TABLE 1.
Strains and plasmids
| Strain or plasmid | Relevant properties | Reference or source |
|---|---|---|
| Strains | ||
| E. coli | ||
| DH5α | E. coli host strain for pBluescript SK(−) | Stratagene |
| DH5α(pTCV-lac) | E. coli DH5α containing plasmid pTCV-lac | 20 |
| TX4577 | E. coli DH5α containing plasmid pTEX4577 | 27 |
| E. faecalis | ||
| OG1RF | Gel+ serine protease positive (Spr+) Rifr Fusr | 13 |
| TX5240 | OG1RF fsrA mutant with pTEX4577 insertion in fsrA; Gel− Spr− Kanr | 22 |
| TX5241 | OG1RF fsrB mutant with pTEX4577 insertion in fsrB; Gel− Spr− Kanr | 22 |
| TX5242 | OG1RF fsrC mutant with pTEX4577 insertion in fsrC; Gel− Spr− Kanr | 22 |
| TX5266 | OG1RF fsrB deletion mutant, deletion from bp 79 to 684 of fsrB; Gel− Spr− | This study |
| Plasmids | ||
| pBluescript SK(−) | Cloning vector; Ampr | Stratagene |
| pTEX4577 | Suicide vector in E. faecalis derived from pBluescript SK(−); Kanr | 27 |
| pTCV-lac | Shuttle vector containing promoterless lacZ; Kanr Eryr | 20 |
| pTEX5267 | pTEX4577 containing fsrB flanking regions (917-bp 5′ region: bp −839 to +78, amplified using BDF1 and BDR1 primers; 1,065-bp 3′ region: 45 bp before stop codon to 1,020 bp after stop codon, amplified using DBF2 and DBR2 primers), used for construction of fsrB deletion mutant; Kanr | This study |
| pTEX5268 | fsrA promoter cloned upstream of lacZ in pTCV-lac, from bp −406 to −6 (401 bp, amplified using APRF1 and APRR1 primers) relative to fsrA start codon; Kanr Eryr | This study |
| pTEX5269 | fsrB promoter cloned upstream of lacZ in pTCV-lac, from bp −110 to −8 (103 bp, amplified using BPRF1 and BPRR1 primers) relative to fsrB start codon; Kanr Eryr | This study |
| pTEX5270 | gelE promoter cloned upstream of lacZ in pTCV-lac, from bp −218 to −16 (203 bp, amplified using EPRF1 and EPRR1 primers) relative to gelE start codon; Kanr Eryr | This study |
| pTEX5298 | fsrB promoter region (bp −90 to −8 relative to fsrB start codon, amplified using BPRF1 and BMP1 primers) cloned upstream of lacZ in pTCV-lac; Kanr Eryr | This study |
| pTEX5299 | fsrB promoter region (bp −72 to −8 relative to fsrB start codon, amplified using BPRF1 and BMP2 primers) cloned upstream of lacZ in pTCV-lac; Kanr Eryr | This study |
| pTEX5300 | fsrB promoter region (bp −85 to −8 relative to fsrB start codon, amplified using BPRF1 and BMP4 primers) cloned upstream of lacZ in pTCV-lac; Kanr Eryr | This study |
| pTEX5301 | fsrB promoter region (bp −91 to −8 relative to fsrB start codon in which bp −70 to −65 were altered from AAGGAA to TTCCTT, amplified using BPRF1 and BMP5 primers) cloned upstream of lacZ in pTCV-lac; Kanr Eryr | This study |
| pTEX5302 | fsrB promoter region (bp −72 to −8 relative to fsrB start codon) with putative gelE promoter regulatory region (bp −188 to −170 relative to gelE start codon), amplified using BPRF1 and BMP6 primers, cloned upstream of lacZ in pTCV-lac; Kanr Eryr | This study |
| pTEX5303 | gelE promoter region (bp −188 to −16 relative to gelE start codon, amplified using EPRF1 and EMP1 primers) cloned upstream of lacZ in pTCV-lac; Kanr Eryr | This study |
| pTEX5304 | gelE promoter region (bp −170 to −16 relative to gelE start codon, amplified using EPRF1 and EMP2 primers) cloned upstream of lacZ in pTCV-lac; Kanr Eryr | This study |
| pTEX5305 | gelE promoter region (bp −188 to −16 relative to gelE start codon in which bp −167 to −161 were altered from AAGGAA to TTCCTT, amplified using EPRF1 and EMP5 primers) cloned upstream of lacZ in pTCV-lac; Kanr Eryr | This study |
| pTEX5306 | gelE promoter region (bp −170 to −16 relative to gelE start codon) with putative fsrB promoter regulatory region (bp −91 to −73 relative to fsrB start codon), amplified using EPRF1 and BMP6 primers, cloned upstream of lacZ in pTCV-lac; Kanr Eryr | This study |