rust et al., figure s1
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Rust et al., Figure S1
A
B
Amino acid sequence comparison and alignment of various bacterial FlgM proteins color-coded according to different physico-chemical properties. Sequence alignments were performed using ClustalW2 (Larkin et al., 2007); the alignment was color-coded and depicted using the software GeneDoc (Nicholas et al., 1997). A) alignment of FlgM of different Campylobacterales species which all possess short N-termini; B) alignment of FlgM of diverse eubacteria.
H.pylori_26695 : H.pylori_J99 : H.hepaticus : HPAG1_1060 : H.acinonychis : H.mustelae : C.jejuni : W.succinogenes : B.subtilis : A.aeolicus : E.coli : S.Typhimurium : Y.enterocolitica : P.mirabilis : V.cholerae : P.putida : B.bacteriovorus :
* 20 * 40 * 60-----------------------------MNIKLKDFTMINAVSSLAPVQSLGNYKRVEK--------------------------------------MINAVSSLTPVQSVGNYKRVEK--------------------------------------MVNGVNTSVKVANMLNRDVLAK-----------------------------MNIKLKDFTMINAVSSLAPVQSLGNYKRVEK--------------------------------------MINAISSLTPTQSWGNYKRVEK--------------------------------------MLNIIGKNPMVANVDSKIHKVG--------------------------------------MINPIQQSYVANTALNTNRIDK--------------------------------------MISPIQSGATLPTAKNVQNREV-----------------MKINQFGTQSVNPYQKNYDKQAVQKTVAQPQDKIEISSQAKEM------------------MVNRIELSRLIGLLLETEKRKNTEQKESGTNKIEDKVTLSKI------------MSIDRTSPLKPVSTVQPRETTDAPVTNTR---AAKTTASTSTSVTLSD------------MSIDRTSPLKPVSTVQTRETSDTPVQKTR---QEKTSAATSASVTLSD------------MSIDRTQPLLPVTPVQPRETSDIAQQTRKP-SAQSKTPVSGTEVKLSD------------MSIERTNPLIPITAISQRNLNEGAQEARKTGNAQTKAMAGDTSVKLSE----MAGIDNIRAGQSLNTTSRTNVRSSDASNSSTQSGAVSRSSASGQDAVSLSSQSKAI----MVIDFSRLNNSPSVTGGVRGNTASGSAEKPAASQEAVKDTSASGEAVHLSQEAQQLKITHNKVGQNLNLTDSSRADNAAGIKGKADNIGAAKADVLTSSNLGESSRVELSPRAQEA
: 31 : 22 : 22 : 31 : 22 : 22 : 22 : 22 : 43 : 42 : 45 : 45 : 47 : 48 : 56 : 56 : 60
H.pylori_26695 : H.pylori_J99 : H.hepaticus : HPAG1_1060 : H.acinonychis : H.mustelae : C.jejuni : W.succinogenes : B.subtilis : A.aeolicus : E.coli : S.Typhimurium : Y.enterocolitica : P.mirabilis : V.cholerae : P.putida : B.bacteriovorus :
