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Journal of Virology, December 1999, p. 9816-9826, Vol. 73, No. 12
Department of Genetics, Stockholm University,
S-106 91 Stockholm, Sweden
Received 24 June 1999/Accepted 27 August 1999
Phage W Temperate phage W Satellite phage P4 is a defective phage that lacks genes encoding
structural proteins and lysis functions; therefore it requires P2 or a
P2-related phage as helper to grow lytically (31). P2 and P4
are unrelated; their sequence homology is less than 1% and is limited
primarily to the phage end regions required for DNA maturation and
packaging. In the absence of a helper, P4 has the capacity to integrate
into the host chromosome and to become a repressed prophage (10,
47) or to establish itself as a multicopy plasmid (15,
24). P4 has the capacity to utilize the P2 helper in a
coinfection, after infection of a P2 lysogenic strain, or after P2
infection of a strain containing P4 as a prophage or a plasmid. P4 can
gain access to the late genes of a repressed P2 helper in two ways,
i.e., transactivation and derepression. Transactivation is mediated by
the P4 Chemicals and enzymes.
All enzymes were purchased from
Pharmacia, except Vent DNA polymerase, which was obtained from New
England Biolabs. [
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Transcriptional Switch of Bacteriophage W
, a
P2-Related but Heteroimmune Coliphage
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
is a member of the nonlambdoid P2 family of temperate
phages. The DNA sequence of the whole early-control region and the
int and attP region of phage W has been
determined. The phage integration site was located at 88.6 min of the
Escherichia coli K-12 map, where a 47-nucleotide sequence
was found to be identical in the host and phage genomes. The W Int
protein belongs to the Int family of site-specific recombinases, and it
seems to have the same arm binding recognition sequence as P2 Int, but the core sequence differs. The transcriptional switch contains two
face-to-face promoters, Pe and Pc, and two repressors, C and Cox,
controlling Pe and Pc, respectively. The early Pe promoter was found to
be much stronger than the Pc promoter. Furthermore, the Pe transcript
was shown to interfere with Pc transcription. By site-directed
mutagenesis, the binding site of the immunity repressor was located to
two direct repeats spanning the Pe promoter. A point mutation in one or
the other repeat does not affect repression by C, but when it is
included in both, C has no effect on the Pe promoter. The Cox repressor
efficiently blocks expression from the Pc promoter, but its DNA
recognition sequence was not evident. Most members of the P2 family of
phages are able to function as helpers for satellite phage P4, which
lacks genes encoding structural proteins and packaging and lysis
functions. In this work it is shown that P4 E, known to function as an
antirepressor by binding to P2 C, also turns the transcriptional switch
of W from the lysogenic to the lytic mode. However, in contrast to
P2 Cox, W Cox is unable to activate the P4 Pll promoter.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
was originally
isolated from Escherichia coli W, where, in the prophage
form, it restricted the growth of phage 
(29). W is
serologically unrelated to phage , but it is closely related to P2,
which it resembles under the electron microscope (29, 40).
In spite of its close antigenic relationship to P2, they are not
coimmune, since W grows on a P2 lysogen and vice versa
(29). A W lysogen restricts not only phage but also
phages T2 and T4, which indicates that it contains genes equivalent to
P2 old and tin (26, 36, 37). Like most
other members of the P2 family, W is not inducible by UV light, and it functions as a helper for satellite phage P4 (6).
protein, which has the capacity to directly activate the P2
late promoters, bypassing the need for P2 DNA replication, and the P2
Ogr protein, which activates the late promoters during lytic P2 growth
(27). Derepression of prophage P2 is mediated by the P4 E
protein (23), and recently E has been shown to act as an
antirepressor by forming a complex with the P2 immunity repressor C,
thereby blocking its capacity to bind to the operator (33).
Derepression is mutual; i.e., infection of a P4 lysogenic strain by P2
induces the P4 lytic cycle. The derepression of prophage P4 is mediated
by the P2 Cox protein, which acts as a transcriptional activator on the
P4 Pll promoter (41, 48). Since P4 is able to derepress P2
as well as W even though they have different immunities, we have
determined the DNA sequence of the transcriptional switch of W,
determined the location of the promoters and operators, compared the
sensitivities of the immunity repressor of the respective phage to the
P4 E protein in vivo, and compared the capacity of P2 and W to
derepress prophage P4.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-32P]ATP and
[14C]chloramphenicol were obtained from Amersham.
