1. No category

Biodrimica et Biop/rysica Acta, 1130 ( 1992) 81-84
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
81
Short Sequence-Paper
BBAEXP 90320
Listeriolysin genes: complete sequence of ilo
from Listeria !,v~novii and of /so from Listeria seeligeri
Albert Haas
1
,
Martina Dumbsky and Jürgen Kreft
Institut fiir Genetik 1md Mikrobiologie.
U~ril'ersität
Wiirzhurg, Würzburg (German_\')
( Received 11 December 1991 )
Key words: Thiol-activated cytolysin; Listeriolysin 0; Cysteim: motif; ( l.. irmwrii ): ( L .\'('t'lig(•ri)
The completc DNA scqucnccs coding for thc thiol-activutcd cytolysins fmm /..i.'itericl inmol'ii. ivanolysin 0 <ILO) and for
sccligerolysin 0 (LSO) from Listeritl seeligel'i havc bccn dctcrmincd. Thc dcduccd amino acid scqucnccs rcvcalcd that: (i) thc
primary translation products comprisc 528 (JLQ) and 530 (LSO) amino acids. rcspcctivcly. (ii) ILO c,mtains two c~·stcincs. L.SO
has u substitution in thc conscrvcd cystcinc motif.
The thiol-activated cytolysins are a family of poreforming toxins from diverse genera of Gram-positive
bacteria [16], most of which are secreted into the
extracellular medium. These toxins include the
species-specific types of Iisteriolysin 0 produced by the
three Listeria species which are hemolytic on blood
agar: Listeria monocytogenes (pathogenic for man anct
animals), Listeria ivanoL·ii (animal pathogen) and Listeria seeligeri (apathogenic). Their Iisteriolysins have previously bet!n characterized biochemically [6,10],and it
has also been shown that they vary in cytolytic activity.
The gene for Iisteriolysin 0 (h/y) from 1.4. monocytogenes has been cloned and sequenced [4,13] and homologous DNA sequences have been detected in the
chromosome of the two other hemolytic Listeria species
[11]. lt has been firmly established, that Iisteriolysin 0
(LLO) is an essential virulence factor of L. monocytogenes (reviewed in Refs. 2 and 3), which enables this
facultative iiltracellular parasite [2,5] to escape from
the phagosome of the invaded mammalian cell, e.g.,
macrophages or other phagocytes. L. ir:anol'ii has also
been shown to replicate intracellularly, whereas the
avirulent L. seeligeri does not [5,8].
The sequence data reported in this paper have been submitted to the
EMBL/Genbank Data Libraries under the accession numbers
X60461 (i/o) and X60462 (/so).
1 Present address: UCLA. Molecular Biology Institute. 405 Hilgurd
Ave., Los Angeles, CA 90024-1570. USA.
Correspondence: J. Kreft, (from 16 March 1992 on) Biozentrum der
Universität Würzburg, Lehrstuhl Mikrobiolosie, Am Hubland. W8700 Würzburg, Germany.
We have established genomic DNA Hbraries of L.
