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Studies of heat-inducible lambda bacteriophage
J. Mol. Biol. (1966) 16, 149-163
Studies of Heat-inducible Lambda Bacteriophage I. Order of Genetic Sites and Properties of Mutant Prophages MARGARET LIEB
Department of Microbiology, University of Southern Oalifornia School of Medicine and Los Angeles Oounty General Hospital Los Angeles, Oalif., U.s.A. (Received 8 July 1965, and in revised form 1 November 1965) When a lysogen containing a heat-inducible A mutant is subjected to an appro. priate heat treatment, phage is usually produced and the bacterium dies. Under certain conditions, the heated bacterium dies although phage production does not take place. Non-lysogenic cells, and lysogens containing wild-type A, do not die when heated. Therefore, death after heating must be due to an activity of the mutant prophage. In this study, killing of the lysogen is called induction, whether or not phage is produced. Mutations resulting in heat-inducible prophage have been mapped at 12 different sites in the A CI cistron, Some of the heat-inducible prophages with mutations in the left side of the cistron are extremely sensitive to ultraviolet induction. A heat-inducible mutant that is simultaneously resistant to ultraviolet induction maps on the left side of CI, as does A isui: , Prophages with mutations in the right side of CI are not induced to kill the lysogen when heated in media containing chloramphenicol or puromycin, or in the absence of a carbon or nitrogen source. Protein synthesis during the heating period appears to be required for induction of these mutants. However, when the mutation resulting in heat-inducibility is in the left part of CI, induction occurs under conditions that block protein synthesis. In a bacterium containing phage genomes of both types, heat induction requires protein synthesis. It is proposed that the CI product is a protein molecule with two functions. The part of the molecule corresponding to the right side of CI represses transcription of A DNA. The part of the cI product corresponding to the left side of the CI is assumed to inhibit a phage-specified enzyme(s) present in the lysogenic bacterium.
1. Introduction Mutations that prevent bacteriophage>. from becoming a stable prophage are located in the cI region of the phage (Kaiser, 1957; Kaiser & Jacob, 1957). The ex product, or "immunity substance" (Bertani, 1958) is necessary for the maintenance of the prophage, and also prevents the replication of superlnfeoting phage in a lysogenic bacterium. It has been suggested that thec! product is a repressor that combines with an operator and thereby prevents transcription of>. genes the products of which are required for>. replication (Jacob & Monod, 1961). As I shall try to show, the product of the ex gene may have more than one function, so I shall refer to it as the "ex product" rather than "repressor". Induction of a lysogenic bacterium was defined by Lwoff (1953) as the "action of provoking the development of bacteriophage ... or the initiation of the vegetative 149
150
M. LIEB
state". In practice, phage production by the lysogen has often been used to measure induction. However, the "initiation of the vegetative state" may not always lead to phage production. In this study I observed that under certain conditions, heatinducible lysogens were killed by heating to 43 to 45°C, and yet did not produce phage. In a non-permissive host, sus mutations can prevent a prophage from producing completed progeny, but the lysogen is still killed by a dose ~f ultraviolet radiation that does not kill non-lysogenic bacteria (Campbell, 1962): A new exonuclease accumulates after ultraviolet radiation in bacteria containing defective ,\ mutants (Radding, 1964). Thus one can detect the induction of some phage functions in a lysogen even when no phage is produced. I propose to define "induction" of a lysogen as the initiation of any event dependent on the presence of the prophage. In the following report, I study the induction of killing of the lysogen. The induction of ,\ mRNA synthesis (Green, 1966) is also discussed. In addition to non-lysogenizing mutants, two other types of Cr mutants are known. Bacteria containing'\ ind- prophages, unlike lysogens containing wild-type A, are not sensitive to ultraviolet killing, and they do not produce phage after ultraviolet irradiation (Jacob & Campbell, 1959). Other mutations in 01 result in the incapacity to maintain the prophage state at high temperatures. For example, At857 prophage is induced when the lysogen is heated at 40 to 45°C in a growth medium (Sussman & Jacob, 1962). Sussman & Jacob observed that there was no induction (killing of the lysogen) when an auxotroph containing At857 was deprived of a growth factor requirement during heating. They suggested that the mutant repressor was not thermolabile, but that the synthesis of repressor was prevented at the high temperature. Presumably, growth of the culture was required for the dilution of repressor and subsequent induction. In this report, I describe the properties of At857 and of a series of new heat-inducible ,\ mutants. Mutations have been located at 12 sites in the 01 gene. Five of the mutants resemble At857, but others result in heat induction of the lysogen in the presence of chloramphenicol or under other non-growing conditions. Such mutants contain alterations mapping in the left side of Cr, while t857 and similar mutations map in the right side. I suggest that the Cr product in all the heat-inducible mutants is heatlabile, and propose a model in which two different parts of the 01 product act, respectively, as an enzyme inhibitor and a repressor of'\ mRNA synthesis.