TABLE 1.
Synthetic oligonucleotides used in this study
Media. Luria-Bertani (LB) broth or LB agar plates were routinely used for growth of bacteria, as described previously (2). When needed, antibiotics were added to the following final concentrations: ampicillin, 50 to 100 µg/ml; kanamycin, 50 to 100 µg/ml; chloramphenicol, 10 to 50 µg/ml.
Bacteria and bacteriophages.
The bacterial strains and
bacteriophages used in this study are listed in Table
2.
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Plasmid constructions. Plasmids were constructed by standard techniques (44). E. coli C-1a was used as the recipient unless otherwise stated. All constructs were verified by automatic DNA sequencing with a Thermo Sequenase fluorescently labelled primer cycle-sequencing kit (Amersham Pharmacia Biotech, Uppsala, Sweden).
(i) pEE900.
The W C gene was cloned into the
expression vector pET16b under the control of the T7 
10 promoter.
The C gene was amplified from phage W DNA by PCR with
primers w-C1his and w-C2, and the generated PCR fragment was
inserted into the filled-in NdeI site of pET-16b. In this
way, a histidine tag was added to the N-terminal end of the C polypeptide.
(ii) pEE901.
The W cox gene was cloned into
the expression vector pET8c under the control of the T7 10 promoter.
The cox gene was amplified from phage W DNA by PCR with
primers w-11R and 79.0L, and the generated PCR fragment was inserted
into the filled-in NcoI site of pET-8c.
(iii) pEE902 and pEE903.
pEE902 and pEE903 are derivatives
of pACYC177 expressing W C and W Cox, respectively, from the
bla promoter of the vector. To avoid interference from
translation of the bla gene, a universal translational
terminator was inserted into the HincII site of pACYC177,
creating plasmid pEE719. The region containing the ribosomal binding
site of T7 10 and the W C gene was amplified by PCR from plasmid pEE900 with primers 7-for and w-C2. The PCR fragment was inserted into the ScaI site of pEE719 generating pEE902.
pEE903 was constructed like pEE902, but the DNA fragment was amplified from pEE901 with primers 7-for and 79.0L.
(iv) pEE904 and pEE905.
pEE904 and pEE905 are derivatives of
the promoter assay plasmid pKK232-8. The W Pe and Pc promoter region
was amplified by PCR from phage W DNA with primers w-8R and
w-9L. The generated fragment was inserted in both orientations into
the SmaI site of pKK232-8, which contains a promoterless
cat gene. In plasmid pEE904, the cat gene is
under the control of the Pc promoter, and in plasmid pEE905, it is
under the control of the Pe promoter.
(v) pEE906.
pEE906 is the derepression assay plasmid. The
W C-Pe-Pc region was amplified from phage W DNA by PCR with
primers w-C2 and w-9L, and the generated fragment was inserted
into the SmaI site of pKK232-8. One clone was selected where
Pe directs cat gene expression, and the cat gene
is repressed due to expression of C from the Pc promoter. Thus,
derepression will lead to expression of the cat gene.
(vi) pEE907 and pEE908.
pEE907 and pEE908 are derivatives of
pEE904 and pEE905, respectively, where the 
10 sequence of Pe promoter
was changed from TATATT to TAGCTT by
recombinant PCR (28). The Pc promoter directs cat
gene expression in pEE907, whereas the Pe promoter controls the
cat gene in pEE908.
(vii) pEE909, pEE910, and pEE911. pEE909, pEE910, and pEE911 are derivatives of pEE905 where the directly repeated sequence TATTGGTGAC, proposed to be the repressor binding site O1 and O2, respectively, was changed to TATTTGTGAC by recombinant PCR (28). The generated plasmid pEE909 contains the mutated base in site O1, pEE910 has site O2 mutated, and pEE911 has both sites mutated.
DNA sequence determination.