see/igeri (SLCC3379) and L. iL·anol'ii (ATCCI9119) by
ligating chromosomai DNA fragments from partial
Sau3A digests ( L. il'allot·ii) or size-fractionated Hindill
digests ( L. seeligeri) into the plasmid vector pTZ 18R
[12]. Transformed recombinant E. coli DH5-a clones
were screened by colony hybridization for homology to
a h/y-specific gene probe (651 bp Hindill fragment
from pLM47, Ref. 11 ). One of the positive clones
yiclded plasmid pAHA9, which by DNA sequcncing
with the didcoxy chain termination mcthod was shown
to contain the completc determinant (/!J·o) for Iisteriolysin 0 from L. seeligeri, termcd seeligerolysin 0
(LSO) (Fig. 1). Among more than 15000 recombinants
from the L. il'anor·ii library, three positive clones were
identified which contained overlapping inscrts of dif·
ferent sizes, but which were too small to span the
entire Iisteriolysin gene. The DNA sequence from all
three recombinants was determined. The largest insert
(from pAHAlO) included 1029 base pairs from the
carboxy-terminal moiety of the gene (i/o) for listeri·
olysin 0 from L. il'anol'ii, ivanolysin 0 (ILO). The
other two independent inserts contained shorter segments from the identical ilo sequence. For both pAHA9
and pAHAlO the Iisterial origin of the inscrted DNA
was confirmed by Southern hybridization. Several at·
tempts to detect the complete ilo gene in the genc
bank failed, but by the polymerase chain-reaction
(PCR) we could isolate, clone into pTZJ8R and sequence the complete detcrminant (Fig. 1). The PCR·
primer for the 5' rcgion was dcduced from a previously
determined upstream sequence (Kreft and Weber, unpublished). in addition the complete lsp gene was
82
mo11ocytogenes [4,13]. ILO, LSO and LLO are highly
homologous: identity ILO /LLO 80%, LSO/LLO 82%,
ILO jLSO 76%; similarity 91%, 90% and 86%, respectively, with rather heterogeneaus N-.termini. ILO shows
one deletion at position 25 and LSO the insertion of
one serine at position 33. The analysis of the predicted
signal peptide of LSQ r~v~~,~~ Jb,,~-;. ~.~- ~i,n~.r~- . ~JllP:,:fi­
cantly frQm th~ cori:~sp()n.g~~g ·~Q. an{J. Lt~>.~·-~~~
quences: LSO Iacks one positive eilarge at position 3
(lle versus Lys) and has one hydrophobic amino acid
less in the core region. Therefore, the LSO signal
peptide might be less effective as an export..directing
sequence.
The most interesting findings from the analysis of
the deduced protein sequences were: (i) that LSO has
a non-conservative amino acid substitution (Phe versus
reisolated by the same method in order to confirm the
sequence data from the library.
The DNA sequence homologies between ilojhly,
lsojhly and üojlso were 78%, 77% and 76%, respectivel)\ The deduced amino acid sequences (Fig. 1)
showed that the primary translation product of ilo is a
