Volume 277, number 1,2, 59-64
FEBS 09236
December 1990
Sequence analysis of the potent mitogenic toxin of PasteureZZamultocida Alistair J. Lax’, Neil Chanter’, Gillian D. Pullingerl, Theresa Higgins2, James M. Staddon2 and
Enrique Rozengm+ 1AFRC Institute for Animal Health, Compton, Newbury, Berkshire, RG16 ONN, UK and 21mperial Cancer Research Fund, PO Box 123, Lincoln’s Inn Fields, London, WCZA 3PX, UK
Received 11 October 1990 Pasteurella multocida toxin is a potent mitogen for cultured Swiss 3T3 cells where it causes an accumulation of inosito’l phosphates and activation
of protein kinase C. The gene sequence described here coded for a 146 kDa protein. The ORF was preceded by a ribosome binding site and followed by a stem loop. There was no evidence for a signal sequence. The gene had a low G + C base ratio which differs from the rest of the Pasteurella genome. There was no significant homology with other known proteins, although a motif found in certain bacterial toxins which are ADP-ribosyl transferases is present. A recombinant expressing only part of the PMT gene was not mitogenic. Mitogen; Toxin; Protein kinase C: Pasteure[ia multoc~da
I. INTRODUCTION Proliferation of eukaryotic cells is controlled by complex pathways of signal transduction, which when activated lead to cell division. Some of the components of this process are known for Swiss 3T3 cells, a model system for studying signal transduction [ 11. Understanding the molecular mechanisms controlling signal transduction in eukaryotic cells is of primary importance in explaining cell proliferation and has been greatly aided by the use of bacterial toxins which intervene at different stages in the signal transduction process [2,3]. A toxin produced by some strains of Puste~re~~~ ~u~toc~da can reproduce the pig disease atrophic rhinitis [4,5]. The main signs of disease are loss of the nasal turbinate bones, twisting of the snout and a reduction in weight gain [6]. The toxin has been purified [7-l l] and the gene has been cloned [12-141. Parenteral injection of piglets with recombinant toxin reproduces clinical atrophic rhinitis and causes damage to liver and kidney, and proliferation of cells in the ureter, bladder and turbinate mucosa [12]. We have recently shown that the P. multocida toxin is the most potent mitogen yet identified for cultured fibroblasts [15]. The toxin stimulates DNA synthesis and cell proliferation as effectively as serum in the absence of other synergistic factors [IS]. The toxin stimulates the production of inositol phosphates and increases the phosphorylation of the 80 kDa protein, a prominent substrate for protein kinase C in 3T3 cells [16]; other proteins are phosphorylated independently Correspondence address: A.J. Lax, AFRC Institute for Animal Health, Compton, Newbury, Berkshire, RG16 ONN, UK
of protein kinase C. The toxin does not increase the cellular content of CAMP [t 51. Characterisation of the mode of action of the toxin could assist in discovering new signal transduction pathways involved in mitogenesis because the activation of protein kinase C alone is not sufficient to elicit a mitogenic response [ 11. The sequence of the toxin could identify potentialIy important domains and motifs, and hence greatly aid molecular analysis. Whilst sequencing the mitogenic toxin gene in this laboratory the sequence of toxin genes isolated from P. multocida in other laboratories became available [17-191. Consequently, it was important to ascertain whether these sequences were related to the mitogenic toxin which we have described [ 151. The sequence of the mitogenic toxin gene is analysed here and compared with the other toxin gene sequences; we have also examined the mitogenic activity of one of the previously sequenced toxins [ 171. 2. METHODS AND MATERIALS 2.1. Strains and growth conditions The E. co/i recombinant TOX2 containing plasmid pAJL 13 and the non-toxigenic recombinant containing pAJL14 (121 were grown in L broth [20]. P. multocida ssp multocida 45/78 was obtained from the National Collection of Type Cultures, Centraf Public Health Laboratory, UK (Accession Number NCTC 12178). and was grown on Bacto-Tryptose broth [21] at 37°C with agitation. All bacteria were stored as cell suspensions at - 7O’C in 12% (v/v) glycerol [20]. 2.2. Toxin purification and assays for toxicity and miiogenicity We have previously described the methods used for purification of toxin [S], measurement of toxicity 122,231and assessing DNA synthesis and cell proliferation [ 151. 2.3. Chemicals and biochernicals Restriction and other enzymes were from BRL, Biolabs or Boehr-
Published by Elsevier Science Publishers 3. K (Biomedical Division)
~~45793/~/$3.50
Q 1990 Federation of European Biochemical Societies
59
Volume 277, number 1,2
FEBS LETTERS
December 1990
-
(2.31 CCCTTGACCTAGAGGGGCTTTTTTATTACATCAAAAAAATAAACCCCAAACACTGCGAAT‘TTTGGGGTTTTATTTATAACCAAAATACATTAATATGTT TATTAAGTAAGCATTATCTTACTTTAGGAATAAACTAACAT~TTATGGAT
AAA Lys
ACT
GCC
GAT
Ala
Asp
GAA Glu
ATT Il.
TTT Ph.
A‘A
s*r
AAA Lys
GAA
GTT
Glu
Val
TTT Ph.
ATT Il.
AAT Asn
CGT Arq
TTT Ph.
GGT Gly
ATT 1le
ATT Il.
AAT Asn
GAA
GGG
GGC
‘1~
AAA Lys
CAA
Giy
CTT L.”
GAA
Glu
Gin
Gly
CTG L.”
TCA
TCT
TCT
ser
ser
GAC Asp
AGC
ser
TCC Ser
GAT Asp
GGG ‘ly
AAC GTT A.,, Val
TTT Ph.
TGC
AAA Lys
GGA
vai AAA tys
TG‘ Trp
AAA Lys
GTA
‘ly
S.r
Arq
AGA Arq
AAA Lys
CAT His
ACT Thr
GAT Asp
CAT
AT‘ M.t
CTA L."
GAT Asp
ACT
CCTAAT
Thr
AAAAAT
Lau
Mst
His
CCT
GAC
Pro
ASP
TTT Ph.
100 AAC Asn
TCA S.r
GAT Asp
TTT Ph.
CAA Gln
TTA L."
ARC Asn
AAT
GTA
Asn
Val
Pro
ASn
CCG Pro
AGA Arq
AAG Lys
ATT Il.
GTA Val
GAA GIU
AAC (2) ACT ACA Thr Thr A.i-l
TAT Tyr
TTC Ph.
AAC ATT A.,, Il.
TTT
ATA Il.
OAT
CCT
A.-
Asp
Pro
GAA Glu
CAT His
TAT Tyr
ARC
TCT
TTA
TCA
GAA
L.”
Set’
Glu
Asn Ser
TTT Phc RAG Lys
Lys
TCC S.1
TAT
CGAGTT
Tyr
Arq
Val
ACT Thr
‘AT
GCC
Asp
Ala
ATG
AAT
ATT
GAG GlU
CGT Arg
TAT Tyr
TTT Ph.
‘AT Asp
OAT Asp
TGG Trp
GAC Asp
AAT Asn
s.r
TTA Leu
AAT
L.u
CTA L.u
GCC AGT
Il.
GTG Val
AGC
Asn
TTT Ph.
TTG
Met
Ala
S.r
Asn
GGA Gly
ATA 110
TAT Tyr
TCA Ser
GTA Val
GGA
AAA LyS
GAA Glu
GGA Gly
GCT Al.
TAT Tyr
TAT Tyr
CCT Pro
GAT Asp
CAT His
GAT Asp
TAT Tyr
GGT Gly
CCA Pro
GAA Glu
TAT
AAC
TY~
Asn
CCT Pro
GTT
Gly
Val
TGG Trp
G‘A Gly
CCA Pro
ARC Asn
GAA Glu
CAA ‘In
ATT Il.
TAC Tyr
CAT His
TCT Ser
AGA Arq
GTG Val
ATT 11.
GCA Ala
GAT Asp
ATC Il.
CTT TAT L.,u Tyr
GCT Al.
CGC Arg
TCC Ser
GTA VS3.
TGG Trp
GAT Asp
GAA Glu
TTT Ph.
AAA Lys
AAA tys
TAC Tyr
TTC Ph.
ATG Met
GAG ‘lu
TAT Tyr
TGG Trp
CAA ‘ln
AAA Lys
TAT Tyr
GCT Ala
CAG Gln
CTT L."
TAT Tyr
ACC Thr
GAA ‘lu
ATG Mst
TTA TCT L.-J 5.r
GAT ASP
ACA Thr
TTT Ph.
CTT L."
GCA Ala
ATG
GCT
N.t
Ala
ATT Il.
CAG Gin
CAA TAT ‘lrl Tyr
ACA Thr
CGA Arg
CAA Gln
ACG Thr
CTT Lau
ACT Thr
GAT Asp
GAA Glu
GGC Gly
TTT Ph.
CTT Leu
ATG "at
GTT va1
TGT cys
AAC ACA ABll Thr
TAT Tyr
TAT Tyr
GGC Gly
AAT Asn
AAG
GAA Glu
GAA ‘lu
GTT Val
CAA Gln
ATA Il.
ACT Thr
CTA La"
CTA Leu
GAT Asp
ATC Il.
TAT Tyr
GGA Gly
TAC Tyr
CCT Pro
TCC 5.x
ACT Thr
GAT Asp
ATA Il.
ATT Il.
TGT Cys
ATA Il.
GAG Glu
CAA Gln
AAA
‘GG ‘ly
CTT L."
CCT Pro
ACT Thr
CCT Pr*
AAA GTG l‘ys Val
ATA Ile
CTT L."
TAC Tyr
ATT If.
CCT Pro
GGA ‘ly
GGA Gly
ACA Thr
CAA Gln
CCA Pro
TTT Ph.
GTT Val
GAA Glu
TTT Ph.
CTT L."
AAT Asn
ACA Thr
GAT
GAT
Asp
Asp
ia) CTG L.u
AAA Lys
CAA Gin
TGG Trp
ATT Ile
GCA Ala
T‘G Trp
CAT His
TTA Lsu
AAA Lys
CAT Asp
AAC Asn
AAA Lys
CAT His
ATG Met
‘TC Val
CGA GCA Ala Arq
(2.3) TTC CGC Ph. Arq
AAA Lys
CAT His
TTC Ph.
TC‘ S.r
CTA Lcu
AAA Lys
Lys
ACT Thr
190
340
Ph.
