JC1 MCSLLKKIVFSLLGPTSFQRVPTENTPHVVSISKRNLSRIIEGGRISSWKFME 61 SK-93-1035 MCSLLKKIVFSLLGPTSFQRVPTENTPHVVSISKRNLSRIIEGGRISSWKFME 61 DGI2 MCSLLKKIVFSLLGPTSFQRVPTTENTPHVVSISKRNLSRIIEGGRISSWKFME 61 PID18 MCSLLKKIVFSLLGPTSFQRVPTTENTPHVVSISKRNLSRIIEGGRISSWKFME 61 PID2059 MCSLLKKIVFSLLGPTSFQRVPTENTPHVVSISKRNLSRIIEGGRISSWKFME 61 MS11 MCSLLKKIVFSLLGPTSFQRVPTENTPHVVSISKRNLSRIIEGGRISSWKFME 61 PID1 MCSLLKKIVFSLLGPTSFQRVPTENTPHVVSISKRNLSRIIEGGRISSWKFME 61 PID332 MCSLLKKIVFSLLGPTSFQRVPTENTPHVVSISKRNLSRIIEGGRISSWKFME 61 NCCP11945 MCSLLKKIVFSLLGPTSFQRVPTENTPHVVSISKRNLSRIIEGGRISSWKFME 61 F19 MCSLLKKIVFSLLGPTSFQRVPTENTPHVVSISKRNLSRIIEGGRISSWKFME 61 MC...LLKKIVFSLLVGPTTSFQRVPTENTPHVVSISKRNLSRIIEGGRISSWKFME 58 JC1 GDLELQKDTTTGQLFVRSLTSNPTNLFVISEEGKTYLLVLKPTSKQGDNIVIDVGNRRE 122 SK-93-1035 GDLELQKDTTTGQLFVRSLTSNPTNLFVISEEGKTYLLVLKPTSKQGDNIVIDVGNRRE 122 DGI2 GDLELQKDTTTGQLFVRSLTSNPTNLFVISEEGKTYLLVLKPTSKQGNNIVIDVGNRRE 122 PID18 GDLELQKDTTTGQLFVRSLTSNPTNLFVISEEGKTYLLVLKPTSKQGNNIVIDVGNRRE 122 PID2059 GDLELQKDTTTGQLFVRSLTSNPTNLFVISEEGKTYLLVLKPTSKQGDNIVIDVGNRRE 122 MS11 GDLELQKDTTTGQLFVRSLTSNPTNLFVISEEGKTYLLVLKPTSKQGDNIVIDVGNRRE 122 PID1 GDLELQKDTTTGQLFVRSLTSNPTNLFVISEEGKTYLLVLKPTSKQGDNIVIDVGNRRE 122 PID332 GDLELQKDTTTGQLFVRSLTSNPTNLFVISEEGKTYLLVLKPTSKQGDNIVIDVGNRRE 122 NCCP11945 GDLELQKDTTTGQLFVRSLTSNPTNLFVISEEGKTYLLVLKPTSKQGDNIVIDVGNRRE 122 F19 GDLELQKDTTTGQLFVRSLTSNPTNLFVISEEGKTYLLVLKPTSKQGDNIVIDVGNRRE 122 MC GDLELQKDTTTGQLFVRSLTSSPTNLFVVSEEGKTYLLVLKPTSKQGDNIVIDVGNRHE 119 JC1 VLSQTGPMPVTMNSTEYVRIKKMMTSMMRGTTGDMGINHSHEYQTVPLWKDTLFIQN 183 SK-93-1035 VLSQTGPMPVTMNSTEYVRIKKMMTSMMRGTTGDMGINHSHEYQTVPLWKDTLFIQN 183 DGI2 VLSQTGPMPVTMNSTEYVRIKKMMSMMRGTTGDMGINHSHEYQTVPLWKDTLFIQN 183 PID18 VLSQTGPMPVTMNSTEYVRIKKMMSMMRGTTGDMGINHSHEYQTVPLWKDTLFIQN 183 PID2059 VLSQTGPMPVTMNSTEYVRIKKMMSMMRGTTGDMGINHSHEYQTVPLWKDTLFIQN 183 MS11 VLSQTGPMPVTMNSTEYVRIKKMMSMMRGTTGDMGINHSHEYQTVPLWKDTLFIQN 183 PID1 VLSQTGPMPVTMNSTEYVRIKKMMSMMRGTTGDMGINHSHEYQTVPLWKNTLFIQN 183 PID332 VLSQTGPMPVTMNSTEYVRIKKMMSMMRGTTGDMGINHSHEYQTVPLWKNTLFIQN 183 NCCP11945 VLSQTGPMPVTMNSTEYVRIKKMMSMMRGTTGDMGINHSHEYQTVPLWKNTLFIQN 183 F19 VLSQTGPMPVTMNSTEYVRIKKMMSMMRGTTGDMGINHSHEYQTVPLWKNTLFIQN 183 MC VLSRTGPMPVTMNSTEYVRTIKKMMTSMMRGTTGDMGINHSHEYQTIPLWKNTLFIQN 180 JC1 GSYTDMQGLSFTLTNLGTKQLEIREQEFYRQGVLVVRKQILQPGEITDVFIISRPGG 244 SK-93-1035 