme Génomique, Département Génomes et Génétique, Institut Pasteur, 28 Rue du Docteur Roux, 75724, Paris cedex 15, France.
authors.forename: Christiane
authors.initials: C
authors.lastname: Bouchier
authors.affiliation: Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, 28 Rue du Docteur Roux, 75724, Paris cedex 15, France; Unité INSERM 786, Institut Pasteur, Paris, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Pédron
authors.affiliation: UMR A PAM, AgroSup Dijon/Université de Bourgogne, 1 Esplanade Erasme, 21000, Dijon, France.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Cavin
authors.affiliation: Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, 28 Rue du Docteur Roux, 75724, Paris cedex 15, France; Unité INSERM 786, Institut Pasteur, Paris, France; Chaire de Microbiologie et Maladies Infectieuses, Collège de France, 11 Place Marcelin Berthelot, 75005, Paris, France. Electronic address: philippe.sansonetti@pasteur.fr.
authors.forename: Philippe J
authors.initials: PJ
authors.lastname: Sansonetti
authors.affiliation: UMR A PAM, AgroSup Dijon/Université de Bourgogne, 1 Esplanade Erasme, 21000, Dijon, France; Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, 28 Rue du Docteur Roux, 75724, Paris cedex 15, France.
authors.forename: Hélène
authors.initials: H
authors.lastname: Licandro-Seraut
mesh_terms: D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D015723:Gene Library; D005827:Genetics, Microbial / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing; D007780:Lacticaseibacillus casei / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000379:methods*
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial
keywords: DNA extraction; Lactic acid bacteria; Lactobacillus casei; Sanger sequencing; bacterial genetics; transposon mutagenesis