实施例10:PagP和PagL在百日咳博德特氏菌中表达之后的脂质A结构的改变
为了在百日咳博德特氏菌株Tohama中表达PagP和PagL,在表达载体pMMB67EH的低拷贝数的广范围宿主中表达支气管炎博德特氏菌的PagL基因(pagL(Bb))和百日咳博德特氏菌的PagP基因(PagP(Bp))。作为对照,还构建了表达大肠杆菌pagP基因(pagP(Ec))的菌株。从野生型百日咳博德特氏菌株Tohama和表达PagP或表达PagL百日咳博德特氏菌株Tohama中分离LPS,并通过Tricine-SDS-PAGE电泳进行分析。分离自PagL(Bb)表达菌株的LPS,在胶上显得未受到影响,然而分离自PagP表达菌株的LPS显得受到可能的修饰,因为检测出比野生型百日咳博德特氏菌LPS更低电泳迁移率的带。此外,与PagP(Bp)表达菌株相比,在PagP(Ec)表达菌株中出现更高的修饰效率(图14)。为了进一步详细地评价可能的LPS修饰,通过ESI-MS以阴离子模式分析菌株本脂质A部分。这种分析表明在野生型LPS中存在四种主要的脂质A类型(图15A)。m/z1557的峰代表通常发现于百日咳博德特氏菌中的特有的五酰化二磷酸酯类型(Caroff等,Microbes.Infect.,1994),而m/z1477的峰相当于五酰化一磷酸酯类型。m/z1307和1251的两种其它峰代表分子离子在m/z1477的脱酰基脂质A类型,它们分别缺失了3位的一级3-羟基癸酸残基或一级3-羟基十四烷酸残基(在2或3'位点)。这些结果表明在野生型百日咳博德特氏菌的脂质A类型之间的高不均一性,即在凝胶分析中表现出的不溶解性(图14)。有趣的是,计算来自相应峰高的各个脂质A类型的相对量,揭示了在野生型百日咳博德特氏菌LPS中,大量的脂质A类型(~50%)由四酰化形式组成。此外,大多数的脂质A类型是一磷酸酯形式(~80%)。为了排除高丰度的低酰基化的和低磷酸化的脂质A类型是由用于分离脂质A的水解方法引起的人工假象这种可能性,我们测试更短或更长的水解周期(介于1至4小时之间)是否影响脂质A类型的相对丰度,然而情况并非如此(数据未显示)。此外,测定已知浓度的野生型百日咳博德特氏菌的纯化LPS的溶液的总磷酸酯含量(totalphospahecontent)。与由ESI-MS检测出的最为普遍的一磷酸脂质A类型一致,当LPS已经完全被磷酸化时,检测到的磷酸酯含量仅仅比预期的磷酸酯含量的一半稍多(数据未显示)。
根据PagL(Bb)的表达(图15B),在m/z1081、1307和1387分别存在三种脂质A类型。在m/z1307的主峰相当于缺失3位的3-羟基癸酸残基的脱酰基的一磷酸酯形式,而在m/z1387的峰相当于m/z1307的二磷酸化形式的分子离子。在m/z1081的峰相当于缺失3-羟基葵酸残基和3-羟基十四烷酸残基的一磷酸酯形式。缺失其3位的3-羟基癸酸残基的脂质A类型的相对含量,从野生型百日咳博德特氏菌LPS中的约37%提高到表达PagL(Bb)的菌株中的92%以上。因此,即使LPS的电泳迁移率不受到影响(图14,泳道2),编码脂质A3-O-脱酰基酶的PagL(Bb)在百日咳博德特氏菌中仍具有活性。
PagP(Ec)(图15C)和PagP(Bp)(图15D)表达后,检测出一些新的脂质A类型(表III)。在m/z1320、1490、1545、1625、1715以及1796的峰,分别相当于分子离子在m/z1081、1251、1307、1387、1477以及1557时的期望PagP介导的棕榈酰化。通过Tricine-SDS-PAGE电泳后(图14)可以观测的在大肠杆菌PagP和百日咳博德特氏菌PagP中的修饰效率的差异,也可在质谱分析中得到展现。在表达大肠杆菌PagP的菌株中,~47%的总脂质A群体被棕榈酰化,与之相反的是,在表达PagP(Bp)的菌株中,只有~9%。有趣的是,与在表达PagP(Ec)的菌株中相反,在表达PagP(Bp)的菌株中,发现缺失3-羟基十四烷酸残基的脂质A类型没有被棕榈酰化。对于这种差异的可能解释是:这是两种PagP酶的特异性中的差异。尽管大肠杆菌PagP在脂质A的2位点添加了酰基链,但百日咳博德特氏菌PagP在3'位点添加了棕榈酸根(Bishop等,EMBOJ.,2000;Preston等,Mol.Microbiol.,2003)。因此,完全没有被棕榈酰化的脂质A类型(缺失了百日咳博德特氏菌PagP的表达菌株的一个3-羟基十四烷酸残基),暗示着缺失3-羟基十四烷酸残基的脂质A分子,是在其3'位点的特异缺失了该残基。由于大肠杆菌PagP的底物库大于百日咳博德特氏菌PagP的底物库,于是这可部分解释在两种PagP酶中所观测到的修饰效率的差异。此外,这种假说与存在体内低酰基化的脂质A类型相一致。
参考文献
1.Raetz,C.R.H.,andWhitfield,C.(2002)Annu.Rev.Biochem.71,635-700
2.Loppnow,H.,Brade,H.,Durrbaum,I.,Dinarello,C.A.,Kusumoto,S.,Rietschel,E.T.,andFlad,H.D.(1989)J.Immunol.142,3229-3238
