TABLE 2.
Plasmids used in this study
| Plasmid | Relevant characteristic or descriptionc | Source or reference |
|---|---|---|
| pCF10 | cCF10-inducible conjugative plasmid | 19 |
| pCF10-101 | pCF10 ΔoriT2 | 49 |
| pPD1 | cPD1-inducible conjugative plasmid | 52 |
| pBK2 | pCI3340 vector containing prgX-Q region of pCF10 with a pheromone-inducible lacZ reporter gene | 34 |
| p043lacZ | pAT18 vector containing prgX-Q region of pCF10 with a pheromone-inducible lacZ reporter gene | 35 |
| pDL278 | Gram-positive cloning shuttle vector; Spectr | 23 |
| pPCR4a | cCF10 expression construct derived from prgQ region cloned into pDL278 | 45 |
| V2L | V→L change at position 2 of cCF10 | 24 |
| T3S | T→S change at position 3 of cCF10 | 24 |
| L4V | L→V change at position 4 of cCF10 | 24 |
| L4I | L→I change at position 4 of cCF10 | 24 |
| F6Y | F→Y change at position 6 of cCF10 | 24 |
| V7I | V→I change at position 7 of cCF10 | 24 |
| pJRC2 | pPCR4 construct altered to express cAM373 signal peptide and pheromone from prgQ promoter | This study |
| pJRC3 | pJRC2 construct altered to express cCF10 | This study |
| pJRC4 | pPCR4 construct altered to express native iCF10 peptide | This study |
| pGEM-T-Easy | AmprlacZ cloning vector | Promega |
| pGEM pJRC2 | pGEM-T-Easy containing ∼250-bp PCR product used for cloning pJRC2 | This study |
| pGEM pJRC3 | pGEM-T-Easy containing ∼250-bp PCR product used for cloning pJRC3 | This study |
| pDL276 | Gram-positive cloning shuttle vector; Kanr | 23 |
| pMSP6043 | prgN, -O, -P, -W, -Z, and -Y cloned into pDL276 | 26 |
| pMSP6049 | prgN, -O, -P, -W, and -Y cloned into pDL276 | 26 |
| pMSP6043-1 | prgN, -O, -P, -W, and -Z cloned into pDL276 | 12 |
| pMSP6043-2 | prgN, -O, -P, and -W cloned into pDL276 | This study |
| pMSP3545 | Nisin-inducible cloning vector | 8 |
| pMSP3545Y | prgY cloned into pMSP3545 | This study |
| pMSP3545Sb | Spectr derivative of pMSP3545 | 12 |
| pMSP3545S-1 | Cloned E. faecalis pCF10 prgY | 12 |
| pMSP3545S-2 | Cloned E. faecalis pPD1 traB | 12 |
| pB/Y1 | Fusion of prgY after pPD1 traB base 726 | This study |
| pB/Y2 | Fusion of pPD1 traB after prgY base 723 | This study |
| pB/Y3 | Fusion of prgY after pPD1 traB base 234 | This study |
| pB/Y4 | Fusion of prgY after pPD1 traB base 375 | This study |
| pB/Y5 | Fusion of pPD1 traB after prgY base 960 | This study |
| pB/Y6 | Fusion of pPD1 traB after prgY base 1050 | This study |
| pB/Y9 | pPD1 traB fusion between bases 234 and 375 of prgY | This study |
| pB/Y10 | pPD1 traB fusion between bases 375 and 1098 of prgY | This study |
a
Constructs expressing mutant cCF10 peptides V2L, T3S, L4V, L4I, F6Y, and V7I were made by oligonucleotide-directed random mutagenesis of the cCF10 coding sequence of the pPCR4 construct, and their construction is described by Fixen et al. (24).
b
Genes and chimeric gene fusions in pMSP3545S-1 and -2 and B/Y1-10 were cloned into NcoI and XbaI of pMSP3545S.
c
Spectr, spectinomycin resistance; Ampr, ampicillin resistance; Kanr, kanamycin resistance.