PID1 GSYTAADMQGLSFTLTNLGTKQLEIREQEFYRQGVLAVAVRKQILQPGEITDVFIISRPGG 244

Transkript

1 JC1 MCSLLKKIVFSLLGPTSFQRVPTENTPHVVSISKRNLSRIIEGGRISSWKFME 61 SK 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 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 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 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.

2 DGI2 MMRLKLSITILILSGCSTLTMSGIGGSEKFRCPNRNSDDPYCESISSNYKSVGV 60 F19 MMRLKLSITILILSGCSTLTMSGIGGSEKFRCPNRNSDDPYCESISSNYKSVGV 60 MS11 MMRLKLSITILILSGCSTLTMSGIGGSEKFRCPNRNSDDPYCESISSNYKSVGV 60 NCCP11945 MMRLKLSITILILSGCSTLTMSGIGGSEKFRCPNRNSDDPYCESISSNYKSVGV 60 PID1 MMRLKLSITILILSGCSTLTMSGIGGSEKFRCPNRNSDDPYCESISSNYKSVGV 60 PID18 MMRLKLSITILILSGCSTLTMSGIGGSEKFRCPNRNSDDPYCESISSNYKSVGV 60 PID332 MMRLKLSITILILSGCSTLTMSGIGGSEKFRCPNRNSDDPYCESISSNYKSVGV 60 SK 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 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 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 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.

3 DGI2 MRVKVNKFKLSRKRQLIGMVGSMIVGSIYVIEGNKKKSEVTQVQNKRQILRTDFTSPQGI 70 NCCP11945 MRVKVNKFKLSRKRQLIGMVGSMIVGSIYVIEGNKKKSEVTQVQNKRQILRTDFTSPQGI 70 PID18 MRVKVNKFKLSRKRQLIGMVGSMIVGSIYVIEGNKKKSEVTQVQNKRQILRTDFTSPQGI 70 SK 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 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 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 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 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 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 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.

4 Plasmid Diagram of male-trak fusions TraK amino acids Mb recognition 2E5 3H4 3B8 C S X pmr12 L44-G pck3 L44-I113, G231-G244 + pck4 G231-G244 S X pck5 D114-G pmr81 D114-I146 N.D. pmr82 D114-D190 N.D. S X pmr84 S152-G 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.

5 TraK GGI MCSLLKKIVFSLLGPTSF TraK F MRKNNTIIFGSL--FFSCSVM VirB9 Ti MTRKLFI--LCLFTGEEDTPMGKLDPRMRYLYNPDQVVRLSTVGTLVVT TraO N MKKLLLSVVLSVLGGTNVMLEVGRNSPYDYRIKSVVYNPVNVVKIDVGVTHIV TraK GGI QRVPTENTPHVVSISKRNLSRIIEGGRISSWKFMEGDLELQKDTTTGQLF TraK F -NGTLPTVVPMVNGGQSIISNTSPNLFTVPGDRIIVNSLDGLTNNEQTSGGVV VirB9 Ti FTNETVTSVVSNSKDLLPRGNYLFFKSQVLTPQPVIVLTSDSGM-RRYVFSISS TraO N VPDETYITHFGDSESRTFHKMNHFFVKPKQMSDTNLVIVT DKRTYNIVL TraK GGI VRSLT SNPTNLFVISEEGKTYLLVLKPTSKQGDNIVIDVGNRREVLSQ TraK F VTVN KKPFTFILETERGLNLSIQVPREGGRTIQLVSDLRGTGEE VirB9 Ti KTLSHLDKEQPDLYYS VQFYPDDRRREQQRVVDRLH TraO N HFIGEETKKNDGTVSKSFIETPWVRHVLQLTYEYPFEQQEKKSDKKRITQKLKQ TraK GGI TGPMPVTMNSTEYVRIKKMMSMMRGTTGDMGINHSHEYQTVPLWKDTLFIQNGSYT TraK F ----GWETSTPYESLLVTISQVRGGKLPGWYQVPVTKETLQPGLSSVDVWTGN VirB9 Ti EQYQRKEDLLDQPVTLGTDSNWHYVQGD---RSLLPLEVFDNGFTTVFHFPGNVR TraO N T FGKNYQYVMSEQPEMRSIQPVHVWDNYRFTRFEFPNE TraK GGI DMQGLSFTLTNLGTKQLEIREQEFYRQGVLVVRKQILQ--PGEITDVFIISRPGG TraK F HLKMVRFVENKTLSLNIRESDFWQPGTRVMFSQPSQLLGRMDVYVIRDGEGN VirB9 Ti IPSIYTINPDGKEVNYSVKGSD---VEISSVSRGWRLRDGHTVLCIWNYDPVGQRP TraO N LPQVYMISSGKETLPNSHVVGENRNIIEVETVKEWRIRLGDKVVGVRNNNFPGRGV TraK GGI TraK F VirB9 Ti QTGTVRPDVKRVLKGKG TraO N TGTSPDVRRVQIGEDN B Hydrophobicity values TraKGGI GGI TraKF F VirB9Ti Ti TraON N 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 ), 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).

