From the Department of Biochemistry, University of Adelaide, Adelaide 5005, Australia
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ABSTRACT |
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The tum gene of coliphage 186, encoded on a LexA controlled operon, is essential for UV induction of a 186 prophage. Primer extension analysis is used to confirm that Tum is the sole phage function required for prophage induction and that it acts against the maintenance repressor, CI, to relieve repression of the lytic promoters, pR and pB, and thereby bring about lytic development. In vitro experiments with purified proteins demonstrate that Tum prevents CI binding to its operator sites. Tum does not compete with CI for binding sites on DNA, and unlike RecA mediated induction of lambda prophage, the action of Tum on CI is reversible. Mechanisms by which Tum may act against CI are discussed.
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INTRODUCTION |
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Following infection of a host bacterium by a temperate bacteriophage, the phage can develop in one of two ways: lytically, whereby progeny phage are produced and released by lysis of the host cell, or lysogenically whereby the phage genome is integrated into the host cell (the prophage state) and replicated along with the host DNA. Although these two life cycles are independent, some temperate phage retain the ability to switch between them. For example, in certain classes of temperate bacteriophage, exposure to UV light or other DNA-damaging agents stimulates the phage to switch from the passive prophage state to the productive lytic cycle, the process of prophage induction.
In the lambdoid family of bacteriophage, commitment to prophage induction after UV irradiation is achieved by inactivation of the prophage maintenance function, CI, through linkage to the complex global stress response system known as the SOS response (1). Upon exposure of a cell to DNA-damaging agents, host RecA protein is converted to an activated form that catalyzes by autoproteolysis the inactivation of host cell-encoded LexA protein, a transcriptional repressor of a series of unlinked genes, the SOS genes (2). Activated RecA also catalyzes the self-cleavage of lambda CI repressor protein, relieving repression of lytic genes to bring about prophage induction (3, 4).
Coliphage 186 is a close relative of the non-inducible P2 family of phage (5). The finding that 186, like lambda, is UV-inducible (6) was therefore unexpected. Induction of a 186 prophage differs from that in lambda. RecA is not directly involved in induction of a 186 prophage, but rather 186 relies upon a phage encoded function tum, whose expression is under host LexA control (7, 8). Thus, prophage induction in 186 can be considered an SOS function. The SOS operon of 186 (Fig. 1) contains two genes under control of the p95 promoter, tum and orf97 (8). The tum gene contains several alternative translational start points, and the product of one of these (Tum) is essential for prophage induction. Expression of the orf97 gene product from a plasmid blocks infection of the cell by 186; however, its absence has little effect on prophage induction (8).
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The prophage state of coliphage 186 is, like lambda, maintained by the product of a single gene, CI (13). 186 CI represses transcription from two promoters, pR and pB (Fig. 1). The lytic promoter pR directs the synthesis of genes required for excision and replication (10, 11), whereas pB is responsible for expression of the B protein, a transcriptional activator required for expression of the late genes (14). In addition, there are two more CI binding sites, FL and FR, whose functions are unknown (12) but which may be involved in DNA looping. Exposure of a 186 lysogen to a DNA-damaging agent leads to production of the activated form of RecA, which in turn catalyzes the irreversible inactivation of host LexA, resulting in a general SOS response, including relief of the LexA-mediated repression of p95. The resulting Tum protein then acts in an unknown fashion to activate lytic development (7, 8).
In the present work, we investigate the mechanism of Tum action and demonstrate that Tum acts against the CI repressor, the sole phage function required for maintenance of lysogeny. By using Tum and CI purified to near homogeneity, we show that Tum specifically prevents effective CI binding to its operator sites and that this Tum-CI interaction is reversible.
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EXPERIMENTAL PROCEDURES |
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Radiolabeled nucleotides and oligonucleotide primers were purchased from Bresagen (Adelaide, Australia). Media and general techniques for 186 are as described (15). General cloning techniques were as described (16).
Oligonucleotides
Primer A is (2872-2843) TAACGATAGGTGCAGGCACTTTGATGATTG. It is used in primer extension to detect pR activity.
