TABLE 1.
Plasmids and Escherichia coli strains used in this study
| Strain or plasmid | Relevant characteristics | Selective marker(s)a | Reference or source |
|---|---|---|---|
| Bacterial strains | |||
| DH5α | supE44 ΔlacU169 (φ80dlacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 | Nal | Laboratory collection (29a) |
| DY330 | W3110 ΔlacU169 gal490 λc1857 Δ(cro-bioA) | Nal or Rif | 69 |
| MC4100 | araD139 Δ(argF-lac)U169 rpsL150 relA1 flbB3501 deoC1 ptsF25 rbsR | Sm | Manoil; laboratory collection |
| RI89 | MC1000 phoR Δara714 leu+ | Sm | 60a |
| RI90 | RI89 dsbA::kan1 | Km, Sm | 60a |
| RI179 | RI89 ΔdsbC::cam | Cm, Sm | 60a |
| XK1200 | F−lacΔU124 Δ(nadA aroG gal attλ bio gyrA) | Nal | K. Ippen-Ihler (54) |
| Plasmids | |||
| PCR4Blunt-TOPO | 4-Kb cloning vector with covalently linked topoisomerase | Amp, Km | Invitrogen |
| pBAD30 | Cloning vector for controlled expression from Para-BAD | Amp | 28 |
| pOX38-Tc | pOX38::mini-Tn10 | Tc | 4 |
| pOX38-Tc ΔtraF::kan | pOX38-Tc with kan replacing codons 34 to 229 in traF | Tc, Km | This study |
| pOX38-Tc ΔtrbB::cat | pOX38-Tc with cat replacing codons 14 to 160 in trbB | Tc, Cm | This study |
| pFTraF | F traF in pBAD30 (DPID)b | Amp | This study |
| pFTraF(CPYC) | F traF (D135C, I137Y, D138C)c in pBAD30 (CPYC)b | Amp | This study |
| pFTrbB | F trbB in pBAD30 (CPYC)b | Amp | This study |
| pFTrbB(Y63H) | F trbB (Y63H)c in pBAD30 (CPHC)b | Amp | This study |
| pFTrbB(SPYS) | F trbB (C61S, C64S)c in pBAD30 (SPYS)b | Amp | This study |
| pHTrhF | R27 trhF in pBAD30 (CQFC)b | Amp | This study |
| pHHtdT | R27 htdT in pBAD30 (CDGC)b | Amp | This study |
| pHDsbC | R27 dsbC in pBAD30 (CGFC)b | Amp | This study |
| pHDsbC(SGFS) | R27 dsbC (C107S, C110S)c in pBAD30 (SDGS)b | Amp | This study |
| pKDsbA | E. coli K-12 dsbA in pBAD30 | Amp | This study |
| pKDsbA(NheI) | E. coli K-12 dsbA in pBAD30 with NheI site at codons 17 and 18 | Amp | This study |
| pFBASS | dsbA signal sequence with A34 to K159 of F trbB in pBAD30 | Amp | This study |
| pFBASS(Y63H) | pFBASS using F trbB (Y63H)c | Amp | This study |
| pHFASS | dsbA signal sequence with Q137 to R324 of R27 trhF in pBAD30 | Amp | This study |
| pHCASS | dsbA signal sequence with A82 to K227 of R27 dsbC in pBAD30 | Amp | This study |
a
Antibiotic resistances: Amp, ampicillin; Cm, chloramphenicol; Km, kanamycin; Nal, nalidixic acid; Rif, rifampin; Sm, streptomycin; Tc, tetracycline.
b
Sequence of the four amino acids at the C-X-X-C motif indicated in Fig. 1 (in parentheses).
c
The first letter indicates the wild-type amino acid; the second letter indicates the mutant amino acid; the number refers to the position of the amino acid within the mature protein.