Table 1.

Strains and plasmids

Bacterial strain or plasmidRelevant phenotypeaDescription and/or source
Bacterial strains
E. coli
  DH5αMCRRecAGibco-BRL
  EM24NRRecA Nalr Rifr, Pir RecA derivative of LE392 (39), used as recipient for integrative Pir requiring vectors
  BW19851RecA, ΩRP4 tra,uidA::pir + R6Kpir integrated into the uidA of S17-1 (50); host strain for R6K replicons (23)
B. thetaiotaomicron
 BT5482
 BT4001Rifr Spontaneous rifampin resistant isolate of BT5482
 BT4004RifrTcr BT4001 containing the conjugative transposon, CTnERL
 BT4004N3Rifr TcrLnr BT4004 with single copy of NBU2 in site 2
 BT4004N6Rifr TcrLnr BT4004 containing two copies of NBU2, one in site 1 and one in site 2
Plasmids
 pUC19Apr E. coli cloning vector (62)
 pGEM-TApr Promega PCR product cloning vector
 pBR328::NBU2CmrTcr Circular intermediate of NBU2 digested withPstI and cloned into the PstI site of the ColE1 replicon vector pBR328 (11), this study
 pUC19::attN2Apr 1.2-kbpHincII of NBU2 containing the joined ends cloned into theHincII site of pUC19, this study
 pUC19::LJ1 Apr A 5-kbpHindIII fragment containing the left junction of NBU2 in one of the chromosomal targets (site 1) in BT4004N6 cloned into theHindIII site of pUC19, this study
 pEP185.2Cmr, R6KoriV Cloning vector that requires pir product in trans for replication; contains the RP4 oriT for mobilization (24)
 pEPECmr 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 bothBacteroides and E. coli hosts, this study
 pEPIntN2Cmr 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
 pEPIntN2DCmr, R6KoriV, Int- (Emr Int)0.9-kbp internal deletion of intN2 in pEPIntN2 cloned into pEPE, this study
 pEPE::N2-J1 and pEPE::N2-J2Cmr(Emr) EcoRV clone of both NBU2-chromosomal junctions from pEPIntN2 insertions in BT4001 chromosome in sites 1 (J1) and 2 (J2), this study
 pNLY1Apr Cmr(Cmr) Bacteroides-E. coli shuttle vector (38)
 pNLY-MLApr Cmr(Cmr Lnr)3.5-kbp HindIII fragment of NBU2 containingmefEN2-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.