Short Communication

DNA Sequencing of a Plasmid
with Virulence from Marine Fish Pathogen Vibrio anguillarum

WU Hai-Zhen, ZHANG Hui-Zhan*,
LÜ Cai-Xia, LIANG Na, JIN Hui-Yi, MA Yue, ZHANG Yuan-Xing

(
State Key Laboratory of Bioreactor Engineering, ECUST, Shanghai
200237, China )

Abstract        DNA
sequence of a plasmid pEIB1 associated with virulence from the marine fish
pathogen Vibrio anguillarum  was
determined using the methods of restriction endonuclease digestion, subcloning,
and primer walking. The whole length of obtained pEIB1 DNA sequence was 66 164
bp, and the overall G+C content of DNA sequence is 42.7%. This sequence encodes
44 open reading frames containing the genes of DNA replication, biosynthesis
and regulation of the siderophore anguibactin and transport of
ferric-anguibactin complexes.

Key
words     Vibrio
anguillarum; virulence plasmid; DNA sequencing; open reading frame

Vibriosis is one
of the most prevalent fish diseases caused by bacteria belonging to the genus
Vibrio  in farmed and wild marine
fish[1]. Outbreaks affecting close to 48 species of salt-water fish have been
reported in more than 14 countries[2]. The most important aetiological agent of
vibriosis occurring in farmed marine fish in the salt or brackish water is
Vibrio anguillarum, a polarly flagellated, Gram-negative, curved rod with a
guanine plus cytosine (G+C) content of 43%－46%[1]. V. anguillarum causes a fatal hemorrhagic septicemic disease
in salmonids and other fish including eels[3], which shows striking
similarities to the septicemic disease in humans caused by Vibrio
vulnificus[4,5].

Iron is an
essential element for nearly all microorganisms, yet in biological fluids it
exists only as a complex with iron-binding proteins, making it essentially
unavailable[3,6]. In the host cells, most iron is not freely available, and
concentrations of free ferric iron are about 10－18 mol/L, which is much lower than that a bacterium needs for
growth. Therefore, invasive microorganisms must have the ability to overcome
this iron limitation in order to grow in their hosts[7]. The key feature that
enables many pathogenic strains of V. anguillarum  seretypes O1 to survive this host imposition is a 65 kb
virulent plasmid. This plasmid encodes a specific iron-uptake system that could
help bacteria overcome nutritional immunity, one of the non-specific defense
mechanisms in the host[3,8－10]. When V. anguillarum loses the virulent plasmid or an essential
component of plasmid in it is mutated, its virulence of to establish infections
decreases enormously[8,11].

In this work,
the whole sequence of the virulence pJM1-like plasmid named pEIB1 from V.
anguillarum  was determined and
analyzed, which provides a good basis for further researching the virulent
mechanism and developing avirulent live vaccine.

1    Materials and Methods

1.1   Plasmids, strains and culture
conditions

Strain: Vibrio
anguillarum  MVM425 (wild type, seretype
O1) was isolated from the marine fish pathogen of vibriosis occurring in the
high density cultured Lateolabrax japonicus in the Huanghai Sea close to
Shangdong province; E. coli JM83 was used as host in our lab.

Plasmid: pEIB1 is pJM1-like plasmid from
Vibrio anguillarum strain MVM425; pUC18 and pSPORT1 are used as vectors in our
lab.

Culture condition: V. anguillarum strains
were grow at 28 ℃ for 24 h on
Luria agar or in Luria broth, respectively containing 2% NaCl; E. coli was
cultured at 37 ℃ for one
night in Luria broth or on Luria agar. when needed ampicillin was added at 100
mg/L.

1.2   General DNA procedures

DNA
purification, restriction endonuclease analysis, DNA ligations and
transformations, PCRs, and agarose gel electrophoresis were performed according
to standard protocols[12].

DNA sequences
were determined using the method of the dideoxy chain-termination[13] and
completed by Bioasia (Shanghai, China).

1.3   Isolation of the virulent plasmid
pEIB1

Purification of
the plasmid pEIB1 was performed using the Large-construct kit of Qiagen
Company.

