title: Specialized transduction designed for precise high-throughput unmarked deletions in Mycobacterium tuberculosis.
abstract: Specialized transduction has proven to be useful for generating deletion mutants in most mycobacteria, including virulent Mycobacterium tuberculosis. We have improved this system by developing (i) a single-step strategy for the construction of allelic exchange substrates (AES), (ii) a temperature-sensitive shuttle phasmid with a greater cloning capacity than phAE87, and (iii) bacteriophage-mediated transient expression of site-specific recombinase to precisely excise antibiotic markers. The methods ameliorate rate-limiting steps in strain construction in these difficult-to-manipulate bacteria. The new methods for strain construction were demonstrated to generalize to all classes of genes and chromosomal loci by generating more than 100 targeted single- or multiple-deletion substitutions. These improved methods pave the way for the generation of a complete ordered library of M. tuberculosis null strains, where each strain is deleted for a single defined open reading frame in M. tuberculosis.
  
  
  OBJECTIVE
  This work reports major advances in the methods of genetics applicable to all mycobacteria, including but not limited to virulent M. tuberculosis, which would facilitate comparative genomics to identify drug targets, genetic validation of proposed pathways, and development of an effective vaccine. This study presents all the new methods developed and the improvements to existing methods in an integrated way. The work presented in this study could increase the pace of mycobacterial genetics significantly and will immediately be of wide use. These new methods are transformative and allow for the undertaking of construction of what has been one of the most fruitful resources in model systems: a comprehensive, ordered library set of the strains, each of which is deleted for a single defined open reading frame.
authors.forename: Paras
authors.initials: P
authors.lastname: Jain
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: Tsungda
authors.initials: T
authors.lastname: Hsu
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Arai
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: Karolin
authors.initials: K
authors.lastname: Biermann
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: David S
authors.initials: DS
authors.lastname: Thaler
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Nguyen
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: Pablo A
authors.initials: PA
authors.lastname: González
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: Joann M
authors.initials: JM
authors.lastname: Tufariello
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: Jordan
authors.initials: J
authors.lastname: Kriakov
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: Bing
authors.initials: B
authors.lastname: Chen
authors.forename: Michelle H
authors.initials: MH
authors.lastname: Larsen
authors.affiliation: jacobsw@hhmi.org.
authors.forename: William R
authors.initials: WR
authors.lastname: Jacobs
mesh_terms: D000483:Alleles; D001483:Base Sequence; D017353:Gene Deletion; D015870:Gene Expression; D023061:Gene Order; D059765:Homologous Recombination; D006801:Humans; D008969:Molecular Sequence Data; D009160:Mycobacteriophages / Q000502:physiology; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000821:virology; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D014161:Transduction, Genetic