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FSAC #92607

Cloning, sequencing and expression of a GroEL-like protein gene of

Vibrio vulnificus

Hin-chung Wong1*, Kai-Hsi Lu1 and James D. Oliver2

1Department of Microbiology, Soochow University, Taipei, Taiwan,

Republic of China and 2Department of Biology, University of North

Carolina at Charlotte, Charlotte, North Carolina, U.S.A.

Running title: groEL gene of Vibrio vulnificus

*To whom correspondence and reprint requests should be addressed.

Mailing address: Department of Microbiology, Soochow University,

Taipei, Taiwan 111, Republic of China. Tel: 8862-8819471 Ext. 6852; Fax

8862-28831193; E-mail: wonghc@ scu.edu.tw

First Draft: Dec. 23, 2003

Revised: Sept. 12, 2004

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ABSTRACT

Vibrio vulnificus causes wound infection and septicemia in

humans with high mortality via the consumption of contaminated

seafood. This study cloned, sequenced and analyzed the general

properties of the GroEL-like protein gene of this pathogen. A 1.6 kb

amplified DNA fragment from V. vulnificus ATCC27562 was cloned

into pGEM-T Vector and nucleotide sequenced. The open reading

frame consisted of 1,644 bp, and encoded for a 57.7 kDa GroEL-like

protein of 547 amino acids. The amino acid sequence of the V.

vulnificus GroEL-like protein showed high identity (82-92%) with

other GroEL proteins, especially those of vibrios, E. coli and S.

enterica Typhi. The amino acid residues 269 to 282 of the V. vulnificus

GroEL-like protein were significantly more hydrophobic than others,

with two basic lysine residues replaced by hydrophobic isoleucine

residues. The groEL-like gene of V. vulnificus was cloned into the

pQE-30 expression vector and expression of a His-tagged GroEL-like

protein was rapidly induced by isopropyl-β-D- thiogalactopyranoside

and confirmed by immunoblotting with anti-GroEL and Penta-His

antibodies. The over-produced GroEL-like protein of V. vulnificus

was present largely in an insoluble form. Results of this report may

facilitate the study of responses of V. vulnificus to environmental

stresses.

Key Words: Vibrio vulnificus, GroEL, heat shock protein, nucleotide

sequence

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INTRODUCTION

Vibrio vulnificus causes human wound infection and septicemia,

largely a result of occupational activities around seawater. V. vulnificus

occurs in high numbers in molluscan shellfish, primarily oysters, and its

ingestion in raw oysters results in a ca. 60% mortality in those persons

who are susceptible to this bacterium (Linkous and Oliver 1999). In

addition to humans, V. vulnificus is also pathogenic to aquatic and other

animals, such as eels, shrimp, mice, etc. (Amaro et al, 1994; Biosca et al,

1999).

The physiology and virulence of pathogenic vibrios are affected by

environmental stresses (Parsot and Mekalanos 1990; Yildiz and Schoolnik

1998). In V. vulnificus, highly variable responses to environmental

stresses are found in different strains. Starvation enhances freeze-thaw

resistance and heat tolerance for some strains (Bang and Drake 2002).

Other than studies on the viable but nonculturable state, the effect of

various stresses on V. vulnificus has not been well characterized. Since the

presence of the environmental stress mediator ToxRS protein has been

shown in V. vulnificus (Lee et al, 2000), V. vulnificus may also have

tightly regulated responses to environmental changes, as do V. cholerae,

V. parahaemolyticus and other vibrios.

Beside their chaperone activity, GroEL proteins are also associated

with the virulence of some pathogenic bacteria. The GroEL-like protein

of Actinobacillus actinomycetemcomitans is found in extracellular

material and is strongly toxic for HaCaT epithelial cells (Goulhen et al,

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1998). A significant amount of GroEL homolog protein is absorbed on

the surface of Helicobacter pylori (Dunn et al, 1997). The GroEL-like

protein of Campylobacter rectus stimulates both interleukin-6 and IL-8

secretion by a confluent monolayer of human gingival fibroblast cells

(Hinode et al, 1998). During infection, V. vulnificus moves from the

environment into the human body and is challenged by a shift-up of

incubation temperature. Heat shock proteins like GroEL may be produced

and play a role in the pathogenesis of this bacterium. In this study, the

groEL-like gene of V. vulnificus was cloned using polymerase chain

reaction (PCR) method with primers derived from the conserved

sequences of the Salmonella enterica serotype Typhi groEL gene (Lindler

and Hayes 1994; Rusanganwa et al, 1992), and the cloned gene was

sequenced and analyzed.