1.4   Sequencing stratagem

DNA sequence of
pEIB1 was determined using the methods of restriction endonuclease digestion
and subcloning and primer walking[13]. DNA sequence splicing was accomplished
with the program SeqMan among the DNA analysis package DNAStar (DNAStar Inc., Madison,
WI). The identification of putative open
reading frames (ORFs) was made using the graphical analysis tool named ORF
finder on NCBI website.

2    Results

2.1   Restriction endonuclease analysis
of plasmid pEIB1

The virulence
plasmid pEIB1 was digested with 11 restriction enzymes separately. The results
are shown in Fig.1. Size of most fragments from BamHI, EcoRI and PstI
digestions were among 1－15 kb. These fragments were suitable to subclone into the vectors
such as pSPORT1.

Fig.1       Physic
map of pEIB1

H, HindIII; X, XhoI; P, PstI; E, EcoRI; Bg,
BglII; Xb, XbaI; Sa, SalI; Sc, SacI; S, SphI; K, KpnI; B, BamHI; M, Marker.

2.2   Construction and identification of
the subclone

The virulence plasmid pEIB1 (>1
μg) was completely digested with BamHI, EcoRI or PstI, and then deposited
respectively. These short or long restriction fragments were cloned into
pSPORT1, and the resulted recombinants containing different fragments are shown
in Table 1.

Table 1   Recombinants
containing the fragments from pEIB1

Recombinants
BII51, EI32, EI12 and P8 containing larger fragments (>7.0 kb) were
cleavaged with different restriction endonucleases and subcloned respectively. These
subclones and the other clones listed in Table 1 were directly sequenced. All
DNA sequences were spliced with program SeqMan. With known DNA fragments as
probes, DNA walking was also performed using the clones from PstI digestion as
templates. At last, the whole DNA sequences of plasmid pEIB1 were assembled
(shown in Fig.2). Among them, the clones EII36-EI32, BII51-BI150 and
EII37-EI7-EI12 were integrated into one part with gap1, gap2-2 and gap3-2,
which were accomplished through directly sequencing the products of PCR. The B4
fragment was not cloned for some reasons and sequenced with primer walking.

Fig.2       Fragment
assembly of the whole DNA pEIB1

2.3   DNA sequence analysis of plasmid
pEIB1

The whole
sequence of plasmid pEIB1, comprising 66164 bp was obtained, (GenBank accession
No: AY255699). The overall G+C content of this DNA sequence is 42.7%, which is
very similar to the Vibrio anguillarum chromosome (G+C) content of 43%－46%[1]. Using ORF finder on NCBI
website, 44 open reading frames (>400 bp) containing the genes of DNA
replication, biosynthesis and regulation of the siderophore anguibactin and
transport of ferric-anguibactin complexes were found. A scheme of pEIB1 gene
organization is shown in Fig.3.

Fig.3       Gene
map of plasmid pEIB1

ORFs are represented by arrows. Among them, ORF 1, 4, 7, 19－26, 29 and 31 are
similar to the proteins in the public databank; 1, 7, 9, 12, 14, 17, 19, 29,
30, 34, 37 and 42 are the putative or identified transposases.

3    Discussion

Shotgun is a
popular method to sequence the genome of large plasmid and virus. The main
advantage is that about 90% DNA sequence of genome can be rapidly achieved. But
the remaining part cannot be easily obtained. In this work, using the methods
of restriction endonuclease digestion, subcloning and primer walking, the whole
DNA sequence of pEIB1 comprising 66 164 bp was finally obtained. And it was
found that there are many overlapping DNA fragments in pEIB1 that are very
useful for researching the virulent mechanism.

As a result,
several features of pEIB1 were also found. (1) Genes fatD, C, B, A of the iron
uptake system of V. anguillarum were included in a transposon-like structure,
as same as angR and angB[14,15]. (2) The proteins encoded by ORF 9 and 34 or
ORF 37 and 42 were identical and similar to the probable transposases (GenBank
accession No: AAN66262.1 & S15163) in public databank, with the identities
of 56% and 66% respectively. But they were dissimilar to the transposases
ISV-A1 and ISV-A2[14,15] which were already found in V. anguillarum. There were
many potential transposon-like structures in pEIB1, which suggests a disturbing
possibility that these virulence determinants may be transposable in some
cases. From these results, it can be deferred that the whole DNA sequences of
virulent plasmid pEIB1 will be great helpful to future revealing the iron
uptake system evolvement in V. anguullarum.