6 * TraV GGI MMRLKLSITILILSGCSTL TMSGIGGSEKFRCPNRNSDDPYCESISS TraV F MKQTSFFIPLLGTLLLYGCGTSTEFECNTTSDTCMTMEQNEKKKLERSSEKPV VirB7 Ti ----MKYCLLCLVVLSGCQTNDTISCKGPIFPLNVGRWQPTPSDLQLRNSGGRYDG TraN N ----MRSLLLMGVLLISCSSGHKPPPEPDWSNTVPVNKTIPV DTQGGRNES TraV GGI NY KSVGVLKDGMKRQVGQPYTDQSVRTLMLTQYSSGT TraV F SLPRLEGNFRTMPVQTVTTTPSGSRPVTHPEQKLLPRPLFTREVKTVVPVSSV VirB7 Ti TraN N TraV GGI PVRSQSEIRIWVPYLDTDGDLNDQSFTYVVLNQGDWLIHNGQQIINEYRP TraV F TPVTPPRPLRTGEQTLWIPYIDNQDVYHQPSSVFFVIKPSWGKPRIN VirB7 Ti TraN N TraV GGI IRLLGDGKTSGSSVETSPTNSNNTNDRIPDVGVNLHLQETSGIPGTVR TraV F VirB7 Ti TraN N B Hydrophobicity values TraVGGI GGI TraVF F VirB7Ti TraNN N 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 ), 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).

