NOTE / NOTE

Over-expression of rexA nullifies T4rII exclusion inEscherichia coli K(λ) lysogens

Roderick A. Slavcev and Sidney Hayes

Abstract: Dosage and relative cellular levels of RexA and RexB proteins encoded by the rexA�rexB genes of a λprophage are important for the Rex+ phenotype, which was nullified when greater RexA or RexB was provided thanwas necessary for the complementation of a rexA� or a rexB� prophage.

Key words: bacteriophage lambda (λ), T4rII exclusion (Rex) phenotype, lambda pM�cI�rexA�rexB�timm operon.

Résumé : Le dosage et les niveaux cellulaires relatifs des protéines RexA et RexB codées par les gènes rexA�rexB duprophage λ jouent un rôle importants dans le phénotype Rex+, qui est annulé par un excès de RexA ou de RexB.Ceux-ci ont été fournis afin de parvenir à la complémentation d�un prophage rexA� ou rexB�.

Mots clés : bactériophage lambda (λ), phénotype d�exclusion Rex T4rII, opéron lambda pM�cI�rexA�rexB�timm.

[Traduit par la Rédaction] Slavcev and Hayes 136

The ability of a repressed lambda lysogen of Escherichiacoli K to inhibit the plating of T4rII mutants, termed theRex (rII exclusion) phenotype (Benzer 1955), is encoded bythe rexA�rexB genes expressed from the pM�cI�rexA�rexB�timmoperon of a repressed λ prophage (Hayes et al. 1997; Matz etal. 1982). Cellular manifestations of the Rex phenotype aretriggered by the infection of a λ lysogen by T4rII and in-clude (i) the loss of cellular membrane potential, proton mo-tive force, and cellular ATP and (ii) the cessation ofmacromolecular synthesis, precluding the vegetative growthof the invading phage (Colowick and Colowick 1983; Parmaet al. 1992; Snyder and McWilliams 1989).Several lines of evidence suggest that the Rex phenotype

is governed by RexA:RexB stoichiometry, although verylittle is known regarding Rex protein function. RexB is ex-tremely hydrophobic, traversing the cell membrane fourtimes (Parma et al. 1992), and has been reported to inhibitClpPX and ClpPA proteolysis of λ O (Schoulaker-Schwarzet al. 1991), P1 Phd, and E. coli MazF proteins (Engelberg-Kulka et al. 1998). In contrast, RexA is hydrophylic and pre-sumably resides in the cytoplasm, though its function re-mains a mystery. Snyder and McWilliams (1989) noted that

plasmid over-expression of rexA relative to rexB results inthe cessation of host macromolecular synthesis in the ab-sence of phage infection. Parma et al. (1992) reported thatthe over-expression of rexB suppresses T4rII exclusion in aλ lysogen. In addition, the over-expression of rexA�rexBfrom a multicopy plasmid excludes both T4 and T4rII(Shinedling et al. 1987). However, since the over-expressionof rexA in a λ lysogen was suspected to result in a lethalmembrane depolarization event (Parma et al. 1992; Snyderand McWilliams 1989), the effect of over-expressing rexArelative to rexB on the Rex+ phenotype remains undeter-mined. We previously reported that greater than half of Rex+lysogens can remain viable following infection with T4rII(MOI 10) but are temporarily arrested for growth (Slavcevand Hayes 2002). We showed elsewhere that Rex+ lysogenstransformed with multicopy rexA plasmids exhibit 11%�20% viability and a similar prolonged growth arrest(Slavcev and Hayes 2003). In this study, we examined theeffect of disrupting RexA:RexB stoichiometric balance onT4rII exclusion activity.We used derivatives of E. coli K, including R594: F� lac-