* 80 * 100 * NEKVENNE----AALDRVAEIKKAIENNQYKINLHETSHKMAKDLLGIS---NEKVENNE----AALDRVAEIKQAIENNQYKINLHETSHKMAQDLLGIS---QNENNEVKEKEQTQLSKVEQIKEQIKNGEYKIDLQQTSEKMASNLLNL----NEKVENSE----AALDRVAEIKKAIENNQYKINLHETSHKMAQDLLGIS---NEEVQNNE----VALDKVAQIKHAIENNQYKINLRETSHKMAQDLLGIS---DQKVQAEK----IQSDKVEKISQAVKSGGYKVDLQATSEKMALNLLGL----ETKTNDTQ---KAENDKASKIAEQIKNGTYKIDTKATAAAIADSLI------ESKTESS-----ASSSKVQAIKESLQKGEYKLDLPATAEKMALNLLNRE---QHASDAVTGS---RQEKIAQLKAQIENGSYKVDANHIAKNMINFYKKQ----AQELSKNDVEEKDLEKKVKELKEKIEKGEYEVSDEKVVKGLIEFFT------AQAKLMQPGSSDINLERVEVLKLAIRNGELKMDTGKIADALINEAQQDLQSNAQAKLMQPGVSDINMERVEALKTAIRNGELKMDTGKIADSLIREAQSYLQSKAQAKLMQPGSQDINVERVETLKQAIRSGQLTMDTGKIADALLKNVADDLKNSAQKKLVQPSNQDINVEKVARLKAAIADGTLTMDSSKIAEALFREAAESITK-GEMHSEMASRPSFDSAKVAAIKEAIANGSYVVDPEKLADNMIKFEKELGGF-QKISDKLRDEPVVNSARVAQLKQAIADGSYQVDAGRVASKLLDFEAQR----KRIKELALATPDVDEAKVAKFRALIDEGKYKVDAKAIADKMVDEHLEF---- 4 6 6 6
: 76 : 67 : 70 : 76 : 67 : 66 : 65 : 66 : 88 : 88 : 97 : 97 : 99 : 99 : 107 : 104 : 108
H.pylori26695 : HPAG1_1060 : H.pylori_J99 : H.acinonychis : H.mustelae : H.hepaticus : W.succinogenes : C.jejuni :
* 20 * 40 * 60 * 80MNIKLKDFTMINAVSSLAPVQSLGNYKRVEKNEKVENNE----AALDRVAEIKKAIENNQYKINLHETSHKMAKDLLGISMNIKLKDFTMINAVSSLAPVQSLGNYKRVEKNEKVENSE----AALDRVAEIKKAIENNQYKINLHETSHKMAQDLLGIS---------MINAVSSLTPVQSVGNYKRVEKNEKVENNE----AALDRVAEIKQAIENNQYKINLHETSHKMAQDLLGIS---------MINAISSLTPTQSWGNYKRVEKNEEVQNNE----VALDKVAQIKHAIENNQYKINLRETSHKMAQDLLGIS---------MLNIIGKNPMVANVDSKIHKVGDQKVQAEK----IQSDKVEKISQAVKSGGYKVDLQATSEKMALNLLGL----------MVNGVNTSVKVANMLNRDVLAKQNENNEVKEKEQTQLSKVEQIKEQIKNGEYKIDLQQTSEKMASNLLNL----------MISPIQSGATLPTAKNVQNREVESKTESS-----ASSSKVQAIKESLQKGEYKLDLPATAEKMALNLLNRE---------MINPIQQSYVANTALNTNRIDKETKTNDTQ---KAENDKASKIAEQIKNGTYKIDTKATAAAIADSLI--- M6n 6 n k k d4v Ik 6 n YK61l Ts k6A L6
: 76 : 76 : 67 : 67 : 66 : 70 : 66 : 65
Rust et al., Figure S2
Amino acid sequence alignment and comparison of H. pylori FliA and FlhA (C-terminal cytoplasmic domain only). Alignment was color-coded according to different physico-chemical properties. The sequence alignment was performed using ClustalW2 (Larkin et al., 2007); the alignment was color-coded and depicted using the software GeneDoc (Nicholas et al., 1997).
