MICROBIOLOGY

LETTERS
ELSEVIER FEMS Microbiology Letters 145 (1996) 17-22

Integration of the DNA of a novel filamentous bacteriophage

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VSK from Vibrio cholerae 0139 into the host chromosomal DNA
Sudeshna Kar ‘, Ranajit K. Ghosh a$*, Amar N. Ghosh b, Amit Ghosh ’
” Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Calcutta-700032, India
” National Institute of Cholera and Enteric Diseases, P-33 C.I T. Road, Scheme XM, Calcutta-700010, India
L Instiiute qf Microbial Technology, Sector 39A, Chandzgarh, India

Received 23 July 1996; revised 2 September 1996; accepted 5 September 1996

Abstract

An unusual lilamentous bacteriophage, VSK, containing single-stranded, circular DNA as its genome was isolated from
Vibuio cholerae 0139 strains PO7 and B04. Unlike other single-stranded DNA phages, VSK can integrate its genome into the
chromosome of the host and enter into a lysogenic state. The double-stranded replicative form (RF) of the single-stranded
phage DNA was isolated. A restriction map of the VSK RF DNA was constructed using HaeII, AvnII, ClaI and XbaI. By
Southern blot analysis of the chromosomal DNA of the lysogen using labeled phage DNA as probe, the attachment site (attP)
on the viral genome was also identified.

Keywords: Vibrio choleroe 0139; Filamentous phage; Single-stranded DNA phage; integration

1. Introduction stranded DNA molecules as their genome, leads

neither to lysis nor to lysogeny of the host cells.
The temperate bacteriophages have a wide range Instead, the infected cell extrudes progeny phage
of lysogenization modes. At one extreme is bacte- particles while continuing to grow and produce vi-
riophage Mu, which gives rise to lysogens by ran- able daughter cells.
domly integrating its DNA into the host chromo- Vibrio cholerae, the causative agent of cholera, has
some. At the other extreme is bacteriophage Pl, been found to be the host for a large number of lytic
which lacks the integration system and phage DNA as well as temperate phages [l]. The most recently
is maintained as a plasmid rather than as a part of discovered temperate choleraphage was isolated from
the host chromosome. In between fall bacteriophages V. cholerae serogroup 0139 strain which had re-
?L and P22 which have highly preferred sites for in- placed V. cholerae serogroup 01 as the epidemic
tegration. On the other hand, infection with filamen- strain in 1992 [2,3]. No single-stranded DNA con-
tous phages like M13, which carry circular single- taining bacteriophage from V. cholerae has been re-
ported so far. We report here the isolation of a fila-
mentous choleraphage containing single-stranded,
* Corresponding author. Tel.: +91 (33) 4730492/4736793; circular DNA molecule as its genome from V. cho-
Fax: +91 (33) 4730284/943333/954321. lerae 0139. However, unlike other single-stranded