GSYTDMQGLSFTLTNLGTKQLEIREQEFYRQGVLVVRKQILQPGEITDVFIISRPGG 244 DGI2 GSYTDMQGLSFTLTNLGTKQLEIREQEFYRQGVLVVRKQILQPGEITDVFIISRPGG 244 PID18 GSYTDMQGLSFTLTNLGTKQLEIREQEFYRQGVLVVRKQILQPGEITDVFIISRPGG 244 PID2059 GSYTDMQGFSFTLTNLGTKQLEIREQEFYRQGVLVVRKQILQPGEITDVFIISRPGG 244 MS11 GSYTDMQGLSFTLTNLGTKQLEIREQEFYRQGVLVVRKQILQPGEITDVFIISRPGG 244 PID1 GSYTDMQGLSFTLTNLGTKQLEIREQEFYRQGVLVVRKQILQPGEITDVFIISRPGG 244 PID332 GSYTDMQGLSFTLTNLGTKQLEIREQEFYRQGVLVVRKQILQPGEITDVFIISRPGG 244 NCCP11945 GSYTDMQGLSFTLTNLGTKQLEIREQEFYRQGVLVVRKQILQPGEITDVFIISRPGG 244 F19 GSYTDMQGLSFTLTNLGTKQLEIREQEFYRQGVLVVRKQILQPGEITDVFIISRPGG 244 MC GSYTDMQGLSFTLTNLGTKQLEIREQEFYRQGVLVVRKQILQPGEITDVFIISRLGG 241 Figure S1. lignment of the amino acid sequence of TraK from eight sequenced Neisseria gonorrhoeae strains carrying the GGI and two N. gonorrhoeae strains (JC1 and PID2059) for which we sequenced the trak locus. representative N. meningitidis strain (MC: α275) is included for reference. In some strains of N. meningitidis, including α275, an insertion sequence is present at the start of the trak gene (1). Non-conserved amino acids are highlighted in gray.
DGI2 MMRLKLSITILILSGCSTLTMSGIGGSEKFRCPNRNSDDPYCESISSNYKSVGV 60 F19 MMRLKLSITILILSGCSTLTMSGIGGSEKFRCPNRNSDDPYCESISSNYKSVGV 60 MS11 MMRLKLSITILILSGCSTLTMSGIGGSEKFRCPNRNSDDPYCESISSNYKSVGV 60 NCCP11945 MMRLKLSITILILSGCSTLTMSGIGGSEKFRCPNRNSDDPYCESISSNYKSVGV 60 PID1 MMRLKLSITILILSGCSTLTMSGIGGSEKFRCPNRNSDDPYCESISSNYKSVGV 60 PID18 MMRLKLSITILILSGCSTLTMSGIGGSEKFRCPNRNSDDPYCESISSNYKSVGV 60 PID332 MMRLKLSITILILSGCSTLTMSGIGGSEKFRCPNRNSDDPYCESISSNYKSVGV 60 SK-93-1035 MMRLKLSITILILSGCSTLTMSGIGGSEKFRCPNRNSDDPYCESISSNYKSVGV 60 MC MMRLKLSITILILSGCSTLTMSGIGGSEKFRCSNWNSDDPYCESISSNYKSVGV 60 DGI2 LKDGMKRQVGQPYTDQSVRTLMLTQYSSGTPVRSQSEIRIWVPYLDTDGDLNDQSFT 120 F19 LKDGMKRQVGQPYTDQSVRTLMLTQYSSGTPVRSQSEIRIWVPYLDTDGDLNDQSFT 120 MS11 LKDGMKRQVGQPYTDQSVRTLMLTQYSSGTPVRSQSEIRIWVPYLDTDGDLNDQSFT 120 NCCP11945 LKDGMKRQVGQPYTDQSVRTLMLTQYSSGTPVRSQSEIRIWVPYLDTDGDLNDQSFT 120 PID1 LKDGMKRQVGQPYTDQSVRTLMLTQYSSGTPVRSQSEIRIWVPYLDTDGDLNDQSFT 120 PID18 LKDGMKRQVGQPYTDQSVRTLMLTQYSSGTPVRSQSEIRIWVPYLDTDGDLNDQSFT 120 PID332 LKDGMKRQVGQPYTDQSVRTLMLTQYSSGTPVRSQSEIRIWVPYLDTDGDLNDQSFT 120 SK-93-1035 LKDGMKRQVGQPYTDQSVRTLMLTQYSSGTPVRSQSEIRIWVPYLDTDGDLNDQSFT 120 MC LKDGMKRQVGQPYTDQSVRTLMLTQYSSGTPVRSQSEIRIWVPYLDTDGDLNDQSFT 