Table 1.
Bacterial strains, plasmids, and primers
| Strain, plasmid, or primer pair | Relevant genotypic or phenotypic propertiesa | Source and/or referenceb |
|---|---|---|
| Strains | ||
| L. lactis | ||
| IL1403 | Gal+, plasmid‐free wild‐type, host strain | INRA (Chopin et al.1984) |
| LL302 | L. lactis MG1363 derivative, RepA+ | (Leenhouts et al.1998) |
| IL1403ΔglaR | Gal‐, ΔglaR, Ems , plasmid‐free, IL1403 derivative | This study |
| IL1403ccpA ‐ | Lac+ , CcpA‐ (ISS1), Ems, plasmid‐free, IL1403 derivative | (Aleksandrzak‐Piekarczyk et al., 2005) |
| IL1403ΔglaR‐pGhost9glaR | Gal+, Emr, IL1403ΔglaR derivative carrying pGhost9glaR | This study |
| E. coli | ||
| TG1 | Δ(hsdMS‐mcrB)5 Δ(lac‐proAB) supE thi‐1 F’(traD36 proAB + lacI q ZΔM15) | (Gibson1984) |
| EC1000 | Kmr, RepA+ MC1000 | (Leenhouts et al.1996) |
| BL21 | B F‐ ompT dcm lon hsdSB(rB‐ mB‐) gal [malB+]K‐12(λS) | (Miroux and Walker1996) |
| Plasmids | ||
| pGEM‐T | Ampr, M13ori, linear T‐overhang vector | Promega |
| pGhost9 | Emr, repA (Ts) | INRA (Maguin et al.1996) |
| pIL253 | Emr, high‐copy number lactococcal vector | (Simon and Chopin1988) |
| ptXB1 | Ampr, M13ori, rop, lacI, Mxe GyrA intein | New England Biolabs |
| Recombinant plasmids | ||
| pGhost9ΔglaR | Emr, pGhost9 carrying glaR upstream, and downstream regions | This study |
| pGhost9glaR | Emr , pGhost9 carrying glaR under the control of its promoter | This study |
| Primersc | ||
| For deletion and complementation of the glaR gene | ||
| glaRUPf/glaRUPr | CCATCGATTCAAGTTCCCAAACGCTCC/GGAGAATTCGCCAAGTATAGGATTCAGC | |
| glaRDOWNf/glaRDOWNr | GGAGAATTCCAAGAGTAGTCTTGAGGTG/AAGATGACATAATCCCACCAACAAC | |
| glaRfor/glaRrev | GCTAAGACCGCAGCTTC/GACCAGAAGGCAATGTC | |
| ptXB1for/glaRBamHrev | GTGAGCGGATAACAATTCC/GGATCCTTATTGTTTTAAAGTATAAATGG | |
| For qPCR amplifications | ||
| LlGlaRaF/LlGlaRaR | TGCAACTTTTCCGTAAGCCC/TTGGGATTTTGTCCTTTGGC | |
| LlLacSaF/LlLacSaR | CTGGAACACCACATGAGGATGC/AAGATGACATAATCCCACCAACAAC | |
| LlGalMaF/LlGalMaR | TGACCATCCTTTCTTGTTAGACCAG/CCATGGTGCACTTGCTTTTTC | |
| LlGalKaF/LlGalKaR | AACAAGCCGGTGTCTTGGG/TCCAACTTTGTTGAACCAGAACC | |
| LlGalTaF/LlGalTaR | AAAAAGACCCCAAAGCCATTG/ATTGGAAGCCCCAGTCTTCG | |
| LlThgAaF/LlThgAaR | CCAAATGTTACGATTGACACGG/AGACTCCCTGCGCCAATCAC | |
| LlLacZaF/LlLacZaR | GAAAGCACTTCTTGTTCGTGGAG/TCACACAATTCATACCAGCGTG | |
| LlGalEaF/LlGalEaR | GCCTGATGGAACTTGTATTCGTG/CCTGTTACTTTTCGTGCGGTTTC | |
| LlYufCaF/LlYufCaR | TTGCAGGAGAAACTTTGACGG/TCTGCCCACGGAATAGCAC | |
| LlPurMaF/LlPurMaR | ATTGCGTAGCCATGTGCGTC/CTGTTTCTCCACCAATCAGCG | |
| LlTufaF/LlTufaR | CGTGACCTCTTGAGCGAATACG/GAGTGGTTTGTCAGTGTCGCG | |
| For amplification of nucleotides for EMSA | ||
| glaRfor/glaRrev | GCCAGAGTCCTAATGAAAG/CATGGCTTACTATGCCC | |
| lacSfor/lacSrev | CTAATTGATGCTTACTCC/CTTTCATGGGAATCCTCC | |
| galMfor/galMrev | GCCTATCCTGGTGCAAC/CCATGATATTTCCTAACT | |
| galTfor/galTrev | GTTGTCGGTTATCCAGC/CAAGTGGCTCAATCGTTCC | |
| thgAfor/thgArev | CAGGAAGCAGTTGGAGAAG/CAGCCAGAGCAACAAATGG | |
| galEfor/galErev | GGACATTGGCATCTACTTG/CTGCCACATCGTAACCACG | |
| yufAfor/yufArev | CTTGAAGTGCTTGAAACC/CCATTACATTTTCATGACG | |
aAmp, ampillicin; Em, erythromycin; Km, kanamycin; r, resistance; s, sensitivityl; CcpA, catabolite control protein AbINRA, Institut National de la Recherche Agronomique (Jouy‐en‐Josas, France).cAll primers were designed on the basis of the L. lactis IL1403 genome nucleotide sequence, NCBI with accession no. AE005176 (http://www.ncbi.nlm.nih.gov/genome). To certain primers, restriction sites were added for digestion with EcoRI or ClaI.