JOURNAL OF BACTERIOLOGY, July 2011, p. 3313–3323 Vol. 193, No. 130021-9193/11/$12.00 doi:10.1128/JB.00119-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

The Lysis-Lysogeny Decision of Bacteriophage 933W: a 933WRepressor-Mediated Long-Distance Loop Has No Role

in Regulating 933W PRM Activity�

Tammy J. Bullwinkle† and Gerald B. Koudelka*Department of Biological Sciences, University at Buffalo, Buffalo, New York

Received 25 January 2011/Accepted 26 April 2011

Our data show that unlike bacteriophage �, repressor bound at OL of bacteriophage 933W has no role inregulation of 933W repressor occupancy of 933W OR3 or the transcriptional activity of 933W PRM. This findingsuggests that a cooperative long-range loop between repressor tetramers bound at OR and OL does not formin bacteriophage 933W. Nonetheless, 933W forms lysogens, and 933W prophage display a threshold responseto UV induction similar to related lambdoid phages. Hence, the long-range loop thought to be important forconstructing a threshold response in lambdoid bacteriophages is dispensable. The lack of a loop requiresbacteriophage 933W to use a novel strategy in regulating its lysis-lysogeny decisions. As part of this strategy,the difference between the repressor concentrations needed to bind OR2 and activate 933W PRM transcriptionor bind OR3 and repress transcription from PRM is <2-fold. Consequently, PRM is never fully activated,reaching only �25% of the maximum possible level of repressor-dependent activation before repressor-mediated repression occurs. The 933W repressor also apparently does not bind cooperatively to the individualsites in OR and OL. This scenario explains how, in the absence of DNA looping, bacteriophage 933W displaysa threshold effect in response to DNA damage and suggests how 933W lysogens behave as “hair triggers” withspontaneous induction occurring to a greater extent in this phage than in other lambdoid phages.

Studies of how the lysis-lysogeny decisions of lambdoid bac-teriophages are regulated have illustrated the importance ofshort- and long-range cooperative DNA binding by proteins incontrolling a complex gene regulatory network. In all lambdoidphages, the repressor protein directs the establishment andmaintenance of the lysogenic state by simultaneously repress-ing transcription of the genes needed for lytic phage growthand activating transcription of a gene needed for lysogen for-mation (30). Based primarily on studies of bacteriophage �,two different cooperative repressor-DNA binding events arethought to be required for regulation of lambdoid phage lyso-gen development. The first involves formation of a repressortetramer between two repressor dimers, one bound to each ofthe adjacent OR1 and OR2 sites. A similar tetramer is alsoformed at the OL1 and OL2 sites. The “side-by-side” cooper-ative binding by a repressor tetramer is prerequisite to forminga stable � lysogens (2, 20, 30).

Additional cooperative interactions between two tetramers,each bound to a pair of adjacent operators that are separatedby �2.5 kb, occur in bacteriophages � and P22 (13–15, 31). In�, a repressor octamer-mediated OL-cI8-OR complex formswith the intervening DNA looping out (3, 13, 15). This long-range cooperative interaction helps modulate the prophage’scompensatory response to low doses of DNA damage (3) andthereby regulates lysogen stability. In all well-studied lambdoid

phages, the amount of phage produced is not linearly relatedto the amount of DNA damage until a particular thresholdamount of DNA damage is absorbed by the lysogen. Thisthreshold is due to the compensatory effect of removing therepressor from a partially occupied OR3, an occupancy that isfacilitated by the long-range loop mediated by a repressoroctamer bound to OL1, OL2, OR1, and OR2 (3). The discoveryof the long-range interaction between repressor tetramersbound at OR and OL in phage � seemingly solved two long-standing puzzles: (i) the function of cI binding at OL3 and (ii)given that the � repressor’s affinity for OR3 is too weak tosignificantly repress its own promoter, how does autogenousnegative control work?

Despite the apparent importance of long-range cooperativ-ity in stabilizing lambdoid phage lysogens, several recent ob-servations indicate that this interaction may not be required.We and others have reported that unlike all other well-studiedbacteriophages, bacteriophage 933W contains only two, notthree, repressor binding sites in OL (16, 23, 36) (Fig. 1). Bac-teriophage 933W is derived from the Shiga toxin-producingEscherichia coli (STEC) strain EDL933. The gene encodingShiga toxin is present on bacteriophage 933W and is part of anoperon directly controlled by the bacteriophage PR� promoter.Shiga toxin is not produced when the bacteriophage is in itslysogenic state, but toxin production increases substantiallyupon phage lysogen induction. As a result of its downstreamposition in the lytic cascade, the activity of PR� and therebyShiga toxin production by 933W are ultimately controlled byfactors that influence 933W repressor DNA binding. Hence, inaddition to providing information on bacteriophage gene con-trol mechanisms, these studies will inform studies aimed atcontrolling Shiga toxin production in infected individuals.

The absence of an OL3 site suggests that 933W repressor

* Corresponding author. Mailing address: Department of BiologicalSciences, University at Buffalo, 109 Cooke Hall, North Campus, Buf-falo, NY 14260. Phone: (716) 645-4940. Fax: (716) 645-2975. E-mail:koudelka@buffalo.edu.

† Present address: Department of Microbiology, Ohio State Univer-sity, Columbus, OH 43210-1292.

� Published ahead of print on 6 May 2011.

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binding to OR3 may not be assisted by formation of a �-likeOL-cI8-OR looped complex. Also, we reported earlier that inintact 933W OR, the 933W repressor does not bind its OR1 andOR2 sites at an identical concentration (23), suggesting that the933W repressor is incapable of binding cooperatively to thesetwo adjacent sites. Cooperativity is not essential to the con-struction a of lysis-lysogeny switch in a lambdoid phage. Forexample, Babic and Little found that cis-acting mutations canallow a � bacteriophage encoding a repressor that is incapableof forming DNA-bound tetramers (and, presumably, octam-ers) to form stable lysogens (2). Taken together, these resultsimply that the lysis-lysogeny regulatory circuitry of bacterio-phage 933W does not involve cooperative binding of repressortetramers or octamers.

Here we characterize the DNA binding and gene regulatorystrategies that allow 933W bacteriophage to function as anoutwardly “normal” temperate phage. Our findings confirmthat the basic transcriptional behavior of 933W’s lysis-lysogenycircuitry resembles that found in other well-characterizedlambdoid phages. However, these data show that 933W repres-sors bound at OL regulate neither OR3 occupancy nor PRM

activity. Since the activity of 933W PRM, the maintenance oflysogeny, is crucial, the stability of 933W lysogens is not regu-lated by a long-range DNA loop. Instead, our data show thatOR3 occupancy and thus PRM activity depend on a small dif-ference in the intrinsic affinities of the 933W repressor for OR2and OR3. It is clear that 933W bacteriophage has evolvedalternative strategies to regulate the lysogen stability decision.Remarkably, some of these strategies overlap those of mutant� phage previously identified by Babic and Little (2).

