Download Molecular analysis of genetic differences between

Microbiology (2000), 146, 999–1009
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Molecular analysis of genetic differences
between virulent and avirulent strains of
Aeromonas hydrophila isolated from diseased
fish
Y. L. Zhang, C. T. Ong and K. Y. Leung
Author for correspondence : K. Y. Leung. Tel : j65 8747835. Fax : j65 7792486.
e-mail : dbslky!nus.edu.sg
Department of Biological
Sciences, Faculty of
Science, National
University of Singapore,
10 Kent Ridge Crescent,
Singapore 119260
Aeromonas hydrophila, a normal inhabitant of aquatic environments, is an
opportunistic pathogen of a variety of aquatic and terrestrial animals,
including humans. A. hydrophila PPD134/91 is defined as virulent whereas
PPD35/85 is defined as avirulent on the basis of their different LD 50 values in
fish. Suppression subtractive hybridization (SSH) was used to identify genetic
differences between these two strains. Sixty-nine genomic regions of
differences were absent in PPD35/85, and the DNA sequences of these regions
were determined. Sixteen ORFs encoded by 23 fragments showed high
homology to known proteins of other bacteria. ORFs encoded by the remaining
46 fragments were identified as new proteins of A. hydrophila, showing no
significant homology to any known proteins. Among these PPD134/91-specific
genes, 22 DNA fragments (21 ORFs) were present in most of the eight virulent
strains studied but mostly absent in the seven avirulent strains, suggesting
that they are universal virulence genes in A. hydrophila. The PPD134/91-specific
genes included five known virulence factors of A. hydrophila : haemolysin
(hlyA), protease (oligopeptidase A), outer-membrane protein (Omp),
multidrug-resistance protein and histone-like protein (HU-2). Another 47 DNA
fragments (44 ORFs) were mainly present in PPD134/91, indicating the
heterogeneity among motile aeromonads. Some of these fragments encoded
virulence determinants. These included genes for the synthesis of O-antigen
and type II restriction/modification system. The results indicated that SSH is
successful in identifying genetic differences and virulence genes among
different strains of A. hydrophila.
Keywords : Aeromonas hydrophila, genomic subtraction, virulence genes
INTRODUCTION
Aeromonas hydrophila is the causative agent of motile
aeromonad septicaemia, which occurs in a wide variety
of freshwater fish species (Thune et al., 1993 ; Austin &
Adams, 1996). Outbreaks usually occur only when the
fish are immunocompromised by stresses such as overcrowding or concurrent disease (Stevenson, 1988). The
pathogenesis of A. hydrophila is multifactorial. A.
hydrophila produces several toxins including haemolysins and enterotoxin (Chakraborty et al., 1984 ; Howard
.................................................................................................................................................
Abbreviations : R/M, restriction/modification ; SSH, suppression subtractive hybridization.
The GenBank accession numbers for the sequences determined in this work
are given in Table 5.
et al., 1996), and a repertoire of enzymes which digest
cellular components, such as proteases, amylases and
lipases (Leung & Stevenson, 1988a ; Pemberton et al.,
1997). Other virulence factors such as the S layer (Dooley
& Trust, 1988), ability to internalize (Tan et al., 1998),
serum resistance (Mittal et al., 1980 ; Janda et al., 1984)
and resistance to phagocyte-mediated killing (Leung et
al., 1995a) are also implicated in the resistance of A.
hydrophila to the host’s non-specific immune defences.
A. hydrophila strains PPD134\91 and PPD35\85 were
isolated from our local environment and used in our
previous studies. A. hydrophila PPD134\91 was defined
as virulent whereas PPD35\85 was defined as avirulent
on the basis of their differences in LD values in tilapia
&! PPD134\91 can
and blue gourami (Leung et al., 1995b).
survive and proliferate in tilapia serum and phagocytes
0002-3723 # 2000 SGM
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Y. L. Z H A N G, C. T. O N G a n d K. Y. L E U N G
(Leung et al., 1995a, b), and it can internalize and induce
severe morphological changes in EPC (epithelioma
papillosum of carp) cells (Tan et al., 1998). On the other
hand, A. hydrophila PPD35\85 is serum-sensitive, unable to invade or produce cytotoxic effects in EPC cells,
and is incapable of surviving in phagocytes after
opsonization.
It is important to determine whether there are any
special virulence genes present in A. hydrophila
PPD134\91 but not in PPD35\85. Subtractive hybridization has been used to identify sequences that
are present in one genome but absent in another (Straus
& Ausubel, 1990 ; Mahairas et al., 1996). The analysis of
the differences between two complex genomes holds
promise for the discovery of unknown virulenceassociated factors and probes useful for genetic studies
(Lisitsyn et al., 1993 ; Quinn et al., 1997). Traditional
subtractive hybridization methods involved several
rounds of hybridization and physical separation of
single-stranded and double-stranded DNA. Recently a
new technique called suppression subtractive hybridization (SSH) overcame some of these limitations.
The step of suppression PCR can prevent undesirable
amplification while enrichment of target molecules
proceeds (Diatchenko et al., 1996 ; Gurskaya et al.,
1996).
The objective of this study was to identify genetic
differences between virulent and avirulent strains of A.
hydrophila. Sixty-nine subtracted genomic regions
unique to PPD134\91 were identified. Among them, 22
fragments (21 ORFs) were present in most of the virulent
strains. These presumptive virulence genes were identified, sequenced, and their biological functions analysed.
