Cre-loxP recombination system for large genome rearrangements in Lactococcus lactis

Abstract

We have used a new genetic strategy based on the Cre-loxP recombination system to generate large chromosomal rearrangements in Lactococcus lactis. Two loxP sites were sequentially integrated in inverse order into the chromosome either at random locations by transposition or at fixed points by homologous recombination. The recombination between the two chromosomal loxP sites was highly efficient (approximately 1 x 10(-1)/cell) when the Cre recombinase was provided in trans, and parental- or inverted-type chromosomal structures were isolated after removal of the Cre recombinase. The usefulness of this approach was demonstrated by creating three large inversions of 500, 1,115, and 1,160 kb in size that modified the lactococcal genome organization to different extents. The Cre-loxP recombination system described can potentially be used for other gram-positive bacteria without further modification.

FIG. 1.

Schematic representation of the Cre-loxP recombination system. A, B, C, and D represent the chromosome order. The letter n represents the number of AU of the integrated plasmids. The loxP sites, represented by triangles, are drawn in inverse orientation in order to yield an inversion after Cre-mediated recombination. The other option, whereby the loxP sites would be oriented as direct repeats, has been omitted from the figure.

FIG. 2.

(A) Structures of the loxP cassettes used in the Cre-loxP recombination system. The loxP sites are represented by triangles. (B) Plasmids used for the Cre-loxP recombination. Abbreviations: N, NotI; C, I-CeuI; A, ApaI; H, HindIII; E, EcoRI; S, SmaI; Ac, AccI; X, XbaI. (C) Schematic representation of the chromosomal organization of strain NZ9000 showing the location of the inversion endpoints. Different gray scales indicate inverted fragments. Arrows indicate the orientation of the loxP sites.

FIG. 3.

(A) Theoretical chromosome structures of strain CL307/1 (panel 1), strain CL307/1-311 (panel 2), and parental- (panel 3) and inverted (panel 4)-type strains. The predicted sizes of the I-CeuI restriction fragments are indicated, and those hybridizing with the tetL probe are indicated in boldface. Fragment sizes are not drawn to scale. C, I-CeuI. (B) Hybridization of I-CeuI-digested chromosomes with the tetL probe after separation by PFGE (electrophoresis conditions were 10 V/cm and 17 s of pulse time for 13 h). Lanes: M, lambda DNA concatemer size standards; L, HindIII-digested lambda DNA; 1, CL307/1-311; 2, CL307/1-311 × pGh-Cre in permissive replication conditions; 3, parental-type strain; 4, inverted-type strain. Hybridization was performed in the presence of ³²P-labeled lambda DNA.

FIG. 4.

Visualization of the pCL307 and pCL311 amplification units in strains CL307/1-311 and CL307/1-311 × pGh-Cre in different conditions by hybridization of PFGE-separated chromosomal DNA after ApaI digestion with pBSIIKS(−) as a probe. The number above each lane indicates the generation number. The signs − and + correspond to the absence and presence, respectively, of Em and Spc in the growth medium.

FIG. 5.

Hybridization of ApaI-digested chromosomes with the tetL probe after separation by PFGE (electrophoresis conditions were 10 V/cm and 7 s of pulse time for 13 h). Lanes: M, lambda DNA concatemer size standards; L, HindIII-digested lambda DNA; 1, CL307/5-311 × pGh-Cre in permissive replication conditions; 2, parental-type strain isolated after pGh-Cre curing; 3, inverted-type strain isolated after pGh-Cre curing. Hybridization was performed in the presence of ³²P-labeled lambda DNA. The letter I corresponds to the ApaI fragment that signs the inverted structure, whereas P corresponds to the ApaI fragment characteristic of the parental structure.