MATERIALS AND METHODS

Bacterial strains, bacteriophages, and DNA. All plasmids were propagated ineither Escherichia coli strain K802 (40) or strain JM101 (27). 933W repressor waspurified from the E. coli strain BL21(DE3)::pLysS (Novagen, Madison, WI)bearing a plasmid that directs its overexpression (p933WR) as described previ-ously (23). Construction of the plasmid containing the 933W OR operator wasdescribed previously (23). Templates for DNase I footprinting and transcriptionwere generated by PCR from these plasmids as described below.

The �imm434 bacteriophage used in lysogen induction experiments was fromour collection. �imm933W (36) was the generous gift of D. Friedman (Universityof Michigan). Lysogens were formed in MG1655.

Deletion of the OL region of �imm933W was effected by using the lambdaRed/Gam homologous recombination system expressed on helper plasmidpKD46 (12). For this process, OL, as well as part of the N gene found onp933WOL (23), was replaced with the spectinomycin resistance gene. Deletion ofthe 933W OL region within the lysogen was confirmed via PCR. As a conse-quence of the deletion of part of the N gene, the �imm933W�OL lysogen is notinducible with mitomycin.

Primers for site-directed mutagenesis, quantitative reverse transcription-PCR(RT-PCR), and the primer for detecting 933W repressor occupancy of OR (seebelow) were obtained from Integrated DNA Technologies (Coralville, IA).

In vitro transcription assays. Template DNA was PCR amplified from theappropriate plasmid by using the standard M13 forward and reverse primers andgel purified. Increasing amounts of 933W repressor were incubated with 8 nM

template DNA for 10 min at 25°C in transcription buffer (0.2 M Tris-HCl [pH7.5], 75 mM KCl, 50 mM MgCl2, 0.05% Triton X-100, 1 mM dithiothreitol[DTT]). E. coli RNA polymerase (2 mg/ml) was added and allowed to form opencomplexes at 37°C for 10 min. Single-round transcription was then initiated byadding a nucleoside triphosphate mix (2.5 mM each for ATP, CTP, and GTP, 0.5mM UTP and [�-32P]UTP, and 1 mM heparin), and the reaction mixture wasincubated at 37°C for 15 min. Transcription was stopped with formamide dye andheated to 95°C for 4 min, and products were separated on a 6% acrylamide–7 Murea gel in Tris-borate-EDTA (TBE; 89 mM Tris, 89 mM borate, 1 mM EDTA).Radiolabeled products were visualized and quantified by phosphorimaging.

UV induction of phage lysogens. Measurements of the UV induction thresholdresponse were performed using methods similar to those described previously(25). Briefly, lysogens were grown in LB to mid-log phase and then chilled,centrifuged, and washed with TMG buffer (10 mM Tris-HCl [pH 8.0], 10 mMMgSO4, 10 �g/ml gelatin). Resuspended cells were irradiated at a distance of 20cm (�1 �W/cm2) by using a 254-nm UV light (Mineralight UVG-54). At variousexposure times, aliquots of the cultures were plated on LB to ascertain viablebacterial cell counts. The UV-exposed culture was then diluted 1:10 in 5 ml of LBand incubated at 37°C shaking for 2 h. The induced culture was treated withCHCl3, and debris was removed by centrifugation at 18,000 � g for 1 min. Phagetiters were measured by plating as described previously (1).

EMSA. Electrophoresis mobility shift assays (EMSA) were performed as es-sentially described (23). DNA containing the individual naturally occurring933W repressor binding sites was obtained by annealing 60-base oligonucleotidescontaining the 15-bp 933W binding site sequence and two additional naturallyoccurring bases on either end of the identified site. The DNA fragments wereradioactively labeled at their 5� ends by incubating the DNA with [-32P]ATP(6,000 Ci/mmol; Perkin-Elmer, Boston, MA) in the presence of T4 polynucle-otide kinase (Epicentre, Inc., Madison, WI). OR1 DNA was also radiolabeledusing [�-32P]dATP (3,000 Ci/mmol; Perkin-Elmer, Boston, MA) in place ofdATP in a PCR mixture with an OR1-containing plasmid as a template toincrease the radioactive signal and detect DNA to concentrations below 0.1 nM.Labeled DNA was incubated with the specified concentrations of 933W repres-sor protein in binding buffer (10 mM Tris [pH 8.0], 50 mM NaCl, 1 mM MgCl2,10% glycerol, 100 �g/ml bovine serum albumin [BSA], 1 mM isopropyl--D-thiogalactopyranoside [IPTG], 1 mM DTT) for 10 min at 25°C. The protein-DNA complexes were resolved on 5% polyacrylamide gels at 25°C. The electro-phoresis buffer was 1� TBE. The amounts of protein-DNA complexes presenton the dried gels were quantified using a Storm imager (GE Lifesciences, Pis-cataway, NJ).

Values of the dissociation constant (KD) were determined by nonlinearsquares fitting of the EMSA data using Prism 4.0 software (GraphPad SoftwareInc.). Each dissociation constant was determined from at least eight replicatemeasurements. Two-tailed t tests were employed to determine the significance ofobserved differences in dissociation constants of the various 933W repressor-DNA complexes.

DNase I footprinting. Template DNA was PCR amplified from the desiredplasmid using 5�-end-labeled standard M13 forward and unlabeled M13 reverseprimers and gel purified. Following phenol-chloroform-isoamyl alcohol extrac-tion and ethanol precipitation, this DNA (�0.05 nM) was incubated without orwith 933W repressor in binding buffer (10 mM Tris [pH 8.0], 50 mM NaCl, 1 mMMgCl2,100 �g/ml BSA, 1 mM DTT) for 10 min at 25°C prior to addition ofsufficient DNase I to generate, on average, one cleavage per DNA molecule with5 min of additional incubation. The cleavage reactions were terminated byprecipitation with ethanol and sec-butanol dehydration, and the DNA was dis-solved in 90% formamide solution containing tracking dyes. The products alongwith chemical sequencing reactions (Maxam Gilbert 1980) derived from thesame templates were resolved on 6% acrylamide gels containing 7 M urea in 1�TBE. Cleavage products were visualized using a Storm imager (GE Lifesciences,Piscataway, NJ).

Values of the dissociation constant (KD) were determined by nonlinearsquares fitting of the measured intensities of repressor-protected bands for eachof the three individual binding sites. Each dissociation constant was determinedfrom measurements made in three independent experiments. The reported dis-sociation constants varied by �20%.