This will form a foundation for further studies in
elucidating how A. hydrophila causes disease in humans
and fish.
METHODS
Bacterial strains, plasmids, and growth conditions. The
bacterial strains and plasmids used in this study are listed in
Table 1. The characteristics and virulence of A. hydrophila
strains studied were determined previously (Mittal et al.,
1980 ; Leung & Stevenson, 1988b ; Leung et al., 1995b).
Virulent strains were defined as having a lower LD value in
blue gourami or rainbow trout ( 10'n&) than the&!avirulent
strains ( 10(n&). A. hydrophila strains were maintained on
tryptic soy agar (TSA) (Difco) or in tryptic soy broth (TSB)
(Difco) at 25 mC. Escherichia coli strains were maintained on L
agar (Difco) or in Luria broth (LB) (Difco) at 37 mC. As
necessary, media were supplemented with antibiotics (Sigma)
at the following concentrations : ampicillin at 50 µg ml−" and
kanamycin at 50 µg ml−". Bacteria were stored as frozen
cultures at k80 mC in either TSB or LB containing 25 % (v\v)
glycerol.
DNA manipulations. Bacterial genomic DNA was extracted
according to the manual of the QIAGEN Genomic DNA Kit
and the Wizard Genomic DNA Purification Kit (Promega).
Plasmid DNA was prepared by using Qiagen and Promega
columns. Restriction endonuclease digestion was accomplished by standard methods (Sambrook et al., 1989).
subtractive hybridization (SSH). Bacterial
genome subtraction was performed following the user manual
of the PCR-Select Bacterial Genome Subtraction Kit
(Clontech). Briefly, the tester (PPD134\91) and driver
(PPD35\85) genomic DNAs were digested with RsaI. The
tester DNA was then subdivided into two portions, each of
which was ligated with a different adaptor provided by the
subtraction kit. Two hybridizations were performed. In the
first, an excess of driver was added to each adaptor-ligated
tester sample. The samples were then heat-denatured and
allowed to anneal. In the second hybridization, the two
primary hybridization samples were mixed together without
denaturing. The entire population of molecules was then
subjected to PCR to amplify the tester-specific sequences. The
PCR amplification product was cloned into pT-Adv and
transformed into E. coli TOP10Fh competent cells according
to the manual of the Advantage PCR Cloning Kit (Clontech).
Positive clones were screened on LB medium supplemented
with X-Gal (Sigma), IPTG (Sigma) and ampicillin.
Southern hybridization. Southern blots were performed to
identify the subtractive clones that contained PPD134\91
unique fragments. At the same time, similar probes were used
to screen genomic DNA of several virulent and avirulent
strains of A. hydrophila. Nick-translated RsaI fragments of
recombinant plasmids were used as probes to hybridize with
RsaI digests of PPD134\91, PPD35\85, and other bacterial
genomic DNA. Transfer of DNA to nylon membrane
(GeneScreen, NEM Research Products) and hybridization
conditions were in accordance with standard methods
(Sambrook et al., 1989). Probe DNAs were labelled by nicktranslation with biotin-14-dATP (BioNick Labelling System,
Gibco-BRL) and visualized with strepavidin–alkaline phosphate conjugate (BluGene Nonradioactive Nucleic Acid Detection System, Gibco-BRL).
DNA sequencing. DNA sequencing was carried out on an
Applied Biosystems PRISM 377 automated DNA sequencer by
the dye termination method. The ABI PRISM dRhodamine
Terminator Cycle Sequencing Ready Reaction Kit was used
(Applied Biosystems). The sequences were edited by using the
manufacturer’s software. Sequence assembly and further
editing were done with  DNA analysis software.
 and  sequence homology analyses were performed by using the National Centre for Biotechnology
Information  network service.
Genome walking. Unknown genomic DNA sequences adjacent to the subtracted DNA fragments (F3, 33, 44, 46, 52,
101, 106, 108, 109 and 121) were identified following the user
manual of the Universal GenomeWalker Kit (Clontech).
GenomeWalker libraries using five restriction enzymes (DraI,
EcoRV, PvuII, ScaI and StuI) were constructed and two PCR
amplifications were performed for the DNA walking. Specific
primers were synthesized by Gibco-BRL for primary (P) and
nested (N) PCR as well as for right (R) and left (L) sides of
each subtracted fragment. PCR was performed using an
Advantage Tth Polymerase Mix purchased from Clontech and
following two-step cycle parameters : 7 cycles of 25 s at 94 mC,
3 min at 72 mC, 32 cycles of 25 s at 94 mC and 3 min at 67 mC.