s.28::'i\11li·p~:~~~.i~.:,;·Jl.f9~~1~<" 9f:x~~.~4J- .Q~,. that of /so comp~~S.!~·S~p;,arot·no~i,ä.qi~$::~($.9./lSltDa>~ We have previously
d~tetmine.di . hie. }'l;ienninal amino. acid sequence of
matufe Jit0::: [:10]~ therefore the signal peptide cleavage
site for lU.C)? was placed after Ala-24. The N-termini of
mature bSO• and LLO are not known, but signal peptides are preferentially cleaved after an alanine, which
is also found in both LSO and LLO at position 24.
The deduced amino acid sequences were compared
(Fig. 2) to the deduced sequence for LLO from L.
ls~
tJo
la~
ATGAAAATATTtGGTTTAGTTATCATGTCGTTGCTATTTGTTAGTTTGGCAATAACACAACAACCTGAAGCGAGGGATGTCCCCGCGTACGATAGAAGCCAGGTGACTATATCTCCTGCT
N 1C 1 r G L V 1 M I
L L r V S L P 1 T 0 0 P E A R D V P A Y D R S E V T I S P A
A~TAATGCTACTTTTAATr.ACATTGTTACTAGTAAGTTTACCGTTAGCACAAGAAGCTCAAGCA•·•~TGCCTCAGTATATAGTTAC•••CAAGGCATAATTTCACACATG
N K K r
N L L L MT
Hpan (Jao}
L.L- LV I
S
Hpan (.Uo)
L p LA 0
Spill (lao)
I
A Q A
CJal (.Uo)
•
DAS
V Y S
Y • Q
G
I
1 S H H
CAAACACCACACTCCCCACCGGCAACACCAAAAACACCTGTAGAGAAAAACCA~CGGAAGAAATTAATAAATATATTTGGGGATTAAACTATGATAAAAATAGTA~GTCTATCAA
I
'I'
A
PP
P
I
P P A lt
P
IC
'I'
P
V E lC
K
ASPPAK
P
IC
T
P
V E K
K HA A
H
A
E
I
1
H
K
Y
Q
I
D
0
Y I
I
N Y
D
IC
H
W G
L
S
I
0
L D Y D IC N H I
L
V Y Q
120
40
114
38
240
80
234
Jlo
GCACCACCAGCGTCTCCGCCTGCAAAGCCTAAGACG~GAAAAATGCAGCTCAAAfCGArcAATATATACAAGGGCTGCA'I'TATGATAAAAACAATATATTAGTGTACGAT
,: $0
GGTGAAGCAcrrTACAAACGTTC:CACCGAMAAGGGCTACAAAGATGGCAG'I'GAATA'!'ATTGTCC'I'TGAAAAAAAGAAAAAAGGTATC.U.TCAAAACAATGCAGACA'l"l'TCTGTCATMA'l'
G I A V 'I' H V P P K K G Y K D G S I Y I V V I IC K K K G I H 0 H H A D I
S V I N
GGAGAAGC'I'CTTAAAAATG'I'TCCACCAAAAGCAGGATACAAAGAAGGAAA'l'CAATATATTC'l'AGTGGAGAAAAAGAAAAAATCTATC.U.TCAAAATAACGCAGATA'l"l'CAAGT'l'ATTAAC
G I A V lC H V P P lC A G Y lC I G N 0 Y I V V E lC IC K 1C S I H 0 H H A D I
Q V I H
360
120
354
118
GCAATTTCGAGCCTTACTTATCCTGGAGCGTTGGTAAAAGCAAATAGAGAATTAGTAGAAAATCAACCTAATGTA~tACCAGTAAAACGAGATTCACTTACATTAAGTGTAGATTTACCA
480
160
.i l o
G
LV
Y D
Ec:oRIUlo)
lao
.ilo
J so
J lo
A 1 S 8 L 'I' Y P G A L V IC A H R I
L V I H 0 P N V L P V lC R D I L 'l' L S V D L P
TCGCTTGCAAGCCTTACTTATCCAGGAGCTTTAGTGAAGGCG.Ur2'CAGAGTTAGTCGAAAATCAACCCGATGTCCTCCCTGTGAAACGAGATTCAGTTACACTTAG'l'ATTGA'l"l'TGCCT
S L A I L T Y P G A L V IC A H S I
L V I H Q P D V L P V lC R D 8 V 'l' L S I D L P
GGAATGACTAAAAAAGATAA'I'AAAATA'l'TC:GTTAAAAACCCTACAAAGTCAAACGTAAATAATGCCGTGAATACATTAGTAGAGCG'I"'J'GGAATGATAAGTATTCAAAAGCGTATCCTAAT
G N 'I' K lC D H IC 1 P V IC N P T K 8 H V H N A V H T L V I R W H D IC Y S K A Y P M
GGAA'J'GGTTAACCATGACAATGAAATAGTCG'l'TOAAAATGCAACTAAATCCAATATAAA'l'GACGGAGTGAATACTTTAGTAGATCG'l'TGGAATAATAAATACTCCGAAGAATACCCAAAT
G N V N H D N I I V V Q N A T IC I H I N D G V N T L V D R W N N IC Y S E B Y P N
lcoU(ilo)
J•o
J.lo
t•o
IJC!