MATERIALS AND METHODS

Bacterial strain and cultivation

The type strain V. vulnificus ATCC27562 was used for the cloning of

the groEL-like gene in this study. It is a hemolytic strain isolated from a

human blood sample (Okada et al, 1987). Bacterial strains were stored in

culture broth with 10% glycerol at–85°C. V. vulnificus was cultured in

Tryptic Soy Agar (TSA, Difco Laboratories, Detroit, MI, USA) with a

supplement of 3% NaCl, or Luria-Bertani Agar Medium (LA, Difco)-3%

NaCl (pH 7.5) at 37C. For the purification of chromosomal DNA, a

single colony of V. vulnificus was inoculated into 100 ml Luria-Bertani

Broth Medium (LB, Difco)-3% NaCl and cultured at 37°C for 16 h.

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Escherichia coli JM109 was cultured in these media without additional

NaCl.

DNA techniques

Purification of plasmids and genomic DNA, analysis of DNA by

agarose gel electrophoresis, preparation of competent cells and plasmid

transformation techniques were as described by Sambrook et al.

(Sambrook et al, 1989). PCR amplified products were separated by gel

electrophoresis and purified by a gel band purification kit (Pharmacia,

Uppsala, Sweden).

Cloning and sequencing

The groEL-like gene of V. vulnificus was amplified by PCR using

primers (5’-CGCGGATCCGCGATGG

ACGCTAAAGACGTAAAATTCGG,3’-CGCGGATCCGCGTTACAT

CATGCCGCCCATGCCAC) designed according to the S. typhi groEL

gene sequence (Lindler and Hayes 1994). PCR was performed by a

Personal cycler 20 (Biometra biomedizinische analytik Gmbh, Gottingen,

Germany) with the following parameters: 94 °C for 1 min, followed by

30 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1.5 min, and

finally, an additional 10 min at 72 oC (Wong et al, 1998).

The 1.6 kb amplified fragment was cloned into pGEM-T Vector

(Promega Corp., Madison, WI, USA) by the TA cloning techniques (Zhou

and Gomez-Sanchez 2000) and transformed into E. coli JM109 recipient

cells (Sambrook et al, 1989).

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The DNA sequence of the insert in the recombinant plasmid was

determined in both strands with primers for the vector and internal

primers. DNA sequencing was performed on an ALFexpress DNA

Sequencer (Pharmacia) by fluorescence-based dideoxy-sequence

reactions. The sequence was confirmed by repeating the determinations

with an ABI Prism dye terminator cycle sequence kit and an ABI model

377-96 DNA Sequencer (Applied Biosystems, Foster City, CA, USA).

The Deep Vents DNA polymerase of high PCR fidelity (New England

Biolabs, Beverly, MA, USA) was used in these sequencing reactions

(Cline et al, 1996).

Sequence analysis

The sequence determined in this study was compared with homologous

sequences from the GenBank Library using BLAST software from the

National Center for Biotechnology Information. The amino acid sequence

was generated and its properties analyzed by the Expert Protein Analysis

System of the Swiss Institute of Bioinformatics. Human leukocyte

antigens (HLA) peptide motifs prediction was done using the world wide

web (http://bimas.dcrt.nih.gov/) based on Parker et al. 1994 (Parker et al,

1994). The B cells epitope prediction of the amino acid sequences were

done manually (Cancino-Diaz et al, 1998).

Expression and analysis of the GroEL-like protein

The insert from the recombinant plasmid was excised by BamH1

digestion, cloned into the QIAexpressionist pQE-30 expression vector

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(Qiagen GmbH, Hilden, Germany) and transformed into E. coli JM109

(Chow et al, 2000). The transformed bacteria were cultured in

Luria-Bertani Broth (Difco) supplemented with ampicillin (200 μg/ml)

and expression of the recombinant protein was induced with 1 mM

isopropyl-β-D- thiogalactopyranoside (IPTG) at 37°C for 30 min to five

hours. The bacterial cells were harvested by centrifugation and lysed by a

lysozyme treatment (Thies et al, 1999). Soluble and insoluble fractions

were separated by centrifugation at 10,000g for 15 min at 4°C. The

GroEL-like protein was analyzed by SDS-polyacrylamide gel

electrophoresis and immunobloting using an anti-GroEL antibody (Sigma

Co., St. Louis, MO, USA) or Penta-His antibody (Qiagen)(Wong et al,

1998; Wong et al, 2002). A prestained protein ladder (Gibco-BRL,

Gaithersburg, MD, USA) was used as molecular size markers.

