Establishment and Maintenance of Repression by Bacteriophage Lambda: the Role of the Ci, Cit, and Ciii Proteins (E

Proc. Nat. Acad. Sci. USA Vol. 68, No. 9, pp. 2190-2194, September 1971 Establishment and Maintenance of Repression by Bacteriophage Lambda: The Role of the ci, cit, and ciII Proteins (E. coli/lysogeny versus lysis/cy- mutants/DNA binding) HARRISON ECHOLS AND LINDA GREEN Department of Molecular Biology, University of California, Berkeley, Calif. 94720 Communicated by A. D. Kaiser, July 8, 1971 ABSTRACT To define the events necessary for the provided by the cI protein; for example, an inhibition of establishment and maintenance of repression in a X- of late proteins might delay the late stage of lytic infected cell, we have studied the requirements for efficient synthesis synthesis of the cI protein ("X-repressor"). Three classes development until cl-mediated repression can take over (3). of X mutants defective in the establishment of repression Another plausible mechanism involves timing the estab- are also defective in the appearance of cI protein activity lishment of repression by the time of synthesis of cI protein; at the normal time. Two of these mutational classes (cII- this possibility is suggested by the finding that the syn- and cIII-) probably result from inactivation of X-specified proteins, but the third class (cy-) may involve a structural thesis of cI protein is subject to regulation (4). defect. We conclude that at least three regulatory elements Since active clI and cII genes are essential for the effective are likely to be required for the normal turn-on of cI pro- establishment of lysogeny in an infected cell, but not for the tein synthesis in an infected nonlysogenic cell: clI and maintenance of lysogeny (5), the cII and cIII proteins are cIII proteins and an "active" y-region of X DNA. From likely to function as critical timing elements in the establish- these and other results, the complete role of cII and cIII proteins in the establishment of repression may involve a ment of repression. As a result of previous experimental ef- bifunctional regulatory activity: positive regulation of the forts to understand the role of the cII and cIII genes, we cI gene and negative regulation of late genes. A possible provided indirect evidence that the cII and cIII proteins molecular model for cII and cIII action is discussed. Since perform both "activities" indicated above: an inhibition of the cII and cIII genes are repressed by the cI protein under conditions of stable lysogeny, a separate mechanism is required for the maintenance of cI protein synthesis. After infection of a lysogen by c11- phage, the rate of in- crease of cI protein activity is substantially greater than after infection of a nonlysogen. From these and other int cM N cIcdHOP Q results, the cI protein may also have a bifunctional regu- Head Tail Recomb Reg DNA Lysis latory activity: positive regulation of the cI gene and * A^0 negative regulation of early lytic genes. _ ____ _ __ -0 After infection by the temperature phage X, the establishment FIG. 1. Genetic and functional map of X DNA. Genes with of lysogeny requires two functionally separate events: a related function exhibit extensive clustering along the X DNA. repression of the capacity of the phage DNA for lytic growth, molecule. This is indicated on the diagram by "Head" for genes recombination event that concerned with the structure of the phage head; "Tail" for genes and a site-specific genetic integrates "Recomb" involved with DNA at a site. The concerned with tail structure; for genes the viral DNA into the host specific general and site-specific recombination; "Reg" for genes exerting maintenance of the lysogenic state is inherently a simpler a regulatory function in lytic development or lysogeny; "DNA". phenomenon than its establishment; the repression of lytic for genes specifying replication proteins; and "Lysis" for genes capacity must simply be maintained, and no recombination concerned with cell lysis. Specific genes of the "regulation re- events are involved. gion"-cIII, N, cI, cII-are indicated above the "X DNA", as The maintenance of repression for phage X is probably ac- are the integrative recombination gene int, the DNA replication complished by one phage protein-the ci protein (or "X- genes OP, and the late regulator gene, Q. repressor")-which acts to repress RNA synthesis from critical Approximate DNA regions transcribed and the direction of early genes required for viral DNA replication and lytic gene transcription during the different stages of lytic growth are also. activation (Fig. 1, see also refs. 1 and 2). The establishment of shown: v represents immediate early RNA synthesis, per- for X a more sit- formed solely by the host transcription machinery; -_ rep- repression phage involves complex regulatory RNA on N at least one viral resents