3350 galK2 galT22 rpsL179 IN(rrnD�rrnE)1 λ� (Bachmann1987) and TC600 supE44 (Hayes collection) from C600(Bachmann 1987). Lysogens of R594 were made usingphages λ, λcI857, λcI857rexB5A, and λcI857rexAamQ. Wild-type bacteriophage λ and λcI857 were from our laboratorycollection, as were rex mutants λcI857rexAamQ andλcI857rexB5A (initially G. Gussin via W. Szybalski). PhageT4rII∆1589 (deletion spans the rIIA and rIIB genes produc-ing an in-frame fusion that is phenotypically RIIB+) was ob-tained from G. Mosig.The plasmids employed are shown in Fig. 1. The con-

struction of plasmids pRS7, pRS10, pRS13, pRS14, pRS15,pRS16, pRS17, and pRλlacZ′ are described in Slavcev and

Can. J. Microbiol. 50: 133�136 (2004) doi: 10.1139/W03-115 © 2004 NRC Canada

133

Received 10 September 2003. Revision received 3 December2003. Accepted 8 December 2003. Published on the NRCResearch Press Web site at http://cjm.nrc.ca on 18 March2004.

R.A. Slavcev1 and S. Hayes.2 Department of Microbiologyand Immunology, University of Saskatchewan, Saskatoon, SKS7N 5E5, Canada.1Present address: Department of Medical Genetics andMicrobiology, University of Toronto, Toronto, ON M5S 1A8,Canada.2Corresponding author (e-mail: hayess@duke.usask.ca).

I:\cjm\cjm5002\W03-115.vpMarch 11, 2004 2:11:27 PM

Color profile: DisabledComposite Default screen

Hayes (2003). Plasmids pRλlacZ′, pRS15, pRS16, andpRS17 are pBR322 derivatives in which the rex genes inpRS15-pRS17 are under the control of the lambda PR pro-moter. Only the multiple cloning site in pRλlacZ′ and inpRS15-pRS17 was taken from pUC19. Plasmids pUC18(used to make pRS13), pUC19 (used to make pRS7 and

pRS14), and pACYC184 (used to make pRS10, pRS18,pRS19) were from New England Biolabs (Beverly, Mass.).The same polymerase chain reaction primers used to con-struct pRS10 were used to make pRS18 and pRS19. λ se-quences pM�cI857�rexA�rexB�timm (in pRS10), pM�cI857�rexAamQ�rexB�timm (in pRS18), and pM�cI857�rexA�rexB5A�timm (in pRS19) (Fig. 1) were inserted downstreamof the promoter for tetR in pACYC184. The orientation ofthe λDNA inserts within the plasmids was confirmed as de-scribed (Slavcev and Hayes 2003). Each of the cloned rexfragments has a terminator, timm, downstream from rexB, ex-cept for pRS13, which is placZ ′�rexA�tlacZ ′.Rex phenotypic activity was measured in cells grown be-

tween 30 and 40 °C. The efficiency of plating (eop) for eachphage was determined by dividing the titer of T4rII on theassayed host cells by the titer obtained in parallel on the per-missive host cells R594. For each assay, the cultures weregrown overnight at 30 °C, diluted tenfold into fresh TBbroth (10 g Bacto Tryptone, 5 g NaCl/L), and grown 4�6 hat the assay temperature. Assays were performed at varioustemperatures by first transferring culture aliquots (about 3 ×108 CFU) to a heated water bath, adding 0.1-mL dilutions ofphage lysates of T4rII, adding 3 mL of TB top agar (TB plus6.5 g Bacto agar/L), and pouring the mixture onto TB bottomagar plates (TB plus 11 g Bacto agar and 1 mg thiamine/L)pre-warmed to the assay temperature. The plates were incu-bated inverted at the assay temperature for 16 h.The Rex+ phenotype was assayed in R594 and R594(λ)

cells that were transformed with multicopy rexA+, rexB+, orrexA+�rexB+ plasmids (Table 1). Cells are designated Rex+if the eop for T4rII on them is <6 × 10�6 compared withplating on R594 cells, and Rex� if the eop approaches unity.Nonlysogenic R594 cells that coordinately expressed RexAand RexB from a prophage or from a plasmid remainedRex+ (Table 1). In contrast, lysogenic R594(λ) cells weremade Rex� by the addition of plasmids expressing rexA(pRS13) or rexB (pRS14). The Rex� phenotype of theR594(λ)[pRS13]3 and R594(λ)[pRS14] isolates was reversed