References

1     Actis
LA, Tolmasky ME, Crosa JH. In: Woo PK, Bruno DW eds. Fish Diseases and
Disorders, Vol. 3. CAB Wallingford, UK:
International Publishing, 1999

2     Austin
B, Austin DA. Bacterial Fish
Pathogens: Disease in Farmed and Wild Fish. 3rd ed. Chichester,
UK: Parxis Publishing
Ltd., 1999

3     Crosa
JH. A plasmid associated with virulence in the marine fish pathogen Vibrio
anguillarum specifies an iron-sequestering system. Nature, 1980, 284(5756): 566－568

4     Linkous
DA, Oliver JD. Pathogenesis of Vibrio vulnificus. FEMS Microbiol Lett, 1999,
174(2): 207－214

5     Strom
MS, Paranjpye RN. Epidemiology and pathogenesis of Vibrio vulnificus. Microbes
Infect, 2000, 2(2): 177－188

6     Bullen
JJ. Griffiths E eds. Iron and
Infection: Molecular, Physiological and Clinical Aspects, 2nd ed. West
Sussex, UK:
John Wiley and Sons Ltd., 1999

7     Crosa
JH. Signal transduction and transcriptional and posttranscriptional control of
iron-regulated genes in bacteria. Microbio Mol Bio Rev, 1997, 61(3): 319－336

8     Crosa
JH, Hodges LL, Schiewe MH. Curing of a plasmid is correlated with an
attenuation of virulence in the marine fish pathogen Vibrio anguillarum. Infect
Immun, 1980, 27(3): 897－902

9     Crosa
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the fish pathogen Vibrio anguillarum. Infect Immun, 1997, 18(2): 509－513

10   Crosa
JH, Walsh CT. Genetics and assembly line enzymology of siderophore biosynthesis
in bacteria. Microliol Mol Biol Rev, 2002, 66(2): 223－249

11    Wertheimer
AM, Verweij W, Chen Q, Crosa LM, Nagasaw M, Tolmasky ME, Actis LA et al.
Characterization of the angR gene of Vibrio anguillarum: Essential role in
virulence. Infect Immun, 1999, 67(12): 6496－6509

12    Ausubel
FM, Brent R, Kingston RE, Moore
DD, Seidman JG, Smith JA, Struhl K. Short Protocols in Molecular Biology, 3rd
ed. Canada:
John Wiley & Sons, Inc., 1995

13    Maniatis
T, Fritsch EF, Sambrook J eds. Molecular Cloning: A Laboratory Manual. Cold
Spring Harbor, NY: Cold Spring
Harbor Laborat ory, 1982

14    Tolmasky
ME, Crosa JH. Iron transport genes of the pJM1-mediated iron uptake system of
Vibrio anguillarum are included in a transposonlike structure. Plasmid, 1995,
33(3): 180－190

15    Welch
TJ, Chai S, Crosa JH. The overlapping angB and angG genes are encoded within
the trans-acting factor region of the virulence plasmid in Vibrio anguillarum:
Essential role in siderophore biosynthesis. J Bacteriol, 2000, 182(23): 6762－6773

_______________________________________

Received: June
2, 2003  Accepted: July
9, 2003

This work was supported by a grant from the
National High-Technology Programs 
of China
(No. 2001AA622020)

* Corresponding author: Tel,
86-21-64252515; Fax, 86-21-64252255; e-mail, [email protected]

Updated at: 2003-10-09

鳗弧菌毒力质粒DNA序列的测定

吴海珍     张惠展*    吕彩霞     梁娜    金慧怡     马悦    张元兴

( 华东理工大学生物反应器工程国家重点实验室, 上海 200237 )

摘要       采用亚克隆法与引物步移法相结合的测序战略, 对海洋鱼类重要病原菌鳗弧菌毒力质粒pEIB1进行序列测定, 测得整个质粒序列长度为66 164 bp。 序列的初步分析结果表明, G＋C含量为42.7%, 共有44个可读框（ORF）, 其中包括与铁载体合成、调节、运输以及质粒复制相关的基因。

关键词   鳗弧菌； 毒力质粒； 序列测定； 可读框