CTA
490
640
790
Lys
CA&
CGT
CAG
GAA
GGA
GAA
ACG
TTT
AAA
GCACTT
CAA
GCA
GAA
GAG
TCC
CCT
GAA
‘1~
Gly
Glu
Thr
Ph.
Ile
Asp
Lya
Al.
L.u
Gin
TAT Tyr
ATT
Gln
GGT Gly
‘AT
Arq
ACA Thr
ATA
Gin
Il.
Al.
Glu
Glu
SOT
Pro
Glu
TGG Trp
CCT Pro
GCC Al.
AAT Asn
AAA Lys
TAC Tyr
ATC Il.
CTT L.u
TAT Tyr
AAT Asn
CCG Pro
ACA Thr
CAT His
TTA Lsu
GAA Glu
ACA Thr
‘AA Glu
AAT Asn
TTA L.u
TTT Ph.
AAC Asn
ATC Il.
ATG Mst
ATG M.t
AAG Lys
CGA Atq
ACA Thr
GAA Glu
CAG Gln
COG Acq
ATG M.t
CTT L.u
GAA Glu
OAT Asp
AGT Ser
GAT Asp
GTA Val
CAG Gln
ATT Ile
AGA Arq
TCA 5.1
AAT TCA Am,, Sbr
GAA Glu
GCT AIA
ACC Thr
COT Arq
GAC Asp
TAT Tyr
‘CT Al.
CTT L."
TCA S.c
TTA L."
CTC L."
GAA GlU
ACC Thr
TTT Ph.
ATT Xl.
TCA S.r
CAG Gln
TTA La"
TCT S.r
GCA Ala
ATA Il.
GAT Asp
ATG M.t
TTA Lou
GTA CCA Vi,1 Pro
GCA Al.
GTA V.1
GGT Gly
ATC Il.
CCA Pro
ATT Il.
AAT Asn
TTT Phe
GCC Ala
CTA LB"
TCA Ser
GCT Al.
ACA Thr
GCA Al.
TTA L.u
GGA Gly
CTT Leu
AGC Sar
TCG S.t
OAT Asp
ATT Il.
GTA V.1
GTT V.1
AAT Aan
GGA Gly
GAT Asp
TCA S.r
TAT Tyr
GAA Glu
AAG Lys
A‘A Arq
AAA Lys
TAT Tyr
GGA Gly
ATT Il.
‘GO Gly
TCC S.r
TTA I."
GTG V.l
CAA Gin
TCT Ser
GCA Ala
TTA L.u
TTC Ph.
RCA Thr
GGA Gly
ATT Il.
AAT A."
CTT L.”
ATT Il.
CCA Pro
GTT V.l
ATT Il.
TCG S.r
GAA ACC ‘1%~ Thr
GCA Ala
GAA Glu
ATT Ile
TTA LB"
TCT S.T
TCT TTC S.t: Ph.
TCT SOT
AGA Arq
ACA Thr
GAA Glu
GAA Glu
GAT Asp
ATT Il.
CCA Pro
GCT Ala
TTT Phe
TTC Ph.
ACT Thr
GAA Glu
GAA Glu
CAA Gin
GCT Ala
TTA L."
GCT Ala
CAA Gin
CGC Arq
TTT Phe
GAA Glu
ATA Il.
GTA GAA '7.1 Glu
GAA Glu
GAA Glu
TTA L.u
CAT His
TCT S.r
ATC Il.
TCA CCT SBS' Pro
GAT Asp
GAT Asp
CCT Pro
CCT Pro
CGA Arq
‘AA Glu
ATT Ile
ACT Thr
GAC Asp
GAA Glu
AAT Asn
TTA Leu
CAT His
AAA Lys
ATT Il.
CGT Arq
CTG L.u
GTA Vel
CGT Arq
CTT Leu
AAC Asn
RAT Asn
GAA Glu
RAT Asn
CAA Gln
CCT Pro
TTA Leu
GTT Val
GTG Val
TTA Lou
CGA Arq
AGA Arq
TTA Leu
GGA Gly
“A Gly
AAT Asn
AAA Lys
TTT Ph.
ATC Il.
AGA Arq
ATC Il.
GAG Glu
CCT Pro
ATA Il.
ACA Thr
TTC Ph.
CAG ‘ln
GAA ‘1~
ATA Il.
AAA Lys
GGT ‘ly
TCT 5.r
TTA Lo"
GTA Val
A‘T S.r
‘AA Glu
GTT Val
ATA Il.
AAT Aen
CCA Pro
GTG Vei
ACT Thr
AAT AAA As,, Lys
ACG Thr
TAC Tyr
TAC Tyr
GTA "al
AGC S.r
AAT GCT As,, Ala
AAA Lys
CTA Lo"
TTA Leu
GGG Gly
GGC Gly
TCT Ser
CCT Pro
TAT Tyr
AGT S.r
CCT Pro
TTC Ph.
CGT Arq
ATT Il.
GGA Gly
TTA L."
GAA Glu
GGT Gly
GTT V.1
TGG Trp
ACA Thr
CCA Pro
GAG Gl"
GTA V.1
TTA LB"
AAA Lya
GCA Ala
AGA Arq
GCT Ala
TCC S.r
GTT V.1
ATT Ila
GGA Gly
AAG Lys
CCT Pro
ATT Il.
GGA Gly
GAA Glu
TCA SOT
TAT Tyr
AAA LyS
AGA Arq
ATA Il.
TTA Leu
GCC Ala
AAA Lyr
CTA Lsu
CAR Gln
AGA Arq
ATA Il.
CAT
AAC
AGT
AAT
ATC
His
Aen
Ser
Asn
Il.
TTA Lcu
‘AT Asp
GAG Glu
CGA Arq
CAR Gin
GGT Gly
TTA Leu
ATG Met
CAT His
GAA Glu
CTC Leu
ATG Met
GAG Glu
CTT Leu
ATT Il.
OAT Asp
CTT Leu
TAT Tyr
GAA Gl"
GAA Glu
TCG 5.r
CAA Gin
CCT Pro
TCT S.r
TCA 5.r
GAG Glu
CGT Arq
TTG Lb"
AAT Asn
GCT Al.
TTT Ph.
CGT Arq
GAA Glu
CTG L.u
CGT Arq
ACT Thr
CAR Gin
TTA Leu
GAA Glu
AAA Lys
GCG Ala
CTT L.u
TAT Tyr
CTT LB"
CCT Pro
GAA Glu
ATG M.t
GAA Glu
GCA Ala
TTA LB"
AAA Lys
AAA Lys
CAA ‘ln
ATA Il.
CTA L."
CAG ‘ln
ATT 11s
CCT Pro
AAC Asn
AAA Lys
GOT ‘ly
940
1090
1240
1390
is40
1.590
1840
1990
2140
of the toxin gene PMTI. (a) marks the end of the insert in pAJL14. Differences between PMTI and other sequences by bases above the PMTl sequence and amino acid changes below. The sequences are: 2 1171; 3 [IX]; 4 [19].
and were used according to the manufacturer’s chemicals were from BDH.
specifications.
2.4. Sequencing strategy The insert in pAJL13 was isolated by digestion with HpaII followed by gel electrophoresis and elution onto silica gel (Geneclean, Statech).
60
ACA Thr
TGT Cys
TTG
CAGATG Gln
AAA Lyr
Cys Leu
Fig. 1. Sequence
inger, Other
ATG Met
are shown
It was digested with either A/u1 or SuuIIIA and ligated into M13mp18 cut with either SmaI or BumHI respectively (prior to ligation the cut vector was treated with phosphatase). DNA from isolated phage containing
inserts
was
purified
and
sequenced
using
an
Applied
Biosystems sequencer. This strategy produced about IO discrete short lengths of sequence. Since these sequences closely matched parts of
Volume
277, number
December
FEBS LETTERS
1,2
TCT
GGT
FCC
GCT
CGA
TTT
CGT
ACA
GCC
AAA
LOU
Arg
Thr
Ala
Glu
Ala
Gly
Lys
AGT Ser
AGC
Phe
ACC Thr
GAA
Arg
ATG Mot
GGA
Ala
AAT ASn
GCT
Ala
ATG Met
GAA
Gly
TTA LOU
CTT
SOT
Glu
S-r
ACG Thr
GCT Ala
GAT Asp
TTA Leu
ATA 110
CGC Arg
TTT Ph.
GCC Ala
TTG Lou
CAA Gin
GAT Asp
ACA Thr
GTA Val
ATT 110
TCA Ser
GCG Ala
CCT Pro
TTT Ph*
CGC Arg
GGA Gly
TAT Tyr
GCT Ala
GGT Gly
GCG Ala
ATT II*
CCA Pro
GAG Glu
GCA Ala
ATA Ile
GAC Asp
TTT Phe
CCT Pro
GTA Val
AAA Lya
TAT Tyr
GTA Val
ATA 11.
GAA Glu
GAC Asp
ATA 110
TCT 5.r
GTA Val
TTT Pho
GAT Asp
AAA Lys
ATA 110
CAG Gln
ACA Thr
AAT As”
TAC Tyr
TOG Trp
GAA Glu
CTT Lcu
CCT Pro
GCT Ala
TAT Tyr
GAA Glu
AGC Ser
TGG Tq,
AAC Asn
GAA Glu
GGA Gly
AGT Ser
AAT Asn
AGC Ser
CGA GCA Ale Arg
12.31 TTA CTG Lou Lsu
CCT
GGT Gly
TTG LeU
TTA CGT LOU Arg
GAA Glu
TCG Ser
CAA Gln
AGC Set
AAG Lys
GGC
ATG
TTA
AGT
AAG
GAA
AAT
TTT
TAC
AGC
Lys
Arg
11e
Glu
TAT GAA
ATG
scr
Asn
AGC Ser
GAA
I&U
ATC Ile
ATA
Met
TGT Cys
COT
Gly
Tyr
Glu
Glu
Met
Phe
Tyr
Ser
ATT Ile
TCT Ser
CCA
Pro
TAT Tyr
TCA Ser
AAC Asn
Gln
GTT Val
GGA Gly
GGG Gly
CCT Pro
GTA V&l
CAA Gln
GGT Gly
OAT Asp
TTA Leu
GGA Gly
TTT Phe
GAG Glu
CAG Gln
GCC Ala
TCC
TTA
ATG
GAA
AAT
Ser
Leu
Met
01”
Asn
AAT As,,
TAT Tyr
GAT Asp
GAA Glu
GCA Ala
TTC Phe
CAA Gin
GAA Glu
ATT Ile
TGG Trp
Pro
CTTTAT
ATT
GGA
CAT
AGC
Leu
Tyr
110
Gly
His
Ser
TAT Tyr
GAA Glu
TTA Leu
TAT Tyr
CCT Pro
TTC Phe
ACT Thr
TTT Ph.
