TABLE 1.
Bacterial strains and plasmids
| Strain or plasmid | Relevant phenotypea | Source and/or description |
|---|---|---|
| E. coli | ||
| DH5αMCR | RecA | Gibco BRL |
| EM24NR | RecA Strr Nalr Rifr | RecA derivative of LE392 with spontaneous mutations to rifampin and nalidixic acid resistance (20) |
| S17-1 | RecA Tpr TraRP4 | Contains the transfer functions of RP4 integrated in the chromosome (28) |
| BW19851 | RecA Tpr TraRP4 Pir+ | S17-1 with pirR6K in uidA; supports the replication of Pir-dependent vectors (13) |
| Bacteroides thetaiotaomicron 5482 | ||
| BT4001 | (Rifr) | Spontaneous rifampin-resistant mutant of BT5482 |
| BT4107N1 | (Thy− Tcr Emr) | Spontaneous thymidine-requiring mutant of BT5482 (BT4100) containing CTnDOT (1) inserted at attDOT-JS 2 site |
| BT4107-1 | (Thy− Tcr Emr) | BT4100 with CTnDOT inserted in attDOT-JS 1 |
| BT4107-3 | (Thy− Tcr Emr) | BT4100 with CTnDOT inserted in attDOT-JS 3 |
| BT4107-4 | (Thy− Tcr Emr) | BT4100 with CTnDOT inserted in attDOT-JS 4 |
| BT4107-5 | (Thy− Tcr Emr) | BT4100 with CTnDOT inserted in attDOT-JS 5 |
| BT4104N1-3 | (Thy− Tcr) | BT4100 with ΩCTnERL |
| BT4004-5 | (Rifr Tcr) | BT4001 with ΩCTnERL |
| BT4004-Ωint | (Rifr Tcr Emr Exc−) | CTnERL with insertion of pCQW1-int; excision minus (this study) |
| BT4004-Ωorf2 | (Rifr Tcr Emr Exc+) | CTnERL with insertion of pCQW1-orf2; excision plus (this study) |
| Plasmids | ||
| pGEM-T | Apr | E. coli PCR cloning vector (Promega) |
| pGEM-T:DOT-JS2 | Apr | PCR product of CTnDOT target site DOT-JS 2 cloned onto pGEM-T (this study) |
| pNLY3 | (Cmr) Cmr Apr | Bacteroides suicide vector contains IS4351-cat and the oriTRK2 cloned into pUC19. It is Cmr in both E. coli and Bacteroides recipients and can be mobilized by IncPα plasmid RP1 or the RP4 transfer functions in E. coli S17-1 |
| pNLY3:DOT-LE | (Cmr Rep−) Apr | 15-kbp CTnDOT left junction on pNLY3 (this study) |
| pDOT5B/SstI | Apr | 5.5-kbp PvuII-SspI fragment subcloned from pNLY3:DOT-LE into SmaI site of pUC19 (this study) |
| pXBU1 | Apr | 1.5-kbp CTnXBU4422 right junction on pUC19 (26) |
| pUC:DRJ | Apr | 6.2-kbp CTnDOT right junction cloned on EcoRI site of pUC19 (this study) |
| pGERM | (Emr Rep−) Apr | Bacteroides pUC19-based suicide vector containing ermG and oriTRK2 that mobilized by RP4 transfer functions in E. coli S17-1 chromosome (35) |
| pDJE1.1 | (Emr Int−) Apr | 1.1-kbp DJE PCR product subcloned from pGEM-T into pGERM (this study) |
| pDJE2.3 | (Emr Int−) Apr | 2.3-kbp DJE PCR product subcloned from pGEM-T into SstI-SphI sites of pGERM; contains intDOT and integrates efficiently into Bacteroides target sites (this study) |
| pDJE2.6 | (Emr Int+) Apr | 2.6-kbp DJE PCR product subcloned from pGEM-T into pGERM; contains intDOT and orf2; integrates but cannot excise (this study) |
a
Phenotypes in parentheses are expressed only in Bacteroides; phenotypes outside the parentheses are expressed in E. coli. Abbreviations: Apr, ampicillin resistant; Rifr, rifampin resistant; Emr, erythromycin resistant; Tcr, tetracycline resistant; Cmr, chloramphenicol resistant; Int+ or Int−, ability or inability to integrate; Rep−, cannot replicate.