Table 1.
Strains and plasmids used in this study.
| Strains/ plasmids |
Description | Source/reference |
|---|---|---|
| Strains | ||
| MC4100 | F- araD139 Δ(argF-lac)U169 rspL150(Strr) relA1flbB5301 deoC1 ptsF25 rbsR | (Casadaban, 1976) |
| MHR480 | MC4100 ΔcsgE | (Hammar et al., 1996) |
| LSR11 | MC4100 Δcsg | This study |
| LSR35 | MC4100 csgEF::kanR | This study |
| NEB3016 | E. coli protein expression strain | New England Biolabs |
| Plasmids | ||
| pBAD33 | arabinose-inducible expression vector | (Guzman et al., 1995) |
| pET11d | IPTG-inducible expression vector | Novagen |
| pTrc99a | IPTG-inducible expression vector | Pharmacia Biotech |
| pAN65 | an ORF encoding the CsgA signal sequence fused to mature CpxP with a C-terminal 6-his tag in pBAD33 (CsgAss-CpxP-his) |
This study |
| pAN66 | an ORF encoding the CsgA signal sequence fused to mature CpxP with a C-terminal 6-his tag in pTrc99a (CsgAss-CpxP-his) |
This study |
| pAN69 | an ORF encoding the PapD signal sequence with a Gly-Thr linker (encoded by a ggtacc KpnI site) fused to mature CpxP with a C-teriminal 6-his tag, inserted into the NcoI-PstI sites of pLR92 (PapDss-[GT]-CpxP-his) |
This study |
| pAN70 | an ORF encoding the PapD signal sequence fused to the mature N-terminus of CsgA (A22) with a Gly-Thr linker (encoded by a ggtacc KpnI site) fused to mature CpxP with a C-terminal 6-his tag in pLR92 (PapDss-A22-[GT]-CpxP-his) |
This study |
| pAN87 | papD2-his cloned into the KpnI-PstI sites of pAN69 (PapDss-[GT]-PapD2-his) | This study |
| pAN88 | papD2-his cloned into the KpnI-PstI sites of pAN70 (PapDss-A22-[GT]-PapD2-his) | This study |
| pAN93 | the NcoI-PstI fragment of pAN87 cloned into pTrc99a (PapDss-[GT]-PapD2-his) | This study |
| pAN94 | the NcoI-PstI fragment of pAN88 cloned into pTrc99a (PapDss-A22-[GT]-PapD2- his) |
This study |
| pLR1 | csgBA promoter cloned into the BamHI-PstI sites of pACYC177 | (Robinson et al., 2006) |
| pLR42 | csgE inserted into the KpnI-PstI sites of pBAD33 | This study |
| pLR50 | an ORF encoding the CsgA signal sequence and mature N-terminus (A22) fused to mature CpxP with a C-terminal 6-his tag inserted into the KpnI-PstI sites of pBAD33 (CsgAss-A22-CpxP-his) |
This study |
| pLR51 | the CsgAss-A22-CpxP-his NcoI-PstI fragment of pLR50 inserted into pLR1 | This study |
| pLR58 | csgF-HA in pBAD33 | (Robinson et al., 2006) |
| pLR70 | The csgE NcoI-PstI fragment of pLR42 inserted into pLR1 | This study |
| pLR71 | csgE inserted into the NcoI-BamHI sites of pTrc99a | This study |
| pLR73 | csgF in pLR1 | (Nenninger et al., 2009) |
| pLR74 | csgF inserted into the NcoI-PstI sites of pTrc99a | This study |
| pLR75 | csgF in pBAD33 | (Nenninger et al., 2009) |
| pLR92 | a modified pBAD33 vector (modification: cmR gene does not contain an NcoI site) containing csgG-HA flanked by SacI-NcoI (5′) and KpnI-BglII-Pst1 (3′) sites |
(Robinson et al., 2006) |
| pLR93 | csgG in pLR1 | (Robinson et al., 2006) |
| pLR116 | the CsgAss-A22-CpxP-his NcoI-PstI fragment of pLR50 cloned into pTrc99a | This study |
| pMC1 | csgG in pTrc99a | (Chapman et al., 2002) |
| pNH27 | gene encoding cytoplasmic CsgE inserted into the NcoI-BamHI sites of pET11d | This study |