AbstractThe structural gene (vmhA) of hemolysin from Vibrio mimicus (ATCC33653) was cloned and sequenced. The vmhA gene contains an open reading frame consisting of 2232 nucleotides which can code for a protein of 744 amino acids with a predicted molecular mass of 83 059. The similarity of amino acid sequence shows 81.6% identity with Vibrio cholerae El Tor hemolysin

Huh, Sung-Hoi

Kim, Gu-Taek

Kong, In-Soo

Lee, Jong-Young

Yu, Ju-Hyun

Elsevier - Publisher Connector 

Nucleotide sequence of the vmhA gene encoding hemolysin from Vibrio mimicus 

ELSEVIER Biochimica et Biophysica Acta 1360 (1997) 102-104 
BIOCHIMICA ET BIOPHYSICA ACTA 
Short sequence-paper  
Nucleotide sequence of the vmhA gene encoding hemolysin from Vibrio 
mimicus 
Gu-Taek  K im a, Jong-Young Lee a, Sung-Ho i  Huh b, Ju -Hyun Yu c, In -Soo Kong a,* 
a RCOID and Department of Biotechnology and Bioengineering, Pusan 608-737, South Korea 
b Department of Oceanography, Pukyong National University, Pusan 608-737, South Korea 
c Bioproducts Research Center, Yonsei University, 134 Shinchon-dong, Sudaemoon-ku, Seoul 120-749, South Korea 
Received 15 October 1996; revised 17 January 1997; accepted 20 January 1997 
Abstract 
The structural gene (vmhA) of hemolysin from Vibrio mimicus (ATCC33653) was cloned and sequenced, The vmhA 
gene contains an open reading frame consisting of 2232 nucleotides which can code for a protein of 744 amino acids with a 
predicted molecular mass of 83 059. The similarity of amino acid sequence shows 81.6% identity with Vibrio cholerae El 
Tor hemolysin. 
Keywords: Vibrio; Hemolysis; Enteropathogenic bacterium; Vm-hemolysin 
V. mimicus is an enteropathogenic ba terium which 
inhabits aquatic environments and apparently causes 
diarrhea, usually after the consumption of uncooked 
seafood [1]. Several pathogenic factors of V. mimi- 
cus, including cholera toxin (CT) [2], CT-related 
enterotoxin [3], Escherichia coli heat-stable ntero- 
toxin (ST)-like toxins [4,5] or protease [6], have been 
reported. Many V. mimicus strains isolated from 
environment are capable of causing diarrhea, even 
though they cannot produce these enterotoxins. 
Therefore, it was postulated that another toxin is 
involved in the bloody diarrhea which is one particu- 
lar clinical symptom of V. mimicus gastroenteritis. In 
addition to the above toxins, hemolysins are sus- 
pected to be the pathogenic factor of the vibrio. Two 
kinds of hemolysin produced by V. mimicus, Vm-he- 
* Corresponding author. Fax: +82 51 6206180; E-mail: 
iskong@dolphin.pknu.ac.kr 
molysin (M r 58000), Vm-rTDH (M r 22000), have 
been reported [7]. The former is immunologically 
cross-reactive with V. cholerae E1 Tor hemolysin and 
the latter is cross-reactive with V. parahaemolyticus 
thermostable direct hemolysin (TDH). The nucleotide 
sequence comparison of Vm-rTDH and Vp-TDH re- 
vealed that they were very homologous and had only 
minor variations but the flanking sequences of the 
hemolysin genes were dissimilar, indicating that they 