TTC Ph.
AGT Ser
ATG Met
CTT mu
CAA Gln
GAA Glu
TTT Phe
GCC Ala
ACA Thr
CGT Arg
AAC Asn
A (4) TTT TTC Phm Pha TY~
AAT Asn
ACT Thr
CTT Lsu
GTT Val
TCT S.r
GAT Asp
CGA Arg
CTA Leu
ACGATG TTA CTTACA
GAA
AGT
Mat
Lcu
LQ”
Thr
Glu
Scr
TTT Phe
GAT Asp
TAT Tyr
ACA
Thr
CAA Gln
TTT Phs
GCT Ala
GCA Ala
ATG Met
TCT Sar
ATT Ile
AAT Asn
GAA Glu
CGC Arg
ATA 11s
AAC Asn
TCT Ser
TCA Sex
AAA Lys
AGT Ser
ATT Ilc
GAC Asp
TTT Phe
TTC Phe
CTA Lou
AAT Asn
GCT Ala
ATA 11s
TCT Ser
GAT Asp
TTA Leu
CCG Pro
TAT Tyr
GAT Asp
ATT Ile
GTC Vsl
CAA Gin
AAA Lys
ACA Thr
CTT LB”
GAT Asp
GCC Ala
ATG Mat
TTA Le”
TTT Phe
AAC Asn
TTA LBU
GAT Asp
TCA Ser
GCA Ala
AAA
Lys
GAT Asp
AAT Asn
AAT Asn
ATA 110
AAA Lys
TTT Phc
AGA Arg
GCT Ala
TAT Tyr
AAG Lys
TCT Ser
TTA Leu
TAT Tyr
TAT Tyr
AAA Lys
GGA Gly
AAT Asn
ATA 11s
GTT Val
GTA Val
TTT Phe
GCT Ala
GAT Asp
AGT Ser
TCT Ser
CTG Le”
CTC Le”
TTA Leu
AAA Lys
ATA Ile
CCA Pro
GCA Ala
ACT Thr
GTA Val
GTA Val
CCT Pro
ATT Ile
GAA Glu
GTT Vsl
ACA Thr
GAG Glu
TTA Leu
TTT Phe
AAG Lys
TTA Leu
ACG Thr
GCA Ala
GGT Gly
AGG Arg
GAA Glu
GAA Glu
TTA L.U
ATG Mot
GGC Gly
TCT Sar
TGT
GAT
TCA
Cys
Asp
TCT Ser
ATT Ila
ACC Thr
GCT Ala
GGT Gly
CTA Leu AAA Lys
CAG
CCTTGGGAT
TAT
Trp
Asp
GCT Ala
ATT
Pro
Ile
Tyr
GGA Gly
CAT Asp
ATT Ile
GAA Glu
AAA LyS
TGT Cys
ATG Met
AAT Asn
ACC Thr
TAT Tyr
AGA Arg
GGT Gly
GTG Val
AAT Asn
CTA LeU
ACC Thr
ACA Thr
TTC Phs
ATT 11s
GAT Asp
AAT Asn
GGA Gly
CTA Leu
ACC Thr
CAA Gln
GAA Glo
ATC Ilc
TCT Ser
CAA Gln
TTC Phe
TTA LFU
CAA Gin
GGA Gly
AGT SeI:
AAT Asn
GAA Glu
AAA LYS
GGA Gly
GAT Asp
ATT Ile
AAT Asn
GGT Gly
GCT Ala
TTC Phe
AGA Arg
AAG Lys
CTT LOU
CTG Le”
CAA Gln
ATA 110
GGG Gly
CAT His
TCA Ser
CAT Asp
ART Asn
TCT Ser
GTT Val
CCG Pro
CCA Pro
TTT Phe
AAT Asn
AAC Asn
CCT Pro
ATA 110
GCT Ala
GAA Glu
GCA Ala
ATT Ilr
GAA Glu
AAA Lys
CTA LOU
GAT Asp
CGA Arg
GAA Glu
GGT Gly
CAA Gln
AAA Lya
TTT Phs
AAC As,,
AGC Ser
ACG Thr
CCT Pro
GGG ACA Gly Thr
GGT Gly
CGT Arq
CCT Pro
ATG Met
CCA Pro
GGA Gly
CTA L*u
GTT Val
CAR Gin
TAT Tyr
GAT Asp
AGC Ser
GAT Asp
GOT Gly
GCA Ala
TGG Trp
CAA Gin
TTT Ph.
CTT La”
CCA Pro
GAT Asp
GTA Val
GCT Ala
TCA Sar
AGC Sar
AGA Arg
GTT Val
GAA Glu
AAT A5n
TGG Trp
CAA Gln
GTC Val
TTA LB”
ACT Thr
CCT Pro
CCA Pro
CAA Gln
GGT Gly
AAG Lys
ATT Ile
CTT Leu
GGA Gly
TTA Le,,
AAG Lys
CAA Gln
TTT Phs
CCA
ACA Thr
GAA Glu
CAA Gln
AGT Ser
CGC Arg
TTA Ls”
CCT
CTT
TTA
GAG
Pro
Lou
Leau Glu
AAT Asn
TCG Sar
GTT Val
TCT Ser
GAA Glu
CAT Asp
TTA Lau
CAA Gin
AAG
ATT
LyS
110
GAT Asp
GCA ADA
ATA II-
AAA Lya
AAT As”
GAT Asp
GTG V11
AAA Lys
ATG Mat
AAT As,,
AGT Ser
TTA L.u
GTG Val
TGT Cys
ATG ,,.t
GAA Glu
GCT Ale
SOT
GTA Val
AGC SOK
CCT Pro
AAG LYS
GTA Val
GCT Ala
GCC Ala
CGT Arg
CTT Leu
AAA Lys
GAT Asp
ATG “et
GGG Gly
TTA GAA LOU Glu
GCT Ala
GGG Gly
ATG “at
G‘T Gly
GCT Ala
TGG Trp
TGG Trp
AGA Arg
CGT Arg
GAA Glu
GGC Gly
GGG ATG Gly net
GAA Glu
TTT Phe
TCA Ser
CAT His
CAG Gln
ATG net
CAT His
ACT Thr
ACT Thr
GCT Ala
TCC ser
TTT Phs
AAA Lys
TTT Phe
AAA
GAG
TTT
GCC
GTG
GAT
GCT
TCA
CAT
TTA
CAA
TTT
GTA
CAC
GAC
CAA
TTA
OAT
ACA
ACT
ATC
CTG
Lys
Glu
Phe
Ala
~a1
Asp
Ala
Ser
His
Leu
Gin
Phe
Val
His
Asp
Gin
Leu
Asp
Thr
Thr
Ile
LPU
ATA Ile
CCT Pro
GTA Val
GAT Asp
GAT Asp
TGG Trp
GCT Ala
TTA Leu
GAA Glu
ATA Ile
GCT Ala
CAA Gln
AGA Arg
AAT Asn
CGG GCT Arg Ala
ATT Ile
AAT As,,
CCT Pro
TTT Phe
GTG Val
GAA Glu
TAT Tyr
GTT Val
AGT Ser
ACA Thr
GGA Gly
AAC AS”
ATG Met
TTA LBU
CTC LOU
TTC Phe
ATG Met
CCT Pro
CCT Pro
CTT Lou
TTC Pha
ACA Thr
CCT Pro
CGC Arg
TTA LB”
ACA Thr
AGA Arg
GCA Ala
CTA LB”
TAA
CTA
Ala
GCA
Pro
Thr
AAG Lys
ATTAAAAACTGTATTAAAGCCTTATATTATAAGGCTTTAATTTTCTTTCAAGAATTATTAAGTAGAAGAATCAAAATCAATGAGATAGATAAAATCAAAT
1990
2290
2440
2590
2740
1890
3040
3190
3340
3490
3640
3790
3940
l
4115
GTTATTACCAATACAACTTTCTTAAGTATACTTTTTGAATTTTTTGCGTTAATAAATTTATAATACCCTTAACTCAATAAAAGAAGTTATTGAGAAGTTT
AAATCTTGTGAGCAAGATGAAGATATAATTTCAGCAATCGATCTTATTAGCGCTTCATATAGAAGGGCTGTGGATGCAGTGGAACAAAGATTCGGTTCTAG
: Shine
Dalgarno
ribosoir
binding
site
_:
4316
terminator
Fig. I contj~~ed
the P. multocidu toxin sequence which had become available [17], a differnt strategy was adopted to obtain the full sequence of the mitogenic toxin. Oligonucleotides based on the available sequence [17] were synthesised on an Applied Biosystems 381A machine at intervals of about 200 bases along the gene and these were used for double-stranded sequencing using the Sequenase kit according to the manufacturer‘s instructions. For reasons of safety, most of the sequencing was done using a non-toxigenic derivative of the gene (pAJL14) which was created by removal of a 394 bp fragment by digestion with NdeI and subsequent religation. Removal of the NdeI fragment induces a frameshift
mutation, resulting in a non-toxigenic construct (see below). Plasmid pAJL13 was used to obtain the sequence of this fragment, but in a containment laboratory. Sequence analysis was carried out using UWGCG programs.