120 DGI2 YVVLNQGDWLIHNGQQIINEYRPIRLLGDGKTSGSSVETSPTNSNNTNDRIPDVGVNL 180 F19 YVVLNQGDWLIHNGQQIINEYRPIRLLGDGKTSGSSVETSPTNSNNTNDRIPDVGVNL 180 MS11 YVVLNQGDWLIHNGQQIINEYRPIRLLGDGKTSGSSVETSPTNSNNTNDRIPDVGVNL 180 NCCP11945 YVVLNQGDWLIHNGQQIINEYRPIRLLGDGKTSGSSVETSPTNSNNTNDRIPDVGVNL 180 PID1 YVVLNQGDWLIHNGQQIINEYRPIRLLGDGKTSGSSVETSPTNSNNTNDRIPDVGVNL 180 PID18 YVVLNQGDWLIHNGQQIINEYRPIRLLGDGKTSGSSVETSPTNSNNTNDRIPDVGVNL 180 PID332 YVVLNQGDWLIHNGQQIINEYRPIRLLGDGKTSGSSVETSPTNSNNTNDRIPDVGVNL 180 SK-93-1035 YVVLNQGDWLIHNGQQIINEYRPIRLLGDGKTSGSSVETSPTNSNNTNDRIPDVGVNL 180 MC YVVLNQGDWLIHNQQQIIDEYRPIRLLGNGKTSDSTVDPSSTNSNNTNDRIPDVGVNL 180 DGI2 HLQETSGIPGTVR 193 F19 HLQETSGIPGTVR 193 MS11 HLQETSGIPGTVR 193 NCCP11945 HLQETSGIPGTVR 193 PID1 HLQETSGIPGTVR 193 PID18 HLQETSGIPGTVR 193 PID332 HLQETSGIPGTVR 193 SK-93-1035 HLQETSGIPGTVR 193 MC NLQETSGIPGTTR 193 Figure S2. lignment of the amino acid sequence of TraV from eight sequenced Neisseria gonorrhoeae strains carrying the GGI. representative N. meningitidis strain (MC: α275) is included for reference. Non-conserved amino acids are highlighted in gray.
DGI2 MRVKVNKFKLSRKRQLIGMVGSMIVGSIYVIEGNKKKSEVTQVQNKRQILRTDFTSPQGI 70 NCCP11945 MRVKVNKFKLSRKRQLIGMVGSMIVGSIYVIEGNKKKSEVTQVQNKRQILRTDFTSPQGI 70 PID18 MRVKVNKFKLSRKRQLIGMVGSMIVGSIYVIEGNKKKSEVTQVQNKRQILRTDFTSPQGI 70 SK-93-1035 MRVKVNKFKLSRKRQLIGMVGSMIVGSIYVIEGNKKKSEVTQVQNKRQILRTDFTSPQGI 70 PID1 MRVKVNKFKLSRKRQLIGMVGSMIVGSIYVIEGNKKKSEVTQVQNKRQILRTDFTSPQGI 70 PID332 MRVKVNKFKLSRKRQLIGMVGSMIVGSIYVIEGNKKKSEVTQVQNKRQILRTDFTSPQGI 70 MS11 MRVKVNKFKLSRKRQLIGMVGSMIVGSIYVIEGNKKKSEVTQVQNKRQILRTDFTSPQGI 70 F19 MRVKVNKFKLSRKRQLIGMVGSMIVGSIYVIEGNKKKSEVTQVQNKRQILRTDFTSPQGI 70 MC MRVKVNKFKLSRKRQLIGMVGSMIVGSIYVIEGNKKKSEVTQVQNKQQILKTDFTSPQGI 70 DGI2 TDNSLWMNTESSKIEYNRKISELETMVQELKEKENSSNPDTSKGLGPDGLGKPPISGIGDNGRLPPP 140 NCCP11945 TDNSLWMNTESSKIEYNRKISELETMVQELKEKENSSNPDTSKGLGPDGLGKPPISGIGDNGRLPPP 140 PID18 TDNSLWMNTESSKIEYNRKISELETMVQELKEKENSSNPDTSKGLGPDGLGKPPISGIGDNGRLPPP 140 SK-93-1035 TDNSLWMNTESSKIEYNRKISELETMVQELKEKENSSNPDTSKGLGPDGLGKPPISGIGDNGRLPPP 140 PID1 TDNSLWMNTESSKIEYNRKISELETMVQELKEKENSSNPDTSKGLGPDGLGKPPISGIGDNGRLPPP 140 PID332 TDNSLWMNTESSKIEYNRKISELETMVQELKEKENSSNPDTSKGLGPDGLGKPPISGIGDNGRLPPP 140 MS11 TDNSLWMNTESSKIEYNRKISELETMVQELKEKENSSNPDTSKGLGPDGLGKPPISGIGDNGRLPPP 