3.Steeghs,L,Berns,M.,tenHove,J.,deJong,A.,Roholl,P.,vanAlphen,L.,Tommassen,J.,andvanderLey,P.(2002)Cell.Microbiol.4,599-611
4.Nikaido,H.,andVaara,M.(1987)inEscherichiacoliandSalmonella:CellularrandMolecularBiology(Neidhardt,F.C.,ed)Vol.1,pp.7-22,AmericanSocietyforMicrobiology,Washington,D.C.
5.Caroff,M.,Karibian,D.,Cavaillon,J-M.,andHaeffner-Cavaillon,N.(2002)MicrobesInfect.4,915-926
6.Guo,L.,Lim,K.B.,Gunn,J.S.,Bainbridge,B.,Darveau,R.P.,Hackett,M.,andMiller,S.I.(1997)Science276,250-253
7.Guo,L.,Lim,K.B.,Poduje,C.M.,Daniel,M.,Gunn,J.S.,Hackett,M.,andMiller,S.I.(1998)Cell95,189-198
8.Gunn,J.S.,Belden,W.J.,Miller,S.I.(1998)Microb.Pathog.25,77-90
9.Gunn,J.S.,Lim,K.B.,Krueger,J.,Kim,K.,Guo,L.,Hackett,M.,andMiller,S.I.(1998)Mol.Micrrobiol.27,1171-1182
10.Gunn,J.S.,Ryan,S.S.,VanVelkinburgh,J.C.,Ernst,R.K.,andMiller,S.I.(2000)Infect.Immun.68,6139-6146
11.Miller,S.I.,Kukral,A.M.,andMekalanos,J.J.(1989)Prroc.Natl.Acad.Sci.U.S.A.86,5054-5058
12.Gunn,J.S.,andMiller,S.I.(1996)J.Bacterriol.178,6857-6864
13.Ernst,R.K.,Guina,T.,andMiller,S.I.(1999)J.Infect.Dis.179,Suppl.2,326-330
14.Emst,R.K.,Yi,E.C.,Guo,L.,Lim,K.B.,Burns,J.L.,Hackett,M.,andMiller,S.I.(1999)Science286,1561-1565
15.Trent,M.S.,Ribeiro,A.A.,Lin,S.,Cotter,R.J.,andRaetz,C.R.H.(2001)J.Biol.Chem.276,43122-43131
16.Lee,H.,Hsu,F.F.,Turk,J.,andGroisman,E.A.(2004)J.Bacteriol.186,4124-4133
17.Gibbons,H.S.,Lin,S.,Cotter,R.J.,andRaetzC.R.H.(2000)J.Biol.Chem.275,32940-32949
18.Karbarz,M.J.,Kalb,S.R.,Cotter,R.J.,andRaetz,C.R.H.(2003)J.Biol.Chem.278,39269-39279
19.Bishop,R.E.,Gibbons,H.S.,Guina,T.,Trent,M.S.,Miller,S.I.,andRaetz,C.R.H.(2000)EMBOJ.19,5071-5080
20.Tanamoto,K.,andAzumi,S.(2000)J.Immunol.164,3149-3156
21.Robey,M.,O'Connell,W.,andCianciotto,N.P.(2001)Infect.Immun.69,4276-4286
22.Trent,M.S.,Pabich,W.,Raetz,C.R.H.,andMiller,S.I.(2001)J.Biol.Chem.276,9083-9092
23.Bhat,U.R.,Forsberg,L.S.,andCarlson,R.W.(1994)J.Biol.Chem。269,14402-14410
24.Moran,A.P.,Lindner,B.,andWalsh,E.J.(1997)J.Bacteriol.179,6453-6463
25.Kumada,H.,Haishima,Y.,Umemoto,T.,andTanamoto,K.(1995)J.Bacteriol.177,2098-2106
26.Tommassen,J.,vanTol,H.,andLugtenberg,B.(1983)EMBOJ.2,1275-1279
27.Sambrook,J.,Fritsch,E.F.,andManiatis,T.(1989)inMolecularCloning:ALaboratoryManual.ColdSpringHarbourLaboratoryPress,ColdSpringHarbour(NY)
