TABLE 1.
Strains and plasmids
| Bacterial strain or plasmid | Relevant phenotypea | Description and/or source |
|---|---|---|
| Bacterial strains | ||
| E. coli | ||
| DH5αMCR | RecA | Gibco-BRL |
| EM24NR | RecA Nalr Rifr, Pir− | RecA derivative of LE392 (39), used as recipient for integrative Pir requiring vectors |
| BW19851 | RecA, ΩRP4 tra, uidA::pir+ | R6K pir integrated into the uidA of S17-1 (50); host strain for R6K replicons (23) |
| B. thetaiotaomicron | ||
| BT5482 | ||
| BT4001 | Rifr | Spontaneous rifampin resistant isolate of BT5482 |
| BT4004 | Rifr Tcr | BT4001 containing the conjugative transposon, CTnERL |
| BT4004N3 | Rifr Tcr Lnr | BT4004 with single copy of NBU2 in site 2 |
| BT4004N6 | Rifr Tcr Lnr | BT4004 containing two copies of NBU2, one in site 1 and one in site 2 |
| Plasmids | ||
| pUC19 | Apr | E. coli cloning vector (62) |
| pGEM-T | Apr | Promega PCR product cloning vector |
| pBR328::NBU2 | Cmr Tcr | Circular intermediate of NBU2 digested with PstI and cloned into the PstI site of the ColE1 replicon vector pBR328 (11), this study |
| pUC19::attN2 | Apr | 1.2-kbp HincII of NBU2 containing the joined ends cloned into the HincII site of pUC19, this study |
| pUC19::LJ1 | Apr | A 5-kbp HindIII fragment containing the left junction of NBU2 in one of the chromosomal targets (site 1) in BT4004N6 cloned into the HindIII site of pUC19, this study |
| pEP185.2 | Cmr, R6KoriV | Cloning vector that requires pir product in trans for replication; contains the RP4 oriT for mobilization (24) |
| pEPE | Cmr R6KoriV (Emr) | 1.8-kbp ermG PCR product from CTn7853 (10) cloned into the NsiI site of pEP185.2 to create a suicide vector that is selectable in both Bacteroides and E. coli hosts, this study |
| pEPIntN2 | Cmr R6KoriV Int+ (Emr Int+) | 1.8-kbp PCR product from NBU2 circular form containing the attN2 and intN2 region cloned into ApaI/SstII site of pEPE (Fig. 1); integrates into the chromosomes of Bacteroides and E. coli hosts, this study |
| pEPIntN2D | Cmr, R6KoriV, Int- (Emr Int−) | 0.9-kbp internal deletion of intN2 in pEPIntN2 cloned into pEPE, this study |
| pEPE::N2-J1 and pEPE::N2-J2 | Cmr (Emr) | EcoRV clone of both NBU2-chromosomal junctions from pEPIntN2 insertions in BT4001 chromosome in sites 1 (J1) and 2 (J2), this study |
| pNLY1 | Apr Cmr (Cmr) | Bacteroides-E. coli shuttle vector (38) |
| pNLY-ML | Apr Cmr (Cmr Lnr) | 3.5-kbp HindIII fragment of NBU2 containing mefEN2-linAN2 cloned into pNLY1, this study |
a
The phenotypes in parentheses are expressed Bacteroides hosts, and the phenotypes outside the parentheses are expressed E. coli. Abbreviations used for antibiotics resistances: Ap, ampicillin; Cm, chloramphenicol; Em, erythromycin; Ln, lincomycin; Nal, nalidixic acid; Rif, rifampin; Sm, streptomycin; Tp, trimethoprim. Other phenotype abbreviations: int+ or int− for the ability to integrate and R6KoriV for the pir-dependent replication origin of R6K.