3. RESULTS AND DISCUSSION 3.1, Sequence of the mifogenic toxin The sequence (Fig. 1) contained an open reading frame (ORF) starting at base 155, which encode a pro61
Volume 277, number 1,2
FEBS LETTERS
tein of 1285 amino acids which would have a molecular mass of 146 kDa (Fig. 1). This is in accord with the apparent molecular mass of the mitogenic toxin as estimated by SDS-PAGE. The most likely start is the methionine shown, since the deduced amino acid sequence from this point is identical to the N-terminal amino acid sequence of PMTI [IS] except for one residue. The difference is likely to be due to experimental error in amino acid sequencing. It has been claimed that the N-terminal was blocked [ 17,181. The difference may be related to the source of the toxin since one was purified from recombinant E. coli (unblocked) and the other from P. multocida (blocked). A ribosome binding site is immediateIy upstream of the start (Fig. I), but no obvious E. coli consensus promoter sequence is present. Petersen [ 181 has presented convincing evidence for the location of the promoter region. The gene is expressed well in E. coli [12], but since little is known about Pasteurella gene organisation, it is not clear whether promoter sequences which function in E. co/i do so in P. m~Ito~ida. There were other methionine residues downstream from the presumed start. Although none had a good consensus with a ribosome binding site it is possible that some of the minor polypeptides produced by the recombinant which are antigenically related to the toxin [12] might have resulted from initiation at these residues. There was a stem loop structure typical of a terminator region starting at base 4027. There was no evidence for a signal sequence at any of the potential start codons, which agrees with the lack of secretion from either the native P. multocida or the recombinant E. coli. A hydrophobicity plot (Fig. 2) indicated that the protein might have several domains; in particular there was a hydrophobic region between amino acids 380 and 470 which was bounded by two hydrophilic domains, and the C terminal of the protein Experiments with the was also hydrophobic. lysosomotrophic agent methylamine, which blocks entry of certain toxins 121,and neutralisation of toxin ac-
December 1990
tion by antibody when added early but not late to toxin treated cells both indicate that the toxin binds to the cell surface, is internalised and subjected to processing in endosomal/lysosomal compartments [ 151, from which it presumably exits to exert its biological effects. The hydrophobic regions revealed in Fig. 2 are likely to play a role in the interaction of the toxin with various cellular membranes. The DNA and deduced amino acid sequences were compared with the sequences on the EMBL, Genbank and Swissprot data bases, but no significant homology was found. The motif His-Glu-Trp which is common to several toxins which ADP-ribosylate their substrate [24] was found near the N-terminus of the toxin (Fig. 3). The spacing between the amino acids matched precisely the His-Glu-Trp motif found in the ADP-ribosylating toxins (Fig. 3). Amino acid motifs thought to indicate the NAD and protein substrate binding site in the ADP ribosylating toxins [25] were not present in this toxin. It should be noted that the His-Glu-Trp motif was also found in a variety of proteins that are not thought to ADP-ribosylate any substrates (unpublished). As predicted from the restriction map [12] the gene had a low G + C content (35%). Interestingly, the G + C content of the Pasteurella genome differs markedly from that of the toxin gene, so it is possible that the gene has been acquired relatively recently and that there might exist a family of closely related mitogenic toxin genes in other bacteria. Indeed, recent reports indicate that the toxin gene is flanked by phage elements [17], may be carried on a prophage [26] and that some Salmonella isolates contain part of the Paste~rel~atoxin gene [27]. 3.2. Mitogenicity of P. multocida toxin from another isolate The sequence (hereafter caIled PMTl) was compared to the other P. m~~tocida toxin sequences available (PMT2 [17]; PMT3 [lS]; PMT4 [19]). There were 4 differences within the open reading frames of the genes, and in each case the PMTl sequence was checked
P. mltocida
His 29
xii
Glu 155
I
TV 160
His 426
jr’6
Glu 553
4
TrP 558
“15 21
116
Glu 148
(I
T[P 153
sordetella mrtussis Sl
His 83
116
GlU 210
4
=P 215
Vibrm cholerae CT
His 44
I*S
Glu 170
?
TIP 174
Escherichia cali LTH
His 44
125
Asp
I
TKP 174
toxin
Pseudomonasaeruqinosa ETA Corvnebacteriumdititheriae
DT
170
Comparison of the His-Glu-Trp motif in PMTl with other taxins. The data for the other toxins are taken from 1241.The numbers
Fig. 3.
Fig. 2. Hydrophobicity plot of the PMTI protein, catculated using a program written by Dr M.E.G. Boursnell. Positive numbers indicate hydrophobicity.
62
between the amino acids indicate the spacing between them; the numbers below indicate the position in each protein of the respective amino acid.
December 1990
FEBSLETTERS
Volume 277, number 1,2
100
600 act
PMT (nglml)
Antiserum (d/ml)
Days
Protein (ng/ml)
Fig. 4. Comparison of the mitogenic activity of PMTl and PMT2. (A) Dose-response curve for the stimulation of DNA synthesis by PMTl (0) or PMT2 (a). Cultures of Swiss 3T3 cells were incubated with PMT and DNA synthesis measured as described [15]. Each point is the mean of two determinations: 10% foetal bovine serum gave a level of incorporation of 704 x lo3 cpm. (B) PMT2 stimulates reinitiation of growth in confluent Swiss 3T3 cells. PMT2 was added directly to the medium of Swiss 3T3 cell cultures 6 days after plating to give a final concentration of 10 ng/ml. 2, 3 and 4 days after this addition, cells from both treated (hatched bars) and untreated (open bars) cultures were trypsinised and counted using a Coulter counter. The values shown are mean k SE (n = 5). (C) Dose-response for the inhibition by PMT antiserum (polyclonal 34B) of DNA synthesis induced by PMTl (0) or PMT2 (0). Various concentrations of immune serum were incubated with toxin and added to confluent quiescent Swiss 3T3 cells as described [15]. Each point is the mean of two determinations: 10% foetal bovine serum gave a level of incorporation of 979 x 10’ cpm. (D) Dose-response curve for the stimulation of DNA synthesis by extracts of E. coli transformed with various constructs. Swiss 3T3 cells were treated as described (151with various concentrations of HBlOl + pAJLl4 ( q), HBlOl + pAT153 (4) or HBlOl + pAJLl2 ( n). Each point is the mean of two determinations: 10% foetal bovine serum gave a level of incorporation of 672 x 10’ cpm.
carefully for errors. There was a single base change between the PMTl and the PMT4 sequences (at base position 2712) which resulted in a conservative amino acid substitution. There were 2 differences between the PMTl gene and that of the gene for PMT3, at base positions 1064 and 2477; both resulted in a change from an alanine in PMTl to an arginine residue in PMT3. There was one other difference between the sequences of PMTl and PMT2 at base position 396. These minor differences among the sequences might be due to errors in sequencing, or reflect variation among isolates. If the latter were true, it was important to investigate whether the potent mitogenicity of the protein was affected. PMT2, purified from P. multocida NCTC12178, and PMTl stimulated DNA synthesis with an identical dose-response relationship in Swiss 3T3 cells (Fig. 4A); there was a similar toxininduced increase in cell proliferation (Fig. 4B). The mitogenic activity of each toxin was completely blocked by antibody to PMTl in a concentration dependent manner (Fig. 4C).
One of the recombinants produced in the original clone bank contained part of the gene, but did not produce toxin active on EBL cells [12]. This recombinant produced polypeptide fragments which reacted with antibody to the toxin. Restriction enzyme analysis and the DNA sequence of this plasmid showed that it contained the N-terminus of the gene, and should code for amino acids l-287 to produce a 33.5 kDa peptide (Fig. 1). A crude preparation from this recombinant was unable to induce DNA synthesis (Fig. 4D). 4. CONCLUSIONS We report the complete sequence of the mitogenic toxin PMTl. The deduced protein sequence did not show any significant homology to known sequences with the possible exception of a motif found in toxins which ADP-ribosylate their substrate. There appear to be two hydrophobic domains in the toxin, which could play a role in the interaction of the toxin with cellular membranes. The sequence was almost identical to other 63
Volume 211, number 1,2
FEBS LETTERS
P. muftocida toxin sequences [ 17-191, and the toxin purified from one of these latter strains was as mitogenic as PMTI in Swiss 3T3 cells. It has been suggested that other bacterial toxin genes originally had a eukaryotic origin, and this might also be the case for the P. multocida toxin. The toxin might then be expected to have an as yet undiscovered eukaryotic homologue involved in signal transduction. It is to be hoped that analysis of the toxin action will not only explain its role in atrophic rhinitis, wlrere it has specific effects on bone morphogenesis, but also yield important information about new signal transduction pathways. Acknowfe~ge~en~s: We wish to thank Dr Ian Goldsmith and Karen Mawditt for synthesis of oligonucleotides and Fara Khurshid for automated DNA sequencing.
REFERENCES 111Rozengurt, E. (1986) Science 234, 161-166. 121Middlebrook, J.L. and Dorland, R.B. (1984) Microbial. Rev. 48, 199-221. [31 Pfeuffer, T. and Helmreich, E.J.M. (1988) Curr. Topics Cell Regul. 29, 129-216. [41 Il’ina, Z.M. and Zasukhin, M.I. (1975) Sbornik nauchnykh rabot, Sibirskii nauchno-Issledovatei’skii Veterinarnyi Institut, Omsk 25, 76-86. VI Rutter, J.M. and Mackenzie, A. (1984) Vet. Record 114, 89-90. El Switzer, W.P. and Farrington, D.O. (1975) in: Diseases of Swine, 4th edn (Dunne, H.W. and Leman, A.D. eds) pp. 687-7 11, Iowa State University Press, Ames. [71 Nakai, T., Sawata, A., Tsuji, M., Samejima, Y. and Kume, K. (1984) Infect. lmmun. 46, 429-434.