2. Materials and Methods (a) Media Tryptone broth (TB): 1% Bacto tryptone, 0·5% NaOI; Hershey broth (HB): 0'5% Bacto peptone, 0'8% Bacto nutrient broth, 0'5% NaOI, 0·1 % glucose. Medium A: Gray & Tatum's synthetic medium (1944) with 0·2% glucose and 0'2% casein hydrolysate; K buffer (Kaudewitz, 1959). Plates (TA) were poured with Tryptone agar (TB + 1·2% Bacto agar); HB plus 0'2% sodium citrate and 0'6% Bacto agar was used as atop layer for phage assays. Cultures were grown in TB on a reciprocating shaker at 34 to 35°0 to a concentration of 2 to 4 X 10 s/ml. (b) Bacteria and bacteriophage etocke Escherichia coli strain M3 is a histidine-requiring derivative of K12S (Lieb, 1953). OR 34P-, a. thymine-requiring strain of K12, was isolated by Okada, Yanagisawa & Ryan (1961). Lysogenic strains were prepared by plating the desired phage on M3 at 33°0
PROPERTIES OF HEAT-INDUCIBLE A MUTANTS
151
and later streaking out from growth in a plaque. The streak was replicated to M3 to determine whether all colonies were lysogenic; if sensitive colonies were also present, one or more lysogenic colonies were restreaked until a pure clone was obtained. Each lysogenic stock is derived from a single lysogenic colony. To obtain phage stocks, lysogenic cultures were grown in broth to 4 X 10 8 bacteriajml., transferred to 45°C for 12 min, and then grown at 36°C until lysis. Stocks were sterilized by the addition of chloroform. A C1 mutants c47 and e7l are described by Kaiser (1957). The heat-inducible CI mutant t857 is described by Sussman & Jacob (1962) and was obtained from S. Silver. This is a double mutant and also contains the ind: mutation in eI which makes the prophage noninducible by ultraviolet (Jacob & Campbell, 1959). A new heat-inducible A mutant and a stock of A containing 5-bromouracil, from which this mutant had been isolated, were furnished by J. J. Weigle. Weigle's mutant was designated as A crt! (Lieb, 1964a), but for brevity will be referred to as tl. One additional mutant, t2; was found in this 5BUt A stock. (c) Isolation of heat-inducible mutants
Four new stocks of 5BU A were prepared, starting with small inocula of CR 34T- (,\+). Ten-ml. cultures were grown in synthetic medium A (0,2 mgjml. thymidine) to about 4 X 10B/mI., and resuspended in medium A without thymidine. After 1 hr at 35°C, 20 JLg/ml. of 5BU was added, and the cultures allowed to lyse. The identifying numbers for mutants obtained from these stocks are preceded by the stock number, e.g., 1.20, 2.22, etc. In both the original stock from Weigle, and in stock 3, large "jackpots" of identical mutants were found. The total numbers of identical mutants in the cultures were 105 or more. It appears unlikely that such jackpots could have arisen as the result of the incorporation of 5BU; therefore, mutants tl and t3·1 are probably spontaneous. Ultraviolet-induced mutants (designated by "U" preceding the identifying number) were obtained from ,\ + irradiated to give a survival of 3 X 10- 3 and then plated on ultraviolet-treated M3S to 50% survival (Weigle, 1953). Phages grown in 5BU were plated on unirradiated bacteria. The plates were incubated at 41 to 43°C; prospective mutants (clear plaques) were replated at the high temperature and at 33 or 37°C. Phages that gave turbid plaques at the low temperature but clear plaques at the high temperatures were selected for further study, and lysogenic stocks were prepared as described above. (d) Induction curves of lysogenic cultures