The oligonucleotides for genome walking were as follows : F3LP, 5h-GCATCCCCGTTTCGCATTATC-3h, F3-LN, 5h-CCGTTTCGCATTATCTGAAC-3h, F3-RP, 5h-GATAATGCGAAACGGGGATGC-3h, F3-RN, 5h-CGGGGATGCCACGGCATCC-3h ; F33-LP, 5h-ATTTGATGCGCTTTTGTCCC-3h,
F33-LN, 5h-CGCTTTTGTCCCATTGACAG-3h, F33-RP, 5hTCGTTGCTTTTGGGTTACCAAG-3h, F33-RN, 5h-GGGTTACCAAGATACTACGTTC-3h ; F44-LP, 5h-ACACTCCCACGTCGTTTTAC-3h, F44-LN, 5h-GCTGTTTTACTCASuppression
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Screening virulence genes of A. hydrophila
Table 1. Bacterial strains and vectors used in this study
Strain or plasmid
Genotype and/or clone description*
Source†
A. hydrophila
ATCC 7966
L15
L31
L36
L37
PPD35\85
PPD11\90
PPD64\90
PPD88\90
PPD45\91
PPD70\91
PPD122\91
PPD134\91
TF7
Xs91\4\1
Wild-type, virulent, type strain
Wild-type, avirulent
Wild-type, virulent
Wild-type, avirulent
Wild-type, avirulent
Wild-type, avirulent
Wild-type, virulent
Wild-type, avirulent
Wild-type, avirulent
Wild-type, avirulent
Wild-type, virulent
Wild-type, virulent
Wild-type, virulent
Wild-type, virulent
Wild-type, virulent
ATCC
BAU, Indonesia
BAU, Indonesia
BAU, Indonesia
BAU, Indonesia
PPD, Singapore
PPD, Singapore
PPD, Singapore
PPD, Singapore
PPD, Singapore
PPD, Singapore
PPD, Singapore
PPD, Singapore
UG, Canada
IHB, China
E. coli
TOP10Fh
Amps Kans
Clontech
Subtracted fragments cloned into the RsaI
sites of the pT-Adv vector
Ampr Kanr LacZ
This study
Plasmids
pSC1–129‡
pT-Adv Vector
Clontech
* Virulent strains were defined as having a lower LD value in blue gourami or rainbow trout ( 10'n&)
&!
than the avirulent strains (10(n&).
† ATCC, American Type Culture Collection ; BAU, Bogor Agricultural University of Indonesia ; IHB,
Institute of Hydrobiology, China ; PPD, Primary Production Department of Singapore ; UG, University
of Guelph, Canada.
‡ The 14 empty clones or false positives were as follows : pSC4, 5, 19, 24, 27, 41, 67, 113, 115, 116, 118,
122, 125 and 127.
TAGCTAC-3h, F44-RP, 5h-GCTTGCATCGTTATGCGTCTGT-3h, F44-RN, 5h-GCGTCTGTAGCTATCAATGTG-3h ;
F46-RP, 5h-GCAGGTGGGGAAATCGATGAAC-3h, F46RN, 5h-TCGATGAACTCAAGGCGGT-3h ; F52-LP, 5h-TGGAGTGATGTCGCGTGCGG-3h, F52-RP, 5h-GTAATCCCCAAAACCCG-3h ; F101-LP, 5h-CATCATCGTCAGAAAATGCG-3h, F101-LN, 5h-AGAAAATGCGTTATTTCTAC-3h,
F101-RP, 5h-CTAAGATTGTGTCTGCGAGTG-3h, F101RN, 5h-GTCTGCGAGTGATAAACAAAAG-3h ; F106-LP, 5hTGC TCT CAT TGC TGG GGG GC-3h, F106-LN, 5hGCTGGGGGGCACCTTGTCCAC-3h, F106-RP, 5h-ACATCTAGCGCACGAGATAAAT-3h, F106-RN, 5h-CACGAGATAAATCAGGCCCAAGC-3h ; F108-LP, 5h-AGACAAGCAGAATAACGCCCCG-3h, F108-LN, 5h-AATAACGCCCCGAAATATAACCG-3h, F108-RP, 5h-CAGCGGATTGGCGAAGGTATT-3h, F108-RN, 5h-TTGGCGAAGGTATTTATGTTG-3h ; F109-LP, 5h-TCGTCCTTATTTCGGGTAGGGATCAAGCGG-3h, F109-RP, 5h-CTCCTTGGAAGGTAGACCCCGAACTCTACT-3h ; F121-LP, 5h-GCCCATAGCATCCACATCGG-3h, F121-LN, 5h-TCCACATCGGCCGTATATTC-3h, F121-RP, 5h-GATTACCAGAGGTTTGGCCAAT-3h,
F121-RN, 5h-GAGGTTTGGCCAATATTGCCC-3h.
Nucleotide sequence accession numbers. Sixteen ORFs,
which derived from 23 subtracted fragments, had homologues
with known sequences in GenBank and were assigned with 16
accession numbers (Table 5). Seven of these (F3, 33, 46, 52,
101, 106 and 121) represented complete ORFs via genome
walking while nine of them represented partial ORFs.
RESULTS
Genomic subtraction between A. hydrophila
PPD134/91 and PPD35/85
SSH was carried out between the RsaI-digested genomic
DNA of virulent strain PPD134\91 (tester) and avirulent
strain PPD35\85 (driver) with the aim of isolating
PPD134\91-specific genes (virulence genes). Subtracted
clones [E. coli TOP10Fh(pSCT)] and the DNA fragments (FT) were given a number (represented here by
T) for identification (Table 1). One hundred and
twenty-nine clones were obtained from one round of
subtraction and 14 of these were found to have no
inserts (Table 1). Therefore, 115 were confirmed as
positive clones (Table 2).
Southern analysis of subtracted clones
Plasmid DNA was extracted from these subtracted
clones and was digested by RsaI. The molecular masses
of subtracted fragments were measured and divided into
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Table 2. Summary of subtraction and the use of subtracted fragments in Southern
hybridization analysis between A. hydrophila PPD134/91 (tester) and PPD35/85 (driver)
Size of subtracted
genomic fragments (kb)
No. of fragments
0n18–0n68
0n70–1n4
Total
Total
Tester only*
Testerdriver†
False positive‡
90
25
52
17
3
1
35
7
115
69 (60 %)
4 (3n5 %)
42 (36n5 %)
* Subtracted fragments that hybridized only to A. hydrophila PPD134\91. Refer to Tables 3 and 4 for
fragment (F) numbers in this group. When using F51, 60 and 111 as probes for hybridization, two bands
were found on tester genomic DNA.
