TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Relevant characteristicsa | Source or reference |
|---|---|---|
| Lactococcus lactis subsp. lactis | ||
| BGMN1-596 | Plasmid-free derivative of L. lactis subsp. lactis BGMN1-5 | 18 |
| BGMN1-596R2/23 | Mutant of BGMN1-596 semiresistant to LsbB up to 625 μg/ml | 16 |
| BGMN1-596T | BGMN1-596 transformed with pMN5 plasmid | 18 |
| Lactococcus lactis subsp. cremoris | ||
| MG7284 | MG1363, Prt− Lac− Bacr Fusr Spcr | 41 |
| MG7284/pAZIL-lsbB | MG7284 transformed with pAZIL-lsbB | 16 |
| Enterococcus faecalis | ||
| BGZLS10-27 | Natural isolate, Agg− | 42 |
| BGZLS10-27/pAZIL | BGZLS10-27 transformed with pAZIL | This study |
| BGZLS10-27/pAZIL-YvjBMN | BGZLS10-27 transformed with pAZIL-YvjBMN | This study |
| BGZLS10-27/pAZIL-YvjBMG | BGZLS10-27 transformed with pAZIL-YvjBMG | This study |
| Escherichia coli | ||
| DH5α | supE44 ΔlacU169 (ϕ80lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 | 43 |
| EC101 | JM101 containing repA gene of pWV01 in chromosome | 44 |
| TOP10 | F− mcrA (mrr-hsdRMS-mcrBC) ϕ80lacZΔM15 lacX74 recA1 ara139 (ara-leu)7697 galU galK rpsL (Strr) andA1 nupG | Invitrogen |
| M15(pREP4) | Nals Strs Rifs Thi− Lac− Ara+ Gal+ Mtl− F− RecA+ Uvr+ Lon+ | Qiagen |
| BL21(DE3) | B F− dcm ampT hsdS (rB− mB−) gal λ(DE3) | Agilent Technologies |
| BL21/p-GEX-6P-3/LsbB | p-GEX-6P-3/LsbB transformant of BL21 | This study |
| BL21/p-GEX-6P-3 | p-GEX-6P-3 transformant of BL21 | This study |
| Plasmids | ||
| pMN5 | Natural plasmid carrying lsbB operon | 18; accession no. NC_004922.1 |
| pAZIL | 7,109 bp, Emr, shuttle cloning vector | 45; accession no. LMBP9596b |
| pAZIL-lsbB | pAZIL carrying lsbB gene | 16 |
| pAZIL-lsbB-W25F | pAZIL carrying lsbB gene with amino acid change Trp25Phe | This study |
| pAZIL-lsbB-A30-STOP | pAZIL carrying lsbB gene with deleted Ala30 amino acid | This study |
| pAZIL-lsbB-K29A-STOP | pAZIL carrying lsbB gene with amino acid change Lys29Ala and deleted Ala30 amino acid | This study |
| pAZIL-lsbB-A30+A31 | pAZIL carrying lsbB gene with addition of Ala31 amino acid | This study |
| pAZIL-lsbB-A30V | pAZIL carrying lsbB gene with amino acid change Ala30Val | This study |
| pAZIL-lsbB-A30G | pAZIL carrying lsbB gene with amino acid change Ala30Gly | This study |
| pAZIL-lsbB-A30S | pAZIL carrying lsbB gene with amino acid change Ala30Ser | This study |
| pAZIL-lsbB-A30P | pAZIL carrying lsbB gene with amino acid change Ala30Pro | This study |
| pAZIL-lsbB-K2N-T3S | pAZIL carrying lsbB gene with amino acid changes Lys2Asn and Thr3Ser | This study |
| pAZIL-lsbB-K17A-K19A | pAZIL carrying lsbB gene with amino acid changes Lys17Ala and Lys19Ala | This study |
| pAZIL-YvjBMN | pAZIL carrying yvjB gene from BGMN1-596 | This study |
| pAZIL-YvjBMG | pAZIL carrying yvjB gene from MG7284 | This study |
| pAZIL-YvjBMGB | pAZIL carrying yvjB gene from MG7284 with amino acid changes Ser257Gly and Asn259Lys | This study |
| pAZIL-YvjBMNB | pAZIL carrying yvjB gene from BGMN1-596 with insertion of BamHI restriction site | This study |
| pAZIL-YvjB1/2MN-1/2MG | pAZIL carrying first half of yvjB gene from BGMN1-596 and second half of yvjB gene from MG7284 (YvjBMN1-259-YvjBMG260-428 aa) | This study |
| pAZIL-YvjB1/2MG-1/2MN | pAZIL carrying first half of yvjB gene from MG7284 and second half of yvjB gene from BGMN1-596 (YvjBMG1-256-YvjBMN257-428 aa) | This study |
| pAZIL-YvjBMGE | pAZIL carrying yvjB gene from MG7284 with insertion of EagI restriction site at position of aa 327 without amino acid changes | This study |