28.Laemmli,U.K.(1970)Nature227,680-685
29.Dekker,N.,Merck,K.,Tommassen,J.,andVerheij,H.M.(1995)Eur.J.Biochem.232,214-219
30.Lesse,A.J.,Campagnari,A.A.,Bittner,W.E.,andApicella,M.A.(1990)J.Immunol.Methods126,109-117
31.Tsai,C.M.,andFrasch,C.E.(1982)Anal.Biochem.119,115-119
32.Westphal,O.,andJann,J.K.(1965)MethodsCarbohydr.Chem.5,83-91
33.Wilm,M.,andMann,M.(1996)Anal.Chem.68,1-8
34.Altschul,S.F.,Gish,W.,Miller,W.,Myers,E.W.,andLipman,D.J.(1990)J.Mol.Biol.215,403-410
35.Nielsen,H.,Brunak,S.,andvonHeijne,G.(1999)ProteinEng.12,3-9
36.Parkhill,J.,Sebaihia,M.,Preston,A.,Murphy,L.D.,Thomson,N.,Harris,D.E.,Holden,M.T.,Churcher,C.M.,Bentley,S.D.,Mungall,K.L.,Cerdeno-Tarraga,A.M.,Temple,L.,James,K.,Harris,B.,Quail,M.A.,Achtman,M.,Atkin,R.,Baker,S.,Basham,D.,Bason,N.,Cherevach,I.,Chillingworth,T.,Collins,M.,Cronin,A.,Davis,P.,Doggett,J.,Feltwell,T.,Goble,A.,Hamlin,N.,Hauser,H.,Holroyd,S.,Jagels,K.,Leather,S.,Moule,S.,Norberczak,H.,O'Neil,S.,Ormond,D.,Price,C.,Rabbinowitsch,E.,Rutter,S.,Sanders,M.,Saunders,D.,Seeger,K.,Sharp,S.,Simmonds,M.,Skelton,J.,Squares,R.,Squares,S.,Stevens,K.,Unwin,L,Whitehead,S.,Barrell,B.G.,andMaskell,D.J.(2003)Nat.Genet.35,32-40
37.Snijder,H.J.,Ubarretxena-Belandia,I.,Blaauw,M.,Kalk,K.H.,Verheij,H.M.,Egmond,M.R.,Dekker,N.,andDijkstra,B.W.(1999)Nature401,717-721
38.McClelland,M.,Sanderson,K.E.,Spieth,J.,Clifton,S.W.,Latreille,P.,Courtney,L.,Porwollik,S.,Ali,J.,Dante,M.,Du,F.,Hou,S.,Layman,D.,Leonard,S.,Nguyen,C.,Scott,K.,Holmes,A.,Grewal,N.,Mulvaney,E.,Ryan,E.,Sun,H.,Florea,L,Miller,W.,Stoneking,T.,Nhan,M.,Waterston,R.,andWilson,R.K.(2001)Nature413,852-856
39.Basu,S.S.,White,K.A.,Que,N.L,andRaetz,C.R.H.(1999)J.Biol.Chem.274,11150-11158
40.Kulshin,V.A.,Zahringer,U.,Lindner,B.,Jager,K.E.,DmitrieV,B.A.,andRietschel,E.T.(1991)Eur.J.Biochem.198,697-704
41.Hwang,P.M.,Choy,W.Y.,Lo,E.I.,Chen,L.,Forman-Kay,J.D.,Raetz,C.R.,Prive,G.G.,Bishop,R.E.,andKay,L.E.(2002)Proc.Natl.Acad.Sci.USA.99,13560-13565
42.Kol,M.A.,vanDalen,A.,deKroon,A.I.,anddeKruijff,B.(2003)J.Biol.Chem.278,24586-24593
43.vonHeijne,G.(1983)Eur.J.Biochem.133,17-21
44.Tommassen,J.(1988)inMembraneBiogenesis(OpdenKamp,J.A.F.,ed)NATOASIseries,Vol.H16,pp.351-373.SpringerVerlag,Berlin,Heidelberg,NewYork.