140 F19 TDNSLWMNTESSKIEYNRKISELETMVQELKEKENSSNPDTSKGLGPDGLGKPPISGIGDNGRLPPP 140 MC TDNSLWMNTESSKIEDNRKISELETMVQELKEKENSSNPDTSKGLGPDGLGKPPISGIGDNGQLPPP 140 DGI2 PGTLPNGPPDRPIERKIVSGSMSEELQPGTTGSVNTGNPNENVRVSPQLKEEWVKSKTPRMNIEV 210 NCCP11945 PGTLPNGPPDRPIERKIVSGSMSEELQPGTTGSVNTGNPNENVRVSPQLKEEWVKSKTPRMNIEV 210 PID18 PGTLPNGPPDRPIERKIVSGSMSEELQPGTTGSVNTGNPNENVRVSPQLKEEWVKSKTPRMNIEV 210 SK-93-1035 PGLPNGPPDRSIERKIVSGSMSEELQPGTTGSVNTGNPNENVRVSPQLKEEWVKSKTPRMNIEV 210 PID1 PGTLPNGPPDRSIERKIVSGSMSEELQPGTTGSVNTGNPNENVRVSPQLKEEWVKSKTPRMNIEV 210 PID332 PGTLPNGPPDRSIERKIVSGSMSEELQPGTTGSVNTGNPNENVRVSPQLKEEWVKSKTPRMNIEV 210 MS11 PGTLPNGPPDRSIERKIVSGSMSEELQPGTTGSVNTGNPNENVRVSPQLKEEWVKSKTPRMNIEV 210 F19 PGTLPNGPPDRSIERKIVSGSMSEELQPGTTGSVNTGNPNENVRVSPQLKEEWVKSKTPRMNIEV 210 MC PGTLPNGVPPDRSIERKIVSGSMSEELQPGTTGVNTGNPNENVRVSPQLKEEWVKSKTPRMNIEV 210 DGI2 VEDGGKVVVSQGKFRRDSYIPSGTFFRSVLLGGVDPTGGEQNSPHPVLMRVTDFQLPNRFKYNFR 280 NCCP11945 VEDGGKVVVSQGKFRRDSYIPSGTFFRSVLLGGVDPTGGEQNSPHPVLMRVTDFQLPNRFKYNFR 280 PID18 VEDGGKVVVSQGKFRRDSYIPSGTFFRSVLLGGVDPTGGEQNSPHPVLMRVTDFQLPNRFKYNFR 280 SK-93-1035 VEDGGKVVVSQGKFRRDSYIPSGTFFRSVLLGGVDPTGGEQNSPHPVLMRVTDFQLPNRFKYNFR 280 PID1 VEDGGKVVVSQGKFRRDSYIPSGTFFRSVLLGGVDPTGGEQNSPHPVLMRVTDFQLPNRFKYNFR 280 PID332 VEDGGKVVVSQGKFRRDSYIPSGTFFRSVLLGGVDPTGGEQNSPHPVLMRVTDFQLPNRFKYNFR 280 MS11 VEDGGKVVVSQGKFRRDSYIPSGTFFRSVLLGGVDPTGGEQNSPHPVLMRVTDFQLPNRFKYNFR 280 F19 VEDGGKVVVSQGKFRRDSYIPSGTFFRSVLLGGVDPTGGEQNSPHPVLMRVTDFQLPNRFKYNFR 280 MC VEDGGKVVVSQGKFRRDSYIPSGTFFRSVLLGGVDPTGGEQNSPHPVLMRVTDFQLPNRFKYNFR 280 DGI2 ECFVTGQYGDISSERYIRLQNLSCVGTDGRIDMPVKGYVGEDGKTGVRGNLVTKQGQLLNLMSG 350 NCCP11945 ECFVTGQYGDISSERYIRLQNLSCVGTDGRIDMPVKGYVGEDGKTGVRGNLVTKQGQLLNLMSG 350 PID18 ECFVTGQYGDISSERYIRLQNLSCVGTDGRIDMPVKGYVGEDGKTGVRGNLVTKQGQLLNLMSG 350 SK-93-1035 ECFVTGQYGDISSERYIRLQNLSCVGTDGRIDMPVKGYVGEDGKTGVRGNLVTKQGQLLNLMSG 350 PID1 ECFVTGQYGDISSERYIRLQNLSCVGTDGRIDMPVKGYVGEDGKTGVRGNLVTKQGQLLNLMSG 350 PID332 ECFVTGQYGDISSERYIRLQNLSCVGTDGRIDMPVKGYVGEDGKTGVRGNLVTKQGQLLNLMSG 350 MS11 ECFVTGQYGDISSERYIRLQNLSCVGTDGRIDMPVKGYVGEDGKTGVRGNLVTKQGQLLNLMSG 350 F19 ECFVTGQYGDISSERYIRLQNLSCVGTDGRIDMPVKGYVGEDGKTGVRGNLVTKQGQLLNLMSG 350 MC ECFVTGQYGDISSERYIRLQNLSCVGTDGRIDMPVKGYVGEDGKTGVRGNLVTKQGQLLNLMSG 350 DGI2 VISGMGKGVSEFKVTNNTFGSTTSIRGSDQYRGISGIGGDRLEYYIKLDKVFPVVEVNGRQ 420 