| pAZIL-YvjBMNE | pAZIL carrying yvjB gene from BGMN1-596 with insertion of EagI restriction site at position of aa 327 without amino acid changes | This study |
| pAZIL-YvjB3/4MG-1/4MN | pAZIL carrying first 3/4 of yvjB gene from MG7284 and 1/4 of yvjB gene from BGMN1-596 (YvjBMG1-327-YvjBMN328-428 aa) | This study |
| pAZIL-YvjB1/2MG-1/4MN-1/4MG | pAZIL carrying first half of yvjB gene from MG7284, 1/4 of yvjB gene from BGMN1-596, and last and 1/4 of yvjB gene from MG7284 (YvjBMG1-256-YvjBMN257-327-YvjBMG328-428 aa) | This study |
| pAZIL-YvjBMG-L351F | pAZIL carrying yvjB gene from MG7284 with amino acid change Leu351Phe | This study |
| pAZIL-YvjBMG-T353A | pAZIL carrying yvjB gene from MG7284 with amino acid change Thr353Ala | This study |
| pAZIL-YvjBMG-Q356Y | pAZIL carrying yvjB gene from MG7284 with amino acid change Gln356Tyr | This study |
| pAZIL-YvjBMG-P396Q | pAZIL carrying yvjB gene from MG7284 with amino acid change Pro396Gln | This study |
| pAZIL-YvjBMG-L351F-T353A | pAZIL carrying yvjB gene from MG7284 with amino acid changes Leu351Phe and Thr353Ala | This study |
| pAZIL-YvjBMG-L351F-Q356Y | pAZIL carrying yvjB gene from MG7284 with amino acid changes Leu351Phe and Gln356Tyr | This study |
| pAZIL-YvjBMG-T353A-Q356Y | pAZIL carrying yvjB gene from MG7284 with amino acid changes Thr353Ala and Gln356Tyr | This study |
| pAZIL-YvjBMG-T353A-P396Q | pAZIL carrying yvjB gene from MG7284 with amino acid changes Thr353Ala and Pro396Gln | This study |
| pAZIL-YvjBMG-L351F-T353A-Q356Y | pAZIL carrying yvjB gene from MG7284 with amino acid changes Leu351Phe, Thr353Ala, and Gln356Tyr | This study |
| pAZIL-YvjBMG-L351F-T353A-P396Q | pAZIL carrying yvjB gene from MG7284 with amino acid changes Leu351Phe, Thr353Ala, and Pro396Gln | This study |
| pAZIL-YvjBMG-L351F-T353A-Q356Y-P396Q | pAZIL carrying yvjB gene from MG7284 with amino acid changes Leu351Phe, Thr353Ala, Gln356Tyr, and Pro396Gln | This study |
| pAZIL-YvjBMG-L351F-T353A-Q356Y-T314A | pAZIL carrying yvjB gene from MG7284 with amino acid changes Leu351Phe, Thr353Ala, Gln356Tyr, and Thr314Ala | This study |
| pBluescript | 2,958 bp, Ampr, cloning vector | Stratagene |
| pGEM-T-Easy | 3,015 bp, Ampr, PCR cloning vector | Promega |
| pGEM-T-YvjBex1 | PCR-amplified fragment carrying region of YvjB protein between first and second transmembrane domains cloned into pGEM-T-Easy | This study |
| pGEM-T-YvjBex2 | PCR-amplified fragment carrying region of YvjB protein between second and third transmembrane domains cloned into pGEM-T-Easy | This study |
| pGEX-6P-3 | 4,900 bp, Ampr, GST expression vector | GE Healthcare Life Sciences |
| pGEX-6P-3/GST-LsbB | Cloned lsbB gene into pGEX-6P-3 vector, GST-LsbB fusion protein | This study |
| pQE30 | Ampr, ColE1 replicon, 6×His expression vector | Qiagen |
| pQE30-YvjBex1 | PCR-amplified fragment carrying region of YvjB protein between first and second transmembrane domains cloned as SacI/HindIII into pQE30 | This study |
| pQE30-YvjBex2 | PCR-amplified fragment carrying region of YvjB protein between second and third transmembrane domains cloned as BamHI/HindIII into pQE30 | This study |
| pNZ8150lacZ1PlcnB | PlcnB promoter cloned into pNZ8150lacZ1 vector | 28 |
a
Agg−, aggregative protein negative; Prt−, proteinase negative; Lac−, lactose utilization negative; Bacr, bacteriocin resistant; Fusr, fusaric acid resistant; Spcr, spectinomycin resistant; Strr, streptomycin resistant; Nals, nalidixic acid susceptible; Strs, streptomycin susceptible; Rifs, rifampin susceptible; Emr, erythromycin resistant; Ampr, ampicillin resistant; aa, amino acid.
b
LMBP9596, BCCM plasmid collection accession number.