64
December 1990
PI Chanter, N., Rutter, J.M. and Mackenzie, A. (1986) J. Gen. Microbial, 132, 1089-1097. PI Rimfer, R.B. and Brogden, K.A. (1986) Am. J. Vet. Res. 47, 730-737. 1101 Foged, N.T. (1988) Infect. fmmun. 56, 1901-1906. [Ill Kamp, E.M., Van der Heijdan, P.J. and Tetenburg, B.J. (1987) Vet. Microbial. 13, 235-248. 1121Lax, A.J. andchanter, N. (1990) J. Gen. Microbial. 136, 81-87. [I31 Petersen, S.K. and Foged, N.T. (1989) Infect. Immun. 57, 3907-3913. [I41 Kamps, A.M.I.E., Kamp, E.M. and Smits, M.A. (1990) FEBS Microbial. Lett. 67, 187-190. 1151 Rozengurt, E., Higgins, T., Chanter, N., Lax, A.J. and Staddon, J.M. (1990). Proc. Natl. Acad. Sci. USA 87, 123-127. 1161 Staddon, J.M., Chanter, N., Lax, A.J., Higgins, T.E. and Rozengurt, E. (1990) J. Biol. Chem. 265, 11841-I 1848. [I71 Patent Application (1989) a Pasteurefta vaccine. VW Petersen, SK. f1990) Mol. Microbial. 4, 821-830. Kamp, E.M. 1191 Buys, W.E.C.M., Smith, H.E., Kamps,A.M.l.E., and Smits, M.A. (1990) Nucleic Acids Res. 18, 2815-2816. WI Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Cold Spring Harbor, Cold Spring Harbor Laboratory, New York. Pll Jones, P.W. and Matthews, P.R.J. (1975) J. Hyg. 74, 57-64. LQI Rutter, J.M. and Luther, P.D. (1964) Vet. Record, 114, 393-396. 1231 Chanter, N., Rutter, J.M. and Rutter, P.D. (1986) Vet. Record, 119, 629-630. [x41 Wozniak, D.J., Hsu, L.-Y. and Galloway, D.R. (1988) Proc. Natl. Acad. Sci. USA 85, 8880-8884. WI Gill, D.M. (1988) in: Bacterial Protein Toxins, Zbl. Bakt. Suppi., vo1. 17 (Fehrenbach, F.J., Alouf, J.E., Faimagne, P., Goebel, W., Jeljaszewicz, J., Jurgens, D. and Rappuoli, F. eds) pp. 315-323. WI Andresen, L.O., Petersen, SK., Christiansen, C. and Nielsen, J.P. (1990) in: Proceedings of the 1Ith Congres of the International Pig Veterinary Society, ~60. ~271 Kamps, A.M.I.E., Buys, W.E.C.M., Kamp, E.M. and Smits, M.A. (1990) J. Clin. Microbial. 28, 1858-1861.
FEBS 09236
December 1990
Sequence analysis of the potent mitogenic toxin of PasteureZZamultocida Alistair J. Lax’, Neil Chanter’, Gillian D. Pullingerl, Theresa Higgins2, James M. Staddon2 and
Enrique Rozengm+ 1AFRC Institute for Animal Health, Compton, Newbury, Berkshire, RG16 ONN, UK and 21mperial Cancer Research Fund, PO Box 123, Lincoln’s Inn Fields, London, WCZA 3PX, UK
Received 11 October 1990 Pasteurella multocida toxin is a potent mitogen for cultured Swiss 3T3 cells where it causes an accumulation of inosito’l phosphates and activation
of protein kinase C. The gene sequence described here coded for a 146 kDa protein. The ORF was preceded by a ribosome binding site and followed by a stem loop. There was no evidence for a signal sequence. The gene had a low G + C base ratio which differs from the rest of the Pasteurella genome. There was no significant homology with other known proteins, although a motif found in certain bacterial toxins which are ADP-ribosyl transferases is present. A recombinant expressing only part of the PMT gene was not mitogenic. Mitogen; Toxin; Protein kinase C: Pasteure[ia multoc~da
I. INTRODUCTION Proliferation of eukaryotic cells is controlled by complex pathways of signal transduction, which when activated lead to cell division. Some of the components of this process are known for Swiss 3T3 cells, a model system for studying signal transduction [ 11. Understanding the molecular mechanisms controlling signal transduction in eukaryotic cells is of primary importance in explaining cell proliferation and has been greatly aided by the use of bacterial toxins which intervene at different stages in the signal transduction process [2,3]. A toxin produced by some strains of Puste~re~~~ ~u~toc~da can reproduce the pig disease atrophic rhinitis [4,5]. The main signs of disease are loss of the nasal turbinate bones, twisting of the snout and a reduction in weight gain [6]. The toxin has been purified [7-l l] and the gene has been cloned [12-141. Parenteral injection of piglets with recombinant toxin reproduces clinical atrophic rhinitis and causes damage to liver and kidney, and proliferation of cells in the ureter, bladder and turbinate mucosa [12]. We have recently shown that the P. multocida toxin is the most potent mitogen yet identified for cultured fibroblasts [15]. The toxin stimulates DNA synthesis and cell proliferation as effectively as serum in the absence of other synergistic factors [IS]. The toxin stimulates the production of inositol phosphates and increases the phosphorylation of the 80 kDa protein, a prominent substrate for protein kinase C in 3T3 cells [16]; other proteins are phosphorylated independently Correspondence address: A.J. Lax, AFRC Institute for Animal Health, Compton, Newbury, Berkshire, RG16 ONN, UK
of protein kinase C. The toxin does not increase the cellular content of CAMP [t 51. Characterisation of the mode of action of the toxin could assist in discovering new signal transduction pathways involved in mitogenesis because the activation of protein kinase C alone is not sufficient to elicit a mitogenic response [ 11. The sequence of the toxin could identify potentialIy important domains and motifs, and hence greatly aid molecular analysis. Whilst sequencing the mitogenic toxin gene in this laboratory the sequence of toxin genes isolated from P. multocida in other laboratories became available [17-191. Consequently, it was important to ascertain whether these sequences were related to the mitogenic toxin which we have described [ 151. The sequence of the mitogenic toxin gene is analysed here and compared with the other toxin gene sequences; we have also examined the mitogenic activity of one of the previously sequenced toxins [ 171. 2. METHODS AND MATERIALS 2.1. Strains and growth conditions The E. co/i recombinant TOX2 containing plasmid pAJL 13 and the non-toxigenic recombinant containing pAJL14 (121 were grown in L broth [20]. P. multocida ssp multocida 45/78 was obtained from the National Collection of Type Cultures, Centraf Public Health Laboratory, UK (Accession Number NCTC 12178). and was grown on Bacto-Tryptose broth [21] at 37°C with agitation. All bacteria were stored as cell suspensions at - 7O’C in 12% (v/v) glycerol [20]. 2.2. Toxin purification and assays for toxicity and miiogenicity We have previously described the methods used for purification of toxin [S], measurement of toxicity 122,231and assessing DNA synthesis and cell proliferation [ 151. 2.3. Chemicals and biochernicals Restriction and other enzymes were from BRL, Biolabs or Boehr-
Published by Elsevier Science Publishers 3. K (Biomedical Division)
~~45793/~/$3.50
Q 1990 Federation of European Biochemical Societies
59
Volume 277, number 1,2
FEBS LETTERS
December 1990
-
(2.31 CCCTTGACCTAGAGGGGCTTTTTTATTACATCAAAAAAATAAACCCCAAACACTGCGAAT‘TTTGGGGTTTTATTTATAACCAAAATACATTAATATGTT TATTAAGTAAGCATTATCTTACTTTAGGAATAAACTAACAT~TTATGGAT
AAA Lys
ACT
GCC
GAT
Ala
Asp
GAA Glu
ATT Il.
TTT Ph.
A‘A
s*r
AAA Lys
GAA
GTT
Glu
Val
TTT Ph.
ATT Il.
AAT Asn
CGT Arq
TTT Ph.
GGT Gly
ATT 1le
ATT Il.
AAT Asn
GAA
GGG
GGC
‘1~
AAA Lys
CAA
Giy
CTT L.”
GAA
Glu
Gin
Gly
CTG L.”
TCA
TCT
TCT
ser
ser
GAC Asp
AGC
ser
TCC Ser
GAT Asp
GGG ‘ly
AAC GTT A.,, Val
TTT Ph.
TGC
AAA Lys
GGA
vai AAA tys
TG‘ Trp
AAA Lys
GTA
‘ly
S.r
Arq
AGA Arq
AAA Lys
CAT His
ACT Thr
GAT Asp
CAT
AT‘ M.t
CTA L."
GAT Asp
ACT
CCTAAT
Thr
AAAAAT
Lau
Mst
His
CCT
GAC
Pro
ASP
TTT Ph.
100 AAC Asn
TCA S.r
GAT Asp
TTT Ph.
CAA Gln
TTA L."
ARC Asn
AAT
GTA
Asn
Val
Pro
ASn
CCG Pro
AGA Arq
AAG Lys
ATT Il.
GTA Val
GAA GIU
AAC (2) ACT ACA Thr Thr A.i-l
TAT Tyr
TTC Ph.
AAC ATT A.,, Il.
TTT
ATA Il.
OAT
CCT
A.-
Asp
Pro
GAA Glu
CAT His
TAT Tyr
ARC
TCT
TTA
TCA
GAA
L.”
Set’
Glu
Asn Ser
TTT Phc RAG Lys
Lys
TCC S.1
TAT
CGAGTT
Tyr
Arq
Val
ACT Thr
‘AT
GCC
Asp
Ala
ATG
AAT
ATT
GAG GlU
CGT Arg
TAT Tyr
TTT Ph.
‘AT Asp
OAT Asp
TGG Trp
GAC Asp
AAT Asn
s.r
TTA Leu
AAT
L.u
CTA L.u
GCC AGT
Il.
GTG Val
AGC
Asn
TTT Ph.
TTG
Met
Ala
S.r
Asn
GGA Gly
ATA 110
TAT Tyr
TCA Ser
GTA Val
GGA
AAA LyS
GAA Glu
GGA Gly
GCT Al.
TAT Tyr
TAT Tyr
CCT Pro
GAT Asp
CAT His
GAT Asp
TAT Tyr
GGT Gly
CCA Pro
GAA Glu
TAT
AAC
TY~
Asn
CCT Pro
GTT
Gly
Val
TGG Trp
G‘A Gly
CCA Pro
ARC Asn
GAA Glu
CAA ‘In
ATT Il.
TAC Tyr
CAT His
TCT Ser
AGA Arq
GTG Val
ATT 11.
GCA Ala
GAT Asp
ATC Il.
CTT TAT L.,u Tyr
GCT Al.
CGC Arg
TCC Ser
GTA VS3.
TGG Trp
GAT Asp
GAA Glu
TTT Ph.
AAA Lys
AAA tys
TAC Tyr
TTC Ph.
ATG Met
GAG ‘lu
TAT Tyr
TGG Trp
CAA ‘ln
AAA Lys
TAT Tyr
GCT Ala
CAG Gln
CTT L."
TAT Tyr
ACC Thr
GAA ‘lu
ATG Mst
TTA TCT L.-J 5.r
GAT ASP
ACA Thr
TTT Ph.
CTT L."
GCA Ala
ATG
GCT
N.t
Ala
ATT Il.
CAG Gin
CAA TAT ‘lrl Tyr
ACA Thr
CGA Arg
CAA Gln
ACG Thr
CTT Lau
ACT Thr
GAT Asp
GAA Glu
GGC Gly
TTT Ph.
CTT Leu
ATG "at
GTT va1
TGT cys
AAC ACA ABll Thr
TAT Tyr
TAT Tyr
GGC Gly
AAT Asn
AAG
GAA Glu
GAA ‘lu
GTT Val
CAA Gln
ATA Il.
