Bacteriophage λ repressor allelic modulation of the Rex exclusion phenotype

NOTE

Bacteriophage λ repressor allelic modulation ofthe Rex exclusion phenotype

Roderick A. Slavcev, Harold J. Bull, and Sidney Hayes

Abstract: The sensitivity of ∆red-gam∆ren mutants of bacteriophage λ to Rex exclusion by λrexA+rexB+ lysogens ismodulated by the prophage cI repressor allele. We show the following: (i) λspi156∆nin5 forms plaques on a cI+–rexA+–rexB+ lysogen with 105-fold higher efficiency than on cI[Ts]–rexA+–rexB+ derivatives (ii) The cI[Ts]857 alleleaugmentation of Rex exclusion is recessive to cI+. (iii) The cI857-mediated increase in Rex exclusion activity involvesthe participation of a genetic element mapping outside of cI–rexA–rexB.

Key words: bacteriophage lambda (λ), CI repressor, Rex exclusion phenotype, cI–rexA–rexB operon, bacteriophageλspi156∆nin5, cI[Ts]857, cI[Ts]2.

Résumé : La sensibilité des mutants �red-gam�ren du bactériophage � à l’exclusion Rex par les lysogènes �

rexA+rexB+ est modulée par l’allèle répressive cI du prophage. Nous démontrons dans ces pages que : (i) �spi156�nin5forme des plages sur un lysogène cI+–rexA+–rexB+ avec une efficacité 105 fois plus élevée que sur des dérivés decI[Ts]–rexA+–rexB+. (ii) L’augmentation de l’exclusion Rex par l’allèle cI[Ts]857 est récessive par rapport à cl+; et (iii)L’augmentation de l’activité d’exclusion Rex par cI857 nécessite la participation d’un élément génétique localisé àl’extérieur de cI–rexA–rexB.

Mots clés : bactériophage lambda (�), répresseur CI, phénotype d’exclusion Rex, opéron cI–rexA–rexB, bactériophage�spi156�nin5, cI[Ts]857, cI[Ts]2.

[Traduit par la Rédaction] Slavcev et al. 229

The Rex exclusion system exhibited by cells lysogenic forbacteriophage λ restricts the growth of rII mutants of phageT4, as well as various derivatives of T5, T7, and λ (Benzer1955; Molineux 1991). The Rex phenotype is encoded bythe rexA–rexB genes (Matz et al. 1982), which are co-expressed with the λcI repressor from the PM–cI–rexA–rexB–timm operon in an established prophage (Landsmann et al.1982). Toothman and Herskowitz (1980b) found thatlambdoid phages mutated for ren and red (exo bet) genes aresensitive to Rex exclusion to varying degrees and that re-striction of phage growth is augmented by the presence ofthe cI[Ts]857 repressor allele (1980a). They concluded thattheir findings were consistent with CI repressor being essen-tial for Rex-dependent exclusion. Later, it was shown thatthe Rex exclusion phenotype toward T4rII phage mutantscould be conferred by multicopy plasmid expression of

rexA–rexB genes in the absence of cI (Shinedling et al.1987), and, thus, the repressor cannot be mechanistically in-volved in Rex exclusion of T4rII. In these studies, it remainsunclear how the RexA RexB levels differ for a multicopyplasmid lacking cI compared with prophage PM–cI–rexA–rexB transcription dependent upon CI.

We sought another phage that is influenced by repressorallelism and found that this condition was met by theλspi156∆nin5 phage, which is ∆red–gam and ∆ren and issimilarly as sensitive to Rex exclusion exhibited by a λcI857lysogen as the classical T4rII indicator phage. Using λspi156∆nin5 (Fig. 1), we investigated repressor modulation of Rexactivity and tested (i) if cI[Ts]857 allelic augmentation ofRex activity was dominant or recessive to cI+ and (ii) ifrepressor modulation of Rex exclusion activity was solelydependent upon the cI857–rexA–rexB genes encoded by theimmunity region of the repressed λcI857 prophage.