Primer B is (349-320) GATAGCGGCTTGTTCGCGCATGTGCGGCAT. It is used in primer extension to detect pB activity.
Primer C is CGTAGTGGAGGTCATATGGATAGAGAGCT. It is used to create the NdeI site (shown in bold) at the start of the tum gene.
Primer D is GTCCCCGCGGTACGAGACGCCAGCTCTCATCTTC. It is used to remove the stop codon and introduce a SacII site (shown in bold) at the 3' end of the tum gene.
Clones
pEC303-- pEC303 is a multicopy p95 tum+ plasmid. The 2.5-kb1 EcoRI fragment (92 to 2.3%) from 186+ was ligated into the EcoRI site of pBR322.
pEC306--
pEC306 is a single copy
p95 tum+ plasmid. The
2.5-kb EcoRI fragment (92 to 2.3%) from
186cI+97 was ligated into the
EcoRI site of pKN1562 (17).
pEC307-- pEC307 is a multicopy p95 tum+ plasmid. The 2.0-kb PstI fragment (8578-10,597) from 186tum+ was ligated into the PstI site of pBR322. The orientation of the insert was such that p95-directed transcription was in the opposite direction to pAmp.
pEC308-- pEC308 is same as pEC307, except the 186 DNA source was 186cI+tum16.
pEC309--
pEC309 is same as pEC307, except the 186 DNA source
was 18695.
pEC324--
pEC324 is a derivative of the pL
expression plasmid pPLc28 containing the 3.1-kb EcoRI
restriction fragment from an M13 Ecol clone (M13
Ecol 95.4
/5
) in which
Orf95.4 and Orf95.5 expression had been abolished by mutation (8). The orientation is such that pL
transcription was in the same direction as
p95.
pEC330-- pEC330 is a plac expression plasmid carrying p15A replicon and confers kanamycin resistance. It was constructed as described (8).
pEC331-- The 1.2-kb SspI (8655) to AccI (1372) fragment (end-filled) from pEC303 was inserted into the ScaI site of pEC330. The insert was oriented for expression of Tum from plac.
pEC342-- The 1.3-kb HaeIII (1721) to HaeIII (2998) restriction fragment from 186cItsp was ligated into the HindIII site of a single copy pKO2-derived plasmid.
pEC363-- pEC363 is a pET3a-Tum expression vector. It was created by ligating the 300-bp NdeI (8765) to BamHI (9104) restriction enzyme fragment from the mutated M13 clone, M13EcorNdeTum+ ligated into the NdeI to BamHI backbone of pET3a. The carboxyl-terminal portion of the tum gene was reconstructed by ligating the XhoI (9104) to XhoII (9730) restriction fragment from M13EcorNdeTum+ into the recreated BamHI site of the pET3a construct.
pEC364-- pEC364 is a pET3a Tum16 expression vector. It is the same as pEC363, except the 186 DNA source was the M13 clone, M13EcorNde tum16.
pEC452-- pEC452 is a CI expression plasmid. It contains the 1.3-kb HaeIII-HaeIII (71.2-75.5%) fragment of 186+ inserted into the EcoRV site of pACYC184 such that CI expression was under the control of its own promoter, pL (14).
pEC458-- pEC458 is a single copy B expression plasmid. It was constructed as described (14) such that B expression was under the control of its own promoter, pB.
pET TumHis6--
The tum gene from pEC324
(Orf95.4,.5
(8)) was amplified by
PCR using primer C to create an NdeI site at the 5' end of
the gene and primer D to create a SacII site at the 3' end
of the gene. The PCR product was digested with NdeI and
SacII and ligated into the NdeI, SacII
backbone of pET3a which had been modified (H. Healy, University of
Adelaide) to fuse the coding sequence for LVPRGSHHHHHH at the carboxyl
terminus of any inserted sequence.
pET Tum16His6-- pET Tum16His6 was the same as pET TumHis6 except the template for the PCR reaction was pEC364.
