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The Rex System of Bacteriophage K: Tolerance and Altruistic Cell Death

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The Rex System of Bacteriophage K: Tolerance and Altruistic Cell Death Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press The Rex system of bacteriophage k: tolerance and altruistic cell death David H. Parma, Marlene Snyder/ Sergey Sobolevski, Myra Nawroz, Edward Brody/ and Larry Gold Department of Molecular, Cellular, and Development Biology, University of Colorado, Boulder, Colorado 80309-0347 USA; ^Department of Biology, Mount St. Vincent University, Halifax, Nova Scotia, Canada B3M 2J6; ^Centre National de la Recherche Scientifique, Centre de Genetique Moleculaire, 91190 Gif-sur-Yvette, France The rexA and rexB genes of bacteriophage A. encode a two-component system that aborts lytic growth of bacterial viruses. Rex exclusion is characterized by termination of macromolecular synthesis, loss of active transport, the hydrolysis of ATP, and cell death. By analogy to colicins El and K, these results can be explained by depolarization of the cytoplasmic membrane. We have fractionated cells to determine the intracellular location of the RexB protein and made RexB—alkaline phosphatase fusions to analyze its membrane topology. The RexB protein appears to be a polytopic transmembrane protein. We suggest that RexB proteins form ion channels that, in response to lytic growth of bacteriophages, depolarize the cytoplasmic membrane. The Rex system requires a mechanism to prevent X itself from being excluded during lytic growth. We have determined that overexpression of RexB in A. lysogens prevents the exclusion of both T4 rll mutants and X ren mutants. We suspect that overexpression of RexB is the basis for preventing self-exclusion following the induction of a \ lysogen and that RexB overexpression is accomplished through transcriptional regulation. [Key Words: Bacteriophage X; Rex exclusion; transmembrane domains; gene fusions; alkaline phosphatase; cell death] Received August 12, 1991; revised version accepted January 13, 1992. The lexA and rexB genes of bacteriophage \ encode a reside in the immunity region of X (Fig. 1). Shortly after two-component system that aborts lytic growth of bac­ X phages infect a sensitive host, these three genes are terial viruses (Benzer 1955; Howard 1967; Toothmann transcribed as a polycistronic message from the pro­ and Herskowitz 1980; Matz et al. 1982). The stringency moter for repressor establishment. PRE (Fig. 1; Astrachan of Rex exclusion depends on the level of lex gene expres­ and Miller 1972; Reichardt 1975; Belfort 1978; Lieb and sion. Whereas X lysogens restrict rll mutants of bacterio­ Talland 1981). In a lysogen, the polycistronic message is phage T4, but not T4 wild type, overexpressing the rex initiated at PRM/ the promoter for repressor maintenance genes restricts T4 wild-type and all other tested phages, (Fig. 1; Hayes and Szybalski 1973a; Hayes and Hayes except X wild type (Toothmann and Herskowitz 1980a; 1978; Landsmann et al. 1982). Regulation of transcrip­ Landsmann et al. 1982; Shinedling et al. 1987). In a real tion from PRE and p^^j^ and their role in the X life cycle sense, Rex exclusion is a defense mechanism, analogous have been studied intensely and reviewed eloquently to the plant hypersensitivity response, in which individ­ (Ptashne 1986). ual cells die rather than propagate an infection that A third, enigmatic transcript in the immunity region, threatens the whole organism (Maclean et al. 1974). LIT RNA, has been described (Fig. 1; Hayes and Szybal­ Rex exclusion is characterized by termination of mac­ ski 1973a). The LIT transcript is a monocistronic rexB romolecular synthesis, loss of membrane potential, hy­ mRNA that is initiated at a promoter, PLIT/ that appears drolysis of intracellular ATP, and cell death (see Results; to be in the rexA-coding sequence (Hayes and Szybalski Garen 1961; Sekiguchi 1966; Sauerbier et al. 1969; 1973a; Landsmarm et al. 1982). After induction of a lyso­ Colowick and Colowick 1983; Snyder and McWilliams gen, transcription from PR^ is shut off and transcription 1989). This physiology resembles the response of cells from PLIT is activated. Ten minutes after thermal induc­ that are sensitive to colicins El and K (for review, see tion, the rate of rexB transcription is about 2-fold higher Luria and Suit 1987). Importantly, an exclusion-like re­ than in a lysogen, and by 15-20 min it is 5- to 10-fold sponse can be elicited in uninfected cells by overex­ higher. pressing RexA in the presence of RexB (Snyder and The regulation of LIT transcription is complex and not McWilliams 1989). thoroughly understood. LIT transcription in an induced rexA and rexB, together with the cl (repressor) gene, lysogen requires the X O and P gene products (Fig. 1) and GENES & DEVELOPMENT 6:497-510 © 1992 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/92 $3.00 497 Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Parma et al. establishment transcript pRE < maintenance