To sequence the transcriptional
switch region, we amplified the pertinent region from W phage DNA
with two P2 primers, PSP3-1 and 79.0L, which are located within the P2
ogr gene and orf78 respectively, using the Advantage Tth
polymerase mix kit (Clontech, Palo Alto, Calif.). The PCR product was
cloned into the SmaI site of pUC18. Two independent clones
were selected for sequencing first with primers on the vector and then
extended by primer walking in both directions.
Primer extension assay.
RNA was prepared and concentrated
from 50-ml cultures of C-1a carrying plasmid pEE904, pEE905, pEE906, or
pEE907, using the QiaGen RNeasy kit. Primers w-8R and w-9L were
5'-end labelled with [-32P]ATP by using T4
polynucleotide kinase. Primer extension assays were carried out with
primer extension system VAM reverse transcriptase, as specified by the
manufacturer (Promega, Madison, Wis.). Extended cDNAs were separated on
5% polyacrylamide-7 M urea denaturing gel in 1× TBE (50 mM Tris, 100 mM boric acid, 5 mM EDTA). The gel was vacuum dried prior to
autoradiography. Sequencing reactions with the same primers as in the
extension were performed, and the products were used as markers.
CAT assay. Chloramphenicol acetyltransferase (CAT) assays were performed as described previously (25). Protein concentrations were determined by the method of Bradford (8). CAT activities were quantified with a PhosphorImager and calculated as the ratio of acetylated chloramphenicol to total chloramphenicol.
Nucleotide sequence accession number. The nucleotide sequence data reported in this paper appear in the EMBL/GenBank/DDBJ Nucleotide Sequence Database under accession no. AJ245959.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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DNA sequence of the transcriptional switch of phage W.
To
sequence the transcriptional switch of phage W, attempts were made
to amplify the pertinent region by PCR with a P2 primer from a region
presumed to be conserved in the P2 family, i.e., ogr,
encoding the transcriptional activator of the late genes that is
located to the left of the attP site (13), and
various primers on the right side of attP. We found that
W had sequences analogous to the P2 ogr gene and P2
orf78, located downstream of cox. The amplified fragment was
cloned, and its DNA sequence was determined. The DNA sequence and the
inferred amino acid sequence of the encoded proteins are shown in Fig.
1b. The transcriptional region seems to
have a similar arrangement to other members of the P2 family, i.e., it
has two face-to-face promoters, Pe controlling the early genes and Pc
controlling the genes believed to be involved in lysogeny (Fig. 1a).
Based on the location of the open reading frames, we have adopted the
nomenclature of phage P2, calling the first gene of the Pe transcript
cox and the two open reading frames of the Pc transcript
C and int respectively.
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The W integrase belongs to the Int family of site-specific
recombinases.
An alignment of 105 site-specific recombinases of
the Int family has demonstrated two conserved boxes and three conserved patches of charged amino acids (39). Box I contains the
invariant Arg residue, and box II contains the His-X-X-Arg motif and
the active site Tyr. As can be seen in Fig.
2, W contains the conserved residues
of box I and box II. Patch I contains a group of acidic amino acid
separated by hydrophobic residues and has the consensus sequence
LT-EEV--LL. Patch II contains a well-conserved Lys flanked by Ser or
Thr in one subgroup and by Gly or Met in another. Minor variations of
this motif are also found. Patch III is rich in Phe and is preceded by
acidic residues and often followed by polar residues. The consensus
sequence is [D/E]-[F/Y/W/L/I/A]3-6[S/T]. The W Int
protein conforms to the general themes of the Int family of
recombinases (Fig. 2), but it differs quite extensively at the
N-terminal part from the integrases of the P2-related phages 186 and
HP1. It should also be noted that the W int gene has two
possible start codons, but since only the second Met codon is preceded
by a ribosome binding site, it most probably constitutes the initiation
codon. An evolutionary analysis has indicated that the HP1 and 186 integrases are more closely related to each other (55% identity) than
to P2 Int (35 and 37% identity, respectively) (20), and we
found in turn that W is more closely related to P2 (56% identity)
than to HP1 and 186 (35 and 39%, respectively).
