Efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosomes via homologous recombination

Abstract

An efficient method is described for the generation of site-specific chromosomal integrations in Lactobacillus acidophilus and Lactobacillus gasseri. The strategy is an adaptation of the lactococcal pORI system (K. Leenhouts, G. Venema, and J. Kok, Methods Cell Sci. 20:35-50, 1998) and relies on the simultaneous use of two plasmids. The functionality of the integration strategy was demonstated by the insertional inactivation of the Lactobacillus acidophilus NCFM lacL gene encoding beta-galactosidase and of the Lactobacillus gasseri ADH gusA gene encoding beta-glucuronidase.

FIG. 1

Stability of pGK12 (♦) and pTRK669 (●) in L. acidophilus NCFM at 37°C (closed symbols) and 43°C (open symbols). The percentage of Cm^r cells in each culture was determined by plating on MRS versus MRS plus chloramphenicol.

FIG. 2

(A) Southern hybridization analysis of L. acidophilus NCFM and NCK1398. Chromosomal DNA was digested with EcoRI (lanes 1, 2, and 3) or HindIII (lanes 4 and 5) and hybridized with the 945-bp internal lacL fragment as a probe. Lane 1, pTRK670; lanes 2 and 4, NCFM; lanes 3 and 5, NCK1398; M, digoxigenin-labeled λ-HindIII marker. DNA sizes are indicated in kilobases. (B) Schematic representation of the relevant regions of the NCFM and NCK1398 chromosomes. Chromosomal DNA is represented by a heavy line, plasmid DNA is represented by a thin line, the lacL gene is represented by an arrow, and the internal lacL fragment is represented by a solid box. The EcoRI (E) and HindIII (H) sites are indicated. The repeating unit represents the plasmid DNA present in various copies (“n”). The diagram is not drawn to scale.

FIG. 3

(A) Southern hybridization analysis of L. gasseri ADH and NCK1423. Chromosomal DNA was digested with EcoRI (lanes 1, 2, and 3) or EcoRV (lanes 4 and 5) and hybridized with the 777-bp internal gusA fragment as a probe. Lane 1, pTRK685; lanes 2 and 4, ADH; lanes 3 and 5, NCK1423; M, digoxigenin-labeled λ-HindIII marker. DNA sizes are indicated in kilobases. (B) Schematic representation of the relevant regions of the ADH and NCK1423 chromosomes. Chromosomal DNA is represented by a heavy line, plasmid DNA is represented by a thin line, the gusA gene is represented by an arrow, and the internal gusA fragment is represented by a solid box. The EcoRI (E) and EcoRV (V) sites are indicated. The diagram is not drawn to scale.