Accession numbers

The groEL-like gene sequence of V. vulnificus reported in this paper

has been submitted in 2000 to the GenBank Data Library under the

accession number AY017169.

RESULTS AND DISCUSSION

A 1.6 kb amplified DNA fragment from V. vulnificus ATCC27562 was

cloned into a pGEM-T Vector and nucleotide sequenced (GenBank

accession number AY017169). The open reading frame consisted of 1,644

bp and encoded a 57.7 kDa GroEL-like protein. The properties of this V.

vulnificus deduced GroEL-like protein were similar to those of other

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Vibrio species, S. enterica serotype Typhi and E. coli. The V. vulnificus

GroEL-like protein consisted of 547 amino acids with a theoretical pI of

ca. 4.71. The GroEL protein of V. cholerae (GenBank accession no

AAF95805) consisted of 544 amino acids, 57.2 kDa, and pI of 4.78,

while the GroEL protein of V. parahaemolyticus (GenBank accession no

AAF27528) consisted of 548 amino acids, 57.6 kDa, and a pI of 4.68. A

typical structure of GroEL molecules, a Gly-Gly-Met repeat at the

C-terminus, was found in the V. vulnificus groEL-like sequence (Fig. 1).

The deduced amino acid sequence of the V. vulnificus GroEL-like

protein was compared to known bacterial HSP60 or GroEL protein

sequences. It is highly similar to the groEL sequences of V. vulnificus

YJ016 and CMCP6 which were released after the completion of our

present study (Table 1). High amino acid identity (82-92%) was found in

all other sequences, and the amino acid sequence of the V. vulnificus

GroEL-like protein showed especially high identity with GroEL proteins

of V. cholerae (91%) and V. parahaemolyticus (92%) (Table 1).

Alignment of the deduced amino acid sequence of V. vulnificus with

GroEL protein sequences of other vibrios, E. coli and S. enterica Typhi

(Fig. 1) showed extensive similarity throughout its length, confirming its

identity as a GroEL homolog.

GroEL proteins are strong human leukocyte antigens (HLA), and are

associated with inflammatory diseases, such as ankylosing spondylitis

(Cancino-Diaz et al, 1998). Epitopes with a strong affinity for HLA have

been identified using computer software (Parker et al, 1994). In this study,

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the GroEL proteins of V. vulnificus, V. cholerae, V. parahaemolyticus, E.

coli, and S. enterica Typhi were compared. These pathogens differ in

pathogenesis, however, when the motifs for different HLA molecules

were analyzed, their GroEL proteins exhibited similar but not identical

patterns. All the motifs for the HLA-B27 molecule have an arginine

residue in the second position of the nonapeptides (Cancino-Diaz et al,

1998). Twenty-three arginine residues were present in the GroEL-like

protein of V. vulnificus; therefore, 23 nonapeptides with affinity for the

HLA-B27 molecule, were identified; among these, three showed high

affinity, namely residues 57-65 (AREIELEDK, score of 2000, score is the

estimation of half time of disassociation of a molecule containing this

sequence), residues 284-292 (RRKAMLQDI, score of 1800) and residues

444-452 (LRAMEAPLR, score of 1000). All the GroEL-like proteins of

these three Vibrio species exhibited similar motif sequences, except for

the nonapeptide residues 117-125 (KRGIDKAVA), in which the V.

cholerae GroEL-like protein had a isoleucine rather than an alanine

residue in the last position and had an affinity score increased from 600 to

1800. Motif sequences of GroEL-like proteins of these three Vibrio

species differed from those of E. coli and S. enterica Typhi by a single

nonapeptide (residues 12-20, ARVKMLEGV) with a glutamic acid as a

substitute for an arginine.

Three B cell epitopes, corresponding to those of E. coli, were identified

in the GroEL-like proteins of V. vulnificus, V. cholerae and V.

parahaemmolyticus species (Cancino-Diaz et al, 1998); they were

residues 283-291 (DRRKAMLQD), 361-369 (DKEKLQERV) and

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390-398 (KEKKDRVED) with only a single amino acid switch in the last

two sequences (Fig. 1). The arginine and alanine in residue 362 and 394

of the E. coli GroEL were replaced by lysine and aspartic acid,

respectively, in the GroEL-like proteins of these three vibrios. The HLA

and B cell epitope analysis showed that the antigenic properties of

GroEL-like protein of V. vulnificus were similar to other GroEL proteins.