HPFlhA : HPFliA :
* 20 * 40 * 60 *ASDYGFLMPQIRIRDNLQLPPTHYEIKLKGIVIGEGMVMPDKFLAMNTGFVNKEIEGIPTKEPAFGMDAL---------------------------------------------MILMMENRMPKGIQKTETSEKNIEK M N4 GI E
: 70 : 25
HPFlhA : HPFliA :
80 * 100 * 120 * 140WIETKNKEEAIIQGYTIIDPSTVIATHTSELVKKYAEDFITKDEVKSLLERLAKDYPTIVEESKKIPTGAVLNAYDKQQHHHQDDLAIQYLPAVRAMAFRLKERLPSSIDFNDLVSIGTEELIKLARRYESALNDSFWGY 6 1K22 Q I 6 L 4 D V E L K G
: 140 : 95
HPFlhA : HPFliA :
* 160 * 180 * 200 *IRSVLQALLHEKIPIKDMLTILETITDIAPLVQNDVNILTEQVRARLSRVITNAFKSEDGRLKFLTFSTDAKTRVNGAMLDYLRSLDVISR-SSRKLIKSIDIEITKHLNEHGKEPSDAYLAQTLGENIEKIKEAKTASD 43 6 6 6 D663 3 I 6 L E 4 6 46K 3D
: 210 : 164
HPFlhA : HPFliA :
220 * 240 * 260 * 280SEQFLLNKLRENGTSKSLLLNVGELQKLIEVVSEEAMKVLQKGIAPVILIVEPNLRKALSNQMEQARIDVIYALVPIDEQFNAIEQDEITKKIEAEELLEHVQKALNQMSEREQILIQLYYFEELNLSEIKEILGITESR 6 N 6 E 2 L6E V 6 24 6 L L 26
: 280 : 234
HPFlhA : HPFliA :
* 300 IVLSHAELDPNSNFEALGTIHINFISQIIKEVIKKVR-KSLGVDHG--I E6 LG H
: 304 : 255
HPFlhA : HPFliA :
* 20 * 40 * 60 *ASDYGFLMPQIRIRDNLQLPPTHYEIKLKGIVIGEGMVMPDKFLAMNTGFVNKEIEGIPTKEPAFGMDAL---------------------------------------------MILMMENRMPKGIQKTETSEKNIEK M N4 GI E
: 70 : 25
HPFlhA : HPFliA :
80 * 100 * 120 * 140WIETKNKEEAIIQGYTIIDPSTVIATHTSELVKKYAEDFITKDEVKSLLERLAKDYPTIVEESKKIPTGAVLNAYDKQQHHHQDDLAIQYLPAVRAMAFRLKERLPSSIDFNDLVSIGTEELIKLARRYESALNDSFWGY 6 1K22 Q I 6 L 4 D V E L K G
: 140 : 95
HPFlhA : HPFliA :
* 160 * 180 * 200 *IRSVLQALLHEKIPIKDMLTILETITDIAPLVQNDVNILTEQVRARLSRVITNAFKSEDGRLKFLTFSTDAKTRVNGAMLDYLRSLDVISR-SSRKLIKSIDIEITKHLNEHGKEPSDAYLAQTLGENIEKIKEAKTASD 43 6 6 6 D663 3 I 6 L E 4 6 46K 3D
: 210 : 164
HPFlhA : HPFliA :
220 * 240 * 260 * 280SEQFLLNKLRENGTSKSLLLNVGELQKLIEVVSEEAMKVLQKGIAPVILIVEPNLRKALSNQMEQARIDVIYALVPIDEQFNAIEQDEITKKIEAEELLEHVQKALNQMSEREQILIQLYYFEELNLSEIKEILGITESR 6 N 6 E 2 L6E V 6 24 6 L L 26
: 280 : 234
HPFlhA : HPFliA :
* 300 IVLSHAELDPNSNFEALGTIHINFISQIIKEVIKKVR-KSLGVDHG--I E6 LG H
: 304 : 255
HPFlhA : HPFliA :
* 20 * 40 * 60 *ASDYGFLMPQIRIRDNLQLPPTHYEIKLKGIVIGEGMVMPDKFLAMNTGFVNKEIEGIPTKEPAFGMDAL---------------------------------------------MILMMENRMPKGIQKTETSEKNIEK M N4 GI E
: 70 : 25
HPFlhA : HPFliA :
80 * 100 * 120 * 140WIETKNKEEAIIQGYTIIDPSTVIATHTSELVKKYAEDFITKDEVKSLLERLAKDYPTIVEESKKIPTGAVLNAYDKQQHHHQDDLAIQYLPAVRAMAFRLKERLPSSIDFNDLVSIGTEELIKLARRYESALNDSFWGY 6 1K22 Q I 6 L 4 D V E L K G
: 140 : 95
HPFlhA : HPFliA :