NCCP11945 VISGMGKGVSEFKVTNNTFGSTTSIRGSDQYRGISGIGGDRLEYYIKLDKVFPVVEVNGRQ 420 PID18 VISGMGKGVSEFKVTNNTFGSTTSIRGSDQYRGISGIGGDRLEYYIKLDKVFPVVEVNGRQ 420 SK-93-1035 VISGMGKGVSEFKVTNNTFGSTTSIRGSDQYRGISGIGGDRLEYYIKLDKVFPVVEVNGRQ 420 PID1 VISGMGKGVSEFKVTNNTFGSTTSIRGSDQYRGISGIGGDRLEYYIKLDKVFPVVEVNGRQ 420 PID332 VISGMGKGVSEFKVTNNTFGSTTSIRGSDQYRGISGIGGDRLEYYIKLDKVFPVVEVNGRQ 420 MS11 VISGMGKGVSEFKVTNNTFGSTTSIRGSDQYRGISGIGGDRLEYYIKLDKVFPVVEVNGRQ 420 F19 VISGMGKGVSEFKVTNNTFGSTTSIRGSDQYRGISGIGGDRLEYYIKLDKVFPVVEVNGRQ 420 MC VISGMGKGVSEFKVTNNTFGSTTSIRGSDQYRGISGIGGDRLEYYIKLDKVFPVVEVNGRQ 420 DGI2 VDVVLTQGIEIDTGETK 437 NCCP11945 VDVVLTQGIEIDTGETK 437 PID18 VDVVLTQGIEIDTGETK 437 SK-93-1035 VDVVLTQGIEIDTGETK 437 PID1 VDVVLTQGIEIDTGETK 437 PID332 VDVVLTQGIEIDTGETK 437 MS11 VDVVLTQGIEIDTGETK 437 F19 VDVVLTQGIEIDTGETK 437 MC VDVVLTQGIEIDTGETK 437 Figure S3. lignment of the amino acid sequence of TraB from eight sequenced Neisseria gonorrhoeae strains carrying the GGI. representative N. meningitidis strain (MC: α275) is included for reference. Non-conserved amino acids are highlighted in gray.
Plasmid Diagram of male-trak fusions TraK amino acids Mb recognition 2E5 3H4 3B8 C S X pmr12 L44-G244 + + + pck3 L44-I113, G231-G244 + pck4 G231-G244 S X pck5 D114-G244 + + pmr81 D114-I146 N.D. pmr82 D114-D190 N.D. S X pmr84 S152-G244 + + N.D. S X pmr85 S185-G244 N.D. pmr92 S152-G193 N.D. 100#bp# Figure S4. Characterization of monoclonal antibodies for TraK. Epitopes for three monoclonal antibodies (2E5, 3H4, and 3B8) were determined using MBP-TraK fusions. series of plasmids was constructed in which different lengths of trak were fused to male. The MBP-TraK fusion proteins were expressed in E. coli, and Western blots were performed on E. coli lysates with each monoclonal antibody. In the plasmid diagrams, the broken black rectangle represents male, while the white rectangle indicates the portion of the trak sequence expressed in each fusion. Several restriction sites in TraK are indicated for reference: ClaI (C), gei (), StuI (S), XmaI (X). The TraK amino acids expressed from each fusion are also indicated. The reactivity of the monoclonal antibodies to each fusion protein was determined by immunoblot. +, detection in immunoblot;, no detection in immunoblot; N.D., reactivity not determined.