ACT Thr
CTA La"
CTA Leu
GAT Asp
ATC Il.
TAT Tyr
GGA Gly
TAC Tyr
CCT Pro
TCC 5.x
ACT Thr
GAT Asp
ATA Il.
ATT Il.
TGT Cys
ATA Il.
GAG Glu
CAA Gln
AAA
‘GG ‘ly
CTT L."
CCT Pro
ACT Thr
CCT Pr*
AAA GTG l‘ys Val
ATA Ile
CTT L."
TAC Tyr
ATT If.
CCT Pro
GGA ‘ly
GGA Gly
ACA Thr
CAA Gln
CCA Pro
TTT Ph.
GTT Val
GAA Glu
TTT Ph.
CTT L."
AAT Asn
ACA Thr
GAT
GAT
Asp
Asp
ia) CTG L.u
AAA Lys
CAA Gin
TGG Trp
ATT Ile
GCA Ala
T‘G Trp
CAT His
TTA Lsu
AAA Lys
CAT Asp
AAC Asn
AAA Lys
CAT His
ATG Met
‘TC Val
CGA GCA Ala Arq
(2.3) TTC CGC Ph. Arq
AAA Lys
CAT His
TTC Ph.
TC‘ S.r
CTA Lcu
AAA Lys
Lys
ACT Thr
190
340
Ph.
CTA
490
640
790
Lys
CA&
CGT
CAG
GAA
GGA
GAA
ACG
TTT
AAA
GCACTT
CAA
GCA
GAA
GAG
TCC
CCT
GAA
‘1~
Gly
Glu
Thr
Ph.
Ile
Asp
Lya
Al.
L.u
Gin
TAT Tyr
ATT
Gln
GGT Gly
‘AT
Arq
ACA Thr
ATA
Gin
Il.
Al.
Glu
Glu
SOT
Pro
Glu
TGG Trp
CCT Pro
GCC Al.
AAT Asn
AAA Lys
TAC Tyr
ATC Il.
CTT L.u
TAT Tyr
AAT Asn
CCG Pro
ACA Thr
CAT His
TTA Lsu
GAA Glu
ACA Thr
‘AA Glu
AAT Asn
TTA L.u
TTT Ph.
AAC Asn
ATC Il.
ATG Mst
ATG M.t
AAG Lys
CGA Atq
ACA Thr
GAA Glu
CAG Gln
COG Acq
ATG M.t
CTT L.u
GAA Glu
OAT Asp
AGT Ser
GAT Asp
GTA Val
CAG Gln
ATT Ile
AGA Arq
TCA 5.1
AAT TCA Am,, Sbr
GAA Glu
GCT AIA
ACC Thr
COT Arq
GAC Asp
TAT Tyr
‘CT Al.
CTT L."
TCA S.c
TTA L."
CTC L."
GAA GlU
ACC Thr
TTT Ph.
ATT Xl.
TCA S.r
CAG Gln
TTA La"
TCT S.r
GCA Ala
ATA Il.
GAT Asp
ATG M.t
TTA Lou
GTA CCA Vi,1 Pro
GCA Al.
GTA V.1
GGT Gly
ATC Il.
CCA Pro
ATT Il.
AAT Asn
TTT Phe
GCC Ala
CTA LB"
TCA Ser
GCT Al.
ACA Thr
GCA Al.
TTA L.u
GGA Gly
CTT Leu
AGC Sar
TCG S.t
OAT Asp
ATT Il.
GTA V.1
GTT V.1
AAT Aan
GGA Gly
GAT Asp
TCA S.r
TAT Tyr
GAA Glu
AAG Lys
A‘A Arq
AAA Lys
TAT Tyr
GGA Gly
ATT Il.
‘GO Gly
TCC S.r
TTA I."
GTG V.l
CAA Gin
TCT Ser
GCA Ala
TTA L.u
TTC Ph.
RCA Thr
GGA Gly
ATT Il.
AAT A."
CTT L.”
ATT Il.
CCA Pro
GTT V.l
ATT Il.
TCG S.r
GAA ACC ‘1%~ Thr
GCA Ala
GAA Glu
ATT Ile
TTA LB"
TCT S.T
TCT TTC S.t: Ph.
TCT SOT
AGA Arq
ACA Thr
GAA Glu
GAA Glu
GAT Asp
ATT Il.
CCA Pro
GCT Ala
TTT Phe
TTC Ph.
ACT Thr
GAA Glu
GAA Glu
CAA Gin
GCT Ala
TTA L."
GCT Ala
CAA Gin
CGC Arq
TTT Phe
GAA Glu
ATA Il.
GTA GAA '7.1 Glu
GAA Glu
GAA Glu
TTA L.u
CAT His
TCT S.r
ATC Il.
TCA CCT SBS' Pro
GAT Asp
GAT Asp
CCT Pro
CCT Pro
CGA Arq
‘AA Glu
ATT Ile
ACT Thr
GAC Asp
GAA Glu
AAT Asn
TTA Leu
CAT His
AAA Lys
ATT Il.
CGT Arq
CTG L.u
GTA Vel
CGT Arq
CTT Leu
AAC Asn
RAT Asn
GAA Glu
RAT Asn
CAA Gln
CCT Pro
TTA Leu
GTT Val
GTG Val
TTA Lou
CGA Arq
AGA Arq
TTA Leu
GGA Gly
“A Gly
AAT Asn
AAA Lys
TTT Ph.
ATC Il.
AGA Arq
ATC Il.
GAG Glu
CCT Pro
ATA Il.
ACA Thr
TTC Ph.
CAG ‘ln
GAA ‘1~
ATA Il.
AAA Lys
GGT ‘ly
TCT 5.r
TTA Lo"
GTA Val
A‘T S.r
‘AA Glu
GTT Val
ATA Il.
AAT Aen
CCA Pro
GTG Vei
ACT Thr
AAT AAA As,, Lys
ACG Thr
TAC Tyr
TAC Tyr
GTA "al
AGC S.r
AAT GCT As,, Ala
AAA Lys
CTA Lo"
TTA Leu
GGG Gly
GGC Gly
TCT Ser
CCT Pro
TAT Tyr
AGT S.r
CCT Pro
TTC Ph.
CGT Arq
ATT Il.
GGA Gly
TTA L."
GAA Glu
GGT Gly
GTT V.1
TGG Trp
ACA Thr
CCA Pro
GAG Gl"
GTA V.1
TTA LB"
AAA Lya
GCA Ala
AGA Arq
GCT Ala
TCC S.r
GTT V.1
ATT Ila
GGA Gly
AAG Lys
CCT Pro
ATT Il.
GGA Gly
GAA Glu
TCA SOT
TAT Tyr
AAA LyS
AGA Arq
ATA Il.
TTA Leu
GCC Ala
AAA Lyr
CTA Lsu
CAR Gln
AGA Arq
ATA Il.
CAT
AAC
AGT
AAT
ATC
His
Aen
Ser
Asn
Il.
TTA Lcu
‘AT Asp
GAG Glu
CGA Arq
CAR Gin
GGT Gly
TTA Leu
ATG Met
CAT His
GAA Glu
CTC Leu
ATG Met
GAG Glu
CTT Leu
ATT Il.
OAT Asp
CTT Leu
TAT Tyr
GAA Gl"
GAA Glu
TCG 5.r
CAA Gin
CCT Pro
TCT S.r
TCA 5.r
GAG Glu
CGT Arq
TTG Lb"
AAT Asn
GCT Al.
TTT Ph.
CGT Arq
GAA Glu
CTG L.u
CGT Arq
ACT Thr
CAR Gin
TTA Leu
GAA Glu
AAA Lys
GCG Ala
CTT L.u
TAT Tyr
CTT LB"
CCT Pro
GAA Glu
ATG M.t
GAA Glu
GCA Ala
TTA LB"
AAA Lys
AAA Lys
CAA ‘ln
ATA Il.
CTA L."
CAG ‘ln
ATT 11s
CCT Pro
AAC Asn
AAA Lys
GOT ‘ly
940
1090
1240
1390
is40
1.590
1840
1990
2140
of the toxin gene PMTI. (a) marks the end of the insert in pAJL14. Differences between PMTI and other sequences by bases above the PMTl sequence and amino acid changes below. The sequences are: 2 1171; 3 [IX]; 4 [19].
and were used according to the manufacturer’s chemicals were from BDH.
specifications.
2.4. Sequencing strategy The insert in pAJL13 was isolated by digestion with HpaII followed by gel electrophoresis and elution onto silica gel (Geneclean, Statech).
60
ACA Thr
TGT Cys
TTG
CAGATG Gln
AAA Lyr
Cys Leu
Fig. 1. Sequence
inger, Other
ATG Met
are shown
It was digested with either A/u1 or SuuIIIA and ligated into M13mp18 cut with either SmaI or BumHI respectively (prior to ligation the cut vector was treated with phosphatase). DNA from isolated phage containing
inserts
was
purified
and
sequenced
using
an
Applied
Biosystems sequencer. This strategy produced about IO discrete short lengths of sequence. Since these sequences closely matched parts of
Volume
277, number
December
FEBS LETTERS
1,2
TCT
GGT
FCC
GCT
CGA
TTT
CGT
ACA
GCC
AAA
LOU
Arg
Thr
Ala
Glu
Ala
Gly
Lys
AGT Ser
AGC
Phe
ACC Thr
GAA
Arg
ATG Mot
GGA
Ala
AAT ASn
GCT
Ala
ATG Met
GAA
Gly
TTA LOU
CTT
SOT
Glu
S-r
ACG Thr
GCT Ala
GAT Asp
TTA Leu
ATA 110
CGC Arg
TTT Ph.
GCC Ala
TTG Lou
CAA Gin
GAT Asp
ACA Thr
GTA Val
ATT 110
TCA Ser
GCG Ala
CCT Pro
TTT Ph*
CGC Arg
GGA Gly
TAT Tyr
GCT Ala
GGT Gly
GCG Ala
ATT II*
CCA Pro
GAG Glu
GCA Ala
ATA Ile
GAC Asp
TTT Phe
CCT Pro
GTA Val
AAA Lya
TAT Tyr
GTA Val
ATA 11.
