TABLE 1.
Strains and plasmids used in this study, their relevant characteristics, and references
| Strain or plasmid | Relevant featuresa | Reference |
|---|---|---|
| E. coli strains | ||
| Top10 | Cloning host | |
| XL1-blue | Cloning host | |
| L. lactis MG1363 | Cloning host | 18 |
| L. plantarum strains | ||
| NZ7100 | L. plantarum WCFS1 with chromosomal integration of plasmid pEMnisRK | 44 |
| NZ7140 | L. plantarum NZ7100 with ΔmelA obtained by double-crossover integration of pNZ5340 | This study |
| NZ1800 | Integration of L. lactis usp45 promoter in front of L. plantarum NZ7100 melA | This study |
| NZ1801 | NZ1800 with substitution mutation in the catalytic site of melA, constructed using the pNZ1801 mutagenesis vector | This study |
| NZ1802 | NZ1800 with substitution mutation in the catalytic site of melA, constructed using the pNZ1802 mutagenesis vector | This study |
| NZ1803 | NZ1800 with 1-bp deletion in the catalytic site of melA, constructed using the pNZ1803 mutagenesis vector | This study |
| NZ1804 | NZ1800 with 2-bp deletion in the catalytic site of melA, constructed using the pNZ1804 mutagenesis vector | This study |
| NZ1805 | NZ1800 with 1-bp insertion in the catalytic site of melA, constructed using the pNZ1805 mutagenesis vector | This study |
| NZ1806 | NZ1800 with 2-bp insertion in the catalytic site of melA, constructed using the pNZ1806 mutagenesis vector | This study |
| NZ1807 | NZ1800 with G stretch and 1-bp insertion in melA, constructed using the pNZ1807 mutagenesis vector | This study |
| Plasmids | ||
| pNZ5340 | Cmr Emr; integration vector with homologous regions up- and downstream of L. plantarum WCFS1 melA | 27 |
| pNZ5516 | Kmr; pCRblunt containing Pusp45-melA | 5 |
| pNZ1825 | Cmr; pNZ5516 derivative containing Pusp45-melA-cat-lox71 downstream region of melA | This study |
| pNZ1826 | Cmr; pNZ1825 derivative containing upstream region of melA | This study |
| pNZ1800 | Cmr; pNZ1826 derivative, insertion of a lox66 region; integration vector for inserting Pusp45 in front of melA based on cre-lox system | This study |
| pNZ5348 | Emr; pGID023 derivative containing cre under control of the lp_1144 promoter | 27 |
| pNZ1801 | Cmr; pNZ1800 derivative; substitution mutation in the catalytic site of melA | This study |
| pNZ1802 | Cmr; pNZ1800 derivative; substitution mutation in the catalytic site of melA | This study |
| pNZ1803 | Cmr; pNZ1800 derivative; 1-bp deletion in the catalytic site of melA | This study |
| pNZ1804 | Cmr; pNZ1800 derivative; 2-bp deletion in the catalytic site of melA | This study |
| pNZ1805 | Cmr; pNZ1800 derivative; 1-bp insertion in the catalytic site of melA | This study |
| pNZ1806 | Cmr; pNZ1800 derivative; 2-bp insertion in the catalytic site of melA | This study |
| pNZ1817 | Cmr; pNZ1800 derivative; G stretch and 1-bp insertion in melA | This study |
| pIL252 | Emr; low-copy-number vector | 45 |
| pNZ8048 | Cmr; high-copy-number vector with inducible PnisA from L. lactis | 12 |
| pNZ8154 | Emr; pIL252 derivative; mutator vector with PnisA-TpepN fragment of pNZ8048 | This study |
| pCRblunt | Kmr; positive-selection cloning vector | 6 |
| pNZ1813 | Emr; pNZ8154 derivative; insertion of L. plantarum WCFS1 hexA | This study |
| pNZ1814 | Emr; pNZ8154 derivative; insertion of L. plantarum WCFS1 truncated hexA | This study |
| pNZ1820 | Emr; pNZ8154 derivative; insertion of L. plantarum WCFS1 dinP | This study |
a
Cmr, chloramphenicol resistant; Emr, erythromycin resistant; Kmr, kanamycin resistant.