All regions amplified by PCR were sequenced to ensure no errors had been introduced.Primer Extension Analysis
RNA isolation was based on the procedure of Reddy and Gilman
(18). The concentration and purity of RNA preparations were determined
spectroscopically, an
A260/A280 ratio of
greater than 1.7 being regarded as acceptable for primer extension
analysis. Primer extension reactions were performed essentially as
described (19). All reactions were extended from oligonucleotides that had been labeled at the 5' end using polynucleotide kinase and [-32P]dATP. Electrophoresis was carried out on a 6%
denaturing polyacrylamide gel, and the gel was fixed, dried, and
autoradiographed.
Nitrosoguanidine Mutagenesis
A lysogenic culture, C600(186cItsp) or
(186cItspSA), was grown overnight at 30 °C
in LB, and then diluted 10
2 in LB. Incubation was
continued at 30 °C until A600 = 0.8, and the
culture was transferred to 39.5 °C for 20 min. Nitrosoguanidine was
added (8 µg ml
1 or 16 µg ml
1), and
incubation was continued for a further 20 min. Following cell lysis,
cellular debris was removed by centrifugation, and aliquots of lysate
were diluted 10
3 into LB and stored over chloroform at
4 °C.
Protein Purification
TumHis6--
Cells (BL21 [DE3] pLysS pET
TumHis6) were grown with vigorous shaking at 37 °C in LB
(500 ml) containing ampicillin (100 µg ml
1) and
chloramphenicol (30 µg ml
1) until an
A600 nm of 0.6-0.8 had been reached. IPTG (0.4 mM) was added to induce Tum expression and growth continued
for a further 3 h. The cells were collected by centrifugation,
washed once in 50 mM Tris-Cl, pH 8.0, 0.1 mM
EDTA, 150 mM NaCl, 10% glycerol (TEG150 buffer), and
stored at
20 °C until use.
Gel Retardation Assays
Radiolabeled DNA fragments for gel retardation assays were
generated via the polymerase chain reaction, by inclusion of
[-32P]dATP in the PCR reaction mix. Fragments were
purified on a 6% polyacrylamide gel before use. Samples (10 µl),
containing 150-300 cpm of labeled fragment, were prepared in binding
buffer (50 mM Tris-Cl, pH 8.0, 0.1 mM EDTA, 150 mM NaCl, 20% glycerol, 0.01 mM dithiothreitol,
50 ng µl
1 salmon sperm DNA) and incubated on ice for 30 min prior to loading. A separate lane of tracking dye was used. Gels
(6% polyacrylamide containing 20% glycerol) were run at 4 °C.
Electrophoresis was carried out at 20 mA for approximately 2 h,
and the gels were dried under vacuum and the distribution of labeled
DNA recorded on a phosphor screen. The screen was analyzed with the
Imagequant program on a Molecular Dynamics PhosphorImager.
Western Blotting
Western blots were performed essentially as described (22), using the Supersignal chemiluminescent detection kit (Pierce).
Analytical Ultracentrifugation
Sedimentation equilibrium experiments were carried out in
a Beckman XL-A analytical ultracentrifuge equipped with an An60Ti rotor and absorption optics. Samples were centrifuged for 24 h at
5 °C and scans taken every 4 h thereafter until equilibrium had
been reached. Data sets were collected at 280 nm with a spacing of
0.001 cm as the average of three scans. Proteins were prepared for
centrifugation by exhaustive dialysis against TEG150 buffer. The
partial specific volume of TumHis6 was calculated using the amino acid partial specific volumes of Zamyatnin (23). This value was
0.721 ml g1. Buffer density at 5 °C was measured in an
Anton-Paar precision density meter to be 1.0378 g
ml
1.
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RESULTS |
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Derepression of pR following UV Induction of Prophage-- No function other than CI is required for maintenance of the prophage state (13), and CI is known to repress transcription of the excision and replication genes from pR (10) and transcription of the late activator gene from pB (14). The action of Tum in prophage induction could be to activate transcription that bypasses the repressed pR and pB, or more likely, it could be that Tum interferes with CI function or production.
