TABLE 1.

Strains and plasmids used in this study, their relevant characteristics, and references

Strain or plasmidRelevant featuresaReference
E. coli strains
    Top10Cloning host
    XL1-blueCloning host
L. lactis MG1363Cloning host18
L. plantarum strains
    NZ7100L. plantarum WCFS1 with chromosomal integration of plasmid pEMnisRK44
    NZ7140L. plantarum NZ7100 with ΔmelA obtained by double-crossover integration of pNZ5340This study
    NZ1800Integration of L. lactis usp45 promoter in front of L. plantarum NZ7100 melAThis study
    NZ1801NZ1800 with substitution mutation in the catalytic site of melA, constructed using the pNZ1801 mutagenesis vectorThis study
    NZ1802NZ1800 with substitution mutation in the catalytic site of melA, constructed using the pNZ1802 mutagenesis vectorThis study
    NZ1803NZ1800 with 1-bp deletion in the catalytic site of melA, constructed using the pNZ1803 mutagenesis vectorThis study
    NZ1804NZ1800 with 2-bp deletion in the catalytic site of melA, constructed using the pNZ1804 mutagenesis vectorThis study
    NZ1805NZ1800 with 1-bp insertion in the catalytic site of melA, constructed using the pNZ1805 mutagenesis vectorThis study
    NZ1806NZ1800 with 2-bp insertion in the catalytic site of melA, constructed using the pNZ1806 mutagenesis vectorThis study
    NZ1807NZ1800 with G stretch and 1-bp insertion in melA, constructed using the pNZ1807 mutagenesis vectorThis study
Plasmids
    pNZ5340Cmr Emr; integration vector with homologous regions up- and downstream of L. plantarum WCFS1 melA27
    pNZ5516Kmr; pCRblunt containing Pusp45-melA5
    pNZ1825Cmr; pNZ5516 derivative containing Pusp45-melA-cat-lox71 downstream region of melAThis study
    pNZ1826Cmr; pNZ1825 derivative containing upstream region of melAThis study
    pNZ1800Cmr; pNZ1826 derivative, insertion of a lox66 region; integration vector for inserting Pusp45 in front of melA based on cre-lox systemThis study
    pNZ5348Emr; pGID023 derivative containing cre under control of the lp_1144 promoter27
    pNZ1801Cmr; pNZ1800 derivative; substitution mutation in the catalytic site of melAThis study
    pNZ1802Cmr; pNZ1800 derivative; substitution mutation in the catalytic site of melAThis study
    pNZ1803Cmr; pNZ1800 derivative; 1-bp deletion in the catalytic site of melAThis study
    pNZ1804Cmr; pNZ1800 derivative; 2-bp deletion in the catalytic site of melAThis study
    pNZ1805Cmr; pNZ1800 derivative; 1-bp insertion in the catalytic site of melAThis study
    pNZ1806Cmr; pNZ1800 derivative; 2-bp insertion in the catalytic site of melAThis study
    pNZ1817Cmr; pNZ1800 derivative; G stretch and 1-bp insertion in melAThis study
    pIL252Emr; low-copy-number vector45
    pNZ8048Cmr; high-copy-number vector with inducible PnisA from L. lactis12
    pNZ8154Emr; pIL252 derivative; mutator vector with PnisA-TpepN fragment of pNZ8048This study
    pCRbluntKmr; positive-selection cloning vector6
    pNZ1813Emr; pNZ8154 derivative; insertion of L. plantarum WCFS1 hexAThis study
    pNZ1814Emr; pNZ8154 derivative; insertion of L. plantarum WCFS1 truncated hexAThis study
    pNZ1820Emr; pNZ8154 derivative; insertion of L. plantarum WCFS1 dinPThis study
  • a Cmr, chloramphenicol resistant; Emr, erythromycin resistant; Kmr, kanamycin resistant.