In vivo dimethylsulfate (DMS) footprinting. Both MG1655::�imm933W andMG16655::�imm933W�OL lysogens were transformed with pGP1-2 (35), whichencodes T7 RNA polymerase and whose synthesis is under the control of atemperature-sensitive lambda repressor. Each of these two strains were subse-quently separately transformed with p933WR or pET17b (EMB Biosciences).

Cultures of the two plasmid-containing lysogens were grown for 16 h at 37°Cin LB with 100 �g/ml ampicillin and 50 �g/ml kanamycin. DMS was added to thecultures at a final concentration of 7.9 �M and incubated with shaking for 5 min

FIG. 1. Structure of the 933W immunity region. The positions ofOR1, OR2, and OR3, of OL1 and OL2, and of the repressor (cI) and crogenes (Cro) are indicated by boxes; the transcription start sites of PL,PRM, and PR are indicated by bent arrows.

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at 37°C. The reactions were stopped by addition of an equal volume of ice-coldLB and immediately placed on ice. Genomic DNA from 1.5 ml of treated cellswas isolated using lysozyme-phenol-RNase A essentially as described previously(32) and resuspended in PCR buffer (0.2 mM deoxynucleoside triphosphates, 1.5mM MgCl2, 50 mM KCl, 0.001% gelatin). Resuspended genomic DNA (20 �g)was used as template for each primer extension reaction.

A primer complementary to a sequence just outside the 933W OR region, inthe cro gene, 5�-CCTTTAATCGGCTCATCAAGATTTTGCAT-3�, was 5� endlabeled with T4 polynucleotide kinase (New England BioLabs, Beverly, MA) and[-32P]ATP (6,000 Ci/mmol; Perkin-Elmer, Boston, MA). Labeled primer wasadded to the dissolved genomic DNA to a final concentration of 1.4 �M, and theextension reaction was initiated with the addition Taq polymerase followed bythermocycling. After 35 rounds of extension, the products of extension reactions(one-half the reaction volume) were loaded onto 6% acrylamide–7 M urea gel(89 mM Tris, 89 mM borate, 1 mM EDTA) after addition of formamide dye andboiling of samples for 4 min. A sequencing ladder was also loaded next to theprimer extension reaction mixtures (Sequenase DNA sequencing kit; USB,Cleveland, OH). Gels were dried and imaged with a Storm imager.

Quantitative RT-PCR. Cultures of the plasmid-containing MG1655::�imm933W

and MG166::�imm933W�OL lysogens were grown to early log phase at 30°C in LBwith 100 �g/ml ampicillin and 50 �g/ml kanamycin. Cultures were shifted to 42°Cand grown until late log phase. RNA was extracted from 0.5 ml of cells by usingthe QuickExtract RNA extraction kit (Epicentre, Madison, WI). RNA was fur-ther purified by acid phenol extraction followed by chloroform-isoamyl alcoholextraction and precipitation. RNA was resuspended in 22 �l of DNase I bufferand RiboGuard, and residual genomic DNA was removed by treatment withRNase-free DNase I (Epicentre, Madison, WI) for 1 h at 37°C. cDNA synthesisreaction mixtures containing purified RNA, Affinityscript buffer, forward primer,and Affinityscript reverse transcriptase/RNase block enzyme mix (Agilent Tech-nologies, Cedarville, TX) were incubated at 25°C for 5 min to allow primerannealing, 45°C for 45 min for cDNA synthesis, and finally at 95°C for 5 min toheat kill the reverse transcriptase. Quantitation of RNA was performed byreal-time PCR. DNA products were detected using Sybr Green I (Invitrogen/Molecular Probes Inc., Carlsbad, CA) in a MiniOpticon real-time PCR detectionsystem (Bio-Rad Laboratories, Hercules, CA). Primers used for amplification ofPRM transcript cDNA were complementary to a portion of the 933W openreading frames 23 and 24. (933W PRM trans FWD, 5�-GCCGCTCTAACACCTAGTATTCTC-3�, and 933W PRM trans RVS, 5�-TAAGGCCGCCTGAACATATC-3�). As an internal control for RNA preparation, separate real-time PCRanalyses were performed on the cDNA preparations of the E. coli UmuC genetranscript from the same RNA preparations as the PRM transcripts. These real-time PCR assays used primers complementary to a portion of the UmuC gene(FWD UmuC, 5�-GATTTATGGGGTAAACCGGTGG-3�; RVS UmuC, 5�-CAGTCAGATCGAGACAATTACG-3�). Standard curves for the real-time PCRanalyses were made using known template amounts of a plasmid containing theregion of interest.

Numerical simulation of transcription data. Occupancies of OR1, OR2, andOR3 at various 933W repressor concentrations were calculated using the disso-ciation constants given in Fig. 5, below, and the following equation: frOx bound

� �{([Ox total

] [933W Rtotal] KD) � {([Ox total] [933W Rtotal] KD)2 �4[Ox total][933W Rtotal]}1/2}/(2[Ox total]), where Ox is either OR1, OR2, or OR3and Rtotal is the total repressor concentration. For the simulations, the DNAconcentration was fixed at the value used in the transcription experiments.Relative activities of PRM transcription were calculated using the experimentallyverified assumptions that 933W repressor bound at OR2 activates PRM and that933W repressor bound at OR3 represses the activity of this promoter. Maximalpromoter activity was set to 60, corresponding to the observed maximal activityof PRM relative to PR, as measured on a template bearing a mutation in OR3 thatprevented repressor binding.

Statistical methods. Where employed, tests for statistical significance of dif-ferences between paired data sets were performed using two-tailed t tests.

RESULTS

Examination of the threshold response to UV light. AnOL3-bound � repressor cooperatively assists binding of re-pressor to OR3 (15). This interaction requires the formationof an octameric � repressor-DNA complex containing twotetramers of � repressor, one bound at OR1 and OR2 andanother at OL1 and OL2 (15). Deletion of OL eliminates the

increased PRM activity in response to low doses of UV (noderepression effect), suggesting the DNA damage “thresh-old response” depends on formation of the OL-cI8-OR loopin phage � (3). The observation that bacteriophage 933Wlacks an OL3 site (16, 23, 36) (Fig. 1) indicates that unlikeother lambdoid phages (3), 933W either does not display athreshold response or that the threshold response of thisphage is constructed differently from that of other lambdoidphages. To distinguish between these alternatives, we com-pared the ability of UV light to induce lysogens of twodifferent hybrid bacteriophages: (i) �imm933W, a bacterio-phage that contains the entire immunity region of 933W andwhose lysis-lysogeny decision is controlled by 933W repres-sor-DNA interactions, and (ii) �imm434, a bacteriophagethat bears the immunity region of bacteriophage 434 andwhose repressor is capable of cooperatively binding its DNAsites. Other than the immunity regions (i.e., DNA betweenthe N gene [immediately upstream of OL] and the end of thecro gene [immediately downstream of OR]), the sequencesof these two phages are identical. Hence, any differences inthe ability of UV light to induce these two phages can bedirectly attributed to differences in gene regulation mecha-nisms encoded within the immunity region.

