High-frequency flp recombinase-mediated inversions of the oriC-containing region of the Pseudomonas aeruginosa genome

Abstract

The genomes of the two clonally derived Pseudomonas aeruginosa prototypic strains PAO1 and DSM-1707 differ by the presence of a 2. 19-Mb inversion including oriC. Integration of two Flp recombinase target sites near the rrn operons containing the inversion endpoints in PAO1 led to Flp-catalyzed inversion of the intervening 1.59-Mb fragment, including oriC, at high frequencies (83%), favoring the chromosome configuration found in DSM-1707. The results indicate that the oriC-containing region of the P. aeruginosa chromosome can readily undergo and tolerate large inversions.

FIG. 1

Genomic maps of P. aeruginosa PAO236 (A) and its inversion derivative PAO238 (B). Position 1 is defined as the first nucleotide of oriC. The locations of FRT sites in the Δ(mexAB-oprM)::FRT and Δ(mexCD-oprJ)::FRT mutants and their orientations are indicated by solid circles. The four rrn operons (16) and their chromosome coordinates are indicated by open circles. Since PAO236 is derived from PAO1, their map coordinates are identical except that PAO236 contains a FRT sequence inserted at the mexAB-oprM locus and a FRT-Gm^r-FRT cassette at the mexCD-oprJ locus.

FIG. 2

Chromosomal maps of strains constructed in this study and PCR analysis. Maps of strain PAO236 [Δ(mexAB-oprM)::FRT Δ(mexCD-oprJ)::Gm^r-FRT] (A), its Gm^s derivative PAO277 (B), and a Gm^s derivative (PAO238) that has undergone a chromosomal inversion between the indicated FRT sites (C) are shown. DNA sequences and their transcriptional orientations from the mexAB-oprM and mexCD-oprJ operons are indicated in white and black boxes, respectively. (D) Genomic Southern analyses of strains isolated in this study. One-microgram samples of chromosomal DNAs isolated from the indicated strains were digested with BamHI and separated by electrophoresis on a 1% agarose gel in Tris-acetate-EDTA buffer (11). The separated fragments were transferred to Immobilon-P membranes (Millipore, Bedford, Mass.) and probed with the biotinylated insert from pPS1088. The probe was biotinylated, and the fragments were detected with the NEBlot Phototype labeling and Photostar detection kits from New England Biolabs (Beverly, Mass.), respectively. Lane M contained biotinylated λHindIII standards from New England Biolabs, and their sizes in kilobases are indicated on the left. Lanes: PAO1, wild-type; PAO236 [Δ(mexAB-oprM)::FRT Δ(mexCD-oprJ)::Gm^r-FRT]; PAO277 and PAO278 [Δ(mexAB-oprM)::FRT Δ(mexCD-oprJ)::FRT]; PAO238 [Δ(mexAB-oprJ)::FRT Δ(mexCD-oprM)::FRT], containing the 1.59-Mb chromosomal inversion; PAO238-F [Δ(mexAB-oprJ)::FRT Δ(mexCD-oprM)::FRT], a strain retaining the inversion after reintroduction of pFLP2 into PAO238; PAO281 and PAO282 [Δ(mexAB-oprM)::FRT Δ(mexCD-oprJ)::FRT], two strains that reverted back to the chromosomal configuration found in strains PAO277 and PAO278 after reintroduction of pFLP2 into PAO238.

FIG. 3

(A) PCR analysis of strains PAO238, PAO277, and PAO281 utilizing the primer pairs indicated in the highlighted box. Aliquots of the PCRs were analyzed on 1.5% agarose gels in Tris-acetate-EDTA buffer (11) and stained with ethidium bromide. (B) Sequence analysis of PCR fragments from the mexAB-oprJ and mexCD-oprJ regions of the PAO chromosome. The PCR fragments from the reaction mixtures obtained from PAO238 (panel A, lanes 4 and 5) were purified from an agarose gel using a Geneclean kit (BIO 101, Vista, Calif.), and their sequences were determined by automated DNA sequencing at the University of Colorado at Boulder sequencing facility employing the same primers used in the PCRs. Top three lines, partial sequence of the PCR fragment obtained with primers AB_down and CD_up. Inverted arrows delimit chromosomal sequences and FRT sequences, respectively. Restriction enzyme cleavage sites found in the FRT site are indicated in lowercase letters, as is the initiation codon of nfxB (top). Bottom three lines, partial sequence of the PCR fragment obtained with primers AB_up and CD_down.

FIG. 4

PFGE analysis of PacI digests of PAO genomic DNAs. Samples were prepared and digested with PacI, and fragments were separated by PFGE as previously described (10, 13). The strains analyzed were PAO236 and PAO237 [Δ(mexAB-oprM)::FRT Δ(mexCD-oprJ)::Gm^r-FRT], PAO238 and PAO239 [Δ(mexAB-oprJ)::FRT Δ(mexCD-oprM)::FRT], PAO1 (a prototrophic strain used for determination of the genomic sequence), and DSM-1707 (a prototrophic strain, clonally derived from the same PAO isolate as PAO1). White asterisks mark fragments that differ in individual strains. The sizes of PacI fragments from strain PAO1 and its derivatives are indicated on the left.