We utilized derivatives of Eco K strains of Escherichiacoli: R594 F– lac-3350 galK2 galT22 rpsL179 IN(rrnD–rrnE)1 λ– (Bachmann 1987) to prepare lysogens. λ wildtype, λbio275imm434∆nin5, λcI[Ts]857, and λcI[Ts]2 werefrom our stocks; λspi156∆nin5 was from G. Smith, FredHutchinson Cancer Research Center, Seattle, Wash., as de-scribed by Smith (1975); λrexB5A, λcI857[Ts]rexB5A,λrexA30A, and λcI857[Ts]rexAQam[allele 301] preparationswere from G. Gussin, University of Iowa, Iowa City, Iowa,via W. Szybalski, as was λrexBgo293, which mapped

Can. J. Microbiol. 49: 225–229 (2003) doi: 10.1139/W03-021 © 2003 NRC Canada

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Received 1 October 2002. Revision received 24 February2003. Accepted 3 March 2003. Published on the NRCResearch Press Web site at http://cjm.nrc.ca on 21 March2003.

R.A. Slavcev, H.J. Bull, and S. Hayes.1 Department ofMicrobiology and Immunology, College of Medicine,University of Saskatchewan, Saskatoon, SK S7N 5E5,Canada.

1Corresponding author (e-mail: [email protected]).

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(Gussin and Peterson 1972) to the same interval left ofbio16–3 endpoint within rexB (Daniels et al. 1983) as therexB5A mutation. Variant cI+ rex– phages were obtained bycrossing λcIsus14 with λrex– cI857 and selecting for turbidplaque recombinants at 42°C. We distinguished the cI[Ts]and cI+ variants of λrexB– and λrexA– phage mutants by pre-paring lysogens and confirming thermolability. The mappingof the rex– mutants was described by Gussin and Peterson(1972), Gussin et al. (1973), and Landsmann et al. (1982).Phage T4rII∆1589 (deletion spanning the rIIA and rIIBgenes generating an rIIA–rIIB fusion that is phenotypicallyRIIA– RIIB+) was obtained from G. Mosig, VanderbiltUniverstiy, Nashville, Tenn.

The endpoints for the E. coli spi156 DNA substitution inλspi156∆nin5 were examined by restriction mapping andDNA sequence analysis (Bull 1995). Restriction mapping re-vealed that the left arm (Nu1-J-b) of λ and λspi156∆nin5were equivalent (data not shown). The left endpoint of thespi156 E. coli DNA substitution was in a 408-bp region be-tween cut sites HindIII (27 479 bp λ) and AvaI (27 887 bp),straddling the att λ site (27 731 bp λ). The right substitutionendpoint was between cut sites AvaI (38 214 bp upstream ofλ gene O at 38 686 bp) and a BglII site (38 814 bp). DNAwas extracted from λ wild type, and the λspi156∆nin5 andthe region upstream of λ gene O was sequenced for severalhundred base pairs by the protocol described with Sequenasekit (version 2.0, United States Biochemical Corp., Cleve-land, Ohio) using primer R21 (38 756 5′-TCTGCCACA-TTACGCTCCTG-3′ 38 737) from λ O gene.

Phage λspi156∆nin5 (Smith 1975) is insensitive to CI re-pression but is Rex-sensitive (Hayes and Hayes 1986). Thisis presumably due to the deletion of the distal end of ren by∆nin5 (Daniels et al. 1983) (removing ~5.5% λ) and thespi156 substitution of exo bet (red) gam genes shown by thephage’s insensitivity to P2 interference (Malone andChattoraj 1975) and its inability to form plaques on a RecA–