TraK GGI MCSLLKKIVFSLLGPTSF------------------------------------- 23 60 TraK F MRKNNTIIFGSL--FFSCSVM------------------------------------- 21 60 VirB9 Ti MTRKLFI--LCLFTGEEDTPMGKLDPRMRYLYNPDQVVRLSTVGTLVVT 58 60 TraO N MKKLLLSVVLSVLGGTNVMLEVGRNSPYDYRIKSVVYNPVNVVKIDVGVTHIV 60 60 TraK GGI -------QRVPTENTPHVVSISKRNLSRIIEGGRISSWKFMEGDLELQKDTTTGQLF 76 120 TraK F -NGTLPTVVPMVNGGQSIISNTSPNLFTVPGDRIIVNSLDGLTNNEQTSGGVV 80 120 VirB9 Ti FTNETVTSVVSNSKDLLPRGNYLFFKSQVLTPQPVIVLTSDSGM-RRYVFSISS 117 120 TraO N VPDETYITHFGDSESRTFHKMNHFFVKPKQMSDTNLVIVT-------DKRTYNIVL 113 120 TraK GGI VRSLT-------SNPTNLFVISEEGKTYLLVLKPTSKQGDNIVIDVGNRREVLSQ 129 180 TraK F VTVN-------KKPFTFILETERGLNLSIQVPREGGRTIQLVSDLRGTGEE----- 128 180 VirB9 Ti KTLSHLDKEQPDLYYS----------------VQFYPDDRRREQQRVVDRLH 161 180 TraO N HFIGEETKKNDGTVSKSFIETPWVRHVLQLTYEYPFEQQEKKSDKKRITQKLKQ 173 180 TraK GGI TGPMPVTMNSTEYVRIKKMMSMMRGTTGDMGINHSHEYQTVPLWKDTLFIQNGSYT 189 240 TraK F ----GWETSTPYESLLVTISQVRGGKLPGWYQVPVTKETLQPGLSSVDVWTGN 184 240 VirB9 Ti EQYQRKEDLLDQPVTLGTDSNWHYVQGD---RSLLPLEVFDNGFTTVFHFPGNVR 218 240 TraO N T-----------------FGKNYQYVMSEQPEMRSIQPVHVWDNYRFTRFEFPNE 216 240 TraK GGI DMQGLSFTLTNLGTKQLEIREQEFYRQGVLVVRKQILQ--PGEITDVFIISRPGG--- 244 300 TraK F HLKMVRFVENKTLSLNIRESDFWQPGTRVMFSQPSQLLGRMDVYVIRDGEGN-- 242 300 VirB9 Ti IPSIYTINPDGKEVNYSVKGSD---VEISSVSRGWRLRDGHTVLCIWNYDPVGQRP 275 300 TraO N LPQVYMISSGKETLPNSHVVGENRNIIEVETVKEWRIRLGDKVVGVRNNNFPGRGV 276 300 TraK GGI ------------------ 244 318 TraK F ------------------ 242 318 VirB9 Ti QTGTVRPDVKRVLKGKG 293 318 TraO N TGTSPDVRRVQIGEDN 294 318 B Hydrophobicity values 0.5 0.0-0.5-1.0-1.5-2.0 TraKGGI GGI TraKF F VirB9Ti Ti TraON N -2.5 0 50 100 150 200 250 300 350 Position in alignment Figure S5. lignment of TraK GGI homologs. () lignment of gonococcal TraK (TraK GGI, NCBI ccession No. W83065) with homologs from F plasmid (TraK F, NCBI ccession No. B97947), pkm101 (TraO N, NCBI ccession No. 86458), and. tumefaciens (VirB9 encoded by pti15955, NCBI ccession No. C29979) using lignme (lignment of Membrane Proteins) (2, 3). Identical residues are highlighted in gray. The arrow indicates the predicted Sec-dependent signal peptide cleavage site (SignalP) (4). Cysteines are in red, with the VirB9 Ti cysteine that contributes to the disulphide bridge with VirB7 Ti also boxed. The first column of numbers on the right is the amino acid number in each protein. The second column of numbers is the position in the overall alignment. (B) Hydrophobicity plot of the alignment (2, 3).