† Subtracted fragments that hybridized to both A. hydrophila PPD134\91 and PPD35\85. More bands
were found in tester than driver. Fragment numbers : F14, 76, 95 and 105.
‡ Subtracted fragments that hybridized to both A. hydrophila PPD134\91 and PPD35\85. One band
was found in both tester and driver. Fragment numbers : F6, 7, 9, 10, 12, 13, 15, 22, 23, 26, 28, 29, 37,
39, 40, 42, 43, 45, 47, 48, 53, 54, 56, 57, 62, 63, 64, 65, 68, 69, 71, 74, 78, 96, 98, 102, 103, 117, 120, 124,
128 and 129.
33
T
66
D
T
51
D
T
60
D
T
95
D
T
105
D
T
10
D
T
40
D
T
D
.................................................................................................................................................................................................................................................................................................................
Fig. 1. Southern hybridization analysis of subtracted fragments on RsaI genomic digest of A. hydrophila PPD134/91 and
PPD35/85. A. hydrophila PPD134/91 (tester strain) (T) and PPD35/85 (driver strain) (D) were hybridized with biotin-labelled
subtracted fragments. Fragment designations are shown at the top.
two groups : less than 0n69 kb and more than 0n69 kb in
size (Table 2). Their RsaI digests were biotinylated and
hybridized to RsaI-digested tester and driver. These 115
clones could be divided into three groups according to
the results of our Southern analysis (Table 2).
In the first group (tester only), subtracted fragments only
hybridized to the tester genome digest. There were 69
clones (60 % of the total population studied) in this
group. Examples of this group included F33, 51, 60 and
66 (Fig. 1). In F51, 60 (Fig. 1) and 111 (data not shown),
double bands were detected on the tester genomic DNA.
Fragments in the second group (tester driver) could
hybridize to both tester and driver. However, multiple
bands were observed during hybridization with the
tester whereas only one or two bands were found when
hybridized with the driver (Fig. 1). Examples of this
group included F14, 76, 95 and 105 and they represented
3n5 % of the total population. These different hybridization patterns may reflect multiple copies of a
unique gene present in PPD134\91 or restriction fragment polymorphism. The bands formed on both tester
and driver genomic DNA could be of either the same size
(F105, Fig. 1) or different sizes (F95, Fig. 1).
Inserts in the last group (false positive) hybridized to
both tester and driver, and had the same hybridization
patterns (F10 and 40 ; Fig. 1). These clones were false
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Screening virulence genes of A. hydrophila
A
B
C
109
D E
I
J
K
L M N
F
G
H
O
A
B
C
101
D E
F
G
121
I
J
K
L M N
O
H
32
A
B
C
D
E
F
I
J
K
L M N
G
H
O
A
B
C
I
J
K
D
E
F
G
L M N
O
H
.................................................................................................................................................................................................................................................................................................................
Fig. 2. Southern hybridization analysis of subtracted fragments on RsaI genomic digest of virulent and avirulent strains of
A. hydrophila. Virulent strains PPD122/91, PPD70/91, PPD11/90, PPD134/91, L31, Xs91/4/1, TF7 and ATCC 7966 (lanes A to
H), and avirulent strains PPD35/85, PPD45/91, PPD64/90, PPD88/90, L15, L36 and L37 (lanes I to O) were hybridized with
biotin-labelled subtracted fragments F109, F101, F121 and F32, as indicated at the top.
Table 3. Distribution of group 1 PPD134/91 (tester)-specific DNA fragments among virulent and avirulent strains of
A. hydrophila
.................................................................................................................................................................................................................................................................................................................
Hybridization results were scored as : j, strains hybridized with PPD134\91-specific DNA fragments ; k, strains not hybridized with
PPD134\91-specific DNA fragments.
Fragment no. … 61
Virulent strains
ATCC 7966
L31
PPD11\90
PPD70\91
PPD134\91
PPD122\91
TF7
Xs91-4-1
Homology\total†
Avirulent strains
L15
L36
L37
PPD35\85
PPD45\91
PPD64\90
PPD88\90
Homology\total*
58/72
88/89/109
93
34
52/108
8
99/106
20/97
92
32
85
87
11
2/3
j
j
j
j
j
j
j
j
8\8
j
j
j
j
j
j
j
j
8\8
j
j
j
j
j
j
j
j
8\8
j
j
j
j
j
j
j
j
8\8
j
j
j
j
j
j
k
j
7\8
j
j
j
j
j
j
k
j
7\8
j
j
j
k
j
j
k
j
6\8
j
j
j
k
j
j
k
k
5\8
k
j
j
k
j
j
k
k
4\8
k
j
j
k
j
k
k
j
4\8
k
j
j
k
j
j
k
k
4\8
k
k
k
j
j
j
k
j
4\8
j
j
k
j
j
k
k
k
4\8
j
k
j
k
j
k
k
j
4\8
k
j
j
j
k
k
k
j
4\8
k
j
k
k
k
j
k
2\7
k
j
k
k
k
j
j
3\7
k
j
k
k
k
j
j
3\7
k
j
k
k
j
j
j
4\7
k
k
k
k
k
j
k
1\7
k
j
k
k
k
j
j
3\7
j
k
k
k
k
j
k
2\7
k
j
k
k
k
k
k
1\7
k
k
k
k
k
k
k
0\7
k
k
k
k
k
k
k
0\7
k
k
k
k
k
j
k
1\7
k
k
k
k
k
k
j
1\7
k
j
k
k
k
k
k
1\7
k
j
k
k
k
j
k
2\7
k
k
j
k
k
j
j
3\7
* No. of hybridized strains\total no. of strains studied.
positive, which is a result of the inefficiency of subtraction hybridization. Our Southern analysis indicated
that we achieved a high level of enrichment of PPD134\
91-specific DNA whereby 60 % of our subtracted clones
were present only in the tester but not in the driver
(Table 2). In the tester-only group, 75 % of the subtracted fragments were less than 0n69 kb.