At low UV dosages, neither the �imm434 nor �imm933W

lysogen produces a significant number of bacteriophage (Fig.2). In contrast, once the UV exposure reached a threshold doseof �3.5 J/m2, the amount of phage produced by both of thesestrains rose sharply and increased linearly with UV dosage(Fig. 2). These findings show that the 933W immunity regioncodes for a threshold response to DNA damage. The UVthreshold set point value for 933W is nearly identical to thatobserved with bacteriophage � lysogens (3, 25), but since 933W

FIG. 2. Demonstration of the �imm933W threshold response to UVinduction, similar to that of �imm434. Lysogens containing either 933W(�imm933W) or 434 (�imm434) immunity regions were subjected toeither no UV light or increasing doses of UV light. Lysogens were thendiluted and incubated with aeration at 37°C for 2 h to allow phageinduction. Phage titers were determined by plating on MG1655. Cellnumbers were determined by plating aliquots of cells on LB platesprior to the 2-h incubation. Phage numbers are expressed as the frac-tion of the maximal amount of phage-forming units (PFU) per cell-forming units (CFU). Error bars represent standard deviations calcu-lated from the averages of 5 replicate experiments.

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lacks an OL3 site, its response must be enabled by a mechanismdifferent from phage � and its related bacteriophages.

Repressor occupancy of OR3 is independent of the presenceof OL. Despite the absence of an OL3 site (16, 23), 933Wrepressor bound at OL could have a role in regulating repres-sor occupancy of OR3, the key element of the threshold re-sponse (Fig. 2). We wished to directly test whether the OL

region of 933W has any role in regulating repressor occupancyof OR3. To do this we first used DMS footprinting to examine933W repressor occupancy in vivo in both a �imm933W lysogen(which bears an intact 933W OR and OL) and a �imm933W�OL

lysogen, in which the OL region of �imm933W has been deleted.These strains were transformed with either a control plasmidor one that directs the synthesis of 933W repressor (see Ma-terials and Methods). These strains were exposed to DMS, andthe DMS modification/protection pattern was detected byprimer extension and using a radioactively labeled primer.

To assist with identification of the 933W repressor-protectedbands in the samples isolated from the cells, genomic DNA wasisolated from untreated cells and treated in vitro with DMS inthe absence or presence of purified 933W repressor. As ex-pected, 933W repressor occupancy of OR1, OR2, and OR3 on

isolated DNA in vitro was detectable by a repressor-dependentdecrease in the intensity of several DMS-reactive guanine res-idues in these sites (Fig. 3A, lanes 7 and 8). For example, theDMS reactivity at positions 5�G, 7�G, and 2G in OR1 and 5�Gin OR2 in wild-type DNA decreased in the presence of 933Wrepressor. The ability of the repressor to protect these basesfrom modification in vitro was unaffected by deletion of the OL

region (Fig. 3A, compare lanes 7 and 8 with lanes 8 and 9).Our major focus with respect to the effect of OL deletion on

933W repressor occupancy of its binding sites in OR concernedOR3. Repressor binding to this site was therefore considered indetail. In the absence of any 933W repressor, the DMS reac-tivities of the four guanine residues on the top strand in OR3(positions 2, 7�, 5�, and 1�) were unaffected by the presence orabsence of the OL region (Fig. 3A, compare lanes 7 and 9).Also, for both these DNAs, added 933W repressor protectedthe guanines at positions 2 and 5� of OR3 from DMS modifi-cation (Fig. 3A, compare lanes 8 and 10). The extent of re-pressor-mediated protection of each of the guanines at posi-tions 2 and 5� was essentially identical in both the �imm933W

and �imm933W�OL DNAs (Fig. 3B). These observations estab-lish the protection DMS modification pattern of the repressor-

FIG. 3. Deletion of 933W OL does not affect repressor occupancy at OR3. (A) Representative DMS footprinting gel. DMS methylation of 933Wgenomic DNA template was used to detect repressor protection of OR sites. DMS was either not added (lanes 1 and 2) to lysogenic cells (lanes3 to 6) or isolated genomic DNA from lysogens was added (lanes 7 to 10) and allowed to react at 37°C for 5 min. Isolated genomic DNA in alllanes was subjected to primer extension using a radiolabeled DNA primer complementary to regions just outside of the 933W OR and Taqpolymerase (see Materials and Methods). Locations of guanine bases in OR3, OR2, and OR1 sensitive to methylation by DMS are indicated. Forthe in vivo DMS treatment (lanes 3 to 6), DMS was added to overnight cultures of �imm933W (wt) or �imm933W�OL (�OL) grown at 37°C. The levelof 933W repressor was determined by either endogenous lysogen levels (lanes 3 and 5) or endogenous levels plus additional repressor expressedfrom p933WR (lanes 4 and 6). No DMS was used in lanes 1 and 2. For the in vitro DMS treatment (lanes 7 to 10), DMS was added to purifiedgenomic DNA isolated from �imm933W (lanes 7 and 8) or �imm933W�OL (lanes 9 and 10) lysogens. Lanes 7 and 9 had no 933W repressor presentupon DMS addition. In lanes 8 and 10, saturating amounts of purified 933W repressor were added to genomic DNA prior to the DMS methylationreaction. (B) Quantification of in vitro DMS methylation intensities of guanines in OR3 (see panel A, lanes 7 to 10). (C) Quantification of in vivoDMS methylation intensities of guanines in OR3 (see panel A, lanes 3 to 6). In panels B and C, intensities were normalized to the reactivity atposition 1�. Error bars in panels B and C represent standard deviations derived from 4 replicate experiments.

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bound OR3 site and provide a basis for understanding the invivo DMS modification patterns.