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Localization of the W attP and attB
sites.
The site of integration of phage W upon lysogenization
has previously not been determined, but the sequence between
ogr and int lacks the 27-nucleotide (nt) core
sequence of P2; thus, it most probably integrates at a different
chromosomal location. Since the DNA sequence between ogr and
int is expected to contain the attachment site, a BLASTN
search was performed against the E. coli K-12 genome. A
47-nt region with only one mismatch was found, located between
cpxR and pfkA at 88.6 min on the E. coli K-12 map (Fig. 3). To confirm
the location of the integration site, the phage host junction fragments
were amplified from a W lysogen with primers located on either side
of the hypothesized integration site of the E. coli genome
in combination with primers from the phage int and
ogr genes. A nonlysogenic strain gave the expected PCR
fragment when the E. coli primers were used, but no fragment
was obtained from the W lysogen. Instead, the lysogen gave PCR
fragments when the E. coli primers were combined with the
pertinent W primers. The generated fragments were sequenced, confirming the integration of W at this location (Fig. 3). The mismatched C residue, located in the center of the dyad symmetry of the
identity region, was not present in the W lysogen, which was an
E. coli strain C derivative; thus, this might constitute a
strain variation.
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and the E. coli genome (Fig. 1b). The arm sequence,
i.e., the DNA sequences flanking the core sequence, was analyzed for
the presence of inverted or direct repeats. As can be seen in Fig. 1b,
besides the inverted repeat in the core, the region contains two
additional inverted repeats, IR1 and IR2. IR1 most probably acts as a
terminator for both the ogr and the int
transcripts, since both transcripts will have a poly(U) tail following
the stem-loop structure. If so, the int transcript will continue into the host chromosome in the prophage state. The region also contains several direct repeats. Interestingly, four of them have
a common consensus sequence, aTGTGGACact, which is identical to the consensus sequence of the arm binding sites of P2 integrase (55). Thus, they most probably constitute the arm binding
site of W integrase as well.
No sequence corresponding to the P2 Cox binding site could be detected
between the core sequence and the presumed arm binding sites of the
attP region of W, which implies that W Cox has a
different recognition sequence from that of P2 Cox.
Localization of the Pe and Pc promoters.
The DNA sequence
between the C and cox genes, which are
transcribed in opposite orientations, is expected to contain the
transcriptional control region. The region contains a potential
rightward promoter (Pe) just upstream of the initiation codon of gene
C, with good 10 and 35 regions spaced by 17 nt and a
potential leftward promoter (Pc) just upstream of the initiation codon
of gene cox, which conforms less well with the canonical
E. coli promoter (Fig. 1b). The location of these promoters
was confirmed by a primer extension analysis. The Pe-Pc promoter region
(nt 1722 to 1942 in Fig. 1b) was cloned into the reporter plasmid, in
both orientations, in front of the cat reporter gene. In
plasmid pEE904 the cat gene is controlled by the Pc
promoter, and in pEE905 it is controlled by the Pe promoter. Total RNA
was extracted, and primers w-8R and w-9L, each located about 150 nt downstream of the proposed promoter Pc and Pe transcriptional start
site, respectively, were used for the primer extension. RNA extracted
from cells containing plasmid pEE905 generated a fragment of the
expected size, i.e. 150 nt (Fig. 4,
promoter Pe). The Pe transcriptional start site was further located to
position 1793, residue C in Fig. 1b, by running a sequencing reaction
beside the primer extension. RNA extracted from cells containing
plasmid pEE904 generated no labelled fragment in the primer extension
analysis, most probably due to interference by transcription from the
stronger Pe promoter. To block transcription from Pe, the 10 region
of the Pe promoter in plasmid pEE904 was changed from TATATT
to TAGCTT, generating plasmid pEE907. By
using RNA extracts from cells containing plasmid pEE907, a weak band of
the expected size was obtained in the primer extension analysis (Fig.
4, promoter Pc, lane 1). The exact start site was located to position
1875, residue G in Fig. 1b, by running a sequencing reaction beside the
fragment generated by primer extension.
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Promoter Pe is much stronger than promoter Pc, and it is controlled
by the C repressor.