strain (Salstrom et al. 1979). We sequenced through therightward spi156-λ juncture for several hundred base pairsinto the spi156 E. coli DNA substitution. The rightwardspi156 E. coli sequence was localized to the moaA chlorateresistance gene mapping at 17.6 min on the chromosome,with the substitution end point (Fig. 1) representing base 333downstream from the moaA ATG start. Therefore, the spi156substitution likely includes E. coli chromosomal DNA be-tween attλ site through bioA–D–uvrB–moaA. The right substi-tution end point was within λ gene cII and downstream ofoop (Fig. 1). Thus, the spi156 substitution deletes λ genes int–cII, including the normally essential PR promoter of λ requiredfor transcription of genes O–P–Q. The substituting E. coliDNA must provide an alternative promoter for PR to permit ex-pression of the essential genes O and P for λspi156∆nin5 repli-cation. Both a sigma-70 promoter modulated by the productof modE (McNicholas et al. 1997) and a sigma-54 promoterfall within 170 bp of the moaA AUG translational start siteand may substitute for the deleted PR promoter.

The plasmids used herein are shown in Fig. 2. PlasmidspACYC184 and pUC18 were from New England BiolabsLtd. (Pickering, Ont.). pRS1 was constructed by digesting λwild-type DNA with BglII and ligating λ DNA 38 103 bpthrough 35 711 bp into pACYC184 tetR BamHI cut site at1869 bp. pRS2 was constructed by digesting pCH1 (Hayeset al. 1997) with BglII and ligating λ DNA 38 103 bpthrough 35 711 bp into pACYC184 tetR BamHI site at1869 bp. pRS3 was constructed by digesting pRS2 withMfeI, removing λ DNA 35 764 bp through 37 186 bp andthen religating. pRS4 was constructed by digesting pRS2with BstYI, yielding λ fragment 38 103 bp through35 711 bp, and ligating λ fragment 38 103 bp through 35 711bp into the BamHI site (486 bp) within the MCS lacZ ′ ofpUC18. pRS11 was constructed by digesting pRS4 withMfeI, removing λ DNA 35 764 bp through 37 186 bp, andthen religating.

The Rex exclusion activity encoded by prophage genesrexA–rexB was assayed at 35°C using host culture cellsgrown at 30°C (Table 1). The plating experiments were car-ried out at 35°C because λspi156∆nin5 forms much largerplaques on nonlysogenic cells at this temperature than at 30or 40°C and because λcI[Ts]857 and λcI[Ts]2 lysogens re-mained viable at 35°C. The relative efficiencies of platingfor λspi156∆nin5, λbio275imm434nin5, and T4rII∆1589phages were determined by dividing the plaque titer on theassayed culture cells by their titer obtained in parallel on thepermissive host cells R594. Assays were performed by firsttransferring culture aliquots (1–2 × 108 CFU) to a 35°C wa-ter bath and then adding 0.1 mL of dilutions of a phagelysate. The mixtures were incubated 5 min, and then 3 mLTB (10 g Bacto tryptone, 5 g NaCl per litre) top agar (TBplus 6.5 g Bacto agar/L) was added and poured onto TB bot-tom agar plates (TB plus 11 g Bacto agar and 1 mg thia-mine/L) prewarmed to the assay temperature. The plateswere incubated at 35°C for 20 h.

We show in Table 1, as previously reported, that lysogenswith either cI[Ts] or cI+ prophages exhibit exclusion depend-ent on rexA–rexB toward T4rII∆1589. We further show thatthe reduction in plating of λbio275imm434∆nin5 and the se-vere (>6 × 105-fold) exclusion of λspi156∆nin5 was depend-ent upon rexA–rexB expression from cI[Ts] prophages,

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Fig. 1. The spi156 substitution endpoint in λspi156∆nin5. Therightward spi156 DNA – λ sequence junction is within the moaAgene (see text) of Escherichia coli K-12 (reference sequenceNC_000913, GenBank Accession No. U00096). The upper se-quence (dashed line) shows the left endpoint sequence of spi156(ending TCG). The arrow indicates the beginning of the lambda(solid line) DNA sequence (beginning with ACC at position38 558 bp, shown in the lower sequence). PO is a promoter forthe 77 nucleotide oop antisense RNA and PR promotes for tran-scription of genes cro–cII–O–P. The promoter PR is deleted bythe spi156 substitution (see text).