Identification of common virulence genes in
A. hydrophila
Southern hybridization was carried out to survey the
distribution of these 69 PPD134\91-specific fragments
among eight virulent and seven avirulent strains of
A. hydrophila. Fig. 2 and Tables 3 and 4 show the
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Table 4. Distribution of group 2 PPD134/91 (tester)-specific DNA fragments among virulent and avirulent strains of
A. hydrophila
.................................................................................................................................................................................................................................................................................................................
Hybridization results were scored as : j, strains hybridized with PPD134\91-specific DNA fragments ; k, strains not hybridized with
PPD134\91-specific DNA fragments.
Fragment no. …
Virulent strains
ATCC 7966
L31
PPD11\90
PPD70\91
PPD134\91
PPD122\91
TF7
Xs91\4\1
Homology\total†
Avirulent strains
L15
L36
L37
PPD35\85
PPD45\91
PPD64\90
PPD88\90
Homology\total†
A* (24) B* (10)
90
38
66
C* (3)
16
94/104
18
73
110
55
k
k
k
k
j
k
k
k
1\8
k
k
k
k
j
k
k
k
1\8
k
k
k
k
j
k
k
k
1\8
k
k
k
k
j
k
k
k
1\8
k
k
k
k
j
k
k
k
1\8
k
k
k
k
j
k
k
j
2\8
k
k
k
k
j
j
k
k
2\8
k
j
k
k
j
k
k
k
2\8
k
k
k
k
j
k
k
j
2\8
k
k
j
k
j
k
k
k
2\8
k
k
k
k
j
k
k
j
2\8
k
j
j
k
j
k
k
k
3\8
k
k
k
k
k
k
k
0\7
k
k
k
k
k
j
k
1\7
k
j
k
k
k
k
j
1\7
k
k
k
k
k
j
k
2\7
k
k
k
k
k
j
j
2\7
k
k
k
k
k
k
k
0\7
k
k
k
k
k
k
k
0\7
k
k
k
k
k
k
k
0\7
k
j
k
k
k
k
k
1\7
k
k
k
k
k
j
k
1\7
k
k
k
k
k
j
k
1\7
k
k
k
k
k
k
k
0\7
* PPDI34\91-specific DNA fragments that showed a similar hybridization pattern were grouped as follows : group A, F1, 21, 30, 31, 33,
35, 46, 50, 59, 60, 70, 77, 79, 80, 81, 82, 83, 91, 100, 101, 107, 112, 114 and 119 ; group B, F25, 36, 44, 51, 75, 86, 111, 121, 123 and 126 ;
group C, F17, 49 and 84.
† No. of hybridized strains\total no. of strains studied.
hybridization results. The fraction of hybridization was
calculated for both virulent and avirulent strains (x\8
for virulent and y\7 for avirulent strains, where x and y
are the number of hybridized strains).
Two groups were derived based on the hybridization
patterns. Group 1 (which represents common virulence
genes, x 4 and y 4, n l 22) consisted of subtracted
fragments that were highly enriched in other virulent
strains of A. hydrophila (Table 3). As seen in F109 and
F32, more bands were found on the genomic digest of
virulent than avirulent strains (Fig. 2). All eight virulent
strains hybridized to F109 while only three of the seven
avirulent strains hybridized to it.
Subtracted fragments that were tester-specific (i.e.
hybridized only to PPD134\91 plus at most two other
strains) were classified as group 2 (Table 4, x 3 and
y 2, n l 47). In F101 and 121, among all the virulent
and avirulent strains, only PPD134\91 and PPD64\90
(for F121 only) hybridized (Fig. 2).
Sequence analysis and gene walking
The 69 subtracted fragments were sequenced and their
DNA sequences were subjected to homology search in
PIR\GenBank. Of the 69 subtracted fragments, 46
(66n7 %) demonstrated no significant matches with
entries in the databases and potentially represented new
and novel virulence determinants in A. hydrophila (data
not shown). In the remaining 33n3 % of the clones, 23
DNA fragments representing 16 ORFs showed high
homology to known proteins of other bacteria and four
identical pairs were found (Table 5).
From the 16 ORFs that had homologues, five belong
to group 1 (F2\3, 52, 106, 108 and 88\109 ; Table 3), 11
to group 2 (30, 33, 36\121, 44\86, 46, 50, 51, 60\
91\101\119, 82, 110 and 126 ; Table 4). Full sequences
were determined by genome walking for F3, 33, 44, 46,
52, 101, 106 and 121 for detailed analysis (Table 5).
Table 5 shows subtracted and full-length fragments
that showed high homology to known sequences in
GenBank.
