JOURNAL OF BACTERIOLOGY,
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Feb. 2002, p. 857–858Vol. 184, No. 3
Roderick A. Slavcev and Sidney Hayes*
Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan,
Saskatoon, Saskatchewan S7N 5E5, Canada
Received 6 September 2001/Accepted 21 October 2001
We asked whether Rex exclusion encoded by a lambda prophage confers a protective or a cell-killing
phenotype. We found that the Rex system can channel lysogenic cells into an arrested growth phase that gives
an overall protective ability to the host despite some associated killing.
The term “Rex phenotype” connotes generalized phage ex-
clusion by ? lysogens, a process that restricts plaque formation
by rII mutants of T4 (4), certain T7 and T5 mutants, and
particular variants of lambdoid phages (12, 17). The rex locus
of coliphage ? encoded by genes rexA and rexB (11) is cotrans-
cribed as part of the pM-cI-rexA-rexB-timmoperon expressed by
a repressed ? prophage (8). The model of Parma et al. (13)
predicts that RexB protein forms an inner membrane pore that
is opened upon direct interaction with at least two RexA pro-
teins, resulting in a cellular apoptotic response termed altru-
istic cell death. The degree of apoptosis was unreported. We
asked if the Rex phenotype confers a protective or a cell-killing
response to phage attack.
We utilized derivatives of Escherichia coli K-12 strains R594
[F?lac-3350 galK2 galT22 rpsL179 IN(rrnD-rrnE)1 ??] (3),
W3350A [F?lac-3350 galK2 galT22 IN(rrnD-rrnE)1 ??] (3),
and SA500 [F?his-87 relA1 strA181 tsx-83 ??] to prepare
lysogens. The ? wild type was from our stock (no. 271), and
?rexB5A and ?rexA30A were from G. Gussin (11) via W. Szy-
balski. The phages T4rIIA (point mutation in the rIIA gene of
T4), T4rII?1589 (deletion spanning the rIIA and rIIB genes),
and T4D were obtained from G. Mosig.
Cellular viability was determined following T4rII infection
(multiplicity of infection, 10) of the Rex?lysogen R594(?), the
Rex?lysogens R594(?rexA30A) and R594(?rexB5A), and nonly-
sogenic R594 cells. Optimal infectivity occurred at temperatures
between 37 and 43°C, with a reduction of ?103-fold infectivity at
30°C, in agreement with the results of earlier studies (2, 6). The
examination of spread plates from mock infections without T4rII
showed that virtually 100% of the CFU arose during the first 24 h
of plate incubation at temperatures between 30 and 43°C. No
survivors were seen among the T4rII-infected cells during the
same interval. We continued incubation for an additional 24 h,
during which the CFU from mock infections increased in size,
and tiny surviving CFU appeared between 36 and 48 h among the
T4rII-infected Rex?lysogens, revealing a prolonged growth ar-
rest. The CFU that survived T4rII infection were examined for
retention of the Rex?phenotype and sensitivity to T4. All CFU
tested remained Rex?and T4 sensitive. The Rex?R594(?) lyso-
gens survived T4rII infections with ?40% viability at tempera-
tures between 37 and 43°C. In contrast, we found that the viabil-
ity of Rex?R594 culture cells infected at temperatures between
37 and 43°C was ?0.001%. Similar results were found for
R594(?rexA) and R594(?rexB) lysogens. Identical infections of
the Rex?lysogens SA500(?) and W3350A(?) yielded the same
level of survivors as that of R594(?), whereas the viability of their
Rex?derivatives was ?0.01%. This experiment revealed that the
Rex?phenotype can confer an enormous (?104-fold) protective
advantage to infected ? lysogenic cells. We also monitored the
viability of Rex?and Rex-defective lysogenic and nonlysogenic
cells infected in solution with T4?rII at a multiplicity of infection
by more than 103-fold (assay minimum) within the first hour, and
surviving CFU were not subsequently detected. By contrast, in-
fected R594(?) cells showed a 10-fold drop in cell titer within the
first hour of infection, a lag in cell growth, and a subsequent
found to be resistant to T4. In all of the infection experiments, we
observed that the surviving cells in aliquots removed from cul-
tures appeared as CFU after a prolonged lag in cell growth and
were considerably smaller than the CFU arising from parallel
Our findings suggest that the rex genes of ? confer symbiotic
protection to the lysogenic host against secondary infection.
Previous studies have shown that high cellular levels of Rex
expression restricts plaque formation by phages T2, T4, T5, T6,
and T7 (15); thus, the advantage of the Rex phenotype in the
wild may be more widespread than is appreciated. However,
mechanistically, it is far from clear that the Rex phenotype
evolved, is maintained, or functions in the wild for the purpose
of host protection against secondary lytic infection. It is our
view that the cellular manifestations of Rex exclusion that are
triggered upon infection may be severe enough to result in cell
death but may also provide the intolerable environment nec-
essary to eliminate invading phage DNA. We found that both
the infection of ?rex?lysogenic cells with T4rII and their trans-
formation with a rexA multicopy plasmid (data not shown)
delayed the emergence of CFU. The prolonged arrest in cell
growth of prexA transformants of R594(?) helped us to ac-
count for the results of Snyder and McWilliams (16).
The question of how a Rex?cell avoids lethal gene expres-
sion from infecting T4rII remains unanswered. The stationary
physiological phase of E. coli host cells has been shown to
prevent the growth of T4 phage (7, 9), and these cells maintain
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, College of Medicine, University of Saskatche-
wan, Saskatoon, Saskatchewan S7N 5E5, Canada. Phone: (306) 966-
4307. Fax: (306) 966-4311. E-mail: email@example.com.
a lower proton motive force (10). Furthermore, during starva- Download full-text
tion, the stringent response prevents macromolecular synthesis
(5, 14) and may lead to bacterial apoptosis (1). The Rex system
acquired by lambda can channel lysogenic cells into an arrested
growth phase resembling the stationary phase or the stringent
response, both of which have levels of cell killing associated
with them; however, on the whole, the responses of this system
exhibit mutualism, conferring a protective ability to the host.
The hypothesis that the ? Rex phenotype triggers an altruistic
response in excluding the plating of T4rII requires that the
rexB-rexA genes function as a suicide module. Our study does
not support this model but rather suggests that Rex exclusion
of invading phage is a protective mechanism which results in
the increased survival of infected cells and in turn defends the
cell population as a whole from subsequent phage exposure.
This study was supported by an NSERC operating grant to S. H.
Roderick Slavcev received teaching fellowships from the Colleges of
Medicine and Graduate Studies and Research, University of
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