45.Haas,D.,andHolloway,B.W.(1976)Mol.Gen.Genet.144,243-251
46.Pace,J.,Hayman,M.J.,andGalan,J.E.(1993)Cell72,505-514
47.Hanahan,D.(1983)J.Mol.Biol.166,557-580
48.Thalen,M.,vandenIJssel,J.,Jiskoot,W.,Zomer,B.,Roholl,P.,deGooijer,C.,Beuvery,C.,andTrampen,J.(1999)J.Biotechnol.75,147-159.
49.Ziegler-Heitbrock,H.W.,Thiel,E.,Futterer,A.,Herzog,V.,Wirtz,A.,andRiethmuller,G.(1988)J.Cancer41,456-461
50.Simon,R.,Priefer,U.,andPuhler,A.A.(1983)Bio/Technol.1,784-791
51.Furste,J.P.,Pansegrau,W.,Frank,R.,Blocker,H.,Scholz,P.,Bagdasarian,M.,andLanka,E.(1986)Gene48,119-131
52.Stibitz,S.,Black,W.,andFalkow,S.(1986)Gene50,133-140
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<210>9
<211>173
<212>PRT
<213>Pseudomonasputida
<400>9
MetLysThrArgLeuAlaAlaSerLeuAlaValAlaValLeuAlaPhe
151015
AlaGlyAlaAspLeuValGlnAlaAlaGlnIleSerGlyAlaValGly
202530
AlaThrGlyGlnGlyAspMetThrTyrArgIleGlyMetSerPheAsp
354045
TrpAspLysLysTrpLeuGluSerSerThrGlyHisValSerGlyTyr
505560
TrpAspAlaAlaTyrThrTyrTrpGluGlyGlyAspAlaSerGlyAla
65707580
HisSerLeuSerPheSerProValPheThrTyrGluPheSerGlyPhe
859095
ThrTyrThrProTyrIleGluAlaGlyIleGlyLeuAlaAlaPheSer
100105110
LysThrAspValGlyAspGlnArgLeuGlySerAlaValAsnPheGlu
115120125
AspArgIleGlyPheGlyLeuLysLeuProGlyGluGlnLysValGly
130135140
IleArgAlaMetHisTyrSerAsnAlaGlyIleLysGlnProAsnAsp
145150155160
GlyIleGluSerTyrSerLeuPheTyrSerThrAlaPhe
165170
<210>10
<211>172
<212>PRT
<213>Pseudomonasputida
<400>10
MetArgLysLeuLeuGlyLeuAlaAlaAlaAlaAlaPheValLeuGly
151015
GlnAlaMetSerAlaGlnAlaAlaAspValSerPheSerValGlyGln
202530
ThrGlyAspSerThrMetValTyrArgLeuGlyLeuGlnSerAsnTrp
354045
AspAlaSerTrpTrpGlnThrSerValGlyArgLeuThrGlyTyrTrp
505560
AspGlyAlaTyrThrTyrTrpAspGlyAspGluThrAlaSerAsnHis
65707580
SerLeuSerPheAlaProValPheValTyrGluPheAlaGlyGluSer
859095
ValLysProTyrIleGluAlaGlyIleGlyValAlaAlaPheSerSer
100105110
ThrGluLeuGluSerAsnGluLeuGlySerAlaPheGlnPheGluAsp
115120125
ArgIleGlyPheGlyLeuArgPheAlaGlyGlyHisGluIleGlyVal
130135140
ArgAlaIleHisTyrSerAsnAlaGlyIleLysGluProAsnAspGly
145150155160
ValGluSerTyrSerLeuHisTyrArgMetAlaLeu
165170
<210>11
<211>187
<212>PRT
<213>Burkholderiafungorum
<400>11
MetAsnAsnLysLysAsnValLeuArgAspLeuAlaLeuLysIleThr
151015
AlaGlyAlaValLeuValGlyAlaSerGlyValAlaSerAlaAspGln
202530
PheGlyValGlnValAlaGlyGlyLeuGlyAspArgHisValLysLys
354045
LeuAspLeuGlyPheValTrpAspProAspLeuAsnTrpTrpGlnIle
505560
GlyAspTrpHisPheSerLeuIleGlyGluAlaHisValAlaTrpTrp
65707580
HisThrAsnGluGlyAsnValHisAspAsnIleGlyGluValGlyVal