have a common ancestor and suggesting that they 
may have been transferred between vibrio species as 
a descrete genetic unit [8]. In this communication, we 
report the nucleotide sequence of gene encoding 
hemolysin similar to V. cholerae El Tor hemolysin. 
A gene bank of the V. mimicus (ATCC33653) 
chromosomal DNA (partially digested Pst I /Sa l  I 
fragments) was prepared in the pUC19. One clone, 
pVMH194, was isolated by /3-hemolysis on TSAII 
medium containing 5% sheep blood and the nu- 
cleotide sequence, containing an ORF of 2232 bp, 
0925-4439/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved. 
PII S0925-4439(97)00008-2 
G.-T. Kim et al. / Biochimica et Biophysica Acta 1360 (1997) 102-104 103 
was determined. Fig. 1 shows the nucleotide se- 
quence and deduced amino acid sequence of the 
hemolysin, consisting of 744 aa with a predicted 
molecular mass of 83 kDa. Typical GAGGT, RBS or 
SD, sequence xists in 5 bp upstream from the ATG 
hemolysin initiation codon. However, the protein size 
(83 kDa) is larger than that of previously purified 
Vm-hemolysin (58 kDa) [7]. In V. cholerae E1 Tor 
hemolysin, the 82 kDa preprotoxin synthesized in the 
cytoplasm is secreted through the membrane into the 
culture medium as the 79 kDa inactive protoxin after 
cleavage of the signal peptide and is then further 
processed into the 65 kDa active hemolysin by re- 
lease of the N-terminal 15 kDa fragment [9]. Thus, 
I CTGCAGATC~'GGTCAGATCTCTCTTGTATTGTGAATGAGAC ATI'FGTCr CGTCAATGAGAAGT~CTGTCATTACTCAGG' i~r~GATC~AT~A~ 
Pst l  
121 ATTCTGTTCATA~I"FGATCTFTATTTI'r CATAI"FAATGGACTTAAGTGATA'ITFAATGATTGAAAGTTAATCACrTAATGAATTTCr A~ATCTATr r  AATr CAATL'r AATITAGTT 
241 C~AATTAAATTAGTGCGAATGAATAATGTAAATATCCA~AAAACAAA~I~I~3C~GAATTAATAGT~TGT~AAGAA1"r ~ C ~ T ~ T ~  H T [ C ~ ~ A T ~ C  
361 CTGTTACr.AI"rTTCTGI"r ACAACrATrTAC13Tr GGCATGTITAG6GATAI ' rCCAATAAGTG'T~AG~TAT~A~T~G~TA~C~ 
481 ACg£~6TATGTGI"r ~AG6CGTAGI-r CTGCCCCAAGCCACACACAACATCAAG6ATGACGACGG'r ~ C C C A ~ A G ~ A ~ ~ A T G A ~ ~  
601 TGA'Fr~J~ATTTATCCTCTrA~AC.,CATAACGCI"r ~ l r  ATGTC, C6TAGTGATGA~TGAAI'i'r C C ~ A G ~ L ' r f ' I ' r C A ~ A ~ ~ G ~  
721 CTAAT~ACT.~TAACrGAATCAGTGAGGTTTATATGCCAAAACTCAAT~GTrGTGCAATCGC~T~GATA~CA~TA~A~ATA~G~G~TATCA 
S.D. ~1P  K L N R C A I A I L T I F S A I S S H T V F A N I 27 
841 GTGATCCCGTTGG1~3AAGCTSTr GAAATTATTAGCCAAGTCGCGGAT~ACCACGC~TAAAATAI" rAC~C~AGA~TCAG~A~A~A~A~ 
S D P V G E A V E I I S 0 v A D N H A I K Y Y N A A D W O S E E S ~ L P S L A E 67 
961 TGCGTGAGCAGGTGATTAACCAGCAGTTGCGTATCTTGGTCG.~,TI'r CAGCCAGATCTCTGA~GA~CTAACCG~T~A~G~AG~AT~G 
Lt t  E Q V I N Cl Cl L R I L V D F S Q I S D P D 1t L r E ~ (1V  K F R K T Y G V G F 107 
1081 CZ~ATACATTCATCGTAATCACTGAC£ATk~6GTGAGCTGCTGITr ACACCGTrrGATCGTC~GGAGGATGT~ATCC~CA~A~ACG~GA~6AGTC 
A N T F I V I T E H K G E L L F T P F D R A E D V D P T L L E A P R S E R V L S 147 
1201 GGTC ACGTCGCTC ~TGTCIV, CC.~T.~TGTC, ACTAACA~CAATGAAACGAACACGTTGCCGCACSTIT~ H i iTACATC~T~C~C~T~A~GA~A~ 
R S R It S V S A N N V T N N N E T N T L P H V A F Y I N V N R S I S D E E C T F 187 