Five ORFs in group 1 were found to correspond to
known bacterial virulence genes of A. hydrophila. These
include haemolysin (hlyA), protease (oligopeptidase),
histone-like protein (HU-2), outer-membrane protein
(Omp) and multidrug-resistance gene. In group 2, five
groups of genes were found to represent the heterogeneity of motile aeromonads. These include genes for
the synthesis of O-antigen of LPS (phosphomannomutase, rhamnosyl transferase, O-acetyltransferase,
O-antigen methyltransferase, GDP-mannose 4,6dehydratase and mannosyltransferase B), type II
restriction\modification system (modification methyltransferase of PstI and BsuBI), CII of bacteriophage P4,
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Screening virulence genes of A. hydrophila
Table 5. Summary of sequence analysis of PPD134/91-specific DNA fragments
Fragment
no.
ORF size
(bp)
Start–stop
(bp)
Predicted
protein (aa)
2, 3
1423
227–1249
341
AF146597
30
454
3–452
150
AF146606
33
1858
566–1600
345
AF146603
36, 121
1691
258–1388
377
Accession
no.
AF146601
44, 86
1452
436–1419
328
AF148126
46
1079
88–972
295
AF146602
50
800
325–798
158
AF146604
51
965
3–398
132
AF146596
52
489
58–351
98
AF146598
60, 91,
101, 119
82
88, 109
1597
895
1791
346–1590
411–893
3–1328
415
AF146609
161
AF146605
442
AF146608
106
1672
267–1493
409
AF146029
108
518
294–518
75
AF146599
110
126
942
934
1–942
262–933
314
224
AF146607
AF146595
Homologies to predicted
encoded protein
E value†
Homologue
accession no.
Omp of A. salmonicida (OmpAI and
OmpAII) (Costello et al., 1996)
CII protein of bacteriophage P4
(Ghisotti et al., 1990 ; Halling et al.,
1990)
Mannosyltransferase B (rfb) of
Synechocystis sp. (Kaneko et al.,
1996)
Mannosyltransferase B (rfb) of E. coli
(Sugiyama et al., 1994)
Mannosyltransferase B (rfb) of
Salmonella choleraesuis (Brown et al.,
1992)
GDP-mannose 4,6-dehydratase of
V. cholerae
GDP-mannose 4,6-dehydratase of
E. coli
Phosphomannomutase (PMM) (rfbK)
of Salmonella choleraesuis
Rhamnosyltransferase of Streptococcus
pneumoniae (Morona et al., 1997)
Regulatory protein (repA) in E. coli
(Scholz et al., 1989)
Regulatory protein (repA) in E. coli
(Dorrington & Rawlings, 1990)
O-specific LPS biosynthesis of E. coli
(O-acetyltransferase, rfb) (Yao &
Valvano, 1994)
HU alpha gene (NS2 or HU-2) of
Serratia marcescens
HU alpha gene (NS2 or HU-2) of
A. proteolytica (Giladi et al., 1992)
Modification methyltransferase of PstI
and BsuBI of B. subtilis (Xu et al.,
1992)
Modification methyltransferase of PstI
and BsuBI of R. leguminosarum
(Rochepeau et al., 1997)
Sensory histidine protein kinase in
Calothrix viguieri
Oligopeptidase A (opdA) of
Salmonella typhimurium (Conlin &
Miller, 1992)
Oligopeptidase A (opdA) of E. coli
(Conlin et al., 1992)
Oligopeptidase A (opdA) of H.
influenzae (Fleischmann et al., 1995)
Multidrug-resistance protein 2 of
B. subtilis (Ahmed et al., 1995)
Haemolysin (hlyA) of A. hydrophila
(Wong et al., 1998)
β-Haemolysin of A. salmonicida
(Hirono et al., 1992)
Glycosyltransferase of V. cholerae
(Zhang et al., 1996)
Glycosyltransferase of Yersinia
enterocolitica (Zhang et al., 1996)
Putative O-antigen methyltransferase
of Burkholderia pseudomallei
(DeShazer et al., 1998)
1ek147
5ek97
2ek06
CAA63036
5ek31
D90901
6ek24
I76776
2ek22
S22619
1ek169
Y07786
1ek166
P32054
1ek110
Q00330
3ek14
2209213
2ek32
P20356
7ek20
A36134
1ek25
P37750
7ek36
P52680
2ek35
P28080
3ek99
P33563
9ek17
X99520
7ek23
Y09899
1ek179
P27237
1ek176
451277
1ek173
P44573
1ek07
P39843
8ek26
U81555
5ek06
X65049
4ek42
AB012956
1ek22
U46859
0n002
P13059
AF064070
* The E value indicates the probability of the match. A match with an E value of 1ek5 and below was taken to be significant (i.e. the
match is not due to chance). An E value of 0n002 (for F126) was also taken into consideration.
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Y. L. Z H A N G, C. T. O N G a n d K. Y. L E U N G
histidine protein kinase and DNA regulatory protein
(repA).
DISCUSSION
A prerequisite to comprehending the pathogenicity
mechanisms of an organism is the identification and
examination of all its virulence genes (Strauss & Falkow,
1997). There are many general procedures to detect
virulence genes such as genomic subtraction (Diatchenko et al., 1996 ; Mahairas et al., 1996), transposon tagging (Berg et al., 1994) and in vivo expression
technology (Mahan et al., 1993). By using SSH in this
study, 60 % of the clones obtained were tester-specific
and 69 PPD134\91-specific fragments were derived from
one round of subtraction. Only 36n5 % of the subtracted
fragments were false positive. The studies described
here show that the SSH technique is simple and efficient
for identifying genetic differences between virulent and
avirulent strains. This method offers a possible alternative to identify presumptive virulence genes of pathogenic bacteria.