859095
ThrProIleIleArgPheIleLysGluSerGlyProIleArgProTyr
100105110
AlaGluLeuGlyAlaGlyIleArgLeuLeuSerSerProArgIleSer
115120125
SerThrPheThrLeuGlyThrAlaPheGlnPheAlaAspMetAlaGly
130135140
ValGlyMetGlnPheGlyAsnArgGlnGlnTyrGlnAlaGlyTyrArg
145150155160
PheGlnHisIleSerAsnGlyGlyIleLysGluProAsnProGlyIle
165170175
AsnPheHisGlnLeuTyrLeuGlnTyrAsnPhe
180185
<210>12
<211>189
<212>PRT
<213>Burkholderiamallei
<400>12
MetAsnAspLysAsnGlyGlyArgValGlyArgAlaIleAlaArgThr
151015
AlaLeuAlaLeuAlaLeuValGlyAlaSerGlySerAlaPheAlaAsp
202530
ArgTrpGlyLeuGlnLeuGlyGlyGlyValAlaAspHisAspMetLys
354045
LysGlyAspIleAlaValValTrpAspProAsnTrpThrTrpTrpGlu
505560
IleGlyGlyTrpHisPheAlaPheValAlaGluGlyHisLeuSerTyr
65707580
TrpArgTyrThrGlyAspArgAlaIleAsnSerSerIleTrpGluVal
859095
GlyAlaThrProIleIleArgPheIleLysSerAlaGlyTyrValArg
100105110
ProPheValGluLeuGlyAlaGlyValArgPheLeuSerHisProThr
115120125
IleSerGlnAsnTyrSerMetSerThrSerPheGlnPheAlaAspMet
130135140
ValGlyValGlyAlaGlnPheGlyAsnHisGlnGlnTyrGlnAlaGly
145150155160
PheArgPheGlnHisValSerAsnAlaGlyIleLysAspProAsnPro
165170175
GlyIleAsnPheSerGlnLeuTyrValGlnTyrAsnPhe
180185
<210>13
<211>189
<212>PRT
<213>Burkholderiapseudomallei
<400>13
MetAsnAspLysAsnGlyGlyArgValGlyArgAlaIleAlaArgThr
151015
AlaLeuAlaLeuAlaLeuValGlyAlaSerGlySerAlaPheAlaAsp
202530
ArgTrpGlyLeuGlnLeuGlyGlyGlyValAlaAspHisAspMetLys
354045
LysGlyAspIleAlaValValTrpAspProAsnTrpThrTrpTrpGlu
505560
IleGlyGlyTrpHisPheAlaPheValAlaGluGlyHisLeuSerTyr
65707580
TrpArgTyrThrGlyAspArgAlaIleAsnSerSerIleTrpGluVal
859095
GlyAlaThrProIleIleArgPheIleLysSerAlaGlyTyrValArg
100105110
ProPheValGluLeuGlyAlaGlyValArgPheLeuSerHisProThr
115120125
IleSerGlnAsnTyrSerMetSerThrSerPheGlnPheAlaAspMet
130135140
ValGlyValGlyAlaGlnPheGlyAsnArgGlnGlnTyrGlnAlaGly
145150155160
PheArgPheGlnHisValSerAsnAlaGlyIleLysAspProAsnPro
165170175
GlyIleAsnPheSerGlnLeuTyrValGlnTyrAsnPhe
180185
<210>14
<211>177
<212>PRT
<213>Azotobactervinelandii
<400>14
MetArgLysTyrLeuSerLeuProAlaValAlaValLeuLeuLeuGly
151015
SerAlaGlyValAlaGlnAlaValGluValGlyAlaAlaValGlyVal
202530
ThrSerGlnAsnAspMetThrTyrArgLeuSerLeuGlyLeuProTrp
354045
GluLysGlnTrpTrpLysSerAspLeuGlyTyrValThrGlyTyrTrp
505560
AspAlaGlyTyrThrTyrTrpGluGlyGlySerGlyAsnAspAsnTyr
65707580
AlaGlyAlaHisSerLeuSerPheSerProValPheThrTyrGluPhe
859095
SerGlyPheSerSerValThrProPheLeuGluLeuGlyValGlyVal
100105110
AlaPhePheSerLysThrArgValGlyGluGlnGlnLeuGlySerSer
115120125
PheAsnPheGluAspArgIleGlyAlaGlyIleLysPheAlaGlyGly