© 2004 NRC Canada

134 Can. J. Microbiol. Vol. 50, 2004

Relative level

Prophage Plasmida RexA RexBT4rII efficiency ofplating at 30 °C

Cellular Rexphenotype

None None None None 1.0 �None pUC19 {rex�} None None 1.0 �None pRS7 {rexA+�rexB+} High High <6×10�6 +None pRS13 {rexA+} High None 0.76 �None pRS14 {rexB+} None High 1.0b �λrexA+�rexB+ None Low Low <6×10�6 +cλrexA+�rexB+ pUC19 {rex�} Low Low <6×10�6 +λrexA+�rexB+ pRS7 {rexA+�rexB+} High High <6×10�6 +λrexA+�rexB+ pRS13 {rexA+} High Low 0.21d �λrexA+�rexB+ pRS14 {rexB+} Low High 0.58b �

aGenotypes are shown in braces{}.bT4rII generated large r-type (rapid lysis plaques) on R594[pRS14] and R594(λ)[pRS14].cThe Rex+ phenotype was observed for R594(λ) and also for two colonies each from ampicillin-sensitive R594(λ) lysogens

cured of plasmid pRS13 or pRS14 (data not shown).dT4rII generated tiny asymmetrical plaques on R594(λ)[pRS13].

Table 1. Rex+ phenotype inactivation by RexA or RexB over-expression in Escherichia coli R594.

Fig. 1. Constitutive and inducible Rex plasmids. Map of λ regionfrom terminator timm through cro. The transcripts of λ genes fromthe phage promoters are shown by broken lines. The λ DNA in-serts within plasmids (solid lines) show the rexA�rexB genotypeand the upstream promoter for gene expression.

3 For strain designations, prophages are shown in parentheses and plasmids in brackets.

I:\cjm\cjm5002\W03-115.vpMarch 11, 2004 2:11:27 PM

Color profile: DisabledComposite Default screen

© 2004 NRC Canada

Slavcev and Hayes 135

in R594(λ) colonies spontaneously cured of either plasmid,indicating that T4rII exclusion was not likely influenced bya host mutation arising during plasmid transformation. Cellswith plasmids pRS13 and pRS14 can be grown up to station-ary phase in TB + 25 µg/mL ampicillin between 30 and37 °C (>20 cell divisions).We constructed temperature-inducible plasmids with

rexA�rexB genes downstream from a λ promoter�operatorsequence, pROR, where pR transcription is regulated by theCI[Ts]857 repressor, so that we could manipulate via tem-

perature the level of rex gene expression in transformedcells (Fig. 1). The R594 cells that coordinately expressedboth rexA+ and rexB+ at high or low levels (Table 2) re-mained Rex+. In contrast, R594 cells carrying a low levelrexA+�rexB+ plasmid (pRS10) plus a second temperature-inducible rexA+ or rexB+ plasmid were made Rex� whencells were shifted to 34 °C (trace derepression of pR tran-scription) or above, although T4rII plaques were pinpoint atthis temperature. Plaque size increased at 37 °C (slightderepression of pR transcription), and the plaques were full

T4rII efficiency of platingPrimary plasmida Secondary plasmida Inducible gene(s) 30 °C 34 °C 37 °C (40 °C)b

None None None 1.0 1.0 1.0pRS10 {ptet�rexA+�rexB+} None None <2×10�6 <2×10�6 <2×10�6pRλlacZ′ {rex�} None lacZ′ 0.40 0.61 0.57 (0.70)pRS15 {rexA+�rexB+}c None rexA�rexB 0.25d 9×10�3d <2×10�6pRS16 {rexA+�rexB�}c None rexA 1.0 1.0 1.0 (1.0)pRS17 {rexA��rexB+}c None rexB 0.74 0.62 1.0 (0.46)pRS10 {ptet�rexA+�rexB+} pRλlacZ′ {rex�} lacZ′ <2×10�6 <2×10�6 <2×10�6pRS10 {ptet�rexA+�rexB+} pRS15 {rexA+�rexB+} rexA�rexB <2×10�6 <2×10�6 <2×10�6pRS10 {ptet�rexA+�rexB+} pRS16 {rexA��rexB+} rexB <2×10�6 0.04e 0.19d (0.47)pRS10 {ptet�rexA+�rexB+} pRS17 {rexA+�rexB�} rexA <2×10�6 0.01e 0.06d (0.40)

apRS10 was a pACY184-derived low copy-level plasmid and the remainder were medium copy-level plasmids derived from pBR322. Genotypes areshown in braces {}.

