Replacement recombination in Lactococcus lactis

Abstract

In the pUC18-derived integration plasmid pML336 there is a 5.3-kb chromosomal DNA fragment that carries the X-prolyl dipeptidyl aminopeptidase gene (pepXP). The gene was inactivated by the insertion of an erythromycin resistance determinant into its coding sequence. Covalently closed circular DNA of pML336 was used for the electrotransformation of Lactococcus lactis. In 2% of the erythromycin-resistant transformants the pepXP gene was inactivated by a double-crossover event (replacement recombination) between pML336 and the L. lactis chromosome. The other transformants in which the pepXP gene had not been inactivated carried a

A general system for generating unlabelled gene replacements in bacterial chromosomes

“…Taken together, the frequencies by which gene replacements were generated in the (Table 2). These frequencies are in agreement with our previous results (Leenhouts et al 1991b) and demonstrate that the pORI system can be used to generate gene replacements in regions with low recombination frequencies.…”

“…lactis using a pUC derivative. Frequencies appeared to be relatively low with values as low as 10\ per generation (Leenhouts et al 1991b). Therefore, the pepXP region was chosen to test the pORI delivery system.…”

“…PepXP-deficient mutants were identified using a PepXP plate assay as described before (Leenhouts et al 1991b). AcmA activity was analysed as described by Buist et al (1995).…”

Characterization of the nisin gene cluster nisABTCIPR of Lactococcus lactis

“…An approximately 1 -kbp EcoRI (blunt ended by Klenow-enzyme treatment)-SphI fragment from pUCl9E, containing the erythromycin-resistance gene, was isolated and ligated in the XbaI-digested (blunt ended by Klenowenzyme treatment) and Sphl-digested vector pNZ9005.1, resulting in the plasmid pNZ9006. This pUC19-derived plasmid, which cannot replicate in L. lactis (Leenhouts et al, 1990), was used to transform the nisin-A-producing L. lactis NZ9700. Erythromycin-resistant transformants were obtained, which contained a single chromosomal copy of plasmid pNZ9006.…”

“…Micrococcusfluvus DSM 1790 (obtained from the German Collection of Microorganisms) was used to determine antimicrobial activity as described previously (Kuipers et al, 1992). The following plasmids were used in cloning experiments in E. coli: pUC19 and MI 3mp18/19 (Yanisch-Perron et al, 1985); pET8c, pET3b, pLysS and pLysE (Rosenberg et al, 1987); pUCl9E, a pUC19 derivative described by Leenhouts et al (1990), containing the erythromycin-resistance gene of pE194 (Horinouchi and Weisblum, 1982); pNZ184, a derivative of pA-CYC184 (Chang and Cohen, 1978), in which the SmaIHind111 multiple cloning site of M13mp19 was cloned into the Si~zaIJHindIII-digested vector, to introduce a unique PstI site (van Alen-Boerrigter et a]., 1991). In L. lactis, the plasmids pIL253 (Simon and Chopin, 1988) and pNZ9010 (containing nisA) and pNZ9013 (containing nis2; Kuipers et al, 1992) were used.…”