1321 CCAAI~rC3"r GGCT.~TGGAAAAATGACAAAGGCAGTCGTCCA~GT.~J~AGATGCCAATATCr CATTGATI ' rACCGTGTG~AGA~CA~AGTA~A~ 
A N S W L W K N D K G S R P F C K D A N I S L i Y It V N L E R S L 0 Y G I V G S 227 
1441 C ~.~CTCCGGATGCCAA.~TTGTGCGTATCAGCTTAGATGAcGACAGcAC~VF~CGcGGGTATCCATcTC~TGA~A~ l r  r C~A~ATATACGAC~ATG 
A T P D A K I V R ! S L D D D S T G A G I H L N D O L G Y R F F K A G Y T T L D 267 
1561 CTTACTTCCGT GAGTGGTCAACGGATGCGATAGC GCAGGATTATCGCTrC.~,GI"ITr.~ACGCrrCGAATGAT.~GGCGCAGAT"i'Fr ~GA~C~TCA~CGTC~C~4~ 
A Y F R E W S T D A I A O D Y R F S F N A S N D K A 0 I L K T F P V T N V N A N 307 
1681 TTGAGCGTAAAGAAGTCT~AGCI"r C43TGTA~GTGTrGAA C, CGGATAAAAATGGCCCGAAAGC' r~G~CA~A~G~GA 
F E R K E V S G F E  LG V T G S  VE  A D K N G P K A K L E A K A S Y T ( I S R W L  347 
1801 CCTACAACACGC~AGATTATCGAATTGAGCGGAGTGCGA~AAATGCGC~AAACGTrA~A~CC~A~A~AC~C~T~C~CC6~C~C~ATG 
T Y N T I:l D Y R 1 E ~ S A K N A O N V S F T I~ N I~ O O Y A T A E S L L N I1 S T D 387 
1921 CA'Fr GTGGGTGG`~e~AC~TATCCTGTTGAT~TGAACCGCATCAGCCCGCTG`~`GTTACGCCAGTrrrGTGCCA;u~)~TGGATGT`~A~ ~G CACCG~GAGAC~A~ACCG 
A L ~; V E T Y P V D V N R I S P L S Y A S F V P K ~1 D v I Y K A S P K E T G S T 427 
2041 r r riT~TCATCGACTCT~Cc`~?TCAATATC~T~C~ATCTATAATGGTGC~AC~ACTAT~ATGT~GT~A~`CATCA~AT~A ~ 6 A T A  C ~ C ~  ~ 
V F V I D S S V N 1 R P I Y N G A Y K H Y Y V V G S H O S Y H G F E D T P R R R 467 
2161 TGACAAAATC~.~_~,~ACTGTGGATT~ATCACCCTGTATTTA~TGI}TCGTCCAGTGAAITr ACA ~GAGL 'Tr  C ~ C ~ A ~ A ~ A ~ T C  
V T K S A S F T V D W D H P V F T G G R P V N L O L A S F N N tl C I E A D D Q G 507 
2281 ~GATGGCAACTACCTGTGACAGTCAGCAAGCGGCGCAA3 c I t 11ATCFATGATCAACAAC, GTC~ACGTGAGT~GT.~CACC~A~G~A~ 
R L ~ A T T C D S 0 {1A A Q S F I Y D Q O G It Y V S A 8 N T K L C L D G E A L D 547 
2401 Cl'C'r GCACAC CTGTAACCACjAAUI~FGACCCAACGTr~A~GTGA~,C, GI"f CTGACG~ACTGAGC~T~TG~A~ACA~AC~C.~G~CG 
S L H T C N Q N L T O R W E If R E G S D E L S N V F N G E V L G H D K Q T G E L 587 
2521 GCCITr ATr CAACAGGCAGT~ATGCA~.T~AGCCTAC~TAC~ATrAC~TCT~ACACCkAT5~I~TCCATGAACAG~AAAG~rCACCCf3TGCTI~AACCCAAGGGA~TGAATCAGC 
GL Y S T G S D A V S L R T  I T S Y TN VF  HE  ~IE  SS  PV  L G LT( I  G K V N Q 627 
2641 AC~CGAGTAGAk~AGGATAA~CAACT~rAC~TAC~TGCAGGT~CATrGATG~T~AGGAACTGCT~CTGAG'rrGTTGG1-rGGTG6TACGGGCGGTAGcATCACAACGG1-rGATCTCA 
0 R VE  KD NO L Y V I I  AG AA I D A L G T A P E L L V G G T G G S I T T V D L 667 
2761 ~ A G T r  CTATCACAGCGAC'I'fCT GGCI}AcrTrA~CTr CGGq'~AC*CAGTTC*GTGC, CATTAACCTTrA~A~GA~GTC~C~T~C~CG~GA~ACA 
T 6 L S S I T A T S G Ill F N F G G 0 Q L V A L T F T Y O D G It Q (1 M V G S K E H 707 
28fll TCAGC&ATGCrr CA TGAAGATC~ACGT ACCGACAGGAGCCAAT ATTACCCAAATGAAAGTTT~AGATA-~.CT~GAAAGG'rGT~A~GAC~ G ~ G A  
I S N A H E D R F D V P T G A N I T Q ~1 K V W A D N W L V K G V 0 F O L N * 
3001 "FF.~TC, CCTGAT AATr CAGGGCATT.~TGTTCATAATAt; H ~ i aATGTTTCI"I'A Tr GACTGATTTGTC GAC 
Fig. I. The complete nt and deduced aa sequence of the vmhA gene. The nt sequence is numbered on the left of the sequence and the 
