TABLE 1.
Bacterial strains and plasmids used in the study
| Strains or plasmid | Purpose and relevant characteristic(s) | Reference(s) or source |
|---|---|---|
| E. faecalis strain(s) | ||
| OG1RF | Used for lsa insertional mutagenesis and to amplify lsa for complementation experiments; Rifr, Fusr | 14 |
| V583 | TIGR-sequenced strain; used to amplify lsa for complementation experiments | 17, 22 |
| TX4107-TX4110 | Q-D- and CLI-susceptible isolates from animal feed | This study |
| TX0263, TX0271 | Q-D- and CLI-susceptible clinical isolates | This study |
| TX5076 | Antigen gene disruption mutant generated with pTEX4577 containing aph(3′)-llla; Kanr | 30 |
| TX5243 | sprE gene disruption mutant [OG1RF sprE::pTEX4577 containing aph(3′)-llla]; Kanr | 18 |
| TX5248 | orf1 gene disruption mutant [OG1RF orf1::pTEX4577 containing aph(3′)-llla]; Kanr | 18 |
| TX5332 | lsa gene disruption mutant [OG1RF lsa::pTEX4577 containing aph(3′)-llla]; Kanr | 25 |
| TX5332(pTEX5333.04) (contains lsaV); Kanr, Chlr | 25 | |
| TX5332(pTEX5333.03) (contains lsaOG); Kanr, Chlr | This study | |
| TX5332(pTEX5333.08) (contains lsaOG5′V3′); Kanr, Chlr | This study | |
| TX5332(pTEX5333.09) (contains lsaV5′OG3′); Kanr, Chlr | This study | |
| TX5332(pTEX5333.12) (contains a mutagenized gene, lsaV-M); Kanr, Chlr | This study | |
| TX5332(pTEX5333.13) (contains a mutagenized gene, lsaOG-M); Kanr, Chlr | This study | |
| L. lactis strains | ||
| LM2301 | Used as a gram-positive host for E. faecalis lsa | 27 |
| LM2301(pTEX5333.04) (contains lsaV5); Chlr | This study | |
| LM2301(pTEX5333.03) (contains lsaOG); Chlr | This study | |
| LM2301(pTEX5333.08) (contains lsaOG5′V3′); Chlr | This study | |
| LM2301(pTEX5333.09) (contains lsaV5′OG3′); Chlr | This study | |
| LM2301(pTEX5333.12) (contains lsaV-M); Chlr | This study | |
| LM2301(pTEX5333.13) (contains lsaOG-M); Chlr | This study | |
| TX5392 | LM2301(pWM401); Chlr | This study |
| Plasmids | ||
| pTEX4577 | pBluescript SK(−) with aph(3′)-IIIa inserted into the ScaI site; Kanr, used for insertional mutagenesis | 24 |
| pWM401 | Shuttle vector; Chlr, Tetr | 29 |
| pCR2.1 vector | PCR product cloning vector | Invitrogen |
| pTEX5333.03 | pWM401::lsaOG, contains a ca. 2-kb lsa fragment from OG1RF, used for complementation of TX5332 and transformation of L. lactis LM2301; Chlr | This study |
| pTEX5333.04 | pWM401::lsav, contains a ca. 2-kb lsa fragment from V583, used for complementation of TX5332 and transformation of L. lactis LM2301; Chlr | 25 |
| pTEX5333.08 | pWM401::lsaOG5′V3′, contains a hybrid gene with the 5′ half of lsaOG and 3′ half of lsav, used for complementation of TX5332 and transformation of L. lactis LM2301; Chlr | This study |
| pTEX5333.09 | pWM401::lsaV5′OG3′, contains a hybrid gene with the 5′ half of lsaV and 3′ half of lsaOG, used for complementation of TX5332 and transformation of L. lactis LM2301; Chlr | This study |
| pTEX5333.12 | pWM401::lsaV-M, contains ca. 2-kb lsaV fragment mutagenized in its upstream region at positions −131 and −133 with A's replaced with a G and a T, used for complementation of TX5332 and transformation of L. lactis LM2301; Chlr | This study |
| pTEX5333.13 | pWM401::lsaOG-M, contains ca. 2-kb lsaOG fragment mutagenized in its upstream region at positions −131 and −133 with G and T replaced by A's, four additional nucleotide changes were found at −109, +1248 (silent), +1572 (downstream), and +1517 (downstream), used for complementation of TX5332 and transformation of L. lactis LM2301; Chlr | This study |