Simple Method for Markerless Gene Deletion in Multidrug-Resistant Acinetobacter baumannii

Appl Environ Microbiol. 2015 May 15;81(10):3357-68. doi: 10.1128/AEM.03975-14. Epub 2015 Mar 6.

Abstract

The traditional markerless gene deletion technique based on overlap extension PCR has been used for generating gene deletions in multidrug-resistant Acinetobacter baumannii. However, the method is time-consuming because it requires restriction digestion of the PCR products in DNA cloning and the construction of new vectors containing a suitable antibiotic resistance cassette for the selection of A. baumannii merodiploids. Moreover, the availability of restriction sites and the selection of recombinant bacteria harboring the desired chimeric plasmid are limited, making the construction of a chimeric plasmid more difficult. We describe a rapid and easy cloning method for markerless gene deletion in A. baumannii, which has no limitation in the availability of restriction sites and allows for easy selection of the clones carrying the desired chimeric plasmid. Notably, it is not necessary to construct new vectors in our method. This method utilizes direct cloning of blunt-end DNA fragments, in which upstream and downstream regions of the target gene are fused with an antibiotic resistance cassette via overlap extension PCR and are inserted into a blunt-end suicide vector developed for blunt-end cloning. Importantly, the antibiotic resistance cassette is placed outside the downstream region in order to enable easy selection of the recombinants carrying the desired plasmid, to eliminate the antibiotic resistance cassette via homologous recombination, and to avoid the necessity of constructing new vectors. This strategy was successfully applied to functional analysis of the genes associated with iron acquisition by A. baumannii ATCC 19606 and to ompA gene deletion in other A. baumannii strains. Consequently, the proposed method is invaluable for markerless gene deletion in multidrug-resistant A. baumannii.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acinetobacter Infections / microbiology
  • Acinetobacter baumannii / drug effects
  • Acinetobacter baumannii / genetics*
  • Acinetobacter baumannii / metabolism
  • Anti-Bacterial Agents / pharmacology
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Drug Resistance, Bacterial
  • Gene Deletion*
  • Genetic Markers
  • Genetic Techniques*
  • Humans

Substances

  • Anti-Bacterial Agents
  • Bacterial Proteins
  • Genetic Markers