We first sought to confirm that, upon UV-induced prophage induction, pR was derepressed rather than an alternative promoter activated. Primer extension analyses were performed on RNA accumulated at various time points after UV irradiation of a 186 lysogen, with an oligonucleotide primer (primer A) complementary to the l strand at the start of the apl reading frame. Thus, transcription of the apl gene from pR, essential for optimal prophage induction (11, 24), would generate a 125-bp product in the primer extension assay. As shown in Fig. 2, a 125-base extension product was first visible 60 min after irradiation, indicating that UV irradiation of a prophage results in activation of transcription from pR. However, a second minor product 180 bases in length also appeared in parallel with the 125-base product. The possibility remained therefore that Tum activated an alternative promoter giving the 180-bp product, with the further possibility of processing to yield the 125-base product.
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The Direct Action of Tum Is Equivalent to CI Inactivation-- To confirm that Tum's action is to inactivate CI rather than activate an alternative promoter, we next compared the transcription response following expression of Tum specifically (from a plasmid rather than via UV induction) with that seen after temperature inactivation of a temperature-sensitive CI mutant.
A two-plasmid system was established with pEC331, a pACYC177-derived plasmid with the tum gene placed in front of the plac promoter (8) so making Tum expression IPTG-inducible, and pEC342, an R1-derived (single copy) plasmid carrying the 1.3-kb HaeIII restriction fragment (9) from 186cItsp encoding the cI gene (tsp allele). Once again primer extension analysis was used to assay pR transcriptional activity. At 30 °C, production of CI from pEC342 completely repressed pR activity (Fig. 3). This was unchanged in the presence of the Tum plasmid pEC331. However, 30 min after addition of IPTG, pR activity was evident but not if the tum gene was mutant. This result indicated that Tum is both necessary and sufficient to derepress CI-repressed pR. pEC342, bearing a temperature-sensitive allele of cI, allowed the removal of repression independently of Tum. Raising the temperature of the culture to 41 °C and thus inactivating CI gave a rightward transcription pattern indistinguishable from that obtained through the agency of Tum.
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Derepression of pB during UV Induction-- A second 186 promoter repressed by CI is pB. To confirm that Tum acts to derepress rather than activate, the start point of the B transcript after exposure of the repressed pB to Tum was determined by primer extension analysis, using a three-plasmid system (Fig. 4). pB B was carried as an insert on a single copy RI-derived plasmid pEC458 (14). Transcription from pB was detected as an 82-base extension product and was repressed by CI supplied in trans from pEC452, which is a pACYC184 derivative (14). The third plasmid pEC307 is pBR322-derived and bears the 2.0-kb PstI fragment spanning p95 and tum (25). When the host carrying the three plasmids was UV-irradiated, exactly this 82-base product was detectable at 20 min after irradiation. If the tum plasmid (pEC307) was replaced by either pEC308 carrying the inactive tum16 mutant allelle or pEC309 bearing a tum deletion mutant (25), then UV-mediated activation of pB was no longer evident. The correspondence in size of the UV-induced extension product with that of the pB transcript was further evidence for an antirepressor role for Tum and evidence against the proposal that Tum acts to bypass repressor by activation of a new transcript.
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Genetic Approach to Studying CI-Tum Interaction--
To probe
further the nature of Tum-mediated prophage induction, an attempt was
made to isolate mutants of CI resistant to the action of Tum. Initial
attempts involved screening for the presence of turbid plaques on media
containing mitomycin C, a DNA-damaging agent that causes prophage
induction, and indicator bacteria hosting a single copy
p95 tum+ plasmid
(pEC306). Thus, in theory, 186 tum mutants
would be complemented in trans by the
tum+ plasmid in the host strain, leaving only
mutants of 186 resistant to Tum as turbid plaque formers. From a sample
of 650,000 plaques, generated by plating a nitrosoguanidine-mutagenized
186cItsp stock (see "Experimental
Procedures") on C600(pEC306), four potential Tum-resistant mutants
were isolated. However, the mutations were mapped to the tum
gene itself, identifying them as dominant tum
mutants. This gave the first indication that Tum may act as an oligomer, and indeed we show Tum in solution to be a dimer (see below).