bIdentical values at 37 and 40 °C are shown as one data entry.cThe CI[Ts]857 repressor activity in cells was measured by plating λcI72 at 30, 34, 37, and 40 °C. The efficiencies of plating were <2.4×10�6 at 30 to

37 °C and 0.27�0.34 at 40 °C.dPlaques were tiny.ePlaques were pinpoint.

Table 2. Inactivation of Rex phenotype by induced rexA or rexB gene expression in Escherichia coli R594.

Prophagea PlasmidbComplementingplasmid gene(s)

T4rII efficiency ofplating at 37 °C

Cellular Rexphenotype

None None None 1.0 �None pACYC184 None 1.0 �None pRS10 rexA+�rexB+ <3×10�7 +None pRS18 rexB+ 0.20c �None pRS19 rexA+ 0.09c �λrexA+�rexB+ None None <3×10�7 +λrexA+�rexB+ pACYC184 None <3×10�7 +λrexA+�rexB+ pRS10 rexA+�rexB+ <3×10�7 +λrexA+�rexB+ pRS18 rexB+ <3×10�7 +λrexA+�rexB+ pRS19 rexA+ 2.6×10�5 +λrexAamQ�rexB+ None None 0.30 �λrexAamQ�rexB+ pACYC184 None 0.67 �λrexAamQ�rexB+ pRS10 rexA+�rexB+ <3×10�7 +λrexAamQ�rexB+ pRS18 rexB+ 0.15c �λrexAamQ�rexB+ pRS19 rexA+ 1.8×10�5 +λrexA+�rexB5A None None 0.50 �λrexA+�rexB5A pACYC184 None 0.60 �λrexA+�rexB5A pRS10 rexA+�rexB+ <3×10�7 +λrexA+�rexB5A pRS18 rexB+ <3×10�7 +λrexA+�rexB5A pRS19 rexA+ 0.22 �

aAll prophages carry the cI[Ts]857 allele of λ.bPlasmids pRS10, pRS18, and pRS19 are each low copy-level derivatives of pACYC184 with inserted λ genes

shown in Fig. 1.cPlaques were tiny.

Table 3. Rex complementation in Escherichia coli R594.

I:\cjm\cjm5002\W03-115.vpMarch 11, 2004 2:11:27 PM

Color profile: DisabledComposite Default screen

size at 40 °C (full derepression of pR transcription at 39 °Cand above). These results indicate that RexA+ or RexB+ pro-vided in trans from a multicopy plasmid will nullify theRex+ phenotype when rexA+�rexB+ is expressed from a λwild-type prophage or from a low copy-level plasmid.We attempted to confirm the ability of RexA and RexB to

act in trans by complementation analysis (Table 3). Weshowed that the provision of rexA+�rexB+ on the low copy-number pACYC184 (pRS15) plasmid fully complementedλrexA� and λrexB� prophages, restoring the Rex+ phenotype.Complementation for Rex was also achieved when a func-tional copy of the defective prophage gene on a low copy-level pACYC184 plasmid (i.e., pRS10, pRS18, or pRS19)was introduced into rexA� or rexB� lysogenic cells. We notedthat the introduction of the rexA plasmid (pRS19) into aλ rexA+�rexB+ lysogen reduced but did not block the cellularRex+ phenotype, while providing RexB expressed frompRS18 did not nullify the T4rII exclusion expressed by theselysogens.We report for the first time that providing excess RexA in