78
474
158
100
2~
594
198
ATTAATGCAAAAA'l'TGATTATTCCGATGAAA'I'GGCTTATAGTGAATCACAAT'l'AA'I'TGCCAAA'l'ftGGGACTGCCTTTAAAGCTCT'l'AATAATAG'1"1'TGAATGTAAAT'l"l"J'GAGGCAATT
l N A IC I D Y I D I II A Y I I S 0 L l A 1t P G 'l' A P lC A V H H I L II V II r E A I
A'M'ACTGCGAAAATTGACTAN.U'CMCAAATGGCCTATAGCGAATCGCAATTAGT'l'GCAAAA'I"'"'''GGTGCAGCATT'fAAACCTGT'l'AATAATAGTTTGAA'l'G'l'AAACTT'l'GGAGCCATT
l I A 1C I D Y D Q I N A Y I I I Q L V A 1C P G A A F K A V H H S L H V II r G A I
714
ACITCIATGOOAAAGTACAAGAACAAC'l'CAT'J'AGT'I'TTAAGCAAA'I"I'TATTATAATAftAACGTTAATGAACCTACAAC:TCCTTCCAAA'l'TCI"1'TGGGGGTAGTGTTACCAAAGAACAAC'l'A
I D G K V Q I I V l
I
P IC Q l Y Y N l
N V H E P 'I' 8 P 8 lC r P G G 8 V '1' lC I
0 L
AGTGAAGGTAAGGTGCAAGAAGAAGTTA'l"''AA'I"''TCAAACAAA'I"'"''ATTATAC'l'GT'l'AATG'l"l'AATGAACCTACAAGCCCTTCCAGA'l"l'CT'Z'TGOTAAAAGTG'M'ACTAAAGAAAACTTG
I I G lt V Q I I V I H P IC Q I Y Y 'I' V M V N I P 'I' 8 P 8 R P P G lC 8 V 'I' K I
N L
840
280
8J4
278
720
240
l38
Nclei(Jlo)
l•C# GATGCTT'I'AGGTGTAAA'I'GCCGAAAA'I'CC'I'CC'l'GCTTACATT'l'C'l'AG'l'GTTGCTTAeGGTCGCCAAG'l'TTATG'l'GAAACTA'l'CCTC'l'AGCTCGCATAG'l'AACAAAGT'l'AAAACTGC'l"rl'C
D A L G V H P I M: P P A Y I I 8 V A Y G R Q V Y V IC L I 8 8 8 H 8 II 1C V I '1' A f
llo CAACCGC'I'GGGCG1'AAA'I'GCGGAAAATCCACCCGCATACA'l'C'lC'l'AC'I'GTTGCAJ'UGGTCGTGACATT'l"l'CCTGAAAT'l'A'I'CGAC'l'AO'l"J'CACACAGCACCAGAGTGAAGGCTGCA'l"l'C
Q A L G V M A I N P P A Y I I 8 V A Y G R D I r V lC L 8 'l' S S H S T R V IC A A r
lao
ilo
GAOCICGGCGATGAG'l'GGCAAA'l'CAG'l'GAAACGGGA'I'G'l'AGAA'l"l'AACAAATA'l"l'ATAAAAAA'l":C'l'TCft'l'TAAAGCAG'l'CATT'l'A'l'GGTGGC'J'CAGCGAAAGAAGAGGT'l'GAAATTATT
. I A A II I G I 8 V I G D V I L 'l' M I I K H I I P lC A V I Y G G 8 A K I B V I
I I
GA'l'GCTGCA'I'ftAAGGC'l'AAATCAGTTAAAGGTGATACAGAA'l"l'AGAAAATATTA'l"lCAAAATGCTTCA'l"l"l'AAAGCGG'l'GA'I"J"''A'l'GGTGGTTCAGCCAAAGATGAAGTAGAAATAATT
D A A P IC G IC S V I G D V I L I II I I Q II A S P IC A V I Y G G S A K D B V I I I
1080
3110
GA'l'GGCAA'l"l"l'AGGCGAAC'l"'"CGAGATAft'l'TGAMAAAGGGKCACTTATGATAGAGAAAAeceTGGc:G'l"''C:CGATCTCGTACACAACTAACT'l"l"tTGAAAGATAA'I'GACTTAGCGGTT
D G M L G I L R D I L IC IC G 8 'I' Y D R I M P G V P I I Y 'l' 'l' M P L 1C D II D L A V