An improved screen for Tum resistance was then employed, using a 186 tum deletion mutant, 186cItsp
SA,
that removes all of tum, as well as
p95. Use of this phage was designed to preclude the possibility of mutation to tum
dominance,
as the only source of Tum is that of pEC306. Nitrosoguanidine-mutated stocks of this phage were plated for single plaques on C600(pEC306) in
the presence of mitomycin C. From a screen of 550,000 plaques, three
further potential Tum-resistant mutants were isolated. One of these
mutants (TumR
) mapped to the left of the cI gene, in a
region concerned with integration and excision of the prophage. Indeed
this mutant displayed a deficiency in prophage induction common to both
heat- and Tum-mediated induction and was not pursued. The remaining two
mutants (TumR G and TumR 3) mapped to the cI gene, each
mutant bearing a double mutation resulting in two amino acid
substitutions within cI (V58A,L98F for TumR G; L115F,P158S for TumR 3). The mutants formed unstable prophage that displayed a
defect in prophage induction by Tum (data not shown), suggesting a
simultaneous loss of sensitivity to Tum and of repressibility. However,
the defect was not specific for Tum induction as the mutants were also
defective in heat induction (the cItsp allele was
used in the mutational screen). We therefore concluded that CI activity
and resistance to Tum were not genetically independent, and development
of improved genetic screens was discontinued in favor of biochemical
studies to study CI-Tum interactions.
Biochemical Approach to Studying CI-Tum Interactions
Isolation of Tum--
The expression of tum-derived
proteins in maxicells (8) indicated that up to four proteins
(Orf95.1, .2, .4, and .5) are expressed from the tum
reading frame. Plasmid-driven expression of these various proteins also
demonstrated that the tum function responsible for prophage
induction corresponded to the 146 amino acid Orf95.1 protein
product (8). Oligonucleotide site-directed mutagenesis was used to
create an NdeI site at the GTG initiation codon of Tum
(Orf95.1), in a construct (pEC324 (8)) in which Orf95.4
and Orf95.5 expression had been abolished. This modified tum gene was initially cloned into the pET3a expression
vector to give pEC363. That this clone produced functional protein was shown by the plaque phenotype of 186 on the bacterial strain
HMS174[DE3] hosting pEC363. Even without IPTG induction there was
sufficient basal expression of Tum to give a clear plaque (no lysogens)
phenotype in the presence of pEC363 but not the tum16
control pEC364. However, SDS-PAGE analysis of cell extracts prepared
from induced cultures of HMS174[
DE3] pEC363 showed that very
little of the Tum protein produced was in a soluble form. To facilitate
purification of an active, soluble form of Tum, the gene was cloned
into a modified expression vector such that the expressed protein
contained a carboxyl-terminal six histidine tag (pET
TumHis6). Again, analysis of the plaque phenotype of 186 when plated on HMS174[
DE3] pET TumHis6 showed that
this vector, but not the corresponding tum16 construct,
produced functional Tum protein. The presence of the histidine tag
enabled the use of a one-step affinity purification procedure under
denaturing conditions, followed by a simple refolding procedure, to
yield milligram quantities of soluble protein (see "Experimental
Procedures").
Tum Prevents CI Binding to DNA in Vitro-- Purified TumHis6 and CI (isolated as described (21)) were used in a series of gel retardation assays to determine whether Tum-specific retardation of CI binding activity could be demonstrated in vitro. Several conclusions could be drawn from this series of experiments as follows. (i) As shown by Dodd and Egan (12), purified CI caused retardation of DNA fragments containing the pR (Fig. 5a) and pB (Fig. 5b) CI operator sequences. The presence of Tum resulted in a loss of the ability of CI to cause retardation of these fragments, confirming the in vivo result that Tum acts to prevent effective CI binding. (ii) Tum by itself did not give rise to retardation of the pR (Fig. 5a) or pB (Fig. 5b) containing fragments, indicating that Tum did not compete with CI for binding sites on the DNA. (iii) The inhibitory action of Tum was specific for CI binding. This was demonstrated using a DNA fragment that contained both the FR CI operator and the CII binding site. CII, a transcriptional activator required to establish lysogeny in 186, is like 186 CI, a helix turn helix protein, and binds to a 7-base pair inverted repeat sequence located at the beginning of the CII gene (19) (Fig. 1). As shown in Fig. 5c, CI binding at FR was prevented by the presence of Tum, whereas at the same Tum concentration, the binding of CII was unaffected. (iv) The inactive Tum mutant, Tum16 (7), did not prevent CI binding to pR (Fig. 5a, final lane). In fact, CI binding was somewhat improved in the presence of Tum16. However, given that substitution of the same concentration of bovine serum albumin for Tum16 also improved CI binding (not shown), this effect was considered to be a nonspecific one. (v) Inhibition by Tum of the binding of CI to pR occurred over a narrow range of Tum concentrations. The fraction of labeled pR DNA bound by 150 nM CI was measured at a series of Tum concentrations (Fig. 6). The transition from no inhibition to complete inhibition of CI binding occurred over a narrow (10-fold) range of Tum concentrations. The data were fitted as shown in Equation 1.