The hydrophobicity of the GroEL-like protein amino acid sequence of

V. vulnificus (Fig. 2A) was compared with the E. coli GroEL (Fig. 2B).

Identical hydrophobicity profile was observed in both sequences,

although amino acid residues 269 to 282 of the V. vulnificus GroEL-like

protein were significantly more hydrophobic than the relative segment of

E. coli GroEL (Fig. 2B). In this segment, two basic lysine residues were

replaced by two hydrophobic isoleucine residues in the V. vulnificus

GroEL (Fig. 1). GroEL proteins are chaperonins forming ring-shaped

oligomeric protein complexes that are of crucial importance for protein

folding in vivo. The interior cavity of GroEL offers a hydrophilic-like

environment to the substrate protein (Betancourt and Thirumalai 1999).

The hydrophobic portion of the GroEL protein sequence is involved in

the recognition and stabilization of the protein and substrate complexes

(Fenton and Horwich 1997; Sparrer et al, 1996). Such enhancement of

hydrophobicity of certain segments of the GroEL protein may affect its

chaperonin activity.

The three-dimensional configuration of the V. vulnificus GroEL-like

protein was also generated by the Expert Protein Analysis System of

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Swiss Institute of Bioinformatics and showed similar spatial

configuration with the GroEL of E. coli (data not shown).

The groEL-like gene of V. vulnificus was excised by BamH1

digestion and cloned into the QIAexpressionist pQE-30 expression vector

and transformed into E. coli JM109. Expression of the His-tagged V.

vulnificus GroEL-like protein was analyzed and confirmed by gel

electrophoresis followed by immunoblotting with anti-GroEL or

Penta-His antibody. It was slightly larger than the deduced 57.7 kDa

GroEL-like protein (Fig. 3). Expression of the V. vulnificus GroEL-like

protein was induced by IPTG for 30 min to two hours, and a high

quantity of GroEL-like protein was detected that reached a maximum

level in about one hour (Fig. 3).

The IPTG-induced bacterial cultures were lysed and the soluble and

insoluble fractions were analyzed. Only small amounts of V. vulnificus

GroEL-like protein were detected in the soluble fraction, while large

amounts were in the insoluble fraction (Fig. 3). GroEL and DnaK are

known to be antagonist controllers of inclusion body formation by

promoting and preventing, respectively, the aggregation of proteins in the

cytoplasm (Carrio and Villaverde 2003). It was thus reasonable to

observe the presence of inclusion bodies of the over-produced V.

vulnificus GroEL-like protein while DnaK protein did not simultaneously

enhance inclusion body formation. The solublized form may be produced

by reducing the IPTG stimulation or by simultaneous over-producing

both GroEL or DnaK proteins to obtain the functional V. vulnificus

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GroEL-like protein for further study.

In conclusion, the GroEL-like protein gene of V. vulnificus was cloned,

sequenced and over-produced by IPTG induction, largely in an inclusion

body form. Properties of this GroEL-like protein were similar to other

corresponding proteins of other bacterial species.

ACKNOWLEDGEMENT

The authors would like to thank the National Science Council of the

Republic of China (Contract No. NSC91-2311-B-031-001) for financially

supporting this research.

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Lee SE, Shin SH, Kim SY, Kim YR, Shin DH, Chung SS, Lee ZH, Lee

JY, Jeong KC, Choi SH and Rhee JH. 2000. Vibrio vulnificus has the

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Lindler LE and Hayes JM. 1994. Nucleotide sequence of the Salmonella

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Table 1. Comparison of the amino acid sequence of the GroEL-like

protein of Vibrio vulnificus with other published bacterial sequences.

SpeciesGenBank Accession

NumberIdentity, %

Vibrio vulnificus YJ016 AP005342 97

Vibrio vulnificus CMCP6 AE016801 96

Vibrio parahaemolyticus AAF27528 92

Vibrio cholerae AAF95805 91

Escherichia coli O157:H7 BAB38547 84

Pasteurella multocida AAK03191 84

Actinobacillus

actinomycetemcomitansBAA05977 84

Salmonella enterica Typhi AAA85277 84

Enterobacter aerogenes BAA25215 83

Haemophilus influenzae AAC22201 83

Klebsiella pneumoniae BAA25225 83

Yersinia enterocolitica S26423 82

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Legends for Figures

Fig. 1. Aligment of the amino acid sequence of the GroEL-like protein of

Vibrio vulnificus with the corresponding published sequences of

V. parahaemolyticus (VP), V. cholerae (VC), Salmonella enterica

serotype Typhi (SE) and Escherichia coli (EC). Positions that are

identical in all five bacterial species are noted with asterisks.