GA'I'GGAGA'l"l"l'AACCAAATTACGAGATA'l"'"l"lAAAACAAGGGGCTAA'l"l'T'l'GATAAGAAAAATCCGt:GCC'l'ACCCl\'I'TGCGI'AS'ACAACTAA'l"l"'fC'l"lGATAA'l'CAGTTAGCAGTT
D G D L I IC L R D I L IC Q G A M P D 1C IC M p G V P I A Y 'l' 'I' II P L 1C D II Q L A V
1200
400
1114
398
AYall(l80)
J•o
JJo
160
320
154
311
Bpaii(llo)
ACCI(Jlo)
lcll(lao) Bpaii(Jlo)
·
1074
351
lao
CTTAAAAACAAC'lCAGAATATA'tCGAAACAAC'l"l'CCAAATCTTATACAGATGGAAAAA'l"l'AATA~A'l"l'CTGGTGG'l"''ATGTAGC:CCAATTCAATATATC:TTGGCATGAACTAAGT
V IC II M I I Y I I 'I' 'l' S IC S Y 'I' D G IC I N I D H S G G Y V A Q P N I 8 W D E V S
1 J20
440
.!lo
GTTAAAAATAATTCGGAATATATCGAAA::!AACTTCTAAGGCTTACTCGGATGGAAAAA'l"l'AAC:CTAGATCATTCCGG"l'GCCTATGT'l'Gc:GAGATTCAATG'l"l'ACT'I'GGGATGAAGTTAGC
V K N M 8 I I I I 'l' T S K A Y I D G K I
N L D H 8 G A Y V A R F H V 'I' W D z· V S
1 Jl4
'fAtcAc:c:AGAAO:GAAATGAAATAAAACTTCATAAGAAATGGCGc:GAAAA'l'TATAAGAGTAAGTTAGCTCA'l"l'TCACTTCTTCTATC'l'ATTTGCCAGGAAATGCGAGAAATA'I"l'AACATC
I D I M G H I I K V M lC IC W G I H Y K 8 1C L A H r T 8 8 I Y L P G N A R N I
H I
TATGATGCTAATGGAAATGAAG'I"''G'''''GAACATAAAM.A~TGATAAAGAT AAGTTAQCTCATTTTACGACATCAATC'l'AT'n'GCCAGGGAATGCAAGGAATA'I"l'AATA'I'T
Y D A N G N I V V K H K K W 8 I H D I D I L A H
T T 8 I Y L p G H A R N I N I
1440
480
TATGCAAGA~'l'i'f~'11GliiiGGGAA~~QMCTCT'l'AtAGA'I'GACAGAAACTTACCAT'I'AGTAAA.AAATACAAA'I'CTATCTATTTGGCGTACAACCCTTTACCCAAGA
15110
520
1554
518
llpall(11o)
l•o
.i lo
Jao
r
BpaU(lao)•·
Y
A
R
l'k- '"
"&"
11:- 'C" )
--~ f"
lr f
ll' 'l'
V
I
D
D
R
N
L
P
L
V
lC
N
R
H
V
I
I
II
G
T
'l'
L
Y
P
R
iJO CANCGAAAGAATG'I'ACTGGCTTGGC'i'TGGGAATGG'l'GGAGAACGGTTGTGCATGATAGAAAC'l'TGCCAT'I'AGTAAAAAATAGAAATG'lTl'GTATC'l'GGGGAACAACGC'lTl'ATCCAGCG
HA Kl,F_g_'l'_,9_~,!_W_§._W_'!,..RJ T ·V V DDR N L P LV lC N R R V C I N G T '1' L Y PA
lao CATTCTAATAATCTACATAATCCGATTCAGTAGTAA
1596
~
H S N H V D N P I 0 EndEnd
530
~lo
TATA~TACTGTAGATAATCCAATTAAGTAA
Y S
D T V D II P l
1t End
438
1434
478
1581
528
Fis. I. Nucleotide and deduced amino acid sequences of lso (34.6% G+C) and ilo (35.5% G+C). A few restriction sites, setving as landmarks,
are indic:ated above the DNA sequence. The presumptive signal peptide is underlined, the conserved undecapetide is boxed with a broken line.
Phe-489 in LSO and Cys-509 in ILO are indicated by arrows.