delayed early synthesis, dependent protein; uation, because early protein (the product - - - -s. represents late RNA synthesis, dependent on Q protein (see of the int gene) is required to catalyze the integrative recom- refs. 23, 33, 34 for recent detailed reviews). The cI protein blocks bination essential for stable lysogeny. Thus, the phage must immediate early RNA synthesis; this provides for complete allow expression of at least some early viral genes and yet repression of lytic genes, because delayed early and late RNA. impose repression before an irreversible commitment to synthesis is dependent upon N protein (see refs. 1, 2 for recent lytic growth. detailed reviews). Mutations affecting the establishment of Two general mechanisms have been considered that might repression have been located in the cHI and clII genes and in the provide for the "properly timed" establishment of repression. "y-region" of X DNA between the cI and clI genes (5, 13)-see One involves a repression of viral functions, in addition to that Results section. 2190 Downloaded by guest on September 28, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Phage Lambda Repression 2191 synthesis of late viral proteins and an activation of synthesis of cI protein (6). This report provides much more sub- stantial evidence for the activation of cI protein synthesis by direct assay of the levels of cI protein in extracts from infected cells. The results reported here also suggest that the site of action of the clI and cIII proteins may be between the cI and C3 cli genes. Similar results and conclusions with a different assay > for cI protein are reported in the accompanying paper by Reichardt and Kaiser (7). Conclusions similar to ours have 0 been derived from other types of experiments by Eisen and z 2 Pereira da Silva (personal communication), and by Kourilsky z (9). z Since the cII and cIII genes are repressed under conditions cam of stable lysogeny (10), the maintenance of cI protein synthesis would be expected to occur by a different mechanism. Our results suggest that the cI protein activates its own syn- 0 L thesis in a lysogen. This conclusion agrees with that derived from RNA synthesis experiments (11, 12) and from other 0o measurements of cI protein (7). MINUTES AFTER INFECTION MATERIALS AND METHODS FIG. 2. Kinetics of production of active cI protein. Infection Bacteriophage and bacteria was at a multiplicity of 5 phage per bacterium. At the The Escherichia coli strains used were W3104. specified times cells were chilled, centrifuged, and extracts were W3102su- and prepared. DNA-binding activity was measured by the retention The X mutations were the following: the ci Amber-mutation of 32P-labeled X (or Ximm434) DNA on a nitrocellulose filter in c114, the clI mutations cII68 and cII28(Amber), the cIII mu- the presence of excess unlabeled "chicken-blood DNA". DNA- tations c11167 and c111611 (Amber), and the cy mutations cy42 binding activity is expressed as DNA-binding units/109 cells. and cy2001. For the genetic characterization of the cI, clI, -0-0- represents cI + infection, assayed with XDNA; -0-0- repre- clII, and cy mutations, see refs. (5) and (13). sents cI- infection, assayed with XDNA; -A-A- represents cI+ Phage growth infection, assayed with Ximm434 DNA. Phage stocks were prepared by lytic growth in W3104 bacteria; the growth medium was T-broth liquid (per liter: measure the level of cI protein in crude extracts. The critical 10 g of Difco Tryptone, 5 g NaCl) or T-broth agar (T-broth control experiments are presented in Fig. 2 (Results). containing 1.2% Difco Bacto-agar for the underlayer and Extracts for assay of cI protein were prepared as follows. 0.7% agar for the soft-agar overlayer). Phage were con- Infected cells (about 6 X 1010) were collected by centrifuga- centrated and partially purified by precipitation with poly- tion and suspended in 3 ml of "lysis buffer" [0.05 M Trist ethylene glycol (10% polyethylene glycol-0.5 Ml NaCl), and HCl (pH 7.9)-2 mM EDTA-0.45 M NH4Cl-14%(w/w) phage stocks were stored in "adsorption buffer" (0.01 M sucrose]. The resultant cell suspension was centrifuged, and MIgSO4-0.01 M Tris HCl, pH 7.4) containing 0.01% gelatin. the pellet was frozen and stored at -20°C. For the prepara- In experiments to measure the synthesis of cI protein, tion of lysates, the frozen cells were suspended in 1.5 ml of W3102 cells were grown at 370C in "supplemented T-broth" lysis buffer and lysozyme was added to a concentration of 330 (T-broth + 0.2% maltose + 0.01% yeast extract) to an A59o ,jg/ml. After incubation for 5 min at 370C, the suspension of about 2, centrifuged, and resuspended in adsorption buffer was chilled, and MgCl2 and 2-mercaptoethanol were added to at an A590 of 4. To this cell suspension, phage were added, at a 50 mM and 14 mM, respectively.

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