To quantitate the strength and regulation of
the face-to-face-located Pe and Pc promoters further, the level of
cat expression in strain C-1a containing plasmid pEE904
(cat under the control of Pc) and pEE905 (cat
under the control of Pe) was determined. First, the capacity of cells
harboring the respective plasmid to grow on LB agar plates supplemented
with 50 µg/ml chloramphenicol was analyzed. Cells containing plasmid
pEE905 grow well, whereas cells carrying pEE904 do not grow at all.
Instead, they grow on plates containing 10 µg of chloramphenicol per
ml, which implies that promoter Pe is stronger than promoter Pc, in
agreement with the results of the primer extension experiment described
above. The strength of the respective promoter was then quantified by determining the level of CAT expression. As can be seen in Table 3, experiment 1, the level of CAT
expression in cells with the cat gene under control of the
Pc promoter (pEE904) was hardly detectable and the activity was at
least 100-fold lower compared to that obtained with the Pe promoter
(pEE905). This situation seems very similar to what has been observed
with phage P2, where transcription from the Pe early promoter
interferes with initiation from the Pc promoter. To analyze the
possible interference of transcription initiated from the strong W
Pe promoter on Pc, as described above, the two conserved bases in the
10 region of the Pe promoter were changed (from TATATT to
TAGCTT) by site-directed mutagenesis. As can be
seen in Table 3, experiment 1, the level of CAT expression from the
mutated Pe promoter (pEE908) was reduced 33-fold compared to that from
the wild-type Pe promoter (pEE905), as expected. At the same time, the
level of expression from the Pc promoter in the presence of the mutated
Pe promoter (pEE907) increased fivefold, supporting the hypothesis that
the strong transcriptional initiation from Pe interferes with the
activity of Pc.
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immunity repressor C is expected to control transcription from
the early Pe promoter, and by analogy to other temperate phages, it
might control its own expression by regulating Pc. Therefore, plasmids
pEE904 and pEE905 were transformed into the W lysogenic strain
C-1920 and the capacity of the respective plasmid to express CAT was
measured as growth on LB agar plates supplemented with 50 µg of
chloramphenicol per ml. Strain C-1920, as opposed to the
nonlysogenic strain C-1a, carrying pEE905 did not grow on
chloramphenicol plates, whereas plasmid pEE904 seemed unaffected by the
C repressor, since the level of CAT expression allowed limited growth
in either host on the plates with a low concentration of
chloramphenicol (data not shown). This confirms that the C repressor
inhibits expression from the Pe promoter (pEE905) whereas the Pc
promoter (pEE904) seems unaffected under the conditions used.
The W immunity repressor is unable to repress phage P2 early
promoter but is sensitive to P4 E.
P2 and W are not coimmune,
as previously shown (29). However, as can be seen in Fig.
5A, the C proteins of P2 and W are related (43 of 97 amino acids are identical). The identities are more
prominent in the C-terminal half of the proteins, but the predicted
secondary structures are also similar in the N-terminal part of the
proteins. The active form of P2 C protein is believed to be a dimer,
and the dimerization domain has been located to the C-terminal part of
the protein (33, 34). To analyze the interaction of the
immunity repressor proteins of P2 and W on the homologous or
heterologous early promoters, we have used reporter plasmids that
contain the respective Pe-Pc region in front of a promoterless
cat gene so that the cat gene is under the
control of the Pe promoter. The expression of the cat gene
was then analyzed in the presence of the respective C protein provided
in trans from lysogenic host cells. As can be seen in Table
3 experiment 2, each promoter is repressed by its homologous repressor
but not by the heterologous repressor.
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-sheets. (A) Repressor C of phage P2 and W10 region of the early promoter (43). W also
contains a direct repeat flanking the 10 region, with the sequence
TATTGGTGAC, which thus might constitute the operator (Fig.