83
Ala) at position 489. Ala-489 is the sixth amino acid
residue in the undecapeptide believed to be absolutely
conserved among all thiol-activated cytolysins sequenced so far [7,9,17,18], irtcluding LLO from L.
monocytogenes [4,13]. By site-directed mutagenesis it
has been demonstrated by others that the integrity of
t.I)J~. gQ.m.ain, and in particular the presence of certain
trypt(>,'ph~öe residues, is crucial for the hemolytic activity of these toxins [1]; this is also true for LLO from L.
monocytogenes [14]. Although the normally conserved
alanine is replaced in LSO by another hydrophobic,
nonpolar, uncharged amino acid, the bulky aromatic
ring of phenylalanine might have some detrimental
effect on the hemolytic activity of LSO, compared to
ILO
LSO
LLO
LLO. This notion is supported by the fact that the
phenylalanine is directly adjacent to the critical tryptophane residues. To ensure that the observed amino
acid substitution in LSO did not result from a cloning
artifact during the construction of pAHA9, the relevant chromosomal region of L. seeligeri was amplified
by the poJymerase chain reaction (PCR). The DNA
sequence determined from the PCR product was identical to the one found for pAHA9. (ii) The conserved
undecapeptide mentioned above is termed the 'cysteine motif', as it contains the single cysteine residue
in all thiol-activated cytolysins analyzed so far. It has
been reported that the presence of this cysteine is not
absolutely requircd for the hemolytic activity of pncu-
MKKIML~LM%L~VSLP~AQ~A-DAS~(-~IS~~­
ILO
LSO
LLO
MK~LVIMSLL VSLP~Q~A00D~S~IS~
M K K IM LV FIT LI LV S L 'PI A QQ TE AI< DAS A F NI<- E N SI S SM
-- · - - - - - - - - - - - - - - - - - - - - - - --A
•
APPASPPArilPK'l'PiVlEKRlHJAJA"OliJl.(Q]YIOGLDYfDlKNJÜilLVYfDJ
~P[i)S PP AllJP K 'l' PbzJE K K H AliJi"I[W'1fY I[i}G L(IDYI!!JK Nl!:.l.JL V Yl.gj
A P P A S P P A S: P lC T P I E K K H A.D E I D K Y I Q G L D Y N lC N N V L V Y H
ILO
LSO
LLO
GrilA V[!)N V P PfR:iJG Y K(!]G_NI'ölY.I V V E K K K K srf1N Q N NA D I..Q.. vrilN
G~AVTNVPP~GYKDG~YIVVEKKKIC~NQNNADIWV~N
G .D A V T N V P P R K G Y K D G N E Y I V V E K K K lC S I N Q N N A D I Q V V N
ILO
mi!) S L T Y P G A LV K A N S E LV E N Q P D V L P V lC R
38
40
39
78
80
79
118
120
119
158
160
LLO
D S (2JT L S I D L P
AISSLTYPGALVKAN~ELVENQP~VLPVKRDSL'l'LS~DLP
AISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLP
LSO
159
ILO
GM~:~DN00tvVmNATKSH00N~~VHTLV~RWH~KY~YPH
198
LSO
LLO
GM'l'
QDNKIVVKNATKSNVNNAVN'l'LVERWNEKYAQAYPN
199
ILO
LSO
~A KID y_Q['Q]E M A Y SES Q L~AK F G(!]A F lC AV N N S LN V N F GA I
~AKIDY~EMAYSESQLIAKFGTAFKAVNNSLNVNF00AI
VSAKIDYDDEMAYSESQLIAKFG'l'AFICAVNNSLNVNFGAI
238
SEGK~QEEVI~FKQIYY~NVNEPT~PSRFFG_K~V'l'l<E~L
- 278
LW
SEGKMQEEVISFKQIYYNVNVNEP'l'RPSRFFGKAVTKEQL
279
ILO
~ALGVNAENPPAYISSVAYGR~VYWKLS~SRS~VlC~AF
320
QALGVNAENPPAYISSVAYGRQVYLKLS'l'NSHSTKVKAAF
319
