TABLE 1.
Strains, plasmids, and primers used in this study and their relevant characteristics
| Material | Relevant features or sequencea | Source or reference |
|---|---|---|
| Strains | ||
| Escherichia coli MC1061 | Cloning host | 9 |
| Lactococcus lactis MG1363 | Cloning host | 18 |
| Lactobacillus plantarum | ||
| WCFS1 | Wild-type for which the genome sequence is available | 28 |
| WCFS1-R | Rifr | This work |
| NZ7109 | Rifr Emr; L. plantarum WCFS1 derivative containing a chromosomally located loxP-ery-loxP-cassette | This work |
| Plasmids | ||
| pUC18 | Apr | 54 |
| pUC19 | Apr | 54 |
| pNZ7105 | Apr; pUC18 derivative containing a 5′-truncated fragment of lp_3504 | This work |
| pNZ7106 | Apr; pUC19 derivative containing a 5′-truncated fragment of lp_3503 | This work |
| pNZ7107 | Apr; pNZ7105 derivative containing lp_3503 originating from pNZ7106 | This work |
| pUC19lox1 | Apr; pUC18 derivative containing a loxP site | This work |
| pUC19lox2 | Apr; pUC18 derivative containing 2 loxP sites in tandem repeat | This work |
| pGhost8 | Tetr | 35 |
| pNZ7103 | Apr Tetr; pUClox2 derivative containing tetR originating from pGhost8 | This work |
| pNZ7108 | Apr Tetr; pNZ7107 derivative containing loxP-tetR-loxP cassette originating from pNZ7103 | This work |
| pUC18ery | Apr Emr | 50 |
| pNZ7109 | Apr Emr; contains ery originating from pUC18ery flanked by two synthetic loxP sites and 5′-truncated fragments of lp_3503 and lp_3504 originating from pNZ7106 and pNZ7105, respectively (Fig. 1) | This work |
| pNZ7110 | Apr, pUC18 derivative containing cre and TpepN | 6 |
| pJIM2246 | Low-copy-cloning vector; Cmr | 44 |
| pNZ7124 | Cmr; pJIM2246 derivative containing cre and TpepN originating from pNZ7110 | This work |
| pNZ7125 | Cmr; pNZ7124 derivative containing Tlox upstream of cre | This work |
| pNZ7126 | Cmr; pNZ7125 derivative containing PldhL1 upstream of cre | This work |
| Primers | ||
| cre-R2 | 5′-GTCCATCAGGTTCTTGCG-3′ | |
| BglII-cre | 5′-ATAGTTTACCCCGTCAGC-3′ | |
| lp_3503F | 5′-TGCTTTCCAAGAGCAAGCTG-3′ | |
| lp_3503R | 5′-ACGTCTGCAGTCAGGTGTGAAGTTGGCACT-3′ | |
| lp_3504F | 5′-ACGTCTGCAGCCACTCACCGACTAACACTC-3′ | |
| lp_3504R | 5′-ACGTAAGCTTGACCCACGAGTTACCAACACG-5′ | |
| flox1 | 5′-AGCTCTGCAGATAACTTCGTATAGCATACATTATACGAAGTTATTTGCA-3′ | |
| flox2 | 5′-AATAACTTCGTATAATGTATGCTATACGTAGTTATCTGCAG-3′ | |
| flox3 | 5′-CATAACTTCGTATAGCATACATTATACGAAGTTATCTGCAG-3′ | |
| flox4 | 5′-AATTCTGCAGATAACTTCGTATAATGTATGCTATACGAAGTTATGAGCT-3′ | |
| hrec3503 | 5′-GCATCGCTGCACTCTTTATG-3′ | |
| hrec3504 | 5′-GAATATGGTCCATGGTCCAC-3′ | |
| eryF | 5′-AAGCAATGAAACACGCC-3 | |
| eryR | 5′-TTAGCCAGTTTCGTCGTT-3′ | |
| lasF2 | 5′-ACGTGGATCCGGACAATATGGGGTAAGCG-3′ | |
| lasR | 5′-AAGAAGATCTCTAAAGCTGACGGGGTAAAC-3′ | |
| PldhL-F | 5′-GAAGATCTTCAATCTTCTCACCGTCTTG-3′ | |
| PldhL-R | 5′-GAAGATCTTCAATAAGTCATCCTCTCGT-3′ |
a
Underlined sequences indicate restriction sites subsequently used in cloning procedures. Apr, ampicillin resistant; Cmr, chloramphenicol resistant; Emr, erythromycin resistant; Rifr, rifampicin resistant; Tetr, tetracycline resistant.