Surveying the distribution of virulence genes in
A. hydrophila
Two strains were chosen for the SSH protocol : A.
hydrophila PPD134\91 as the tester strain and
PPD35\85 as the driver strain. In order to confirm that
subtracted fragments were unique not only in the tester
strain but also among other virulent strains, Southern
hybridization analysis was used to probe eight virulent
and seven avirulent strains of A. hydrophila (Tables 3
and 4). Two groups of clones were derived based on the
hybridization patterns. Presumptive universal virulence
genes (21 ORFs including five predicted proteins) were
classified in group 1. Genes encoding heterogeneity or
strain variations (44 ORFs including 11 predicted
proteins) are in group 2 and these also include some
known virulence genes. Genes in group 2 may have
undergone extensive modifications such that they are
not even conserved among different strains of the same
species. Many of these genes are described for the first
time in motile aeromonads. Studying the distribution of
these gene fragments provides insight to their relative
importance.
Group 1 (common virulence genes)
Haemolysin (F108). F108 is highly homologous to the hlyA
gene of an A. hydrophila strain, A6 (Wong et al., 1998),
and a β-haemolysin gene of A. salmonicida (Hirono &
Aoki, 1993). Haemolysin is one of the virulence factors
produced by motile aeromonads and has been cloned
from different strains of aeromonads (Howard et al.,
1996 ; Wong et al., 1998). A. hydrophila secretes at least
two types of haemolysins. One of them is aerolysin
(AerA) which can oligomerize on erythrocyte cell
membranes, form channels, and lead to cell lysis
(Wilmsen et al., 1990). The second is a non-channelforming haemolysin (HlyA) and is proposed to be a
Vibrio cholerae-HlyA-like haemolysin (Wong et al.,
1998). These two types of haemolytic toxins are low in
homology and believed to be distinct. It was suggested
that these two haemolytic toxins contribute to virulence
in A. hydrophila and are widespread within virulent
strains of motile aeromonads (Vadivelu et al., 1995 ;
Hirono et al., 1992). Therefore, it is not a surprise to
detect the absence of the hlyA gene in four of our
avirulent strains including PPD35\85. In addition, our
hybridization results confirm that hlyA is present in
most of our virulent strains (7\8).
Histone-like protein (F52). F52 is homologous to HU-2
genes of Aeromonas proteolytica (Giladi et al., 1992),
Salmonella typhimurium (Higgins et al., 1988) and E.
coli (Kano et al., 1987). These are histone-like proteins
of prokaryotes (such as HU proteins and integration
host factors) that are small, basic, heat-stable and
bind to single- and double-stranded DNA (Drlica
& Rouviere-Yaniv, 1987). Although the biological
functions of histone-like proteins are not fully understood, they are known to alter DNA recognition by
changing DNA dynamic flexibility and accessibility
(Flashner & Gralla, 1988). The resulting alterations in
DNA structure and topology affect several cellular
processes, including initiation of DNA replication, DNA
partitioning and cell division, and transposition of
bacteriophage Mu (Huisman et al., 1989 ; Jaffe et al.,
1997). In addition to the physiological functions, secreted histone-like protein has been demonstrated to
have a potential role in the pathogenesis of Streptococcus-induced tissue inflammation (Stinson et al.,
1998). The biological function of the HU-2 gene in A.
hydrophila will be elucidated in future experiments.
Protease (oligopeptidase A) (F88, 109). A number of
extracellular proteases of A. hydrophila (metallopeptidases and serine peptidases) have been described
(Howard et al., 1996) and correlations have been made
between the production of proteases and virulence
(Leung & Stevenson, 1988b). Extracellular proteases
may aid the organism in overcoming initial host defences
such as resistance to serum killing, and provide amino
acids for cell proliferation (Leung & Stevenson, 1988b).
Furthermore, proteases are needed for the maturation of
exotoxins such as aerolysin (Howard & Buckley, 1985).
For F88 and 109, the predicted protein showed similarity
to the OpdA sequence of S. typhimurium, Haemophilus
influenzae and E. coli (Conlin & Miller, 1992 ; Conlin et
al., 1992 ; Fleischmann et al., 1995). Oligopeptidase A is
the major soluble enzyme in E. coli which is able to
hydrolyse free lipoprotein signal peptide in vitro (Novak
et al., 1986). Fragments F88 and F109, encoding the
putative OpdA protein, were present in all the virulent
strains of A. hydrophila we tested and may be involved
in peptide processing of virulence factors which in turn
affect pathogenicity.
Omp (F2 and 3). A homologue of the outer-membrane
protein OmpA was identified in our tester strain. The
OmpA protein is one of the major outer-membrane
proteins of a wide range of Gram-negative bacteria such
as A. salmonicida (Costello et al., 1996), Shigella
dysenteriae (Braun & Cole, 1982) and E. coli (Beck &
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Screening virulence genes of A. hydrophila
Bremer, 1980). Major physiological functions include
maintenance of the structural integrity and morphology
of the cells and porin activity, as well as a role in
conjugation and bacteriophage binding. A role in
bacterial virulence has been implicated in increased
serum resistance as in E. coli (Weiser & Gotschlich,
1991) and Neisseria gonorrhoeae (Rice et al., 1986).