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(Eq. 1) |
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Tum Reversibly Inactivates CI-- Prophage induction in lambdoid phages is achieved through a specific proteolytic inactivation of the maintenance repressor via cleavage of the repressor at an Ala-Gly site (27). Although 186 CI does not contain such a sequence (9), the possibility remained that Tum may cause proteolytic degradation of CI. To test this, purified Tum (5.7 µM), CI (150 nM), and a 437-bp 32P-labeled DNA fragment containing pR were incubated together under solution conditions identical to those used in the gel shift assay of Fig. 5a. Following a 1-h incubation, samples were subjected to SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and probed with an anti-CI polyclonal antibody. As shown in Fig. 7a, the Western blot showed no evidence of CI degradation and hence no evidence for proteolytic attack by Tum.
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Characterization of Tum-- The genetic experiments aimed at finding Tum-resistant mutants of CI revealed the existence of dominant negative tum mutants, indicative of oligomerization of Tum. To quantitate the extent of any self-association, purified TumHis6 (monomeric molecular weight 17,958) was subjected to sedimentation equilibrium (Fig. 8). Analysis of the data, obtained at two rotor speeds, gave a weight average molecular weight of 34,310 ± 700, indicating that Tum was predominantly dimeric in solution. Inclusion of additional species in the fitting procedure (tetramer or octamer) provided no evidence of higher order association, although we cannot rule out the possibility that the histidine tag may prevent association beyond a dimer.
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DISCUSSION |
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We have confirmed that the Tum protein of 186, produced by the phage as part of the SOS response following damage to the DNA of the host cell, is the only phage function required to derepress the CI controlled promoters pR and pB, an event known to lead to prophage induction. In particular, we have shown that Tum directly interferes with the ability of the CI repressor to bind to its operators and so maintain the lysogenic state.
By what mechanism might Tum inactivate CI? The sequence of Tum provides no clue as to its mechanism of action, as it displays significant homology to only two other sequences (8, 25), the functions of which are unknown. UV induction of lambdoid phages involves proteolytic cleavage of the maintenance repressor by activated host cell RecA protein. We have shown, however, that Tum does not cleave or catalyze the self-cleavage of 186 CI and that in fact CI retains the ability to bind DNA following incubation with Tum. The Tum protein sequence contains no recognized DNA binding motifs and, consistent with this, displays no ability to bind DNA in gel retardation assays. Thus, Tum does not act by competing with CI for its operator sites. All the available evidence suggests that Tum directly but reversibly inactivates the CI repressor.
This mechanism of action is reminiscent of functions described for
other bacteriophage systems. The Ant protein of Salmonella phage P22 for example interferes with the activity of the c2 repressor (28). The Coi antirepressor protein of phage P1 inactivates P1 C1
repressor by forming a noncovalent complex with it, at a molar ratio of
about 1:1 (29, 30), whereas a second P1 antirepressor, Ant, can
specifically coprecipitate the P1 C1 repressor from cellular extracts
(31). In addition, it has recently been reported that the gene
product of the satellite phage P4 efficiently derepresses a P2
prophage, possibly by direct interaction with the P2 C repressor function (32).