1b). To test this possibility, the respective direct repeat in the
reporter plasmid was changed to TATTTGTGAC and
the capacity of the W C protein supplied in trans from
the W-lysogenized host cell to repress cat expression was
analyzed. As can be seen in Table 3, experiment 3, the mutation in
either site O1 (pEE909) or site O2 (pEE910) did not affect the capacity
of repressor C to block the early promoter, since the CAT activity was
reduced by C to the same level as that obtained with the wild-type Pe
promoter, although the mutation in site O1 made the promoter slightly
more sensitive to repression by C compared to the results obtained with
the mutation in site O2. Mutations in both sites (pEE911), however,
rendered the early promoter insensitive to the C protein, which
indicates that this direct repeat contains the recognition sequence of
the C protein and that both sites are required for the C repressor to
bind and block the activity of the Pe promoter. It should also be noted
that in the absence of the C repressor, the mutation on either or both
operator sites increased the activity of Pe twofold (Table 3,
experiment 3). This implies that the changes made in the DNA sequence
affect the binding of RNA polymerase to the promoter region.
P4 has the capacity to form plaques on a W lysogen with about the
same plating efficiency as on a P2 lysogen (data not shown). Thus, P4
has the capacity to activate the late genes of prophage W. Whether
the activation occurs by a direct transactivation of the late promoters
by the P4 protein or by derepression mediated by the P4
antirepressor E or both is not known. To analyze if P4 E acts as an
antirepressor on the C repressor of W, the whole C-Pe-Pc region was
cloned into a reporter plasmid in such a way that the cat
gene was under the control of the Pe promoter, which in turn was
repressed by the C protein (pEE906). This plasmid thus mimics the
lysogenic condition. The addition of P4 E in trans from a
compatible plasmid (pEE804) leads to expression of the cat
reporter gene (Fig. 6), which implies
that E derepresses the W Pe promoter by interacting with the
immunity repressor C, as in phage P2.
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W Cox protein is a negative regulator of its own Pc promoter,
but it cannot functionally replace P2 Cox during excision of prophage
P2 or derepress the P4 Pll promoter.
The Cox protein of phage P2
is a multifunctional protein. It is a negative repressor of the P2 Pc
promoter (42), an activator of the Pll promoter of the
unrelated satellite phage P4 (41), and an architectural
protein required for excision of P2 prophage (55). A
comparison of the Cox proteins of W to other members of the P2
family, i.e., P2, 186, and HP1, reveals very few sequence identities
(Fig. 5B). However, as with the C protein, P2 and W Cox are more
closely related to each other than to HP1 Cox and 186 Apl, and the
predicated secondary structures indicate similarities only at the N
terminal, believed to contain the 
-turn--helix mediating DNA binding.
Cox is able to functionally replace P2 Cox, a P2
cox-defective lysogen (C-6005) carrying plasmid pGP1-2,
which contains a temperature-inducible T7 polymerase, was transformed
with the Cox-expressing plasmid (pEE901) and the capacity of the
lysogen to produce P2 was determined after growth at 30°C overnight
since the overexpression of Cox at 42°C is lethal. As can be seen in
Table 4, the cox defective lysogen was unable to produce P2 spontaneously but the P2
cox-containing plasmid (pEE720) readily complemented the
cox-defective prophage at 30°C to a level comparable to a
wild-type P2 lysogen (C-117) transformed with the same plasmid.
However, when the lysogen was transformed with a plasmid containing the
W cox gene (pEE901), no complementation was observed.
Thus, the W Cox protein cannot replace P2 Cox and promote
spontaneous phage production during growth of a cox
defective P2 lysogenic strain.