DAA~GKSV;GDVEL~NII~N~SFRAVIYGGSAKDEV~II
358
LLO
ILO
LSO
LSO
LLO
ILO
GMTKKDNKI~VKN00'l'KSNVNNAVNTLVERWN~lCY~AYPN
S~GK~QEEVISFKQIYYNINVNEP'l'~PS~FFG~V'l'KEQL
Q AL G V NA E NP PA Y I S SV A Y G RüiJLVt K L S~ S H S ~V lC A A F
200
240
239
280
318
LLO
~AAM
GKSVI<GDVELYNIITNSSFKAVIYGGSAK00EV~II
TAA
SGKSV
GDVELTNIIKNSSFRAVIYGGSAKDEVQII
IU.,
DG~L~LRDILK~GA~PGVPIAY'l'TNFLKDN;LAV
LSO
LLO
DGNL~LRDILKTGS~~PGVPI~YTTNFLICDNDLAV
400
DGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDN
LAV
399
~l<NNSEYIE~'l'SKAYmDGV.IN~DHSG~YVA~FN~WDEV~
438
LSO
ILO
LSO
LLO
ILO
LSO
LLO
ILO
LSO
LLO
~LO
LSO
~l<NNSEYI~'l''l'SK~YTDGRINIDHSGGYVAQFN~WDEVS
IlCNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEV
YD~GNE~HK~WSEN~l<~KLAHFT00SIYLPGNARNINm
YD~GNE~HK~W~EN~KSKLAHFTSSIYLPGNARNINW
Y D P E G N E I V Q H K N W S E N N lC S K L A H F T S S I Y L P
~
y
[!!JA Kr-E-CT-G-LA_W_E_W'i"i1T vl!]o D RN L P LV K N R NfV':gi
y A R:E C T G L[!) W E W W R•T V I D D RN L P LV K N R N lY.fS I
Y A K~_C_'l'_q_ !!~.W_J_w_'!,~jT V I D D RN L P LV K N RN I S I
Y S ~V D N P I
m:J S
N N V D N P I
lil
lQJ
G NA R N I NV
W G T TL Y Pf'Äl
W G T T L Y PL!J
WG T T L Y P K
360
359
398
440
439
478
480
479
518
520
519
528
530
LLO
YS NKV DNP I E
529
Fig. 2. Comparison of the deduced amino acid sequences for ILO and LSO with the LLO sequence (Refs. 4. 13). Regions in ILO und/tlf LSO
which are not identical to LLO are boxed. The presumptive signal peptide is indicated hy a broken line. lhe pnsilinn wh.:re LSO Iacks one
positive charge is marked. The additional Ser-33 in LSO is indicatcd by a black dot below the sequence. Phe-489 in LSO and Cys-~119 in ILO by
arrows above.
84.
molysin; Ul streptolysin 0 [15] and also Iisteriolysin 0
(LLOl from L. monocytogenes [14]. Our results show
that lLO contains two cysteine residues, one in the
conserved region and another one 26 amino acids
distal from there (position 509 in Figs. 1 and 2). Although all members of this group of toxins are oxygentabileFandriJtbl~baetivat~d;·-: only,; IL(),,. migbt be able to
f~i.nl,,:·i~tQmöi~~t•r' ··d.i$~l(id~ bonds upon· oxidation. ·
Furtli,~t· sttidl~s~~itf~::tlie. purlfted~ proteiil and ·with the
isolated' lso· and:; ilo g~nes will show the significance of
the differences described here to LLO and to the other
toxins in this group.
We are grateful to W. Goebel for his continuing
interest and support and to K. Brehm for discussions
and technical advice. M. Leimeister-Wächter and T.
Chakraborty are thanked for kindly providing the h/yspecific gene probe, M. Wuenscher for his help in
preparing the manuscript. This work was supported by
grants from the Bundesministerium für Forschung und
Technologie (BMFT 01 Kl 88059) and the Fonds der
Chemischen Industrie (to J.K.). A.H. was recipient of a
fellowship from the Boehringer lngelheim Fonds.
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