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Open Access Highly Accessed Methodology article

A rapid and simple method for constructing stable mutants of Acinetobacter baumannii

Jesús Aranda1, Margarita Poza1, Belén G Pardo2, Soraya Rumbo1, Carlos Rumbo1, José R Parreira1, Patricia Rodríguez-Velo1 and Germán Bou1*

Author Affiliations

1 Servizo de Microbioloxía-INIBIC, Complexo Hospitalario Universitario A Coruña, As Xubias s/n, 15006. A Coruña, Spain

2 Departamento de Xenética. Facultade de Veterinaria. Universidade de Santiago de Compostela. Campus de Lugo. 27002 Lugo, Spain

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BMC Microbiology 2010, 10:279  doi:10.1186/1471-2180-10-279

Published: 9 November 2010

Abstract

Background

Acinetobacter baumannii is a multidrug-resistant bacterium responsible for nosocomial infections in hospitals worldwide. Study of mutant phenotypes is fundamental for understanding gene function. The methodologies developed to inactivate A. baumannii genes are complicated and time-consuming; sometimes result in unstable mutants, and do not enable construction of double (or more) gene knockout mutant strains of A. baumannii.

Results

We describe here a rapid and simple method of obtaining A. baumannii mutants by gene replacement via double crossover recombination, by use of a PCR product that carries an antibiotic resistance cassette flanked by regions homologous to the target locus. To demonstrate the reproducibility of the approach, we produced mutants of three different chromosomal genes (omp33, oxyR, and soxR) by this method. In addition, we disrupted one of these genes (omp33) by integration of a plasmid into the chromosome by single crossover recombination, the most widely used method of obtaining A. baumannii mutants. Comparison of the different techniques revealed absolute stability when the gene was replaced by a double recombination event, whereas up to 40% of the population reverted to wild-type when the plasmid was disrupting the target gene after 10 passages in broth without selective pressure. Moreover, we demonstrate that the combination of both gene disruption and gene replacement techniques is an easy and useful procedure for obtaining double gene knockout mutants in A. baumannii.

Conclusions

This study provides a rapid and simple method of obtaining stable mutants of A. baumannii free of foreign plasmidic DNA, which does not require cloning steps, and enables construction of multiple gene knockout mutants.