The most likely models for Tum-mediated inactivation of CI repressor seem to be that Tum prevents CI binding (i) by blocking the DNA binding domain of CI, (ii) by preventing CI oligomerization, or (iii) by blocking the cooperative interactions between CI subunits required for effective binding of CI to its operators. The CI repressor of 186 exists in solution in a monomer-dimer-tetramer-octamer equilibrium, the formation of octamer from dimers being a concerted process (21). The stoichiometry of CI binding to its operators is currently under investigation but, given the nature of the binding sites (12), must involve at least tetramers. This rather complex set of linked equilibria provides ample opportunity for Tum to interfere with cooperative interactions.
The isolation of dominant tum mutations
indicates that Tum itself exists as a multimer of at least two
monomers. The ability of Tum to dimerize was confirmed by sedimentation
equilibrium. The phenotypes of different tum mutants (7)
suggests that the protein has two regions of differing functional
importance. Those phage mutants with the most severe loss in the
ability to be induced, both spontaneously and through DNA damage, all
had specific base pair changes within the amino-terminal portion of the
Tum protein. 186tum13, for example, involves a conservative
alanine to valine change at the 34th amino acid, yet has a
tum null phenotype. The Orf95.2 product differs from
Tum only in the absence of the first nine amino acids, yet it possesses
no discernible ability to induce a prophage (8). In contrast, less
severe tum mutants have alterations further downstream
toward the carboxyl half of the protein. Indeed, the 186
95C mutant
has the carboxyl half of Tum removed, yet it is still able to induce a
prophage, albeit at 5% efficiency compared with wild type. These
observations suggest that the active site of Tum involved in
inactivating CI is located in the amino-terminal half of the protein.
Determination of the precise mechanism of action of Tum will require
further structural information on both Tum and CI.
What are the advantages to 186 of the Tum-mediated system of prophage induction? It is in the interest of the prophage that if induction is to occur, then it should do so effectively, such that the process should be essentially irreversible and, once undertaken, should involve a total commitment to bacteriophage production. 186 ensures this by having p95 strongly repressed by LexA (7, 33). Tum will only be expressed following extensive DNA damage, conditions under which the survival of the host bacterium is threatened. Furthermore, once Tum synthesis begins, intermediate concentrations of Tum that derepress pR and pB ineffectively must be avoided. One way to address this problem would be to use cooperative interactions, either between Tum polypeptides or between Tum and CI to enhance inactivation of CI. In this way, relatively small changes in Tum concentration would be magnified to give a more efficient induction signal. This is illustrated in Fig. 9. Although CI binding to its operators is itself a cooperative process (n > 1), we have shown that inactivation of CI by Tum occurs over quite a narrow range of Tum concentrations. The data of Fig. 9 show that this transition (solid line) is sharper than the binding isotherm for CI (dashed line). This would appear to be a necessary requirement for efficient induction, such that inactivation of CI is not negated by rebinding of free CI.
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Once Tum-mediated inactivation of CI has taken place, the phage must also ensure that newly synthesized CI does not contribute to the CI pool and allow reestablishment of repression of the lytic promoters. Further synthesis of CI by the prophage is avoided through Apl-mediated repression of pL (24). In addition, by linking Orf97 production to Tum expression (Fig. 1), superinfection of the host cell is avoided (8), and bacteriophage production can proceed without the risk of an infecting phage adding to the CI pool.
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ACKNOWLEDGEMENTS |
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We thank Professor D. J. Winzor for use of the analytical ultracentrifuge and Dr. Ian Dodd for helpful discussions.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by an Australian Research Fellowship.
§ Supported by an Australian Postgraduate Research Grant. Present address: Dept. of Genetics, University of Adelaide, Adelaide, South Australia 5005, Australia.
¶ Supported by the Australian Research Council. To whom correspondence should be addressed. Tel.: 61 8 8303 5361; Fax: 61 8 8303 4348; E-mail: jegan{at}biochem.adelaide.edu.au.
1
The abbreviations used are: kb, kilobase pair;
bp, base pair; PAGE, polyacrylamide gel electrophoresis; PCR,
polymerase chain reaction; IPTG,
isopropyl--D-thiogalactoside; LB, Luria broth; Ni2+-NTA, nickel-nitrilotriacetic acid; Orf, open reading
frame.
2 R. Schubert and I. Dodd, personal communication.
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REFERENCES |
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