whereas the exclusion of λspi156∆nin5 by a rexA+–rexB+–cI+ prophage was slight. This new result distinguishing ef-fects of cI+ and cI[Ts] alleles raises the question: Why doesmaking the cI repressor temperature-sensitive increase Rex-dependent exclusion by a factor of >105 toward thelambdoid phage without altering Rex exclusion towardT4rII? We asked whether cI[Ts] or cI+ was dominant for thisphenotype. This was assayed by introducing (i) a low copynumber plasmid encoding the cI+ gene into a cell lysogen-

ized by λcI[Ts]857 and (ii) a multicopy plasmid encodingcI[Ts]857 into a cell lysogenized by λcI+. The introductionof either allele had no suppressive effect on Rex exclusionof T4rII, but the combination of the cI+ and cI[Ts] alleleswithin a cell served to remove Rex exclusion towardλspi156∆nin5 and λbio275imm434∆nin5. This result suggeststhat the prophage cI+ allele was dominant to cI[Ts]857, com-pletely suppressing the exclusion of λspi156∆nin5. Next, weasked if genes within the cI–rexA–rexB operon were exclu-

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Fig. 2. Plasmid maps. Map of λ genes rexB–cro with promoters (curved arrows), tImm terminator ( ), and transcripts (dotted arrows)above boxed genes. λ DNA insertions (solid lines) into plasmid pACYC184 cloned downstream of the tetR promoter were pRS1(PtetR–PM–cI+–rexA–rexB–timm), pRS2 (PtetR–PM–cI857–rexA–rexB–timm), and pRS3 (PtetR–PM–cI+). λ DNA insertions (solid lines) intoplasmid pUC18 cloned downstream from Plac Z′ promoter were pRS4 (Plac Z′–PM–cI–rexA–rexB–timm), and pRS11 (Plac Z′–PM–cI857).

Efficiency of plating phageb

Host cellsa λspi156∆nin5c λbio275imm434∆nin5 T4rII∆1589

NonlysogenR594 1.0 1.0 1.0

Lysogens of R594(λcI[Ts]2) <10–6 0.006 <10–6

(λcI[Ts]857) <10–6 0.003 <10–6

(λrexB5A cI857) 0.9 0.3 0.5(λrexAamQ cI[Ts]857) 0.6 0.2 0.3(λcI+) 0.1 0.5 <10–6

(λrexB5A cI+) 0.3 0.3 0.6(λrexA30A cI+) 0.2 0.4 0.5(λimm434) 0.4 ndd 1.0

Plasmid transformants of R594 lysogens(λcI[Ts]857) [pACYC184] 5×10–6 0.9 <10–6

(λcI[Ts]857) [cI+ in pRS3] 0.1 0.7 <10–6

(λcI+) [pUC18] 0.1 0.7 <10–6

(λcI+) [cI[Ts]857 in pRS11] 0.2 0.6 <10–6

Plasmid transformants of R594[pACYC184] or [pUC18] 0.9 1.0 1.0[cI[Ts]857–rexA+–rexB+ in pRS2] 0.2 0.3 <10–6

[cI+–rexA+–rexB+ in pRS1] 0.1 0.4 10–6

[cI[Ts]857 in pRS11] 0.1 ndd 1.0[cI+ in pRS3] 0.2 ndd 1.0aStrain R594 was used as nonlysogen and host for making lysogens.bThe efficiency of plating is expressed relative to plaque-forming units on host strain R594 at 35°C.cPhage λspi156∆nin5 forms tiny, irregular plaques on λ lysogenic hosts but appeared larger and

circular on the R594(λrexBgo293), R594(λrexB5A cI857), and R594(λrexAamQ cI857) hosts.dnd, not done.