GAA Glu
GAC Asp
ATA 110
TCT 5.r
GTA Val
TTT Pho
GAT Asp
AAA Lys
ATA 110
CAG Gln
ACA Thr
AAT As”
TAC Tyr
TOG Trp
GAA Glu
CTT Lcu
CCT Pro
GCT Ala
TAT Tyr
GAA Glu
AGC Ser
TGG Tq,
AAC Asn
GAA Glu
GGA Gly
AGT Ser
AAT Asn
AGC Ser
CGA GCA Ale Arg
12.31 TTA CTG Lou Lsu
CCT
GGT Gly
TTG LeU
TTA CGT LOU Arg
GAA Glu
TCG Ser
CAA Gln
AGC Set
AAG Lys
GGC
ATG
TTA
AGT
AAG
GAA
AAT
TTT
TAC
AGC
Lys
Arg
11e
Glu
TAT GAA
ATG
scr
Asn
AGC Ser
GAA
I&U
ATC Ile
ATA
Met
TGT Cys
COT
Gly
Tyr
Glu
Glu
Met
Phe
Tyr
Ser
ATT Ile
TCT Ser
CCA
Pro
TAT Tyr
TCA Ser
AAC Asn
Gln
GTT Val
GGA Gly
GGG Gly
CCT Pro
GTA V&l
CAA Gln
GGT Gly
OAT Asp
TTA Leu
GGA Gly
TTT Phe
GAG Glu
CAG Gln
GCC Ala
TCC
TTA
ATG
GAA
AAT
Ser
Leu
Met
01”
Asn
AAT As,,
TAT Tyr
GAT Asp
GAA Glu
GCA Ala
TTC Phe
CAA Gin
GAA Glu
ATT Ile
TGG Trp
Pro
CTTTAT
ATT
GGA
CAT
AGC
Leu
Tyr
110
Gly
His
Ser
TAT Tyr
GAA Glu
TTA Leu
TAT Tyr
CCT Pro
TTC Phe
ACT Thr
TTT Ph.
TTC Ph.
AGT Ser
ATG Met
CTT mu
CAA Gln
GAA Glu
TTT Phe
GCC Ala
ACA Thr
CGT Arg
AAC Asn
A (4) TTT TTC Phm Pha TY~
AAT Asn
ACT Thr
CTT Lsu
GTT Val
TCT S.r
GAT Asp
CGA Arg
CTA Leu
ACGATG TTA CTTACA
GAA
AGT
Mat
Lcu
LQ”
Thr
Glu
Scr
TTT Phe
GAT Asp
TAT Tyr
ACA
Thr
CAA Gln
TTT Phs
GCT Ala
GCA Ala
ATG Met
TCT Sar
ATT Ile
AAT Asn
GAA Glu
CGC Arg
ATA 11s
AAC Asn
TCT Ser
TCA Sex
AAA Lys
AGT Ser
ATT Ilc
GAC Asp
TTT Phe
TTC Phe
CTA Lou
AAT Asn
GCT Ala
ATA 11s
TCT Ser
GAT Asp
TTA Leu
CCG Pro
TAT Tyr
GAT Asp
ATT Ile
GTC Vsl
CAA Gin
AAA Lys
ACA Thr
CTT LB”
GAT Asp
GCC Ala
ATG Mat
TTA Le”
TTT Phe
AAC Asn
TTA LBU
GAT Asp
TCA Ser
GCA Ala
AAA
Lys
GAT Asp
AAT Asn
AAT Asn
ATA 110
AAA Lys
TTT Phc
AGA Arg
GCT Ala
TAT Tyr
AAG Lys
TCT Ser
TTA Leu
TAT Tyr
TAT Tyr
AAA Lys
GGA Gly
AAT Asn
ATA 11s
GTT Val
GTA Val
TTT Phe
GCT Ala
GAT Asp
AGT Ser
TCT Ser
CTG Le”
CTC Le”
TTA Leu
AAA Lys
ATA Ile
CCA Pro
GCA Ala
ACT Thr
GTA Val
GTA Val
CCT Pro
ATT Ile
GAA Glu
GTT Vsl
ACA Thr
GAG Glu
TTA Leu
TTT Phe
AAG Lys
TTA Leu
ACG Thr
GCA Ala
GGT Gly
AGG Arg
GAA Glu
GAA Glu
TTA L.U
ATG Mot
GGC Gly
TCT Sar
TGT
GAT
TCA
Cys
Asp
TCT Ser
ATT Ila
ACC Thr
GCT Ala
GGT Gly
CTA Leu AAA Lys
CAG
CCTTGGGAT
TAT
Trp
Asp
GCT Ala
ATT
Pro
Ile
Tyr
GGA Gly
CAT Asp
ATT Ile
GAA Glu
AAA LyS
TGT Cys
ATG Met
AAT Asn
ACC Thr
TAT Tyr
AGA Arg
GGT Gly
GTG Val
AAT Asn
CTA LeU
ACC Thr
ACA Thr
TTC Phs
ATT 11s
GAT Asp
AAT Asn
GGA Gly
CTA Leu
ACC Thr
CAA Gln
GAA Glo
ATC Ilc
TCT Ser
CAA Gln
TTC Phe
TTA LFU
CAA Gin
GGA Gly
AGT SeI:
AAT Asn
GAA Glu
AAA LYS
GGA Gly
GAT Asp
ATT Ile
AAT Asn
GGT Gly
GCT Ala
TTC Phe
AGA Arg
AAG Lys
CTT LOU
CTG Le”
CAA Gln
ATA 110
GGG Gly
CAT His
TCA Ser
CAT Asp
ART Asn
TCT Ser
GTT Val
CCG Pro
CCA Pro
TTT Phe
AAT Asn
AAC Asn
CCT Pro
ATA 110
GCT Ala
GAA Glu
GCA Ala
ATT Ilr
GAA Glu
AAA Lys
CTA LOU
GAT Asp
CGA Arg
GAA Glu
GGT Gly
CAA Gln
AAA Lya
TTT Phs
AAC As,,
AGC Ser
ACG Thr
CCT Pro
GGG ACA Gly Thr
GGT Gly
CGT Arq
CCT Pro
ATG Met
CCA Pro
GGA Gly
CTA L*u
GTT Val
CAR Gin
TAT Tyr
GAT Asp
AGC Ser
GAT Asp
GOT Gly
GCA Ala
TGG Trp
CAA Gin
TTT Ph.
CTT La”
CCA Pro
GAT Asp
GTA Val
GCT Ala
TCA Sar
AGC Sar
AGA Arg
GTT Val
GAA Glu
AAT A5n
TGG Trp
CAA Gln
GTC Val
TTA LB”
ACT Thr
CCT Pro
CCA Pro
CAA Gln
GGT Gly
AAG Lys
ATT Ile
CTT Leu
GGA Gly
TTA Le,,
AAG Lys
CAA Gln
TTT Phs
CCA
ACA Thr
GAA Glu
CAA Gln
AGT Ser
CGC Arg
TTA Ls”
CCT
CTT
TTA
GAG
Pro
Lou
Leau Glu
AAT Asn
TCG Sar
GTT Val
TCT Ser
GAA Glu
CAT Asp
TTA Lau
CAA Gin
AAG
ATT
LyS
110
GAT Asp
GCA ADA
ATA II-
AAA Lya
AAT As”
GAT Asp
GTG V11
AAA Lys
ATG Mat
AAT As,,
AGT Ser
TTA L.u
GTG Val
TGT Cys
ATG ,,.t
GAA Glu
GCT Ale
SOT
GTA Val
AGC SOK
CCT Pro
AAG LYS
GTA Val
GCT Ala
GCC Ala
CGT Arg
CTT Leu
AAA Lys
GAT Asp
ATG “et
GGG Gly
TTA GAA LOU Glu
GCT Ala
GGG Gly
ATG “at
G‘T Gly
GCT Ala
TGG Trp
TGG Trp
AGA Arg
CGT Arg
GAA Glu
GGC Gly
GGG ATG Gly net
GAA Glu
TTT Phe
TCA Ser
CAT His
CAG Gln
ATG net
CAT His
ACT Thr
ACT Thr
GCT Ala
TCC ser
TTT Phs
AAA Lys
TTT Phe
AAA
GAG
TTT
GCC
GTG
GAT
GCT
TCA
CAT
TTA
CAA
TTT
GTA
CAC
GAC
CAA
TTA
OAT
ACA
ACT
ATC
CTG
Lys
Glu
Phe
Ala
~a1
Asp
Ala
Ser
His
Leu
Gin
Phe
Val
His
Asp
Gin
Leu
Asp
Thr
Thr
Ile
LPU
ATA Ile
CCT Pro
GTA Val
GAT Asp
GAT Asp
TGG Trp
GCT Ala
TTA Leu
GAA Glu
ATA Ile
GCT Ala
CAA Gln
AGA Arg
AAT Asn
CGG GCT Arg Ala
ATT Ile
AAT As,,
CCT Pro
TTT Phe
GTG Val
GAA Glu
TAT Tyr
GTT Val
AGT Ser
ACA Thr
GGA Gly
AAC AS”
ATG Met
TTA LBU
CTC LOU
TTC Phe
ATG Met
CCT Pro
CCT Pro
CTT Lou
TTC Pha
ACA Thr
CCT Pro
CGC Arg
TTA LB”
ACA Thr
AGA Arg
GCA Ala
CTA LB”
TAA
CTA
Ala
GCA
Pro
Thr
AAG Lys
ATTAAAAACTGTATTAAAGCCTTATATTATAAGGCTTTAATTTTCTTTCAAGAATTATTAAGTAGAAGAATCAAAATCAATGAGATAGATAAAATCAAAT
1990
2290
2440
2590
2740
1890
3040
3190
3340
3490
3640
3790
3940
l
4115
GTTATTACCAATACAACTTTCTTAAGTATACTTTTTGAATTTTTTGCGTTAATAAATTTATAATACCCTTAACTCAATAAAAGAAGTTATTGAGAAGTTT
AAATCTTGTGAGCAAGATGAAGATATAATTTCAGCAATCGATCTTATTAGCGCTTCATATAGAAGGGCTGTGGATGCAGTGGAACAAAGATTCGGTTCTAG
: Shine
Dalgarno
ribosoir
binding
site
_:
4316
terminator
Fig. I contj~~ed
the P. multocidu toxin sequence which had become available [17], a differnt strategy was adopted to obtain the full sequence of the mitogenic toxin. Oligonucleotides based on the available sequence [17] were synthesised on an Applied Biosystems 381A machine at intervals of about 200 bases along the gene and these were used for double-stranded sequencing using the Sequenase kit according to the manufacturer‘s instructions. For reasons of safety, most of the sequencing was done using a non-toxigenic derivative of the gene (pAJL14) which was created by removal of a 394 bp fragment by digestion with NdeI and subsequent religation. Removal of the NdeI fragment induces a frameshift
mutation, resulting in a non-toxigenic construct (see below). Plasmid pAJL13 was used to obtain the sequence of this fragment, but in a containment laboratory. Sequence analysis was carried out using UWGCG programs.