130135140
GlnLysValGlyIleArgAlaIleHisTyrSerAsnAlaGlyIleLys
145150155160
GlnProAsnAspGlyIleGluSerPheSerAlaTyrTyrSerHisAla
165170175
Phe
<210>15
<211>195
<212>PRT
<213>Ralstoniametallidurans
<400>15
MetProProAlaAsnLeuSerArgLysLeuProSerAlaArgLeuLeu
151015
AlaIleAlaAlaLeuValAlaGlyAlaSerSerAlaAlaSerAlaGlu
202530
GluLeuValGlyTrpAlaHisProAlaValGlnAlaAlaPheAlaArg
354045
AspThrAspHisGlyIleAsnLysTyrGluIleAlaValAsnPheAsn
505560
ThrProIleGlnTyrGlyAsnProAspGlyTrpLeuPheArgLeuGln
65707580
AlaGluAlaAsnMetGlyTyrTrpAspAlaArgSerGlyThrAsnArg
859095
GlnAsnLeuMetGluPheGlyLeuThrProIleLeuArgValGluLys
100105110
ArgGlyGlyTyrPheValProPheLeuGluAlaGlyValGlyLeuArg
115120125
LeuLeuThrHisThrSerThrSerAspGlnHisAsnPheSerThrAla
130135140
PheGlnPheGlyAspMetValGlyLeuGlyValGlyPheGlyLysAsn
145150155160
AlaAlaThrGluValGlyMetArgPheGlnHisIleSerAsnAlaGly
165170175
IleLysGluProAsnProGlyThrAsnLeuTyrThrGlyTyrValArg
180185190
TyrArgPhe
195
<210>16
<211>187
<212>PRT
<213>Ralstoniasolanacearum
<400>16
MetThrArgSerAlaLeuProArgSerAlaLysLeuLeuAlaAlaAla
151015
ValSerAlaAlaThrLeuLeuAlaAlaAlaProAlaGlnAlaAspPro
202530
SerValArgAlaIleTyrGlyArgAspAsnArgHisGlyIleGluLys
354045
TyrGlyValAspIleAspPheAspSerGlyPheHisCysGlyAsnPro
505560
GlnGlyTrpPheLeuAsnLeuAspTrpGluIleAlaLeuGlyGlnTrp
65707580
ArgSerThrLysGlyThrAsnArgGlnAsnLeuThrGluPheGlyVal
859095
ThrProLeuPheArgLeuGluLysArgGlyGlySerTrpValProPhe
100105110
IleGluAlaGlyIleGlyProArgLeuLeuSerHisThrArgThrSer
115120125
AspGluHisAsnPheSerThrAlaPheGlnPheSerAspMetIleGly
130135140
ValGlyValAlaPheGlySerArgGlnGlnPheGlnValGlyTyrArg
145150155150
PheGluHisLeuSerAsnAlaSerIleLysArgProAsnProGlyThr
165170175
AspLeuAsnGluLeuTyrLeuArgTyrThrPhe
180185
<210>17
<211>187
<212>PRT
<213>Salmonellatyphimurium
<400>17
MetTyrMetLysArgIlePheIleTyrLeuLeuLeuProCysAlaPhe
151015
AlaCysSerAlaAsnAspAsnValPhePheGlyLysGlyAsnLysHis
202530
GlnIleSerPheAlaAlaGlyGluSerIleArgArgGlyGlyValGlu
354045
HisLeuTyrThrAlaPheLeuThrTyrSerGluProSerAspPhePhe
505560
PheLeuGlnAlaArgAsnAsnLeuGluLeuGlyGlyPheLysAlaLys
65707580
GlySerAspAspCysSerLysHisSerGlySerValProCysAsnLys
859095
TyrAsnGlnGlyValLeuGlyIleSerLysAspValAlaLeuValHis
100105110
PheAlaGlyIleTyrThrGlyIleGlyLeuGlyAlaTyrIleLysSer
115120125
LysSerArgAspAspMetArgValAsnSerAlaPheThrPheGlyGlu
130135140
LysAlaPheLeuGlyTrpAsnPheGlyAlaPheSerThrGluAlaTyr
145150155160
IleArgHisPheSerAsnGlySerLeuThrAspLysAsnSerGlyHis
165170175
AsnPheValGlyAlaSerIleSerTyrAsnPhe
180185