cells normally expressing rexA�rexB from a λ prophage nul-lified the Rex+ phenotype. We also reproduced the results ofParma et al. (1992) who blocked T4rII exclusion by over-expressing rexB in a λ lysogen. The suppression of the Rex+phenotype required a greater amount of RexA or RexB thanwas necessary for Rex complementation of rexA� or rexB�prophages. Clearly the normal intracellular stoichiometricbalance between RexA:RexB is important to the Rex pheno-type and can be perturbed by even a slight imbalance.Understanding the influences of altering RexA:RexB stoi-chiometry on T4rII exclusion is yet another piece of the puz-zle toward understanding the nature of the elusive Rexexclusion mechanism. However, it is important to recognizethat the molecular activities of RexA and RexB remain to beelucidated. It also remains to be determined whether RexAinteracts with RexB and whether either interact with a phageor a host protein(s).

Acknowledgements

These studies were supported by an NSERC operatinggrant to S.H., and R.S. was supported in part by a teachingfellowship from the College of Medicine and scholarshipfrom the College of Graduate Studies and Research, Univer-

sity of Saskatchewan, Saskatoon, Sask. We thank J. Stehrand A. Urmson for assistance and C. Jackel and M. Horbayfor unpublished information.

References

Bachmann, B.J. 1987. Derivations and genotypes of some mutantderivatives of Escherichia coli K-12. In Escherichia coli and Sal-monella typhimurium: cellular and molecular biology. Vol. 2.Edited by F.C. Neidhardt, J.I. Ingraham, K.B. Low, B. Magasanik,M. Schaechter, and H.E. Umbarger. American Society for Micro-biology, Washington, D.C. pp. 1192�1219.

Benzer, S. 1955. Fine structure of a genetic region in bacteriophage.Proc. Natl. Acad. Sci. U.S.A. 41: 344�354.

Colowick, M.S., and Colowick, S.P. 1983. Membrane ATPase acti-vation on infection of E. coli K(λ) cells with phage rII mutants.Trans. N.Y. Acad. Sci. 28: 35�40.

Engelberg-Kulka, H., Reches, M., Narasimhan, S., Schoulaker-Schwarz, R., Klemes, Y., Aizenman, E., and Glaser, G. 1998.rexB of bacteriophage λ is an anti-cell death gene. Proc. Natl.Acad. Sci. U.S.A. 95: 15 481 � 15 486.

Hayes, S., Bull, H., and Tulloch, J. 1997. The rex phenotype of al-truistic cell death following infection of a λ lysogen by T4rIImutants is suppressed by plasmids expressing OOP RNA. Gene,189: 35�42.

Matz, K., Schmandt, M., and Gussin, G. 1982. The rex gene ofbacteriophage λ is really two genes. Genetics, 102: 319�327.

Parma, D.H., Snyder, M., Sobolevski, S., Nawrox, M., Brody, E.,and Gold, L. 1992. The Rex system of bacteriophage λ: toler-ance and altruistic cell death. Genes Dev. 6: 497�510.

Schoulaker-Schwarz, R., Dekel-Gorodetsky, L., and Engelberg-Kulka, H. 1991. An additional function for bacteriophage λ rex:the rexB product prevents degradation of the λO protein. Proc.Natl. Acad. Sci. U.S.A. 88: 4996�5000.

Shinedling, S., Parma, D., and Gold, L. 1987. Wild-type bacterio-phage T4 is restricted by the lambda rex genes. J. Virol. 61:3790�3794.

Slavcev, R.A., and Hayes, S. 2002. Rex-centric mutualism. J.Bacteriol. 184: 857�858.

Slavcev, R.A., and Hayes, S. 2003. Stationary phase-like propertiesof the bacteriophage λ Rex exclusion phenotype. Mol. Gen.Genomics, 269: 40�48.

Snyder, L., and McWilliams, K. 1989. The rex genes of bacterio-phage lambda can inhibit cell function without phage super-infection. Gene, 81: 17�24.

© 2004 NRC Canada

136 Can. J. Microbiol. Vol. 50, 2004

I:\cjm\cjm5002\W03-115.vpMarch 11, 2004 2:11:27 PM

Color profile: DisabledComposite Default screen