TABLE 1.
Bacterial strains and plasmids used in this work
| Strain or plasmid | Genotype or characteristic(s) | Source or reference(s) |
|---|---|---|
| Strains | ||
| E. coli | ||
| JM109 | recA1 endA1 gyrA96 thi hsdR17(rK− mK+) relA1 supE44 Δ(lac-proAB) [F′ traD36 proAB lacIqZΔM15] | Promega |
| XL1-Blue supercompetent | recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15 Tn10 (Tetr)] | Stratagene |
| RV | Δlac | Laboratory stock |
| Neisseria gonorrhoeae | ||
| F62 | Parental strain | Laboratory stock |
| JCGC10 | ΔaspC-lctP::PlacI-cycA+ | This work |
| JCGC11 | ΔaspC-lctP::PlacI-cycB+ | This work |
| JCGC100 | fnr::erm | 21 |
| JCGC800 | cycA::erm | This work |
| JCGC802 | cycA::kan | This work |
| JCGC805 | cycA::FLAG | This work |
| JCGC810 | cycA::kan | This work |
| JCGC820 | cycA::kan ΔaspC-lctP::PlacI-cycA+ | This work |
| JCGC813 | cycA::FLAG fnr::erm | This work |
| JCGC850 | cycB::erm | This work |
| JCGC851 | cccA::kan | This work |
| JCGC852 | cycB::erm cccA::kan | This work |
| JCGC853 | cycA::erm cccA::kan | This work |
| JCGC855 | cycB::FLAG | This work |
| JCGC860 | cycB::chl | This work |
| JCGC870 | cycB::chl ΔaspC-lctP::PlacI-cycA+ | This work |
| JCGC880 | cycA::kan cycB::chl ΔaspC-lctP::PlacI-cycA+ | This work |
| Plasmids | ||
| pGEM T-Easy | Commercially available vector for cloning of PCR products | Promega |
| pET20bhc | Derivative of pET20 | GlaxoSmithKline |
| pET20bhc-c2 | pET20bhc containing the gonococcal cccA gene on an NdeI-BamHI fragment | This work |
| pSMT2 | ccmABCDEFGH NcoI-SalI fragment cloned into pACYC184 | 47 |
| pLES940 | Source of the cat gene | 40 |
| pSUB11 | Source of the kan gene | 3 |
| pAH105 | cat gene ligated into the AgeI site of pYL6 | This work |
| pAH106 | Kanr gene ligated into the AgeI site of NT100 | This work |
| pNT100 | Gonococcal cycA gene cloned into pGEM T-Easy | This work |
| pNT101 | ermC gene flanked by AgeI sites in pGEM T-Easy | This work |
| pNT102 | ermC gene ligated into the AgeI site of pNT100 | This work |
| pYL4 | kan gene flanked by AgeI sites in pGEM T-Easy | This work |
| pYL6 | 584 bp of DNA upstream of cycB linked by an AgeI site to 448 bp of DNA downstream of cycB in pGEM T-Easy | This work |
| pYL7 | ermC gene ligated into the AgeI site of pYL6 | This work |
| pYL12 | 430 bp of DNA upstream and 331 bp of DNA downstream of the cccA gene in a continuous sequence containing an AgeI restriction site at the end of the upstream DNA ligated into pGEM T-Easy | This work |
| pYL13 | kan gene ligated into the AgeI site of pYL600 | This work |
| pGCC4 | Neisserial insertional complementation system; ermC KanrlctP asp lacIqlacP | 23, 24, 41 |
| pGCC4_cycA | pGCC4 containing the cycA gene inserted into the PacI site | This work |
| pGCC4_cycB | pGCC4 containing the cycB gene inserted between the PacI and FseI sites | This work |