Similar to the pattern of DMS reactivity seen in vitro, onceinside cells DMS modifies guanines at positions 2, 7�, 5�, and 1�of OR3. However, compared to the results obtained in vitro inthe absence of repressor, the relative DMS reactivities of theguanine residues at positions 2 and 5� in OR3 are lower thanseen at positions 7� and 1� (Fig. 3A, compare lane 3 to lanes 7and 8 and lane 5 to lanes 9 and 10; see also B and C). In theabsence of repressor in vitro, the intensities of these bands areessentially equal, indicating near-identical accessibility to DMS(Fig. 3A, lanes 7 and 9, and B). Quantification of the results inlane 3 showed that the DMS reactivities of the guanines atpositions 2 and 5� were 2-fold lower than that at position 1�(Fig. 3C). This finding suggests that at the level of 933Wrepressor found in the �imm933W lysogen, OR3 is partiallyoccupied by the 933W repressor. Consistent with this sugges-tion, the reactivities of the bases at positions 2 and 5� werereduced by an additional 2-fold when cells were transformedwith a plasmid that directs synthesis of an additional amount of933W repressor (Fig. 3A, compare lanes 3 and 4 and lanes 5and 6). Moreover, in the presence of the excess repressor, thelevels of DMS reactivity of position 2 and 5� guanines werereduced to the same level as that seen when this DNA reactedwith DMS in the presence of saturating levels of 933W repres-sor in vitro (see Fig. 5B and C, below).

Importantly, we found that both the pattern and degree ofprotection of the bases in OR3 in the �imm933W�OL lysogen areidentical to that seen in the �imm933W lysogen (Fig. 3A, com-pare lanes 3 and 5, and B and C). This result indicated that theabsence or presence of the OL region has no effect on the abil-ity of the 933W repressor to occupy OR3 in vivo at the levels ofrepressor found in a lysogen. We also found that the degree towhich excess (plasmid-derived) repressor enhanced the protec-tion of the guanines at positions 2 and 5� inside �imm933W�OL

lysogens was identical to that found with the �imm933W lysogen(Fig. 3C). Taken together, these results show that 933W re-pressor binding to sites in OL does not influence repressoroccupancy of OR3. These observations are consistent with thesuggestion that a repressor-mediated DNA loop between OL

and OR does not form in bacteriophage 933W. The DMSmodification patterns in the region around OR1 and OR2 ob-tained in vitro differ slightly from those found in vivo. Wesuspect these differences do not stem from differences in re-pressor occupancy under these two conditions, but rather arisefrom the partial occupancy of this region by RNA polymerasebound at PR and/or PRM. Regardless, inspection of the resultsshown in Fig. 3A, lanes 4 and 6, shows that the DMS modifi-cation pattern of these sites was unaffected by the absence orpresence of OL, indicating deletion of OL does not affect 933Wrepressor binding to these sites. This finding is consistent withour observation that deletion of OL does not affect repressoroccupancy of OR3.

PRM transcriptional activity is not dependent on OL. Tofurther examine the potential role of OL-bound 933W repres-sor on OR3 occupancy, we determined if the presence or ab-sence of the OL region affected the transcriptional activity ofPRM in vivo. For these experiments, we compared the amountof PRM transcript produced in both the �imm933W and

�imm933W�OL lysogens in the absence and presence of addi-tional 933W repressor.

Quantification of PRM transcripts found in �imm933W and�imm933W�OL showed that the differences in the steady-stateamounts of PRM-derived mRNA in these two lysogens werestatistically insignificant (P � 0.34) (Fig. 4). Production ofadditional 933W repressor inside each of these lysogens re-pressed PRM transcript levels by an identical amount (Fig. 4).Since repressor occupancy of OR2 and OR3 positively andnegatively regulates PRM activity, respectively (see Fig. 7, be-low), these results showed that OL has no effect on repressoroccupancy of OR2 and OR3. These findings are completelyconsistent with the results of our in vivo DMS footprintingstudy (Fig. 3), which showed that deletion of the repressorbinding sites in OL did not have any effect on 933W repressoroccupancy of OR3. These observations do differ dramaticallyfrom what was found with bacteriophage �. In that case, de-letion of � repressor binding sites in �OL increased tran-scription from �PRM as a consequence of the decreasedoccupancy of �OR3 by 2- to 3-fold (3, 13). Thus, our resultsshow that occupancy of the binding sites in 933W OR by the933W repressor is by interactions between repressors boundat OR and OL.

Affinities of the 933W repressor for naturally occurring933W OR binding sites in separate sites and intact OR. In theabsence of OL-mediated regulation of OR3 occupancy, howthen might the UV threshold response of bacteriophage 933W(Fig. 2) be determined? To begin to answer this question, wemeasured the affinity of the 933W repressor for its naturallyoccurring sites in OR and OL, both on the individual sites andin the context of the intact operator. The intrinsic affinity of the

FIG. 4. Deletion of 933W OL does not affect in vivo levels of PRMtranscripts. Quantitative real-time PCR was used to determine PRMcDNA levels produced from reverse transcription of total RNA ex-tracted from �imm933W and �imm933W�OL lysogens producing wild-type levels of 933W repressor (containing pET17b and pGP1-2) orexcess 933W repressor ( -containing p933WR and pGP1-2).Amounts of PRM transcripts were normalized to the amount of UmuCtranscripts, determined in parallel reverse transcription reactions (seeMaterials and Methods). Differences between the amounts of tran-script without or with excess repressor were significant (P � 0.0001).The amount of transcripts from OL

and �OL lysogens were notsignificantly different (P � 0.34), regardless of the absence or presenceof the repressor-producing plasmid. Data are derived from eight rep-licate experiments.

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933W repressor for the individual sites was measured in anelectrophoresis mobility shift assay. Figure 5 shows that the933W repressor displayed a hierarchy of affinities for the sitesin OR, as it bound with highest affinity to OR1 and with �100-and 15-fold-lower affinities to OR2 and OR3, respectively. Thedifferences in affinities of the 933W repressor for OR1, OR2,and OR3 were significant (P � 0.005), and the relative affinitiesof the 933W repressor for its sites in OR were qualitatively andquantitatively different than what was seen with other well-studied lambdoid bacteriophage repressors, e.g., � (OR1 of 1,OR2 of 75, and OR3 of 30), 434 (OR1 of 1, OR2 of 14, andOR3of 5.5), and P22 (OR1 of 1, OR2 of 14, and OR3 of 6) (5,6, 19, 21, 33, 34, 37).