Gaps have been introduced to maximize similarity. The

hydrophobic amino acid residues 269 to 282 are in bold. The

underlined amino acid sequences are the predicted B cell

epitopes.

Fig. 2. Hydrophobicity of the amino acid sequences of the GroEL-like

protein of Vibrio vulnificus (A) and Escherichia coli (B). The

score represents the hydrophobicity of the amino acid residue in

the sequence. Higher the number represent higher hydrophobicity

of the residue.

Fig. 3. Expression of the recombinant GroEL-like protein of Vibrio

vulnificus in the soluble (A) and insoluble fraction (B). The

bacterial culture was induced by ITPG for 30 min (lane 1), 1 h

(lane 2) and 2 h (lane 3), and the proteins separated into soluble

and insoluble fractions (B) and visualized by immunoblotting

with Penta-His antibody. Lane M contains the prestained protein

ladder markers (Gibco-BRL), 79.6, 61.3 and 49.0 kDa, from top.

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VV MAAKDVKFGNDARVKMLEGVNILADAVKVTLGPKGRNVVLDKSFGAPTITKDGVSVAREIVP .....................V......................................VC ......R.....................................................SE .................R...V......................................EC .................R...V......................................

****** ********** *** **************************************VV ELEDKFQNMGAQMVKQVASQANDVAGDGTTTATVLAQAIVNEGLKAVAAGMNPMDLKRGIVP ...............E...K...A....................................VC ...............E.......A....................................SE ......E........E...K...A.............S.IT...................EC ......E........E...K...A...............IT...................

****** ******** *** *** ************* * *******************VV DKAVAAAVEELKAMSKDCSTSTEIEQVGTISANSDSSVGKIIAEAMEKVGRDGVITVEEGVP .........Q..EL.VE.NDTKA.AQ.............N.......R............VC ....I........L.VP.ADTKA.A..............N....................SE .............L.VP..D.KA.A..........ET...L.....D...KE......D.EC ....T........L.VP..D.KA.A..........ET...L.....D...KE......D.

**** **** *** * * * ********* ** ***** ** ****** *VV QALHDELDVVEGMQFDRGYLSPYFINNQESGSVELESPFFLLVDKKISNIRELLPALEAVVP ...Q.........................A......N.......................VC ...Q...............................DN..I...............V..G.SE TG.Q......................KP.T.A.......I..A.........M..V....EC TG.Q......................KP.T.A.......I..A.........M..V....

* ********************** * * *** ** ** ********* ** ** *VV AKASRPLLIIAEDVEDEALATLVVNNMRGIVIVAAVIAPGFGDRRKAMLQDIAILTGGTVVP ...............................K............................VC .........V.....G...............K....K.....................V.SE ...GK..........G.........T.....K....K................T......EC ...GK..........G.........T.....K....K................T......

*** **** ***** ********* ***** **** **************** **** *VV ISEEVGLELEKATLEDLGQAKRVSITKENTTIIDGVGEEAMIQGRVAQIRQQIEDATSDYVP ....I..............................A........................VC ....I........................S.....A.DQ.A.............E.....SE ....I.M................V.N.DT...........A.............E.....EC ....I.M................V.N.DT...........A.............E.....

**** * **************** * * ***** * * ************* *****VV DKEKLQERVAKLAGGVAVIKVGAATEVKMKEKKDRVEDALHATRAAVEEGIVAGGGVALIVP ...........................E......................V.........VC ...........................E......................V.........SE .R.........................E.....A................V.........EC .R.........................E.....A................V.........

* ************************* ***** **************** *********VV RAASKIVDLQGDNEEQNVGIRVALRAMEAPLRQITKNAGDEESVVANNVRAGEASYGYNAVP .........E...........................................G......VC .....LSS.V........................V..................GN.....SE .V....A..K.Q..D.....K.............VL.C.E.P.....T.KG.DGN.....EC .V...LA..R.Q..D.....K.............VL.C.E.P.....T.KG.DGN.....

* *** * * ** ***** ************* * * * ***** * * *****VV ATGVYGDMLEMGILDPTKVTRSALQFAASVAGLMITTEAMVTDLPQKDS----GMPDMGGVP ...E...........................................E.AGMPD.GG...VC ........I...............................I.E..K..AP---A....-.SE ..EE..N.ID...............Y............C......KS.A------..L.AEC ..EE..N.ID...............Y............C......KN.AADLGAAGG...

** ** * *************** ************ * * **VV MGGMGGMVP .......VC .......SE A......EC .......

******

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Fig. 1

21

A

B

Fig. 2

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A

M 1 2 3

B

M 1 2 3

Fig. 3