TABLE 1.
Bacterial strains and plasmidsb
| Strain or plasmid | Relevant properties | Source or reference |
|---|---|---|
| E. coli | ||
| SM10 | F−tonA21 lacY1 supE44 Mu λ− [RP4-2 (Tc::Mu)]Km Tra+ | 43 |
| Top10 | F−mcrA Δ(mrr-hsdRMS-mcr BC) φ80lacZΔM15 ΔlacX74 deoR rec araD139 Δ(ara-leu)7697 galU galK rpsL endA1 nupG | Invitrogen |
| recA::RP4::Δnic | chrRP4::Δnic Km DAP− | 1 |
| C1a | rep+ilv+ Nal | 40 |
| BL21λ DE3 | F−ompT hsdSB(rB− mB−) gal dcm (DE3) | Invitrogen |
| E. faecalis | ||
| JH2-2::Tn1549 | Fus Rif Vm | 28 |
| Plasmids | ||
| pACYCGm | Derivative of pACYC184 in which the cat-tet fragment was replaced by aac(3)-IV; Gm Apra | Laboratory |
| pCM105 | pACYCGm Ω250-bp EcoRI-HindIII PCR intergenic fragment orf29-orf30 from Tn1549 | This study |
| pCM105-1 | pCM105 (C141→T)a | This study |
| pCM105-2 | pCM105 (G152G154→ T152C154) | This study |
| pCM105-3 | pCM105 (G159C160→ T159G160) | This study |
| pCM105-4 | pCM105 (G194C195→ T194G195) | This study |
| pCM105-5 | pCM105 (C216C218→ G216G218) | This study |
| pCM106 | pACYCGm Ω28-bp oriTTn1549 | This study |
| pCM107 | pACYCGm Ω130-bp EcoRI-HindIII PCR part of the PCR intergenic fragment orf29-orf30 from Tn1549 | This study |
| pBAD/HisA | Expression vector, araBAD promoter; Amp | Invitrogen |
| pCM108 | pBAD/HisA Ω1,328-bp NcoI-XhoI PCR fragment (rlxTn1549) | This study |
| pUC18Gm | Derivative of pUC18 in which blaT is replaced by aac(3)-IV; Gm Apra | This study |
| pCM109 | pUC18Gm Ω1,879-bp EcoRI-XbaI PCR fragment (intergenic orf30-orf29 region, orf29, and orf28) of Tn1549 | This study |
| pCM110 | pUC18Gm Ω2,664-bp EcoRI-XbaI PCR fragment (intergenic region orf30-orf29, orf29, orf28, orfz, and orf27) of Tn1549 | This study |
| pGB2 | Spc Str | 15 |
| pCM111 | pGB2 Ω372-bp EcoRI-HindIII PCR fragment (mobC) of Tn1549 | This study |
| pET28a(+) | His tag expression vector; Km | Invitrogen |
| pCM112 | pET28a+ Ω354-bp EcoRI-NdeI PCR fragment (mobC) of Tn1549 | This study |
| pCM113 | pET28a+ Ω1,325-bp NcoI-XhoI PCR fragment (rlx) of Tn1549 | This study |
| pCM114 | pET28a+ Ω741-bp NcoI-XhoI PCR fragment (N247rlx) of Tn1549 | This study |
| pCR2.1 | PCR cloning vector; Amp Km | Invitrogen |
| pCM115 | pCR2.1 Ω2,374 bp PCR fragment (intergenic region orf30-orf29 mobC rlx orfz) of Tn1549 | This study |
a
Sequence was from GenBank accession number AF192329.1, but numbering refers to the orf30 termination codon.
b
Amp, ampicillin; Apra, apramycin; Fus, fusidic acid; Gm, gentamicin; Km, kanamycin; Nal, nalidixic acid; Rif, rifampin; Str, streptomycin; Vm, vancomycin.