Our previously reported DNase I footprinting results alsosuggested that the 933W repressor does not bind cooperativelyto OR1 and OR2 when these sites are present within intact OR

(23). However, the conditions under which those experimentswere performed are not directly comparable to those used inthe EMSA determinations reported here. Also, those experi-ments were performed at concentrations of DNA that, basedon the data in Fig. 5, were either within or near the stoichio-metric range for repressor binding to OR1 and OR2, respec-tively. Therefore, we repeated the footprinting experiments

under conditions identical to those used in the EMSA deter-minations (i.e., low DNA concentration and absence of com-petitor DNA) to provide a comparable measure of the affinitiesof 933W repressor for its individual sites in intact OR. Unlikethe case with other lambdoid bacteriophage repressors, the933W repressor binds the three sites in intact OR at distinctlydifferent concentrations (Fig. 6). The dissociation constantsdeduced from a series of footprinting experiments were asfollows: OR1, 0.22 nM; OR2, 3.0 nM; OR3, 5.8 nM (� �20%).Hence, the affinities of the 933W repressor for OR1, OR2, andOR3 in intact OR are essentially identical to the affinities of the933W repressor for the individual, separated sites shown inFig. 5. The similarities between 933W repressor affinities forthe separated sites and the sites present in intact OR led us tomodify our earlier suggestion that the 933W repressor maybind with weak cooperativity to adjacent sites within the intactoperators (23). Instead, our data indicate that the 933W re-pressor does not cooperatively bind to adjacent sites in OR.

Our measurements revealed that the 933W repressor bindsto OL1 and OL2 with similar affinities (Fig. 5). We showedpreviously that in intact 933W OL, the 933W repressor binds itsrespective OL1 and OL2 with nearly equal affinity (23). Similarmeasurements under comparable conditions to our EMSA

FIG. 5. Sequences and intrinsic affinities of 933W for separate naturally occurring 933W operators. (A) Binding of 933W repressor to individualnaturally occurring binding sites. Various concentrations of 933W repressor were mixed with radiolabeled binding site-containing DNA, andprotein-DNA complex formation was detected as described in Materials and Methods. Points represent averages of �8 replicate experiments.Lines represent best nonlinear least-squares fits to the data based on a hyperbolic equation. (B) The sequences and dissociation constants (KD[lswb]� standard deviation]) of 933W repressor binding to OR1, OR2, OR3, OL1, and OL2. Dissociation constants were derived from data shownin panel A and determined as described in Materials and Methods. Standard deviations were calculated from the averages of �8 replicateexperiments.

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studies confirmed this finding (data not shown). Thus, theequal affinities of 933W repressor for OL1 and OL2 seen inintact OL are apparently not a result of cooperative binding bythis protein.

Regardless of whether 933W repressor binds DNA cooper-atively, our data confirm the earlier finding that its affinities forOR2 and OR3 differ by 1.5- to 2-fold. This observation providesa potential explanation as to how the UV threshold in bacte-riophage 933W is generated. Instead of being regulated by along-range loop, 933W repressor occupancy of the sites in OR

is instead governed solely by the intrinsic affinities of the re-pressor for the individual sites in OR.

Positive and negative regulation of PRM transcription by the933W repressor. To further explore how the small differencesin the 933W repressor’s intrinsic affinities for sites in OR mightcontribute to the UV threshold, we determined how the bac-teriophage 933W repressor regulates transcription from PRM.In vitro transcription analysis of a 933W wild-type OR template,containing the 933W OR region and its PR and PRM promoters,showed that in the absence of 933W repressor, no PRM tran-script was observed, while PR was robustly transcribed. As the933W repressor concentration was increased to up to �30 nM,an increasing amount of PRM transcript was synthesized andtranscription of PR was repressed (Fig. 7). Transcription fromPRM was inhibited as the concentration of 933W repressor wasincreased from 30 nM to 120 nM (Fig. 7). Consistent withprevious observations (23), these findings indicate that, similarto other bacteriophage repressors, the 933W repressor bothactivates and represses PRM transcription and functions as arepressor of PR transcriptional activity.

In the other well-studied bacteriophages, the repressor-OR2complex is needed to activate transcription of PRM. To inves-tigate the role of the 933 repressor-OR2 complex in stimulating933W PRM transcription, we created a transcription template

bearing two base substitutions in OR2 at positions 5� and 4�(G5�T4�3 T5�C4�) (see Fig. 1 for wild-type sequences). Controlexperiments established that these sequence changes pre-vented 933W repressor binding to OR2, without detectably

FIG. 6. DNase I footprinting of complexes between the 933W re-pressor and 933W OR. Shown is a representative phosphorimage of arepresentative gel. DNA templates containing 933W OR radioactivelylabeled were partially digested with DNase I in the presence of in-creasing amounts of the 933W repressor. Lane 1 shows the DNase Icleavage pattern of the DNA in the absence of added repressor. Inlanes 2 to 13, repressor concentrations were increased in 1.5-fold stepsstarting at 0.17 nM protein. The arrows identify positions of protectedbands used in measuring site occupancies of OR1, OR2, and OR3.

FIG. 7. OR2 and OR3 are required for regulation of PRM transcriptionby repressor. (A) Representative transcription gels. 933W DNA tem-plates containing wild-type (wt) OR or OR regions bearing mutations ineither OR2 (OR2�) or OR3 (OR3�) were transcribed in vitro in the ab-sence of repressor (lane 1) and at repressor concentrations increased in2-fold steps (lanes 2 to 7), starting with 4.5 nM protein. Positions oftranscripts initiated from PR and PRM are indicated. The 933W repressorwas incubated with DNA template at 25°C for 10 min, followed by addi-tion of E. coli RNA polymerase. The reaction mixture was transferred to37°C for 10 min before the transcription reaction was initiated by theaddition of nucleotides and heparin. (B) Graphical representation of theamount of PRM transcript synthesized as a function of 933W repressorconcentration from the template bearing wild-type OR or templates bear-ing a mutation in OR3 or OR2. The transcript amounts are quantified asa percentage of maximal PR transcription as a function of 933W repressorconcentration. Error bars represent standard deviations calculated fromthe averages of at least three replicate experiments. (C) Numerical sim-ulation of transcription data using OR occupancy data calculated fromdissociation constants given in Fig. 5 (see also Materials and Methods).The lines represent simulated data. Points are the measured PRM activity,as described for panel B.

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altering binding of this protein to OR1 or OR3 or the concen-tration of repressor needed to repress PR transcription (datanot shown). As opposed to the case with wild-type template,933W repressor was incapable of activating PRM transcriptionfrom the OR2� template in vitro (Fig. 7A). This finding showedthat, similar to other lambdoid phages, a 933W repressorbound to OR2 is needed to activate transcription from PRM.

In all other bacteriophages, the repressor-OR3 complex in-hibits transcription initiated at PRM. We investigated themechanism by which the 933W repressor negatively regulatesPRM, and thus its own synthesis, by examining the effects ofmutations that prevent 933W repressor binding to OR3 onPRM transcription in vitro. Mutations that blocked 933W re-pressor binding to OR3 prevented the 933W repressor fromrepressing transcription from PRM (Fig. 7A). These resultsshow that 933W repressor binding to OR3 mediates repressionof transcription from PRM.