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Cox protein on the homologous Pc
promoter and the heterologous Pc promoter of P2, we used the reporter
constructs described above, where the cat gene is under the
control of W Pc (pEE907) or P2 Pc (pSS39-6) and Cox was supplied in
trans from the compatible plasmid pEE903. As described
above, the W Pc promoter was very weak compared to the Pe promoter,
and this phenomenon is due in part to the interference of the strong Pe
promoter. Even though the activity of the Pc promoter was increased
when the 10 region of Pe promoter was mutated, we still found that it
was too weak for analysis of the effects of Cox by using CAT expression
in the reporter system. However, since bacteria containing pEE907 were
able to grow on plates supplemented with 10 µg of chloramphenicol per ml, the effects of Cox on Pc could be analyzed on plates. As shown in
Table 5, in the absence of either W
Cox or P2 Cox, the host cells expressing CAT under the control of W
phage Pc promoter (pEE907) can grow well on plates containing 10 µg
of chloramphenicol per ml but not on plates containing 25 µg/ml and
they do not grow at all in the presence of W phage Cox (pEE903),
whereas the heterologous P2 Cox (pSS27-4) does not inhibit the growth
of the cells. Conversely, the cells harboring pSS39-6, in which the P2
Pc promoter directs CAT expression, grew poorly in the presence of P2
Cox but they grew well when the heterologous W phage Cox was
supplied in trans. This indicates that the W phage Cox
acts as a repressor to control the expression of its own Pc promoter
and that the respective Cox protein is unable to act on the
heterologous Pc promoter, in support of our previous results indicating
that the Cox proteins have different DNA binding sites.
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Cox to activate the P4 Pll promoter was
analyzed. Cells carrying reporter plasmids containing either the
wild-type or the vir1-mutated (pSS61-4) P4 Pll promoter controlling the expression of cat gene were transformed with
a compatible plasmid expressing the cox gene. As shown in
Table 6, with the P4 vir1 Pll
promoter, the activity of CAT was increased 16-fold in the presence of
P2 Cox. In contrast, the W Cox did not seem to have any effect on
the activity of the Pll promoter. Similar results were obtained with
the wild-type promoter, but the enzyme levels were reduced. These
data are in agreement with the finding that W infecting a P4 lysogen
does not lead to any significant P4 production (48a).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The P2 family of bacteriophages were originally placed in a common
group of temperate phages on the basis of host range, noninducibility by UV light (with the exception of phage 186), serologic unrelatedness to phage , and capacity to act as helpers for the unrelated
satellite phage P4 (7). The members of the family have
similar morphology, i.e., an icosahedral head, a base plate, and a
contractible tail with tail fibers, which classifies them as belonging
to the Myoviridiae family of phages. By DNA sequence
homology, phages with different host ranges have been found to belong
to the P2 family, i.e., Haemophilus influenzae phage HP1
(19) and Pseudomonas aeruginosa phage CTX
(38). In this work we have analyzed the lysogenic-lytic control region of phage W and compared it to that of P2 and 186, which are the two best-characterized members of this family.
Prophage location and the site-specific recombination system.
The integration site was located to 88.6 min on the E. coli
K-12 map, and it differs from any known integration sites of phage P2
(1) and 186 (53). The site of integration is in
the 1,880-nt spacer between cpxR and pfkA. The
spacer region contains a 504-nt open reading frame of unknown function,
but since the 47-nt sequence common to E. coli and W is
located outside this open reading frame, integration does not seem to
interrupt any coding part of the host genome. An alignment of the amino
acid sequence of the W integrase clearly showed that it belongs to
the Int family of recombinases, since it contains all the conserved
motifs including the catalytic site in the C-terminal end
(39). Interestingly, a repeated sequence identical to the P2
Int arm binding sites was found on either side of the 47-nt core
sequence. This indicates that even though the core binding domains of
W and P2 Int differ, they have identical arm binding domains. In
fact, the arm binding domain of P2 Int has been suggested to be located
at the N-terminal end, where 18 of the first 20 nt are identical in the
two proteins (54). The arrangements of the presumed Int
binding sites in W are very similar to those in P2; i.e., the core
sequence contains an inverted repeat while the arm binding sites
contain direct repeats.
attP region, and since W is unable to complement a
cox defective P2 lysogen to allow spontaneous phage
production, we believe that W Cox and P2 Cox do not recognize the
same DNA sequence. However, it cannot be excluded that W has another
gene encoding a specific excisionase.
Structure of the transcriptional switch and specificity of the
repressors for their target sites.
Sequence analysis of the
transcriptional switch region shows that phage W has a similar gene
organization to other phages of the P2 family; i.e., it contains two
face-to-face promoters (Pe and Pc) that control phage development, and
the first gene of each transcript is a repressor of the other
transcript (C and Cox) (Fig. 1) (17, 22, 43). In this way,
the promoters are mutually exclusive, and if Pe takes command after
infection, Cox represses Pc and lytic growth is ensured. If, on the
other hand, Pc takes command, C represses Pe and the phage enters the
lysogenic cycle.
promoter Pe is much stronger than the Pc promoter and that transcription initiated at Pe inhibits Pc. Similar findings have been
reported for P2 and 186 (17, 42, 43).