Drug-resistance gene (F106). Bacteria have developed a
number of mechanisms to protect them from environmental toxins and antibiotics, including degradation
and inactivation of drugs by enzymic modifications,
alteration of the drug target, and the production of
multidrug transporters (Lewis, 1994 ; Nikaido, 1994).
The predicted protein of F106 showed similarity to
multidrug-resistance protein 2 of Bacillus subtilis
(P39843, E l 1ek07) (Ahmed et al., 1995). It is possible
that this multidrug transporter in A. hydrophila
PPD134\91 and other virulent strains is used for the
transport of other antibiotics or toxic substances. This
presumptive virulence factor will be studied in future
experiments.
Group 2 (PPD134/91-specific sequences)
O-antigen/polysaccharide (F33, 36/121, 44/86, 46, 51, 110 and
126). LPS is a major component of the outer membrane
of Gram-negative bacteria. It consists of three regions :
the lipid A, the core oligosaccharide and the O-antigen
(Schnaitman & Klena, 1993). Serotypes are distinguished on the basis of O-antigens, and heterogeneity is
known to exist among motile aeromonads (MacInnes et
al., 1979 ; Leblanc et al., 1981). The unique set of Oantigen modification enzymes (such as rfb) carried by
various strains of bacteria contributes to the diversity of
O-antigen. A. hydrophila PPD134\91 and PPD35\85
belong to different serogroups (Leung et al., 1995b).
This is probably the reason for the absence of some
O-antigen modification enzymes in A. hydrophila
PPD35\85. These include mannosyltransferase B (F33),
GDP-mannose 4,6-dehydratase (F36, 121), phosophomannomutase (F44, 86), rhamnosyltransferase (F46),
O-acetyltransferase (F51), glycosyltransferase (F110)
and O-antigen methyltransferase (F126).
The O-antigen is thought to be important in the
pathogenesis of many bacteria. It is involved in serum
resistance and protecting bacteria from phagocytosis
(Stinavage et al., 1989). The O-antigen of A. hydrophila
is also an important adhesin (Merino et al., 1996). When
aeromonads were devoid of O-antigen, their LD value
increased by 100-fold (Merino et al., 1991). &!
Further
studies will be conducted to investigate the differences
between the LPSs of these two strains and their roles in
pathogenicity.
The
ORF encoded by F60, 91, 101 and 119 showed high
homology match to site-specific DNA-methyltransferase
BsuBI of B. subtilis (Xu et al., 1992) and Rhizobium
leguminosarum (Rochepeau et al., 1997). These enzymes
belong to a type II restriction\modification (R\M)
Restriction/modification system (F60, 91, 101 and 119).
system that functions to destroy foreign DNA (Xu et al.,
1992). Thus, it is reasonable to postulate the existence of
a similar type II R\M system in A. hydrophila
PPD134\91. This is the first report of a R\M system in
A. hydrophila. Its presence may be the reason why
previous attempts to introduce plasmids and transposons into the tester strain (PPD134\91) failed (data
not shown). Understanding and crippling this R\M
system will allow easier genetic manipulation of the
bacteria.
Conclusions
Elucidating the genetic differences between virulent and
avirulent strains of A. hydrophila provides insight into
the types of virulence genes. In our previous studies,
many virulence factors present in PPD134\91 but not in
PPD35\85 were characterized (Leung et al., 1995a, b ;
Tan et al., 1998). Attempts are made to correlate some of
these phenotypes with the presumptive virulence genes
we obtained in this study. Features such as resistance to
serum and to phagocyte-mediated killing in PPD134\91
can be explained by the detection of Omp and Oantigen-modification enzymes. The previous failure to
introduce transposons into PPD134\91 has also been
explained by the detection of a type II R\M system.
Furthermore, the presence of haemolysin, histone-like
protein, multidrug-resistance protein and other proteins
is also important in virulence. Elucidation of other
virulence characteristics such as adhesion and invasion
awaits further study of the novel genes and examination
of all the subtracted clones. Although the subtraction
was successful, the library isolated is by no means
complete. This was shown by the presence of only four
pairs of identical clones (2\3, 44\86, 60\101, 91\119).
New rounds of SSH will be carried out to complete the
mapping of genetic differences between virulent and
avirulent strains. Strains PPD64\90 and L36 drew
particular attention. Although avirulent, they contained
many tester-specific fragments (Tables 3 and 4). There
might exist some regulatory mechanism or other key
virulence factors that silenced their expression.
It is hoped that the identification of critical genetic
differences between virulent and avirulent A. hydrophila
can provide insights into the pathogenic mechanisms,
thus supplying the groundwork for the development of
new therapies for A. hydrophila infections in both fish
and humans. On the other hand, the presence of HlyA
and a bacteriophage protein CII among the subtracted
fragments may signify the presence of pathogenicity
island(s) in A. hydrophila (Hacker et al., 1997). Experiments are currently under way to address this possibility.
ACKNOWLEDGEMENTS
The authors are grateful to the National University of
Singapore for providing a research grant for this work. They
would like to thank Drs T. T. Ngiam and H. Loh (the Primary
Production Department of Singapore) for providing the
A. hydrophila isolates. They also wish to thank Dr P. Tang
and Ms S. H. M. Ling for helpful constructive criticism.
1007
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Y. L. Z H A N G, C. T. O N G a n d K. Y. L E U N G
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Received 31 August 1999 ; revised 2 December 1999 ; accepted
11 December 1999.
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