TABLE 1.
Strains and plasmids used in the current study
| Strain or plasmid | Phenotype (genotype)a | Reference or source |
|---|---|---|
| E. coli TOP10 | F−mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 deoR araD139 Δ(ara-leu) 7697 galU galK rpsL (Strr) endA1 nupG | Invitrogen |
| N. gonorrhoeae strains | ||
| FA19 | Wild type | 28 |
| FA6747 | TbpA− (tbpA::mTn3cat) | 14 |
| FA6905 | TbpB− (ΔtbpB) | 16 |
| FA6815 | TbpA− TbpB− (tbpB::Ω) | 1 |
| MCV515 | L9HA TbpA Lbp− (tbpA∇HA lbp::Ω) | 49 |
| MCV516 | L9HA TbpA Lbp−(tbpA∇HA ΔtbpB lbp::Ω) | 49 |
| MCV601 | FA19; Lbp− (lbp::Ω) | 22 |
| MCV801 | HA1(37) TbpB Lbp− (tbpB∇HA lbp::Ω) | This study |
| MCV802 | HA2(97) TbpB TbpA− Lbp− (tbpB∇HA lbp::Ω) | This study |
| MCV803 | HA1(37) TbpB Lbp− (tbpB∇HA tbpA::mTn3 lbp::Ω) | This study |
| MCV804 | HA2(97) TbpB TbpA− Lbp− (tbpB∇HA tbpA::mTn3 lbp::Ω) | This study |
| MCV812 | HA3(175) TbpB Lbp− (tbpB∇HA lbp::Ω) | This study |
| MCV813 | HA3(175) TbpB TbpA− Lbp− (tbpB∇HA tbpA::mTn3 lbp::Ω) | This study |
| MCV814 | HA4(293) TbpB Lbp− (tbpB∇HA lbp::Ω) | This study |
| MCV815 | HA4(293) TbpB TbpA− Lbp− (tbpB∇HA tbpA::mTn3 lbp::Ω) | This study |
| MCV816 | HA5(327) TbpB Lbp− (tbpB∇HA lbp::Ω) | This study |
| MCV817 | HA5(327) TbpB TbpA− Lbp− (tbpB∇HA tbpA::mTn3 lbp::Ω) | This study |
| MCV818 | HA6(424) TbpB Lbp− (tbpB∇HA lbp::Ω) | This study |
| MCV819 | HA6(424) TbpB TbpA− Lbp− (tbpB∇HA tbpA::mTn3 lbp::Ω) | This study |
| MCV820 | HA7(453) TbpB Lbp− (tbpB∇HA, lbp::Ω) | This study |
| MCV821 | HA7(453) TbpB TbpA− Lbp− (tbpB∇HA tbpA::mTn3 lbp::Ω) | This study |
| MCV822 | HA8(607) TbpB Lbp− (tbpB∇HA lbp::Ω) | This study |
| MCV823 | HA8(607) TbpB TbpA− Lbp− (tbpB∇HA tbpA::mTn3 lbp::Ω) | This study |
| MCV824 | HA9(660) TbpB Lbp− (tbpB∇HA lbp::Ω) | This study |
| MCV825 | HA9(660) TbpB TbpA− Lbp− (tbpB∇HA tbpA::mTn3 lbp::Ω) | This study |
| MCV826 | HA1(37) TbpB L9HA TbpA Lbp− (tbpB∇HA tbpA∇HA lbp::Ω) | This study |
| MCV827 | HA2(97) TbpB L9HA TbpA Lbp− (tbpB∇HA tbpA∇HA lbp::Ω) | This study |
| MCV828 | HA3(175) TbpB L9HA TbpA Lbp− (tbpB∇HA tbpA∇HA lbp::Ω) | This study |
| MCV829 | HA4(293) TbpB L9HA TbpA Lbp− (tbpB∇HA tbpA∇HA lbp::Ω) | This study |
| MCV830 | HA5(328) TbpB L9HA TbpA Lbp− (tbpB∇HA tbpA∇HA lbp::Ω) | This study |
| MCV831 | HA6(424) TbpB L9HA TbpA Lbp− (tbpB∇HA tbpA∇HA lbp::Ω) | This study |
| MCV832 | HA7(453) TbpB L9HA TbpA Lbp− (tbpB∇HA tbpA∇HA lbp::Ω) | This study |
| MCV833 | HA8(607) TbpB L9HA TbpA Lbp− (tbpB∇HA tbpA∇HA lbp::Ω) | This study |
| MCV834 | HA9(660) TbpB L9HA TbpA Lbp− (tbpB∇HA tbpA∇HA lbp::Ω) | This study |
| MCV836 | HA5(328) HA7(453) TbpB TbpA− Lbp− (tbpB∇HA tbpA::mTn3 lbp::Ω) | This study |