The W immunity repressor C efficiently blocks the early Pe promoter,
and the proposed operator sequence was identified by site-directed
mutagenesis as directly repeated 10-nt sequences on either site of the
10 region of Pe spaced by 24 nt; thus, the two operators do not seem
to be in phase with each other. A point mutation in one or the other
operator has no effect on the capacity of C to repress Pe, but when
contained in both operators, the Pe promoter becomes insensitive to the
C repressor. The P2 operator region also contains a directly repeated
sequence, but it is slightly shorter, 8 nt, and the sequence differs
from W, as expected since the two phages are heteroimmune
(35). In both phages the operators are located on either
side of the 10 region of Pe, but the P2 operators are separated by 22 nt only (43). The C proteins of P2 and W are of about the
same size, 99 and 97 amino acids, respectively, and they have 42%
identity and very similar predicted secondary structures, indicating
that they are functionally similar (Fig. 5A). The P2 C protein has a
strong dimerization domain, which is believed to be located at the
C-terminal end (33), but since neither P2 nor W C has an
-turn--helix structure typical of prokaryotic repressors, the
DNA-interacting epitope remains unknown. The immunity repressors of
phage 186 and HP1 are larger (188 amino acids) and have no homology to
W or P2; their operators seem to contain inverted repeats (16, 19, 22, 30).
The Cox/Apl proteins of phages P2, HP1, and 186 are small (79 to 91 amino acids) and have dual functions; i.e., they act both as
transcriptional repressors and mediators of excisive recombination (18, 21, 22, 30, 42, 55). In this work, we showed that W
Cox acts as a repressor of the Pc promoter and that it has no activity
on the P2 Pc promoter, in agreement with the finding that they do not
seem to have a common DNA recognition sequence. Since the involvement
of W Cox in excision has not yet been demonstrated, it is not clear
whether W Cox also has dual functions.
Interaction with satellite phage P4.
The defective phage P4
needs a helper to grow lytically, since it lacks genes encoding
structural proteins and DNA-packaging and lysis functions
(31). P4 has the capacity to derepress a P2 prophage to gain
access to the P2 late genes, but it is unable to derepress a 186 prophage (46, 48). Derepression of prophage P2 is mediated
by P4 E, which acts as an antirepressor by binding to the P2 C protein
(23, 33). The antirepressor function of P4 E seems to occur
by formation of multimeric complexes of E and P2 C that prevent the
binding of C to its operator. In this work, we have shown that P4 E
efficiently also turns the W transcriptional switch from the
lysogenic to the lytic state. The region of P2 C that is believed to
interact with E has been located to amino acids 62 to 72 (33). As can be seen in Fig. 5A, this region is well
conserved between the two phages, which might explain why E can bind to
both of them.
Cox
and the P4 Pll promoter, in accordance with the finding that W
infection of a P4 lysogen will not lead to any significant P4 phage
production (48a). Thus, the interactions of P4 with its
helpers differ. For P2, the derepression capacity is mutual; i.e., P4
will derepress a P2 prophage and vice versa. With W, it works only
one way; i.e., P4 is able to derepress prophage W but not the other
way around. Finally, phage 186 can function as a P4 helper only in a
coinfection. The P2-related phage HP1, which has a different host range
from P2 and 186, is not expected to function as a P4 helper since its
cohesive ends differ substantially (19).
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by grant 72 from the Swedish Medical Research Council. T. Liu was supported by the Sven and Lilly Lawski Foundation.
We thank J. M. Eriksson for plasmid pEE720 and Erich Six for unpublished observations and helpful discussions.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Genetics, Stockholm University, S-106 91 Stockholm, Sweden. Phone: 46 8 161270. Fax: 46 8 164315. E-mail: Elisabeth.Haggard{at}genetics.su.se.
| |
REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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