037X-1097/96/$12.00 Copyright :C: 1996 European Federation of Microbiological Societies. Published by Elsevier Science B.V.
PIISO378-1097(96)00369-2
18 S Kur et al. I FEMS
DNA phages, the one from V. cholerae 0139 was
found to be capable of integrating its genome,
more specifically the replicative form, into the host
chromosome.
2. Materials and methods
2.1. Bacterial strains, media and growth conditions
V. cholerae 01 strains 569B, Mak 751 and V. cho-
lerae 0139 strains PO7 and B04 have been described
before and were obtained from Dr. G.B. Nair, Na-
tional institute of Cholera and Enteric Diseases, Cal-
cutta [2]. General bacteriophage techniques and iso-
lation of bacteriophage DNA were essentially as
described [4].
2.2. Electron microscopy
Electron microscopy of the phage and DNA was
done according to the procedures described by
Chakraborty et al. [5].
2.3. Restriction enzyme digestion, electrophoresis
staining techniques
Restriction enzyme digestion and analysis were
performed as described [6]. Acridine orange staining
of agarose gel was done according to the method
described by McMaster and Carmichael [7].
2.4. Southern blotting and DNA hybridization
Conditions for chromosomal DNA preparation
and Southern transfer have already been described
[4]. Purified single-stranded VSK DNA was s2P-la-
beled using a random primer labeling kit (NEB,
USA). Southern hybridization and washing of the
blots were done as described previously [4].
3. Results and discussion
3. I. Isolation of the single-stranded DNA phuge
Previously, we had reported the identification of a
temperate phage from I’. cholerae 0139 strains [4].
Microbiolog_y Letters 14j (1996) 17-22
While working on this phage it was observed that
the phage DNA isolated from 0139 strain PO7 always
produced an additional band of about 7 kb in size
apart from the expected 35 kb band characteristic of
0139 phage DNA. A similar band was absent in the
phage DNA prepared from 0139 strain B02. As the
crude phage preparation was treated with DNase
and RNase prior to DNA isolation, the small
DNA must have been encapsulated and must hence
be of phage origin. Secondly, the 7 kb band was
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somewhat diffuse in nature, resistant to all restriction
enzymes tested and sensitive to Sl nuclease suggest-
ing the single-stranded nature of the DNA. To con-
firm the single-stranded nature of this DNA, DNA
preparations were electrophoresed on agarose gel
and stained with acridine orange as described in Sec-
tion 2. The 7 kb band stained orange, characteristic
of single-stranded DNA, while the 35 kb band was
green, as expected of double-stranded DNA (data
not shown). Thirdly, strain PO7 carries a plasmid
identical in size to that of the single-stranded DNA
which may be speculated to be the double-stranded
replicative form of the single-stranded phage DNA
(see below for homology). Agarose gel electrophore-
and sis followed by acridine orange staining showed that
this plasmid preparation also carried some single-
stranded DNA. These observations strongly sug-
gested that l’. cholerae 0139 strain PO7 carries an
additional phage (named VSK, hereafter) containing
single-stranded DNA as its genome. Due to the pres-
ence of two temperate phages (see below for tempe-
rate nature of VSK) in strain PO7 it was difficult to
isolate phage VSK in pure form from the same
source. It was observed that another plasmid bearing
V. cholerae 0139 strain, B04, also contains phage
VSK and was devoid of the large temperate phage.
So strain B04 was used for phage isolation. This
strain never released any detectable amount of the
phage VSK spontaneously. However, it was found
that phage production could be induced significantly
with mitomycin C at a concentration of 0.2 @ml
under identical conditions described for 0139 tempe-
rate phage isolation [4]. The significant lysis observed
following mitomycin C induction (data not shown)
strongly suggested that the phage VSK genome may
be integrated in the host chromosome, an observa-
tion unique for a single-stranded DNA phage. The
phage VSK was able to infect V. cholerae serogroup
 S. Km ef al. I FEMS Microbiology Levers 145 (1996) 17-22
Fig. 1. Electron micrograph of phage VSK and its DNA. Arrow indicates pointed end of the filamentous
01, biotype Eltor strain (viz. Mak 7.57) while the
serogroup 01 classical biotype strains were resistant.
3.2. Phage morphology
Electron microscopy of phage VSK revealed the
filamentous nature of the particle with an estimated
width of 16 nm. However, a very minor population
of filaments with a width of 8 nm, more akin to the
pili of V. cholerae, was also observed (Fig. 1).
3.3. Phage genomr
It was observed that the phage VSK carried single-
stranded DNA of about 7 kb in size as its genome
(discussed in Section 3.1). Moreover, it was possible
to make radioactive probe from the phage DNA by
random primer labeling without denaturation of the
template DNA prior to the DNA primer annealing
reaction confirming the single-stranded nature of the
phage DNA. Electron microscopy of the DNA re-
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phage
vealed the circular nature of the phage DNA (Fig.
1). Thus, phage VSK has a single-stranded circular
DNA as its genome.
3.4. Replicative form of the phage DNA
It is known that during replication the single-
stranded DNA of filamentous phages is first con-
verted to double-stranded replicative forms [8]. Since
strain B04 carries a plasmid similar in size to that of
the VSK phage DNA we wanted to examine whether
this plasmid represents the replicative intermediates
of the single-stranded viral genome. So, the plasmid
from strain B04 was digested with restriction en-
zymes, electrophoresed on agarose gel and subjected
to Southern blot [9] analysis using “2P-labeled phage
DNA as probe. The probe was prepared from the
purified VSK single-stranded phage DNA by ran-
dom primer labeling without denaturation of DNA.
All the restriction fragments from the plasmid hy-
bridized with the probe suggesting that the plasmid
20 5. Kur ct al. I FEMS Microbiology Letters 145 11996) 17-22

closer to the CluI site has been established from their

map positions relative to HaeII (Fig. 3A, lanes 7 and
5), where both sites were found to be contained in a
1.6 kb Hue11 fragment (see below). Digestion of the
plasmid with Hue11 produced six fragments, 2.0, 1.6,
1.1 (doublet), 0.82 and 0.48 kb in size. The primary
cleavage map of Hue11 (Fig. 3B) was established by
analyzing the partial digestion products produced by
this enzyme - partial digestion products were puri-
fied from a preparative gel and subjected to complete