* 160 * 180 * 200 *IRSVLQALLHEKIPIKDMLTILETITDIAPLVQNDVNILTEQVRARLSRVITNAFKSEDGRLKFLTFSTDAKTRVNGAMLDYLRSLDVISR-SSRKLIKSIDIEITKHLNEHGKEPSDAYLAQTLGENIEKIKEAKTASD 43 6 6 6 D663 3 I 6 L E 4 6 46K 3D
: 210 : 164
HPFlhA : HPFliA :
220 * 240 * 260 * 280SEQFLLNKLRENGTSKSLLLNVGELQKLIEVVSEEAMKVLQKGIAPVILIVEPNLRKALSNQMEQARIDVIYALVPIDEQFNAIEQDEITKKIEAEELLEHVQKALNQMSEREQILIQLYYFEELNLSEIKEILGITESR 6 N 6 E 2 L6E V 6 24 6 L L 26
: 280 : 234
HPFlhA : HPFliA :
* 300 IVLSHAELDPNSNFEALGTIHINFISQIIKEVIKKVR-KSLGVDHG--I E6 LG H
: 304 : 255
HPFlhA : HPFliA :
* 20 * 40 * 60 *ASDYGFLMPQIRIRDNLQLPPTHYEIKLKGIVIGEGMVMPDKFLAMNTGFVNKEIEGIPTKEPAFGMDAL---------------------------------------------MILMMENRMPKGIQKTETSEKNIEK M N4 GI E
: 70 : 25
HPFlhA : HPFliA :
80 * 100 * 120 * 140WIETKNKEEAIIQGYTIIDPSTVIATHTSELVKKYAEDFITKDEVKSLLERLAKDYPTIVEESKKIPTGAVLNAYDKQQHHHQDDLAIQYLPAVRAMAFRLKERLPSSIDFNDLVSIGTEELIKLARRYESALNDSFWGY 6 1K22 Q I 6 L 4 D V E L K G
: 140 : 95
HPFlhA : HPFliA :
* 160 * 180 * 200 *IRSVLQALLHEKIPIKDMLTILETITDIAPLVQNDVNILTEQVRARLSRVITNAFKSEDGRLKFLTFSTDAKTRVNGAMLDYLRSLDVISR-SSRKLIKSIDIEITKHLNEHGKEPSDAYLAQTLGENIEKIKEAKTASD 43 6 6 6 D663 3 I 6 L E 4 6 46K 3D
: 210 : 164
HPFlhA : HPFliA :
220 * 240 * 260 * 280SEQFLLNKLRENGTSKSLLLNVGELQKLIEVVSEEAMKVLQKGIAPVILIVEPNLRKALSNQMEQARIDVIYALVPIDEQFNAIEQDEITKKIEAEELLEHVQKALNQMSEREQILIQLYYFEELNLSEIKEILGITESR 6 N 6 E 2 L6E V 6 24 6 L L 26
: 280 : 234
HPFlhA : HPFliA :
* 300 IVLSHAELDPNSNFEALGTIHINFISQIIKEVIKKVR-KSLGVDHG--I E6 LG H
: 304 : 255
pCJ10014603 bps
BamHI
EcoRI
flhAc
T25
pCJ10014603 bps
BamHI
EcoRI
flhAc
T25
pCJ10043239 bps
BamHIEcoRI
flgM long
T18
pCJ10043239 bps
BamHIEcoRI
flgM long
T18
A B
Rust et al., Figure S3
BACTH plasmid constructs coding for HPFlhAC-T25 (A) and HPFlgM-T18 (B) fusion proteins in plasmids pKNT25 and pUT18, respectively.
FlhAC T25 FlgM T18
pCJ1001+pUT18
pKNT25-ZIP+pUT18-ZIP
pCJ1001+pCJ1004
pKNT25+pUT18
C) Screening of transformants containing BACTH plasmids in E. coli BTH101 on LB-IPTG-x-Gal plates. pCJ1001+pCJ1004 are transformants expressing both FlhAC-T25 and FlgM-T18 fusion proteins.pCJ1001+pUT18 and pKNT25+pUT18 represent two different negative controls (see also table 3). pKNT25-ZIP+pUT18-ZIP are the positive control transformants expressing two leucin zipper domain fusions (for the latter see Karimova et al., Proc.Natl.Acad.Sci. 1998, and BACTH manual).
C
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