3. RESULTS AND DISCUSSION 3.1, Sequence of the mifogenic toxin The sequence (Fig. 1) contained an open reading frame (ORF) starting at base 155, which encode a pro61
Volume 277, number 1,2
FEBS LETTERS
tein of 1285 amino acids which would have a molecular mass of 146 kDa (Fig. 1). This is in accord with the apparent molecular mass of the mitogenic toxin as estimated by SDS-PAGE. The most likely start is the methionine shown, since the deduced amino acid sequence from this point is identical to the N-terminal amino acid sequence of PMTI [IS] except for one residue. The difference is likely to be due to experimental error in amino acid sequencing. It has been claimed that the N-terminal was blocked [ 17,181. The difference may be related to the source of the toxin since one was purified from recombinant E. coli (unblocked) and the other from P. multocida (blocked). A ribosome binding site is immediateIy upstream of the start (Fig. I), but no obvious E. coli consensus promoter sequence is present. Petersen [ 181 has presented convincing evidence for the location of the promoter region. The gene is expressed well in E. coli [12], but since little is known about Pasteurella gene organisation, it is not clear whether promoter sequences which function in E. co/i do so in P. m~Ito~ida. There were other methionine residues downstream from the presumed start. Although none had a good consensus with a ribosome binding site it is possible that some of the minor polypeptides produced by the recombinant which are antigenically related to the toxin [12] might have resulted from initiation at these residues. There was a stem loop structure typical of a terminator region starting at base 4027. There was no evidence for a signal sequence at any of the potential start codons, which agrees with the lack of secretion from either the native P. multocida or the recombinant E. coli. A hydrophobicity plot (Fig. 2) indicated that the protein might have several domains; in particular there was a hydrophobic region between amino acids 380 and 470 which was bounded by two hydrophilic domains, and the C terminal of the protein Experiments with the was also hydrophobic. lysosomotrophic agent methylamine, which blocks entry of certain toxins 121,and neutralisation of toxin ac-
December 1990
tion by antibody when added early but not late to toxin treated cells both indicate that the toxin binds to the cell surface, is internalised and subjected to processing in endosomal/lysosomal compartments [ 151, from which it presumably exits to exert its biological effects. The hydrophobic regions revealed in Fig. 2 are likely to play a role in the interaction of the toxin with various cellular membranes. The DNA and deduced amino acid sequences were compared with the sequences on the EMBL, Genbank and Swissprot data bases, but no significant homology was found. The motif His-Glu-Trp which is common to several toxins which ADP-ribosylate their substrate [24] was found near the N-terminus of the toxin (Fig. 3). The spacing between the amino acids matched precisely the His-Glu-Trp motif found in the ADP-ribosylating toxins (Fig. 3). Amino acid motifs thought to indicate the NAD and protein substrate binding site in the ADP ribosylating toxins [25] were not present in this toxin. It should be noted that the His-Glu-Trp motif was also found in a variety of proteins that are not thought to ADP-ribosylate any substrates (unpublished). As predicted from the restriction map [12] the gene had a low G + C content (35%). Interestingly, the G + C content of the Pasteurella genome differs markedly from that of the toxin gene, so it is possible that the gene has been acquired relatively recently and that there might exist a family of closely related mitogenic toxin genes in other bacteria. Indeed, recent reports indicate that the toxin gene is flanked by phage elements [17], may be carried on a prophage [26] and that some Salmonella isolates contain part of the Paste~rel~atoxin gene [27]. 3.2. Mitogenicity of P. multocida toxin from another isolate The sequence (hereafter caIled PMTl) was compared to the other P. m~~tocida toxin sequences available (PMT2 [17]; PMT3 [lS]; PMT4 [19]). There were 4 differences within the open reading frames of the genes, and in each case the PMTl sequence was checked
P. mltocida
His 29
xii
Glu 155
I
TV 160
His 426
jr’6
Glu 553
4
TrP 558
“15 21
116
Glu 148
(I
T[P 153
sordetella mrtussis Sl
His 83
116
GlU 210
4
=P 215
Vibrm cholerae CT
His 44
I*S
Glu 170
?
TIP 174
Escherichia cali LTH
His 44
125
Asp
I
TKP 174
toxin
Pseudomonasaeruqinosa ETA Corvnebacteriumdititheriae
DT
170
Comparison of the His-Glu-Trp motif in PMTl with other taxins. The data for the other toxins are taken from 1241.The numbers
Fig. 3.
Fig. 2. Hydrophobicity plot of the PMTI protein, catculated using a program written by Dr M.E.G. Boursnell. Positive numbers indicate hydrophobicity.
62
between the amino acids indicate the spacing between them; the numbers below indicate the position in each protein of the respective amino acid.
December 1990
FEBSLETTERS
Volume 277, number 1,2
100
600 act
PMT (nglml)
Antiserum (d/ml)
Days
Protein (ng/ml)
Fig. 4. Comparison of the mitogenic activity of PMTl and PMT2. (A) Dose-response curve for the stimulation of DNA synthesis by PMTl (0) or PMT2 (a). Cultures of Swiss 3T3 cells were incubated with PMT and DNA synthesis measured as described [15]. Each point is the mean of two determinations: 10% foetal bovine serum gave a level of incorporation of 704 x lo3 cpm. (B) PMT2 stimulates reinitiation of growth in confluent Swiss 3T3 cells. PMT2 was added directly to the medium of Swiss 3T3 cell cultures 6 days after plating to give a final concentration of 10 ng/ml. 2, 3 and 4 days after this addition, cells from both treated (hatched bars) and untreated (open bars) cultures were trypsinised and counted using a Coulter counter. The values shown are mean k SE (n = 5). (C) Dose-response for the inhibition by PMT antiserum (polyclonal 34B) of DNA synthesis induced by PMTl (0) or PMT2 (0). Various concentrations of immune serum were incubated with toxin and added to confluent quiescent Swiss 3T3 cells as described [15]. Each point is the mean of two determinations: 10% foetal bovine serum gave a level of incorporation of 979 x 10’ cpm. (D) Dose-response curve for the stimulation of DNA synthesis by extracts of E. coli transformed with various constructs. Swiss 3T3 cells were treated as described (151with various concentrations of HBlOl + pAJLl4 ( q), HBlOl + pAT153 (4) or HBlOl + pAJLl2 ( n). Each point is the mean of two determinations: 10% foetal bovine serum gave a level of incorporation of 672 x 10’ cpm.
carefully for errors. There was a single base change between the PMTl and the PMT4 sequences (at base position 2712) which resulted in a conservative amino acid substitution. There were 2 differences between the PMTl gene and that of the gene for PMT3, at base positions 1064 and 2477; both resulted in a change from an alanine in PMTl to an arginine residue in PMT3. There was one other difference between the sequences of PMTl and PMT2 at base position 396. These minor differences among the sequences might be due to errors in sequencing, or reflect variation among isolates. If the latter were true, it was important to investigate whether the potent mitogenicity of the protein was affected. PMT2, purified from P. multocida NCTC12178, and PMTl stimulated DNA synthesis with an identical dose-response relationship in Swiss 3T3 cells (Fig. 4A); there was a similar toxininduced increase in cell proliferation (Fig. 4B). The mitogenic activity of each toxin was completely blocked by antibody to PMTl in a concentration dependent manner (Fig. 4C).
One of the recombinants produced in the original clone bank contained part of the gene, but did not produce toxin active on EBL cells [12]. This recombinant produced polypeptide fragments which reacted with antibody to the toxin. Restriction enzyme analysis and the DNA sequence of this plasmid showed that it contained the N-terminus of the gene, and should code for amino acids l-287 to produce a 33.5 kDa peptide (Fig. 1). A crude preparation from this recombinant was unable to induce DNA synthesis (Fig. 4D). 4. CONCLUSIONS We report the complete sequence of the mitogenic toxin PMTl. The deduced protein sequence did not show any significant homology to known sequences with the possible exception of a motif found in toxins which ADP-ribosylate their substrate. There appear to be two hydrophobic domains in the toxin, which could play a role in the interaction of the toxin with cellular membranes. The sequence was almost identical to other 63
Volume 211, number 1,2
FEBS LETTERS
P. muftocida toxin sequences [ 17-191, and the toxin purified from one of these latter strains was as mitogenic as PMTI in Swiss 3T3 cells. It has been suggested that other bacterial toxin genes originally had a eukaryotic origin, and this might also be the case for the P. multocida toxin. The toxin might then be expected to have an as yet undiscovered eukaryotic homologue involved in signal transduction. It is to be hoped that analysis of the toxin action will not only explain its role in atrophic rhinitis, wlrere it has specific effects on bone morphogenesis, but also yield important information about new signal transduction pathways. Acknowfe~ge~en~s: We wish to thank Dr Ian Goldsmith and Karen Mawditt for synthesis of oligonucleotides and Fara Khurshid for automated DNA sequencing.
REFERENCES 111Rozengurt, E. (1986) Science 234, 161-166. 121Middlebrook, J.L. and Dorland, R.B. (1984) Microbial. Rev. 48, 199-221. [31 Pfeuffer, T. and Helmreich, E.J.M. (1988) Curr. Topics Cell Regul. 29, 129-216. [41 Il’ina, Z.M. and Zasukhin, M.I. (1975) Sbornik nauchnykh rabot, Sibirskii nauchno-Issledovatei’skii Veterinarnyi Institut, Omsk 25, 76-86. VI Rutter, J.M. and Mackenzie, A. (1984) Vet. Record 114, 89-90. El Switzer, W.P. and Farrington, D.O. (1975) in: Diseases of Swine, 4th edn (Dunne, H.W. and Leman, A.D. eds) pp. 687-7 11, Iowa State University Press, Ames. [71 Nakai, T., Sawata, A., Tsuji, M., Samejima, Y. and Kume, K. (1984) Infect. lmmun. 46, 429-434.
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