Table 1. CI repressor modulation of Rex exclusion phenotypic activity.



sively involved in the Rex exclusion phenotype towardλspi156∆nin5 and λbio275imm434∆nin5. We found thatplasmids carrying either the cI[Ts] or cI+ versions of the cI–rexA–rexB operon conferred the Rex exclusion phenotypetoward T4rII but could not impart exclusion towardλbio275imm434∆nin5 and λspi156∆nin5 phages. This resultsuggests that a DNA target sequence or gene function out-side of the cI–rexA–rexB operon participated in the Rexexclusion phenotype toward λbio275imm434∆nin5 andλspi156∆nin5 phages.

Our results agree with Toothman and Herskowitz (1980a,1980b), who found that λ phages mutated for the ren and redgenes varied in their sensitivity to Rex exclusion and thatRex exclusion of λ Red– Ren– phages was augmented bycI[Ts] alleles of the Rex+ prophage. They proposed the dif-ference in exclusion was due to higher levels of cI–rexA–rexB transcription from the cI[Ts] prophage. However,Hayes (1978) found identical levels of cI–rexA–rexB tran-scription at 30°C from lysogenic W3350 cells with either re-pressed λcI857 or λcI+ prophages. The greater repressoractivity of CI+ to CI[Ts857] at 35°C (Mandal and Lieb 1976;Toothman and Herskowitz 1980a) would suggest that the CI-dependent PM–cI–rexA–rexB transcription (and thus Rex ex-clusion activity) should be higher in the CI+ lysogen than inthe CI[Ts857] lysogen. We assumed that if differential levelsof PM–cI–rexA–rexB expression were responsible for Rexexclusion toward λ∆red∆ren phages, then host cellsR594[pRS2] (Rex+ CI[Ts]) and R594[pRS1] (Rex+ CI+)would markedly differ in exclusion of λspi156∆nin5. How-ever, this was not the case, suggesting that Rex exclusion ofλspi156∆nin5 is not solely dependent upon the cI[Ts]857–rexA–rexB genes encoded by the immunity region of theλcI857 prophage. Accordingly, we suggest that the dominanteffect of CI+ results from its ability to prevent gene expres-sion outside of the cI–rexA–rexB operon at 35°C. One hy-pothesis currently being examined predicts that theincomplete repression of PR transcription from λcI[Ts], butnot from cI+ lysogens at 35°C, should result in some CII-dependent transcription of cI–rexA–rexB from the PE pro-moter (Echols and Green 1971; Eisen et al. 1970) to increasecI[Ts]-rex expression. Over-expression of rexA–rexB genesfrom a multicopy plasmid has been reported (Shinedling etal. 1987) to augment Rex activity, restricting growth ofλimm434∆nin5 and λimm434ren51 derivatives, but not λ wildtype or λimm434 (both Ren+) phages. How the ren and redgenes of λ confer protection to the phage against Rex exclu-sion has yet to be determined.

In summary, our results show the following: (i) Rex exclu-sion of λspi156∆nin5 requires that the λ lysogenic host pos-sesses intact rexA–rexB genes. (ii) Rex exclusion of T4rII islikely distinct from the requirement for Rex exclusion ofλ∆red∆ren phages. (iii) Augmentation of Rex exclusion ac-tivity by the cI[Ts]857 allele is recessive to cI+. (iv) ThecI857 augmentation of Rex exclusion activity involves theparticipation of a genetic element mapping outside of the cI–rexA–rexB operon.

AcknowlegementsThese studies were supported by a Natural Sciences and

Engineering Research Council of Canada (NSERC) oper-ating grant to S. Hayes. R. Slavcev received teaching

fellowships from the College of Medicine and the College ofGraduate Studies and Research, University of Saskatchewan.We are grateful to Dr. Hillory Socket for sequencing λspi-156-∆nin5 DNA.

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Bacteriophage λ repressor allelic modulation of the Rex exclusion phenotype

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