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

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.

Figure 1

omp33 replacement. (a) Schematic representation of the linear DNA constructed for the omp33 gene replacement, which was completely deleted. The oligonucleotides used (small arrows) are listed in Table 2. (b) Screening of omp33 A. baumannii mutants generated by gene replacement. The numbers at the top are bacterial colony numbers. WT, Wild-type control with 2115 bp. Colonies 5 and 7 (lanes 5* and 7*) with 2214 bp (2115 bp - 834 bp [from omp33 deletion] + 933 bp [from kanamycin insertion]) were sequenced to confirm gene replacement. Lambda DNA-Hind III and ϕX174 DNA-Hae III Mix (Finnzymes) was used as a size marker (M). The lengths of PCR products and of some molecular size marker fragments are also indicated.

Figure 2

omp33 disruption. (a) Schematic representation of the strategy used to construct the omp33 mutant by gene disruption (omp33::TOPO). The oligonucleotides used (small arrows) are listed in Table 2. The boxes indicated by A and A' represent the original and the cloned internal fragment of the omp33 gene, respectively. See Materials and Methods for details. (b) Screening of omp33 A. baumannii mutants generated by gene disruption. The numbers at the top are bacterial colony numbers. All PCR products with 697 bp and 798 bp (amplified with primer pairs 33extFW + SP6 and T7 + 33extRV, respectively) were sequenced to confirm omp33 gene disruption. Lambda DNA-Hind III and ϕX174 DNA-Hae III Mix (Finnzymes) was used as a size marker (M). The wild-type strain (WT) was used as a negative control. The lengths of PCR products and of some molecular size marker fragments are also indicated.

Figure 3

Omp33 detection. (a) 2-DE gels showing A. baumannii proteins from the wild-type strain (ATCC 17978), Δomp33::Km mutant, and Δomp33::Km mutant complemented with pET-RA-OMP33 plasmid (+33). The black circles indicate the Omp33 protein. (b) Western blot analysis showing the detection of the Omp33 protein in the protein extracts obtained from the wild-type and the pETRA-OMP33- complemented mutant strains. (+33): Strains complemented with the pETRA-OMP33 plasmid. C-: Δomp33::Km mutant containing the pET-RA vector (without the omp33 gene) as a negative control. The last lane (C+) indicates detection of the purified Omp33 protein used as a positive control. (c) Reversible staining of the membrane containing the transferred protein extracts from the indicated strains showing similar amounts of the majority protein (43 kDa) prior to Western blot analysis.

Figure 4

oxyR and soxR replacement. (a) Schematic representation of the linear DNA constructed for the oxyR gene replacement. The oligonucleotides used (small arrows) are listed in Table 2. (b) Screening of oxyR A. baumannii mutants generated by gene replacement. The numbers at the top are bacterial colony numbers. WT; Wild-type control showing 1600 bp. Colonies 4 and 7 (lanes 4* and 7*) showing 2275 bp (1600 pb - 258 bp [from oxyR deletion] + 933 bp [from kanamycin insertion]) were sequenced to confirm gene replacement. Lambda DNA-Hind III and ϕX174 DNA-Hae III Mix (Finnzymes) was used as a size marker (M). (c) Schematic representation of the linear DNA constructed for the soxR gene replacement. The oligonucleotides used (small arrows) are listed in Table 2. (d) Screening of soxR A. baumannii mutants generated by gene replacement. WT: Wild-type control with 1300 bp. Colonies 1, 2, and 3 (lanes 1*, 2*, and 3*) with 2093 bp (1300 bp - 140 bp [from soxR deletion] + 933 bp [from kanamycin insertion]) were sequenced to confirm gene replacement. Lambda DNA-Hind III and ϕX174 DNA-Hae III Mix (Finnzymes) was used as a size marker (M).

Figure 5

Transcriptional analysis. RT-PCR analysis of RNA extracted from the wild-type, ΔoxyR::Km, ΔsoxR::Km, ΔoxyR::Km-omp33::TOPO, and ΔsoxR::Km-omp33::TOPO strains showing the lack of oxyR and soxR transcription in the corresponding mutants. The gyrB gene was used as a housekeeping gene. The lengths of cDNAs obtained are indicated.

Figure 6

Gene replacement. (a) Schematic representation of the strategy used to construct mutants by gene replacement. Small, red and shaded arrows represent the primers, the target gene, and the kanamycin (Km) resistance cassette, respectively. The three PCR products obtained (PCR1, PCR2, and PCR3) were mixed at equimolar concentrations and subjected to a nested overlap-extension PCR to generate the desired linear DNA (see Materials and Methods for details). (b) Diagram showing the integration of the linear DNA via two recombination events. (c) Representation of the original genetic material replaced by the recombinant DNA on the A. baumannii chromosome.

Figure 7

pET-RA construction. Schematic representation of the construction of the pET-RA plasmid. The GenBank accession numbers of the plasmids are indicated in parenthesis. Rif, rifampicin; Amp, ampicillin; GFP, green fluorescent protein.