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digestion by the same enzyme. By incorporation of
AvuII, X&I or CluI as the second enzyme with
Hue11 the positions of their cleavage sites relative
to the Hue11 map were determined. AvuII cleaved
Fig. 2. Southern blot analysis of the plasmid from V. cholerae
0139 strain B04. A: Restriction endonuclease digestion pattern of
the plasmid digested with various enzymes. B: Southern hybridi- A
zation of the same endonuclease digests with a 32P probe pre- I234567
pared by random prime labeling of VSK ss DNA. Lane 2: Undi-
gested phage DNA from V. cholerae 0139 strain P07. Lanes 335:
B04 plasmid DNA digested with restriction endonucleases. Lane
3: Ha&, lane 4: HincII, lane 5: HpaII. Lane 1: h-Hind111 and
$x174-HaeIII molecular size markers (NEB). Arrow indicates the
position of single-stranded VSK DNA.

is the replicative form (RF) of the single-stranded

DNA (Fig. 2). A small 0.4 kb HaeIl fragment could
not be identified due to its small size. The 7 kb band
that hybridized with the probe was the single-
stranded viral DNA contaminant in the plasmid pre-
paration. Incidentally, plasmid DNA isolated from
PO7 also hybridized with single-stranded DNA probe
(data not shown). However. the phage DNA probe
did not hybridize with the RF of Ml3 demonstrating
their difference.

3.5. Restriction mup of VSK RF DNA

A restriction map of the RF of the VSK phage IKb =

DNA was constructed using AvaII, Cl& XbaI and
HaeII. The first three enzymes had single sites on the
RF DNA and generated a linear double-stranded
DNA 7.0 kb in length (data not shown). Digestion
with AvaIl + ClaI or AvaII + XhaI generated sets of
two fragments with sizes of 4.7 and 2.3 kb (Fig.
3A: lane 3) and 4.2 and 2.8 kb (Fig. 3A, lane 6)
respectively. The X&I site thus may be closer to
ClaI site with a distance of about 0.5 kb or it may
be located 1.9 kb away from it. That the X&I site is
Y
a
Fig. 3. Restriction endonuclease digestion pattern analysis of
phage VSK RF DNA. A: Lane 1: undigested RF DNA. Lanes
2-7: RF DNA digested with HurII, CM+ AwlI, HaelI+ Avall.
Haell+ C/at. .%a1 + AvuII and Hue11 + Xbal, respectively. Arrow
indicates the position of single-stranded phage DNA. Positions
of h-Hind111 molecular size markers ran on the same gel are in-
dicated by bars. From top downwards (in kb): 6.64, 4.36, 2.26,
1.98 and 0.56. Hue11 restriction fragments are indicated by letters
of the alphabet in order of decreasing molecular size. B: Linear-
ized restriction map of phage VSK RF DNA.
 S. Km et al. I FEMS Microbiology
Junction
Junction
A tachment site. When HaeII digested chromosomal
(att PI8
LD
E absent in the chromosomal
Fig. 4. Southern blot analysis of phage VSK DNA integration in
T-Ccholerne 0139 strain B04 chromosome. VSK RF DNA (lane
1) and chromosomal DNA from strain B04 (lane 2) were di-
gested with Ha&, electrophoresed on 1% agarose gel. transferred
onto a nylon membrane (Hybond N, Amersham, USA) and
probed with “‘P-labeled VSK phage DNA (as above). Arrow in-
dicates the position of the single-stranded phage DNA.
HaeIIE generating a 0.75 kb fragment (Fig. 3A, lane
4). The smaller 70 bp fragment could not be detected
in the gel due to its size. As mentioned above, both
CluJ and XbaI cleaved only HaeIIB. The former pro-
duced a 1.5 kb fragment (Fig. 3A. lane 5) and a
small 100 bp fragment which could not be detected
due to its small size. Considering the minimum dis-
tance between C/a1 and AvaII as 2.3 kb, the cleavage
sites for these two enzymes were mapped near the
left end of HueIIB and HaeIIE respectively (Fig. 4B).
XbaI generated two distinct fragments of 1.05 and
0.65 kb in size from HueIIB (Fig. 3B, lane 7) and
following an identical logic its position was also
mapped. Thus, a restriction map was constructed
for the RF of the phage VSK (Fig. 3B). The RF
DNA had no sites for Bg/II, HindIII, BarnHI,
KpnI, X/roI, SalI and SphI.
Letters 145 11996) 17-22 21
3.6. Analysis of the lysogenic state
Poor liberation of the free phage from strain B04
as well as significant induction by mitomycin C in-
dicated a lysogenic state of the phage VSK. If the
above conjecture is correct, then one would expect to
find out the integrated state of the phage DNA by
Southern blot analysis whereby digested chromoso-
ma1 DNA from the lysogen probed with labeled
phage DNA should reveal a change in restriction
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fragment pattern. Based on the physical map of
VSK RF DNA, we elucidated a region for the at-
DNA from strain B04 was compared with Hue11
digested VSK RF DNA in a hybridization experi-
ment with labeled VSK phage DNA as probe, it
was observed that HaeIIA (2.0 kb). HueIIC+D (1.1
kb doublet) and HueIIE (0.82 kb) fragments were
preserved; the smallest HaeIIF (0.48 kb) fragment
presumably ran out of the gel. HueIIB (1.6 kb) was
DNA digest while two
new fragments of size 3 kb and one greater than 24
kb were produced (Fig. 4, lane 2). This confirmed
that VSK can incorporate its genome into that of
its host and the attachment site of the phage (attP)
is located on the HueIIB fragment and contains the
sequence for integrative recombination.
The results presented above describe, for the first
time, the identification of a filamentous single-
stranded DNA phage VSK from I/ cholerue 0139.
This phage is unique in the sense that, despite being
single-stranded, the phage genome integrates into the
host chromosome, entering into a lysogenic state.
The only other report that is available in the litera-
ture to date of a filamentous bacteriophage capable
of integrating its genome into the host chromosome
is that of phage Cflt of Xunthomonus canzpestri [lo].
Evidence has been presented that VSK replicates via
the formation of double-stranded replicative forms,
as observed for other filamentous phages like M13,
fd and fl. It is the RF that integrates into the host
chromosome to produce the lysogenic state. The RF
isolated from another V. cholerur 0139 strain,
namely P07, exhibited some interesting differences.
The CluI site on the uttP fragment HueIIB was ab-
sent while an additional XbuI site could be identified
on this fragment (data not shown). Whether this in-
dicates a random attachment needs further examina-
22 S. Kur rf al. I FEMS Microbiology Letters 145 11996) 17-22

