Rapid 96-well plates DNA extraction and sequencing procedures to identify genome-wide transposon insertion sites in a difficult to lyse bacterium: Lactobacillus casei

Autor: Philippe J. Sansonetti, Hélène Scornec, Magali Tichit, Jean-François Cavin, Thierry Pedron, Hélène Licandro-Seraut, Christiane Bouchier
Přispěvatelé: Procédés Alimentaires et Microbiologiques (PAM), Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, Génomique (Plate-Forme) - Genomics Platform, Institut Pasteur [Paris] (IP), Pathogénie Microbienne Moléculaire, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Collège de France - Chaire Microbiologie et Maladies infectieuses, Collège de France (CdF (institution)), This study was supported by the European Research Council Advanced grant HOMEOEPITH to PJS, Grant agreement no: 232798. PJS is a Howard Hughes Medical Institute Foreign Scholar. HS was supported by a grant from the Ministère de l’Enseignement Supérieur et de la Recherche., European Project: 232798,EC:FP7:ERC,ERC-2008-AdG,HOMEOEPITH(2009), Département de Génomes et Génétique - Department of Genomes and Genetics, Institut Pasteur [Paris], Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Chaire Microbiologie et Maladies infectieuses
Jazyk: angličtina
Rok vydání: 2014
Předmět:
DNA
Bacterial
Genetics
Microbial
Microbiology (medical)
Transposable element
transposon mutagenesis
Lactobacillus casei
Sanger sequencing
Mutant
Microbiology
Genome
Insertional mutagenesis
03 medical and health sciences
Bacterial genetics
MESH: Gene Library
Lactic acid bacteria
Molecular Biology
DNA extraction
MESH: High-Throughput Nucleotide Sequencing
Gene Library
030304 developmental biology
Genetics
0303 health sciences
biology
MESH: Lactobacillus casei
030306 microbiology
High-Throughput Nucleotide Sequencing
MESH: Genetics
Microbial
biology.organism_classification
MESH: DNA
Bacterial
[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology
Lacticaseibacillus casei
Mutagenesis
Insertional
genomic DNA
MESH: DNA Transposable Elements
MESH: Mutagenesis
Insertional
DNA Transposable Elements
Transposon mutagenesis
Zdroj: Journal of Microbiological Methods
Journal of Microbiological Methods, 2014, 106, pp.78-82. ⟨10.1016/j.mimet.2014.08.001⟩
Journal of Microbiological Methods, Elsevier, 2014, 106, pp.78-82. ⟨10.1016/j.mimet.2014.08.001⟩
ISSN: 0167-7012
1872-8359
DOI: 10.1016/j.mimet.2014.08.001⟩
Popis: International audience; 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.
Databáze: OpenAIRE
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