title: Generation and Screening of an Insertion Sequencing-Compatible Mutant Library of Campylobacter jejuni.
abstract: The advent of next-generation sequencing technology has enabled experimental approaches to characterize large, complex populations of DNA molecules with high resolution. Included among these are methods to assess populations of transposon insertion libraries for the fitness cost of any particular mutant allele after applying selection to a population. These approaches have proven invaluable for identifying genetic factors that influence survival of bacterial pathogens within different environments, including animal hosts. One such method, termed insertion-site sequencing (INSeq), was designed to generate a 16 bp fragment of transposon-flanking genomic DNA captured during the protocol, which then serves as the substrate for massively parallel sequencing. Here we describe the generation of a transposon mutant library of Campylobacter jejuni amenable to INSeq and its use in identifying colonization determinants in a day-of-hatch chicken colonization model.
authors.affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, 567 Wilson Road, East Lansing, MI, 48824, USA.|Department of Microbiology, University of Tennessee, 1414 Cumberland Avenue, Knoxville, TN, 37996, USA.
authors.forename: Jeremiah G
authors.initials: JG
authors.lastname: Johnson
authors.affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, 567 Wilson Road, East Lansing, MI, 48824, USA. diritavi@msu.edu.
authors.forename: Victor J
authors.initials: VJ
authors.lastname: DiRita
mesh_terms: D000483:Alleles; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002169:Campylobacter Infections / Q000382:microbiology / Q000473:pathology; D016123:Campylobacter jejuni / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity*; D002645:Chickens; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D056084:Genetic Fitness; D059014:High-Throughput Nucleotide Sequencing; D016254:Mutagenesis, Insertional; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial
keywords: Apo-metalloregulator; Bacterial metalloregulator; Fur; Strep-tag affinity chromatography; Transcription factor