tion of DNA from various independently isolated 121Mitra, S.N., Kar, S., Ghosh, R.K., Pajni, S. and Ghosh, A.
(1995) Presence of lysogenic phage in the outbreak strains of
lysogens.
Vibrio cholerae 0139. J. Med. Microbial. 42, 399403.
The identification of the filamentous phage from
[31 Reidl, J. and Mekalanos, J. (1995) Characterization of Vibrio
V. cholerae has also an important implication that it cholerae bacteriophage K139 and use of a novel mini-transpo-
may allow construction of stable vectors. It is well son to identify a phage-encoded virulence factor. Mol. Micro-
known that plasmids are very unstable in I’. chole- biol. 18, 685-701.

rae. The presence of copious amount of plasmids in [41 Pajni, S., Raychowdhury, N., Ghosh, A.. Kar, S. and Ghosh,
R.K. (1995) Characterization of phage $1139, a Vibrio cholerae
I/ cholerae 0139 strains B04 and PO7 strongly sug-
0139 temperate bacteriophage with cohesive DNA termini.
gests that vectors developed from this phage can FEMS Microbial. Lett. 131, 69974.
provide a useful tool in gene manipulation in this PI Chakrabarti, B.K.. Chattopadhyay, D.J. and Ghosh, A.N.

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organism. (1993) Vibriophage DlO contains non-permuted DNA with
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161 Chattopadhyay, S. and Ghosh, R.K. (1988) Localization of
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Acknowledgments e4 genome. Virology 162, 337-345.
[71 McMaster, G.K. and Carmichael, G.G. (1977) Analysis of
We thank the Department of Biotechnology, Gov- single and double-stranded nucleic acids in polyacrylamide
and agarose gels by using glyoxal and acridine orange. Proc.
ernment of India, for financial assistance (Grant BTI
Natl. Acad. Sci. USA 74, 48354838.
TF/9/13/91). S.K. is the recipient of a Senior Re-
PI Baas, P.D. (1985) DNA replication of single stranded E. coli
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