* TraV GGI MMRLKLSITILILSGCSTL---------TMSGIGGSEKFRCPNRNSDDPYCESISS 51 60 TraV F MKQTSFFIPLLGTLLLYGCGTSTEFECNTTSDTCMTMEQNEKKKLERSSEKPV 60 60 VirB7 Ti ----MKYCLLCLVVLSGCQTNDTISCKGPIFPLNVGRWQPTPSDLQLRNSGGRYDG- 55 60 TraN N ----MRSLLLMGVLLISCSSGHKPPPEPDWSNTVPVNKTIPV-------DTQGGRNES- 48 60 TraV GGI NY-------------KSVGVLKDGMKRQVGQPYTDQSVRTLMLTQYSSGT------- 91 120 TraV F SLPRLEGNFRTMPVQTVTTTPSGSRPVTHPEQKLLPRPLFTREVKTVVPVSSV 120 120 VirB7 Ti ------------------------------------------------------------ 55 120 TraN N ------------------------------------------------------------ 48 120 TraV GGI -------PVRSQSEIRIWVPYLDTDGDLNDQSFTYVVLNQGDWLIHNGQQIINEYRP 144 180 TraV F TPVTPPRPLRTGEQTLWIPYIDNQDVYHQPSSVFFVIKPSWGKPRIN--------- 171 180 VirB7 Ti ------------------------------------------------------------ 55 180 TraN N ------------------------------------------------------------ 48 180 TraV GGI IRLLGDGKTSGSSVETSPTNSNNTNDRIPDVGVNLHLQETSGIPGTVR 193 229 TraV F ------------------------------------------------- 171 229 VirB7 Ti ------------------------------------------------- 55 229 TraN N ------------------------------------------------- 48 229 B Hydrophobicity values 0.5 0.0-0.5-1.0-1.5-2.0-2.5 TraVGGI GGI TraVF F VirB7Ti TraNN N 0 50 100 150 200 250 Position in alignment Figure S6. lignment of TraV GGI homologs. () lignment of gonococcal TraV (TraV GGI, NCBI ccession No. W83068) with homologs from F plasmid (TraV F, NCBI ccession No. B97952), pkm101 (TraN N, NCBI ccession No. 86456), and. tumefaciens (VirB7 encoded by pti15955, NCBI ccession No. CC15164) using lignme (lignment of Membrane Proteins) (2, 3). Identical residues are highlighted in gray. Cysteines are shown in red, with the N-terminal lipidated cysteines indicated by an asterisk. Cys24 in VirB7 Ti, which contributes to the disulphide bridge with VirB9, is boxed (5). The first set of numbers on the right is the amino acid number in each protein. The second set of numbers of the position in the overall alignment. (B) Hydrophobicity plot of the alignment generated using lignme (2, 3).
α1 TraB GGI ------------------MRVKVNKFKLS----RKRQLIGMVGSMIVGSIYV 38 60 TraB F ---------------------------MSINTIVKRKQYLWLGIVVVGTSIGGLYL 33 60 VirB10 Ti ---------MNNDSQQHEVDSGSLVSDKHRRRLSGSQKLIVGGVVLLSLSLIWLG- 50 60 TraF N MRKSVDVDQELDENTGDGEFESERGGFNGSNRRSPRMKFVILMLLLVFIGITVMG 60 60 TraB GGI I-----EGNKKKSEVTQVQNKRQILRTDFTSPQGITDNSLWMNTESSKIEYNRKISE 93 120 TraB F -SDVDMSGNGETVEQEPVPDMTGVVDTTFDDKVRQHTTEMQVTQMQKQYEEIRREL 92 120 VirB10 Ti ------------------------------------------------------------ 50 120 TraF N ------------------------------------------------------------ 60 120 TraB GGI LETMVQELKEKENSSNPDTSKGLGPDGLGKPPISGIGDNGRLPPPPGTLPNGPPD 153 180 TraB F DVLNKQRGDDQRRIEKLGQDNLEQVKLGNPVTTGEPVPQMPSPPGPEGEPQPG 152 180 VirB10 Ti ------------GRQKKVNDNSPSTLINTKPFHPPIEVPPDTPVQEVQPTVPQP 98 180 TraF N KIRPKEDKDGGKQQNTLPNYSFNSDPDVNKPTQNSPTDRVQQDD 120 180 TraB GGI RSIERKIVSGSMS--EELQPGTTGSVNTGNPNENVRVSPQLKEEWVKSKTPRMNIEVV 211 240 TraB F NTPVSFPPQGSVVPPPTFYPGNGVTPPPQVTYQSVPVPNRIQRKVFTRN--------- 203 240 VirB10 