Single-crossover integration in the Lactobacillus sake chromosome and insertional inactivation of the ptsI and lacL genes

Abstract

Single-crossover homologous integration in Lactobacillus sake was studied. Integration was conducted with nonreplicative delivery vector pRV300. This vector is composed of a pBluescript SK ؊ replicon for propagation in Escherichia coli and an erythromycin resistance marker. Random chromosomal DNA fragments of L. sake 23K ranging between 0.3 and 3.4 kb were inserted into pRV300. The resulting plasmids were able to integrate into the chromosome by homologous recombination as single copies and were maintained stably. The single cross-over integration frequency was logarithmically proportional to the extent of homology between 0.3 and 1.2 kb and reached a maximum value of 1.4 ؋ 10 3 integrants/g of DNA. We used this integration strategy to inactivate the ptsI gene, encoding enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system, and the lacL gene, which is one of the two genes required for the synthesis of a functional ␤-galactosidase. The results indicated that our method facilitates genetic analysis of L. sake.

Efficient transformation of Lactococcus lactis IL1403 and generation of knock‐out mutants by homologous recombination

“…3). The integrations were apparently stable in the presences of antibiotics, as has been noted previously in other organisms (Leloup et al 1997).…”

Enzyme I and HPr from Lactobacillus casei: their role in sugar transport, carbon catabolite repression and inducer exclusion

“…Disruption of ptsI and cloning and sequencing of DNA fragments containing flanking regions of L. casei ptsH and ptsI A 865 bp EcoRI fragment, which contained an internal part of the ptsI gene, (Fig. 1) was obtained from plasmid pUCR±H1 and subcloned into the suicide vector pRV300 (Leloup et al, 1997), providing plasmid pVME800. This plasmid was used to transform the L. casei wild-type strain BL23 and integration of the plasmid at the correct location (ptsI::pVME800) was verified by PCR and Southern blot.…”

Use of green fluorescent protein to monitor Lactobacillus sakei in fermented meat products

“…In order to use GFP as a marker expressed in L. sakei, two plasmids were constructed, both expressing GFPuv. One was based on the replicative plasmid pG+host5 [19], the second was derived from pRV300 [24] and pRV80 [13], two plasmids designed for integration in the L. sakei chromosome. The gfp uv gene was isolated from plasmid pGFPuv as an HindIII/EcoRI fragment.…”

“…2). Recombination at the ldhL locus with the 234-bp pldhL fragment was not expected as the minimum size of homology required for recombination was estimated to be 300 bp [24]. The L. sakei 23K strain was transformed with pRV86 for erythromycin resistance (Fig.…”