Abstract
BackgroundAcinetobacter 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.ResultsWe 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.ConclusionsThis 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.
Highlights
Acinetobacter baumannii is a multidrug-resistant bacterium responsible for nosocomial infections in hospitals worldwide
Acinetobacter baumannii is a Gram-negative coccobacillus that is increasingly recognized as a major pathogen causing nosocomial infections worldwide, in patients admitted to intensive care units [1,2]
The method most commonly used to generate A. baumannii mutants involves integration of a plasmid into the chromosome by single crossover recombination. This method requires an internal fragment homologous to the target gene cloned into a suicide vector carrying resistance cassettes [10], which is a major limitation for systematic construction of mutants in post-genomic studies of A. baumannii
Summary
Acinetobacter baumannii is a multidrug-resistant bacterium responsible for nosocomial infections in hospitals worldwide. Study of mutant phenotypes is fundamental for understanding gene function. Acinetobacter baumannii is a Gram-negative coccobacillus that is increasingly recognized as a major pathogen causing nosocomial infections worldwide, in patients admitted to intensive care units [1,2]. The method most commonly used to generate A. baumannii mutants involves integration of a plasmid into the chromosome by single crossover recombination. This method requires an internal fragment homologous to the target gene cloned into a suicide vector carrying resistance cassettes [10], which is a major limitation for systematic construction of mutants in post-genomic studies of A. baumannii. The gene replacement method is a useful way of overcoming these limitations
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