deduced aa sequence on the right. Asterisk represents the stop codon. Rho-independent stop region of mRNA is indicated by arrows. The 
GenBank accession number is U68271. 
104 G.-T. Kim et al. / Biochimica et Biophysica Acta 1360 (1997) 102-104 
VmhA 
HlyA 
-50 
-50 
-150 
'=~" ........................................................... -250 
• - ,~  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  -247 
 2;/;iUi1211 il; /_ Zi i 211 i Ui ii 
" " . . . .  " ~ -397  400 
T~ . . . .  4~ al;~ -500 
Pm . . . . . . . . . . . .  J]~ ~,m,m, -~ . . . . . . . . . . . . . . . . . . . . .  6 . ~ N ~  -597 
-647 
Fig. 2. Alignment of predicted amino acid sequences of VmhA 
from V. mimicus and HIyA from V. cholerae 01 Biotype El Tor. 
Identical amino acid residues in the two hemolysins are boxed. 
Conserved cysteine residues are marked by asterisks. 
we assume that Vm-hemolysin may also have very 
high possibility of the same two-step processing. 
The hemolysin shared 81.6% identity with V. 
cholerae E1 Tor hemolysin, consisting of 741 aa with 
a predicted molecular mass of 82 kDa, having only a 
major variation of three amino acid deletion from 148 
to 150 [9-11] (Fig. 2). However, no sequence homol- 
ogy with other hemolysins/cytolysins was found. 
The placements and numbers of cystine residues for 
Vm-hemolysin and E1 Tor hemolysin were identical, 
reflecting the similarity of their secondary structures. 
According to the comparison of cysteine residues, 
Vm-hemolysin might be divided into three regions; 
the N-terminal region (Met-1 to Cys-185), the central 
region (Cys-185 to Cys-552) and the C-terminal re- 
gion (Cys-552 to Asn-744). The N-terminal region, 
showing relatively lower homology and having the 
variation with E1 Tor hemolysin, seemed to be less 
important for hemolytic activity. It was supported by 
the fact that V. cholerae E1 Tor hemolysin is pro- 
cessed twice at Asn-26 and Asn-158, and the mature 
hemolysin has higher activity than the precursors by 
removing the N-terminal region [9]. The central re- 
gion (Cys-185 to Cys-552) with six cystines had 
higher homology than other regions. The suggestion 
had been reported that the C-terminal region of E1 
Tor hemolysin may be involved in the proper config- 
uration of the protein for maximal hemolysin activity 
[12,13]. Thus, it might have an important role for the 
hemolysin activity although the C-terminal region of 
Vm-hemolysin has less homology than the central 
region. In addition, it will be required to explain the 
biochemical differences between Vm-hemolysin and 
E1 Tor hemolysin. The function of the central and 
C-terminal region of Vm-hemolysin will be eluci- 
dated by further study. 
This research was supported in part by a grant 
from Engineering Research Center and by Non Di- 
rected Research Fund, Korea Research Foundation. 
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Nucleotide sequence of the vmhA gene encoding hemolysin from Vibrio mimicus

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