Our results show that the effects of 933W repressor bindingto OR2 and OR3 on PRM transcriptional activity are similar tothose seen in other bacteriophages. However, inspection of theresults shown in Fig. 7 reveals that the activity of 933W PRM,in response to varied 933W repressor concentrations, differsconsiderably from that seen in other lambdoid bacteriophages.In 933W, the maximal repressor-stimulated PRM transcriptionfrom the OR3� template was nearly 4-fold higher than thatobserved from the wild-type template. In contrast, in bacterio-phage �, the maximal amount of repressor-stimulated PRM

transcript obtained on wild-type and OR3� templates is thesame (17, 28). Similar results were obtained with bacterio-phage 434 (10, 42). Based on the observation that the 933Wrepressor affinity for OR2 is less than 2-fold higher than itsaffinity for OR3 (Fig. 5), the results in Fig. 7A indicate that at933W repressor concentrations needed to completely occupyOR2, the 933W repressor partially occupies OR3. This conclu-sion was verified by accurately simulating these transcriptionresults, using operator occupancy data derived from the disso-ciation constants shown above (Fig. 7C).

On the wild-type OR template, complete repression of PRM

transcription occurred with the addition only 4-fold more933W repressor than is needed maximally for this promoter(Fig. 7). The difference in 933W repressor concentrationneeded to activate versus repress PRM transcription was muchlower than that seen in other lambdoid phages. In bacterio-phage �, �20-fold more � repressor is required to repress PRM

transcription from an OR template than is needed to maximallystimulate this promoter’s activity (17, 29). Similar results wereobtained with bacteriophage 434 (10, 43).

DISCUSSION

Our data clearly show that in vivo, deletion of the 933W OL

region does not influence repressor occupancy of OR3 or theactivity of the PRM promoter. Hence, bacteriophage 933Wregulates PRM activity, and therefore repressor levels, by amechanism that apparently does not involve long-range coop-erative interactions between 933W repressors. Had a long-range DNA loop formed in 933W and had such a loop beenrequired for modulating 933W repressor occupancy of sites inOR, we would also have anticipated that the affinities of 933Wrepressor for the sites in OL should be equal to or greater than

its affinities for OR1 and OR2. However, the intrinsic affinitiesof 933W repressor for OL1 and OL2 were �7-fold lower thanits affinities for OR1 (Fig. 5). It is possible that 933W repressorbinding at OL1 and OL2 stabilizes 933W repressor binding atOR. However, this view is inconsistent with the finding thatdeletion of OL apparently does not impact 933W repressoroccupancy of any sites in OR (Fig. 3).

How then does bacteriophage 933W regulate repressor lev-els so that it can form a stable and yet inducible lysogen? Ingeneral, 933W repressor-mediated regulation of PRM activityin bacteriophage 933W employs a strategy similar to that ofother lambdoid phages. Like those phages, 933W repressorbound to OR2 is responsible for activation of PRM, and repres-sor bound to OR3 is required for repression. However, unlikeother lambdoid phages, in bacteriophage 933W negative reg-ulation of PRM transcriptional activity by the repressor is gov-erned solely by differences in the 933W repressor’s intrinsicaffinities OR2 and OR3. The �2-fold difference between theaffinities of 933W repressor for 933W OR2 and 933W OR3 (asopposed to the 15- to 30-fold differences in affinities of the �repressor for �OR2 and �OR3 in intact � OR) results in tightrepressor autoregulation of PRM activity. The small differencesin relative affinities of the 933W repressor for 933W OR2 and933W OR3 are sufficient to allow repressor occupancy at OR3in vivo for negative regulation of PRM in 933W. That is, theclosely matched affinities of OR2 and OR3 allow for the coun-terbalancing of the autogenous positive- and negative-controlactivities of the 933W repressor-DNA complexes formed at933W OR2 and 933W OR3, respectively, on the transcriptionalactivity of PRM.

Interestingly, repression of 933W PRM transcription in vitro,which is mediated by the 933W repressor binding to OR3,occurs at 933W repressor concentrations where full occupancyof OR2, and thus maximal activation of PRM transcriptionalactivity, has not yet been reached. Furthermore, complete re-pression of PRM occurs at levels of repressor that are only4-fold higher than is needed for activation, further arguing forthe partial occupancy of OR3 at concentrations where OR2 isnot yet completely filled. The lack of repressor-mediated in-teractions between OL and OR suggests that the in vitro tran-scription findings accurately represent the situation in vivo.

The narrow range of 933W repressor concentrations neededto activate and repress 933W PRM transcription is strikinglydifferent from that seen in vitro with other lambdoid phages. Inbacteriophages � and 434, repression of PRM (via occupancy ofOR3) does not occur until a repressor concentration at whichPRM is fully activated (or OR2 is maximally filled) is reached.This is because in these bacteriophages, at least 20-fold morerepressor is required to fully repress PRM than that needed tomaximally stimulate transcription. Therefore, in both � and434 bacteriophages, the activity of maximal repressor-stimu-lated PRM activity on wild-type OR and OR3 mutant templates,where repressor cannot repress PRM transcription, is essen-tially identical (10, 29).

It is important to note that to enable repression of PRM invivo, the weak intrinsic affinity of � repressor for OR3 must beovercome by the long-range DNA loop mediated by repressorbound at OL and OR sites (15). As a consequence, repressionof PRM in bacteriophage � in vivo requires only 4-fold more �repressor than is needed for activation, as opposed to the

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�20-fold needed in vitro (15, 29). Therefore, the range of �repressor concentrations needed to activate and repress � PRM

in vivo transcription matches that seen for 933W. However, asa consequence of the cooperative binding of � repressor toOR1 and OR2, repressor-stimulated transcriptional activity ofPRM in vivo reaches �70% of the maximum amount seen in anOL3� or OR3� � bacteriophage. Hence, in �, repression ofPRM only occurs at repressor concentrations higher than wherePRM is essentially fully activated. This observation contrastswith our findings for intact 933W OR, where PRM reached only�25% of the maximum activation possible.