Ti ------PRGEPERHEPRPEETPIFYSSGDQGVSKRSQGDMGRRQEDKRDDNSLPNGEV 152 240 TraF N GSSNTGRTSNKRKEPSPEELMQRRLGGELQTNQTSNSPGQ-------PQDNETS 173 240 TraB GGI EDGGKVVVSQGKFRRDSYIPSGTFFRSVLLGGVDPTGGEQNSPHPVLMRVTDFQL 271 300 TraB F ---------EGKQGPSLPYIPSGSFKMLIEGD-NSVTGNESTVPMQLRITGLVEM 253 300 VirB10 Ti SGENDLSIRMKPTELQPSRTLLPHPDFMVTQGTIIPCILQTIDTNLGYVKCVLPQDI 212 300 TraF N EGSSLKNLTPRLKSRGVMNPSLTVPKGKMIPCGTGTELDTTVPGQVSCRVSQDV 233 300 * TraB GGI PNRFKYNFRECFVTGQYGDISSERYIRLQNLSCVGTDGRIDMPV--KGYVGEDGKT 329 360 TraB F PNSKTYDTGCFVGLEWGDVSSERIVRTRNISCL-KDGKTIDMPI--KGHVSFR-GKN 309 360 VirB10 Ti RGTTNNIVLLDRGTTVVGEIQRGLQQGDERVFVLWDR-ETPDHMISLTSPSDELGRP 271 360 TraF N YSDGLVRLIDKGSWVDGQITGGIKDGQRVFVLWERIRNDQDGTIVNIDSGTNSLGS 293 360 * * α2 α3 TraB GGI GVRGNLVTKQGQLLNLMSGVISGMGKGVSEFKVTNNTFGSTTSIRGSDQYRGIS 389 420 TraB F GIKGEVVMRNGKILGWWGGFVDGIGQGMERSQP--VGLGTYGGDVLKMGIGG 367 420 VirB10 Ti GLPGSVDSHFWQRFSGMLLSVQGFQS--TYGSSGGG---MSFNSFQNNGEQTTE 326 420 TraF N GIPGQVDHMWERLGIMISLFSDTLTLV--NQTQSNN-----IQYNSTENSGGQLS 346 420 α3 TraB GGI GIGGDRLEYYIKLDKVFPVVEVNGRQVDVVLTQGIEIDTGETK------------ 437 480 TraB F GSKQTLSDYYIKREQYHPVIPIGGNEVTVVFQDGFQLKTVEEMLERTQSREED 427 480 VirB10 Ti TLKTINIPPTLKKNQGDTVSIFVRDLDFFGVYQLRLTGGRGRNRRS--------- 377 480 TraF N ELRSYMSIPPTLYDQQGDVSIFVRDLDFSGVYTLDN-------------------- 386 480 TraB GGI ------------------------------------------------ 437 528 TraB F NPESPVPVPPSESHLNGFNTDQMLKQLGNLNPQQFMSGSQGGGNDGK 475 528 VirB10 Ti ------------------------------------------------ 377 528 TraF N ------------------------------------------------ 386 528
B Hydrophobicity values 0.5 0.0-0.5-1.0-1.5-2.0-2.5-3.0 0 100 200 300 400 500 Position in lignment TraBGGI TraBF F VirB10 Ti TraFN N Figure S7. () lignment of gonococcal TraB (TraB GGI, NCBI ccession No. W83066) with homologs from F plasmid (TraB F, NCBI ccession No. B97948), pkm101 (TraF N, NCBI ccession No. 86459), and. tumefaciens (VirB10 encoded by pti15955, NCBI ccession No. C29981) using lignme (lignment of Membrane Proteins) (2, 3). Identical residues are highlighted in gray. The alpha helix predicted to span the inner membrane (α1) is labeled and underlined (predicted using the TMHMM Server v. 2.0) (6). The highly conserved GxxGxxG motif involved in channel gating in. tumefaciens is indicated with asterisks (7). The alpha helices that form the outer membrane pore (α2 and α3) in the TraF N crystal structure are indicated by boxes (8). The predicted alpha helical regions in VirB10 Ti, TraB GGI, and TraB F are underlined (Phyre2) (9). The alignment was generated by manually diving the amino acid sequences into two regions (after the α1 helix) and aligning the two regions separately. (B) Hydrophobicity plot of the alignment generated using lignme (2, 3).
DPI DN stain FITC merge MS11 (WT) MR600 (trak-flg3 + ) 1 µm Figure S8. Immunofluorescence microscopy for TraK-FLG3 in MS11 (WT) and MR600 (trak-flg3 + ). The DPI DN stain was used to counterstain the cells. FITC-conjugated secondary antibody was used to detect TraK-FLG3.
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