transcript pRM < lit transcript <P pLIT -I rexB I rexA cl - :r;ro|i - ell - O Figure 1. Map of bacteriophage k, drawn PR tR2 to physical scale, showing relevant genes, transcripts, promoters, and transcriptional early rightward transcript terminators. the Escherichia coh dnaB and dnaG functions (Hayes sequence suggests that the lexB gene encodes a polytopic and Szybalski 1973a), but plasmid-based, O- and P-inde­ transmembrane protein. pendent LIT transcription has been inferred (Pirrotta et Cell fractionation and alkaline phosphatase fusion al. 1980; Landsmann et al. 1982). LIT transcription may technology show that RexB is a transmembrane protein occur in an uninduced lysogen (Matz et al. 1982) but at a with four transmembrane domains. Overexpression of level that is undetectable by classic hybridization tech­ RexB in \ lysogens prevents the exclusion of T4 rll mu­ niques (Hayes and Szybalski 1973a). It is not known tants and \ ren mutants. This result suggests that the whether LIT transcription occurs during X lytic infec­ postulated "additional function" of RexB (Landsmann et tion, nor has the precise location of the LIT promoter al. 1982) is to prevent X from excluding itself (self-exclu­ been determined. Pirrotta et al. (1980) have shown that sion) following induction of a lysogen (Toothman and the 150-nucleotide Haelll-Hinil restriction fragment Herskowitz 1980a). that includes the carboxyl terminus of the rexA gene and the amino terminus of the lexB gene has promoter ac­ tivity when cloned in a plasmid and a strong RNA poly- Results merase-binding site in vitro. Nuclease protection exper­ Physiology of Rex exclusion iments place the binding site within the carboxyl termi­ nus of rexA. However, the fragment does not promote in In T4 wild-type infections of the Rex overproducing vitro transcription, which is consistent with the reported strain NAPIV/prex20, RNA, protein, and DNA synthesis requirement for activation of LIT transcription in a lyso­ abort —7-9 min postinfection, as measured by the incor­ gen. poration of radioactive precursors into TCA-precipitable Landsmann et al. (1982) concluded that "the observed material (Fig. 2a-c). Correlated with the termination of temporal shift from coordinate to discoordinate expres­ macromolecular synthesis is the loss of the ability of the sion of rexB relative to rexA, together with an elaborate infected cells to actively transport p-D-thiomethylgalac- structural organization (especially the PLIT promoter) to topyranoside (TMG; Fig. 2d). Active transport of TMG is make such a shift possible, argues strongly that rexB has dependent on the electochemical potential of the inner some additional function, independent of rexA." The re­ membrane (Kashet and Wilson 1973); the inability of in­ cent observation that RexB stablizes the \ O protein duced cells to accumulate TMG is indicative of a loss of (Schoulaker-Schwarz et al. 1991) may be a manifestation membrane potential. These results resemble the re­ of the mechanism for amplifying LIT transcription. sponse of sensitive cells to colicin El and K (Luria and In an effort to decipher the enigma of Rex exclusion, Suit 1987). By analogy, they can be explained by the for­ we have focused our attention on the rexB gene product, mation of ion channels that depolarize the inner mem­ investigating its cellular location, its topology, and ef­ brane (Crammer et al. 1990). fects of its overexpression. The predicted RexB protein It is unlikely that the physiology of exclusion in (Landsmann et al. 1982) has a molecular mass of 16 kD NAPIV/prex20 is an artifact of membrane damage result­ with alternating hydrophilic and hydrophobic domains ing from overexpression of the Rex proteins. The growth and, by inference, is thought to be an integral membrane rate and colony morphology of NAPIV/prex20 are essen­ protein. Further examination of the predicted amino acid tially identical to that of NAPIV, and its physiology of 498 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Rex-mediated cell death 70000 T T T r I 25000 ' • • ' »•» »i ..^..^ .» ..^ • • ' • 40 40 30000 ' T ' f T T ' I " " ' • I 6000 • C (0 S 25000 (n o <b ^ 20000 •C <b X tJ 15000 - CO g 5 X 10000 - -|- 5000 - ' • 40 Time after T4 Infection (minutes) Figure 2. {a-d) Macromolecular synthesis and active transport in T4 wild-type infections. RNA, DNA, and protein synthesis in T4 wild-type infections of NAPIV and NAPIV/prex20 were measured by following the incorporation of radioactive precursors, from 1-min pulses, into TCA-precipitable material and measuring (see Materials and methods). Counts are plotted at the mid-point of their pulse. Active transport was monitored by measuring the intracellular accumulation of ^"^C-labeled TMG that was added to the culture 1 min after infection (see Materials and methods). The TMG data are plotted at the time of sampling. (O) T4 wild-type infections of NAPIV; (•) T4 wild-type infections of NAPIV/prex20. exclusion is very similar to that in a lysogen where the rex gene is expressed at normal levels.
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