| MCV838 | HA5(328) HA9(660) TbpB TbpA− Lbp− (tbpB∇HA, tbpA::mTn3 lbp::Ω) | This study |
| MCV847 | TonBHA(160) Lbp− (tonB∇HA lbp::Ω) | This study |
| MCV848 | HA4(293) HA8(607) TbpB TbpA− Lbp− (tbpB∇HA tbpA::mTn3 lbp::Ω) | This study |
| MCV849 | HA5(328) HA8(607) TbpB TbpA− Lbp− (tbpB∇HA tbpA::mTn3 lbp::Ω) | This study |
| Plasmids | ||
| pCR2.1 | Kanr Ampr | Invitrogen |
| pHSS6GCU | Vector containing gonococcal uptake sequence (Kanr) | 42 |
| pVCU521 | pCR2.1 containing tbpA gene fragment; HA in L9 | 49 |
| pVCU690 | pCR2.1 containing tonB(Δ160-194) | 22 |
| pVCU801 | pCR2.1 containing tbpB gene fragment amplified by PCR with primers oVCU136 and oVCU141; HA1 | This study |
| pVCU802 | pCR2.1 containing tbpB gene fragment amplified by PCR with primers oVCU136 and oVCU141; HA2 | This study |
| pVCU803 | pHSS6GCU containing EcoRI fragment from pVCU801 | This study |
| pVCU804 | pHSS6GCU containing EcoRI fragment from pVCU802 | This study |
| pVCU813 | pCR2.1 containing tbpB gene fragment amplified by PCR with primers GC553 and oVCU216; HA3 | This study |
| pVCU814 | pHSS6GCU containing EcoRI fragment from pVCU813 | This study |
| pVCU815 | pCR2.1 containing tbpB gene fragment amplified by PCR with primers GC555 and oVCU219; HA4 | This study |
| pVCU816 | pHSS6GCU containing EcoRI fragment from pVCU815 | This study |
| pVCU817 | pCR2.1 containing tbpB gene fragment amplified by PCR with primers GC555 and oVCU219; HA5 | This study |
| pVCU818 | pHSS6GCU containing EcoRI fragment from pVCU817 | This study |
| pVCU819 | pCR2.1 containing tbpB gene fragment amplified by PCR with primers GC561 and oVCU226; HA6 | This study |
| pVCU820 | pHSS6GCU containing EcoRI fragment from pVCU819 | This study |
| pVCU821 | pCR2.1 containing tbpB gene fragment amplified by PCR with primers GC561 and oVCU226; HA7 | This study |
| pVCU822 | pHSS6GCU containing EcoRI fragment from pVCU821 | This study |
| pVCU823 | pCR2.1 containing tbpB gene fragment amplified by PCR with primers GC561 and oVCU226; HA8 | This study |
| pVCU824 | pHSS6GCU containing EcoRI fragment from pVCU822 | This study |
| pVCU825 | pCR2.1 containing tbpB gene fragment amplified by PCR with primers GC556 and Tfbp12; HA9 | This study |
| pVCU826 | pHSS6GCU containing EcoRI fragment from pVCU825 | This study |
| pVCU836 | pCR2.1 containing tonB gene amplified by PCR with primers oVCU142 and oVCU143; TonBHA | This study |
a
Phenotype listed includes position of the last amino acid prior to HA insertion (parentheses). Residues are numbered starting from the first amino acid in the mature protein. ∇, insertion of the HA epitope.