7 α1 TraB GGI MRVKVNKFKLS----RKRQLIGMVGSMIVGSIYV TraB F MSINTIVKRKQYLWLGIVVVGTSIGGLYL VirB10 Ti MNNDSQQHEVDSGSLVSDKHRRRLSGSQKLIVGGVVLLSLSLIWLG TraF N MRKSVDVDQELDENTGDGEFESERGGFNGSNRRSPRMKFVILMLLLVFIGITVMG TraB GGI I-----EGNKKKSEVTQVQNKRQILRTDFTSPQGITDNSLWMNTESSKIEYNRKISE TraB F -SDVDMSGNGETVEQEPVPDMTGVVDTTFDDKVRQHTTEMQVTQMQKQYEEIRREL VirB10 Ti TraF N TraB GGI LETMVQELKEKENSSNPDTSKGLGPDGLGKPPISGIGDNGRLPPPPGTLPNGPPD TraB F DVLNKQRGDDQRRIEKLGQDNLEQVKLGNPVTTGEPVPQMPSPPGPEGEPQPG VirB10 Ti GRQKKVNDNSPSTLINTKPFHPPIEVPPDTPVQEVQPTVPQP TraF N KIRPKEDKDGGKQQNTLPNYSFNSDPDVNKPTQNSPTDRVQQDD TraB GGI RSIERKIVSGSMS--EELQPGTTGSVNTGNPNENVRVSPQLKEEWVKSKTPRMNIEVV TraB F NTPVSFPPQGSVVPPPTFYPGNGVTPPPQVTYQSVPVPNRIQRKVFTRN VirB10 Ti PRGEPERHEPRPEETPIFYSSGDQGVSKRSQGDMGRRQEDKRDDNSLPNGEV TraF N GSSNTGRTSNKRKEPSPEELMQRRLGGELQTNQTSNSPGQ PQDNETS TraB GGI EDGGKVVVSQGKFRRDSYIPSGTFFRSVLLGGVDPTGGEQNSPHPVLMRVTDFQL TraB F EGKQGPSLPYIPSGSFKMLIEGD-NSVTGNESTVPMQLRITGLVEM VirB10 Ti SGENDLSIRMKPTELQPSRTLLPHPDFMVTQGTIIPCILQTIDTNLGYVKCVLPQDI TraF N EGSSLKNLTPRLKSRGVMNPSLTVPKGKMIPCGTGTELDTTVPGQVSCRVSQDV * TraB GGI PNRFKYNFRECFVTGQYGDISSERYIRLQNLSCVGTDGRIDMPV--KGYVGEDGKT TraB F PNSKTYDTGCFVGLEWGDVSSERIVRTRNISCL-KDGKTIDMPI--KGHVSFR-GKN VirB10 Ti RGTTNNIVLLDRGTTVVGEIQRGLQQGDERVFVLWDR-ETPDHMISLTSPSDELGRP TraF N YSDGLVRLIDKGSWVDGQITGGIKDGQRVFVLWERIRNDQDGTIVNIDSGTNSLGS * * α2 α3 TraB GGI GVRGNLVTKQGQLLNLMSGVISGMGKGVSEFKVTNNTFGSTTSIRGSDQYRGIS TraB F GIKGEVVMRNGKILGWWGGFVDGIGQGMERSQP--VGLGTYGGDVLKMGIGG VirB10 Ti GLPGSVDSHFWQRFSGMLLSVQGFQS--TYGSSGGG---MSFNSFQNNGEQTTE TraF N GIPGQVDHMWERLGIMISLFSDTLTLV--NQTQSNN-----IQYNSTENSGGQLS α3 TraB GGI GIGGDRLEYYIKLDKVFPVVEVNGRQVDVVLTQGIEIDTGETK TraB F GSKQTLSDYYIKREQYHPVIPIGGNEVTVVFQDGFQLKTVEEMLERTQSREED VirB10 Ti TLKTINIPPTLKKNQGDTVSIFVRDLDFFGVYQLRLTGGRGRNRRS TraF N ELRSYMSIPPTLYDQQGDVSIFVRDLDFSGVYTLDN TraB GGI TraB F NPESPVPVPPSESHLNGFNTDQMLKQLGNLNPQQFMSGSQGGGNDGK VirB10 Ti TraF N

8 B Hydrophobicity values 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 ), 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).

9 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.

10 References 1. Woodhams KL, Benet ZL, Blonsky SE, Hackett KT, Dillard JP Prevalence and detailed mapping of the gonococcal genetic island in Neisseria meningitidis. J. Bacteriol. 194: Stamm M, Staritzbichler R, Khafizov K, Forrest LR lignment of helical membrane protein sequences using lignme. PLoS One 8:e Khafizov K, Staritzbichler R, Stamm M, Forrest LR study of the evolution of inverted-topology repeats from LeuT-fold transporters using lignme. Biochemistry 49: Petersen TN, Brunak S, von Heijne G, Nielsen H SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Meth. 8: Spudich GM, Fernandez D, Zhou XR, Christie PJ Intermolecular disulfide bonds stabilize VirB7 homodimers and VirB7/VirB9 heterodimers during biogenesis of the grobacterium tumefaciens T-complex transport apparatus. Proc. Natl. cad. Sci. U. S.. 93: Jakubowski SJ, Kerr JE, Garza I, Krishnamoorthy V, Bayliss R, Waksman G, Christie PJ grobacterium VirB10 domain requirements for type IV secretion and T pilus biogenesis. Mol. Microbiol. 71: Banta LM, Kerr JE, Cascales E, Giuliano ME, Bailey ME, McKay C, Chandran V, Waksman G, Christie PJ n grobacterium VirB10 mutation conferring a type IV secretion system gating defect. J. Bacteriol. 193: Chandran V, Fronzes R, Duquerroy S, Cronin N, Navaza J, Waksman G Structure of the outer membrane complex of a type IV secretion system. Nature 462:

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