The narrow window of repressor concentrations between theamount needed to activate versus repress 933W PRM transcrip-tion explains why 933W bacteriophage displays a “thresholdresponse” to DNA damage. The observation of a thresholdresponse has been attributed to the response of PRM activity tovarious levels of repressor. As discussed above, in both 933Wand � lysogens, the transcriptional activity of PRM is partiallyrepressed due to partial occupancy of OR3 by repressor. At lowUV doses, as a consequence of RecA-mediated repressor deg-radation, OR3 occupancy decreases, leading to increased PRM

activity and a consequent increase in repressor levels. At highUV doses, the rate of RecA-mediated repressor degradationexceeds the rate of new repressor synthesis, and the repressorconcentration falls below the level needed to occupy OR1 andOR2, leading to lysogen induction. Hence, the key feature ofthe threshold response is partial occupancy of OR3 by therepressor. In bacteriophage �, OR3 binding is facilitated by arepressor-mediated loop between OL and OR that allows anOL3-bound repressor to closely approach OR3 and to help arepressor bind that site via cooperative interactions that arethought to mimic those used by � repressor bound at adjacentsites (3). In bacteriophage 933W, OR3 occupancy is facilitatedby the closely matched affinities of 933W repressor for thebinding site that activates (OR2) and the one that represses(OR3) PRM transcription.

This proposed strategy for generating a UV threshold re-sponse in bacteriophage 933W not only eliminates the need forlong-distance cooperative interactions between two DNA-bound repressor tetramers but also the subsequent role forcooperative binding between spatially adjacent OR3- and OL3-bound repressor dimers in the looped complex. Since the in-teraction between repressors bound at OR3 and OL3 is antic-ipated to use the “side-by-side” cooperativity interface, thisconclusion is consistent with the indication that the 933Wrepressor is incapable of cooperatively binding to adjacent siteson OR (Fig. 5 and 6). Together with the fact that bacteriophage933W forms stable lysogens, this suggestion indicates that the�9-fold difference in relative affinity of the 933W repressor forOR2 and OR1 is sufficient to enable efficient occupancy of OR2and positive regulation of PRM in the absence of side-by-sidecooperative repressor interactions. In contrast, the � phagerepressor’s intrinsic affinity for OR1 is 30 times higher than itsaffinity for OR2 (21, 22, 34). It is known that efficient occu-pancy of OR2 in wild-type � phage, and therefore stable �lysogen formation, requires cooperative repressor binding toOR1 and OR2 (2, 11).

The different levels of maximal PRM activity in vivo in � and933W bacteriophages and the apparent inability of the 933Wrepressor to cooperatively bind adjacent sites may help explain

the observation that 933W lysogens behave as a “hair trigger,”i.e., the frequency of DNA damage-independent (spontane-ous) induction is much higher in 933W than in other relatedbacteriophages (26). The increased induction frequency inthese stx-encoding phages has been attributed to the require-ment for a lower concentration of active RecA necessary forinduction (26). The critical level of RecA needed for inductionof 933W phage could be determined by an increased sensitivityof the 933W repressor to RecA, a decrease in the strength ofthe 933W repressor’s binding interactions with its operators,and/or a decreased total amount of 933W repressor present inthe lysogen.

Our findings show that the absolute affinities of the 933Wrepressor for its DNA sites are not dramatically different thanthe affinities of other lambdoid phage repressors for their cog-nate operators (Fig. 5). We have not directly assessed thesensitivity of 933W to RecA-mediated autocleavage. Results ofin vitro transcription assays indicated that the maximal activityof 933W repressor-stimulated PRM is 60% of the value of PR

(Fig. 7), whereas similar experiments have indicated that themaximal activity of � repressor-stimulated PRM is �3-foldlower, i.e., �20% of � PR activity (18, 39, 41). Our data alsoindicate that the in vivo activity of 933W PRM is only 25% of itsmaximal level. Together these observations suggest that theamount of repressor is lower in 933W lysogens than in � lyso-gens. Therefore, the increased sensitivity of 933W lysogens tospontaneous induction could be due, at least in part, to asmaller amount of repressor in the 933W lysogen than in �lysogens.

Babic and Little (2) were able to isolate cis-acting mutantsthat allow a � bacteriophage encoding a repressor that is in-capable of cooperatively forming DNA-bound tetramers toform stable lysogens. The mutant phages identified by Babicand Little compensated for the repressor cooperativity defect,in part, by increasing the relative affinity of OR2 for the �repressor. We showed here that the affinity of the 933W re-pressor for its OR2 is 4- to 6-fold higher, relative to 933W OR1,than seen in wild-type �. Therefore, the compensatory OR2mutation found in the mutant � phages mimics a key nativefeature of the 933W bacteriophage lysis-lysogeny circuitry.

The increased affinity of repressor for OR2 in the mutant �phage creates a scenario where, akin to bacteriophage 933W,the � repressor binds OR3 and the mutant OR2 with similaraffinities. Consequently, the smaller difference between OR2and OR3 affinities would mean that at � repressor concentra-tions where full occupancy of OR2 has not yet been reached,OR3 would be partially occupied, and thus PRM partially re-pressed. With PRM partially repressed, the mutant � phagerequires a more active PRM promoter to maintain requiredlevels of � repressor to form stable lysogens. This observationis consistent with the inference that the maximal stimulatedactivity of 933W PRM is greater than that of wild-type �PRM.

The 933W repressor is apparently unique among the lamb-doid bacteriophage repressors in its native inability to bindcooperatively to either adjacent or widely separated sites. TheC-terminal domain (CTD) of lambdoid phage repressors isresponsible for mediating any cooperative binding (30). Wehypothesize that the 933W repressor is incapable of bindingDNA cooperatively due to critical sequence differences be-tween its CTD and the CTDs of other lambdoid repressors. A

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sequence comparison of phage repressor CTDs revealed onlya weak (10 to 11%) similarity between the sequence of the933W CTD and those of �, 434, and P22 phages (Fig. 8), Incontrast, the sequences of the CTDs of �, 434, and P22 werehighly similar (36 to 60%) to each other. Strikingly, the se-quence alignment showed many of the � repressor residuesfound in the tetramer interface and those tested for mediatingcooperative interactions, such as E188, K192, S198, G199,Q209, Y210, and M212, differ from those in analogous posi-tions in 933W in terms of both identity and character (4, 7–9,38). In contrast, most of the residues in these regions of the 434and P22 CTDs, which are known to bind DNA cooperatively,are similar to those found in the � repressor. Hence, we sug-gest that these sequence differences underlie the inability ofthe 933W repressor to cooperatively bind DNA.

ACKNOWLEDGMENT

The work described in the paper was supported in part by a grantfrom the National Science Foundation (MCB-0956454) to G.B.K.

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FIG. 8. Alignments of the CTDs of the 933W repressor with three other lambdoid phage repressors known to cooperatively bind DNA.Multiple sequence alignments were performed using ClustalW (24). Shaded boxes were used to demonstrate the output based on residue matches(black) and functional similarities (gray). This program was written by Kay Hofmann and Michael D. Baron. Numbers indicate the residue withinthe protein that defines the beginning of the CTD. Black dots indicate residues in the � repressor that contribute to cooperative interactionsbetween repressor dimers.

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