Lysogenic bacteria. were grown in TB on a reciprocating shaker to about 2 X 108/m I. and then chilled to O°C. Heat induction was initiated by diluting (usually 1 : 100) into TB at the desired temperature. Samples were removed from the warmed medium at intervals and either first chilled to O°C or assayed immediately at room temperature. For ultraviolet induction, bacteria were diluted or resuspended in cold 0'9% saline or K buffer at O°C. Small volumes were irradiated in Petri dishes with constant shaking. The ultraviolet source was 2 Westinghouse Sterilamps each delivering 4 ergs/mm 2jsec to the target area, After irradiation, the bacteria were kept chilled, and plating for eolony-formera and plaque-formers was done under yellow lights. For photoreactivation, bacteria. were placed in covered Petri dishes under a bank of G.E. Blacklight lamps. (e) Mapping of
mutants To increase recombination frequencies, phage stocks were irradiated with an ultraviolet dose allowing about 30% survival. An M3S culture was infected with 10 phages from each of two mutant phages, treated with antiserum and then allowed to lyse. Lysates were plated with M3S at 43°C; wild-type ,\ forms turbid plaques at this temperature, while the heat-inducible mutants give clear plaques. Since there is considerable variation in reo combination frequency observed for the same markers in repeated crosses, the map distances are only approximate. In complementation studies, about 2 X 10 7 bacteria containing a mutant prophage were spread on a TA plate. After drying, drops of phages to be tested (about 10 7jdrop) were spotted on the lawn of lysogenic bacteria, and the plates were incubated at 41 to
t
C1
Abbreviations used: 5BU, 5-bromouracil; CAP, chloramphenicol.
1152
Y. LIEB
43°C. After 12 to 18 hr, the background had lysed almost completely, and the areas corresponding to the phage spots were examined for bacterial growth. When only a few isolated colonies appeared in tho spots, these were considered to be lysogenic bacteria containing wild-type recombinant phage. A continuous film of growth was considered to indicate complementation.
3. Results (a) Heat-induction and cell killing
Sensitive bacteria, or bacteria lysogenic for wild-type A, are not killed when heated in nutrient medium at 45°C for more than an hour; at 37 to 44°C, they divide about every 35 minutes. When bacteria containing a heat-inducible>. mutant are heated in B nutrient medium, the cells are killed and the fraction of survivors at any time depends on the temperature and the genotype of the mutant. Cells of M3 (tl) heated to 43°C start to die after a five-minute lag (Fig. 1). If the lysogens are plated for plaque formers, these are found to increase, as shown. At least 99% of the lysogenic bacteria can become plaque formers.
0-001'---~--~----;c!;:------:;;--~ 10 20 30 40 Time (min) at 43° C
FIG. 1. Induction of M3 ("tIl at 43°C. Procedure given in Materials and Methods. Colonies; --0--0--, plaques.
-e-e-,
When lysogens carrying a heat-inducible prophage are heated at 45°C instead of 43°C, the number of plaque formers first increases, and then decreases after about 10 minutes. The number of surviving bacteria continues to decrease with time. Since lysogens with wild-type prophage are not killed at 45°C, I conclude that the presence of heat-inducible prophage is the cause of the death of the cells at high temperature, even when they produce no phage. I shall call this killing "induction" . (b) Mapping and "lwt spots"
In Fig. 2, the location of 12 heat-inducible A mutations and mutants c47 and e7l is given. The data are in Table 1. The recombination frequency of two distant markers was usually less than the sum of recombination distances between the intervening
~
PROPERTIES OF HEAT-INDUCIBLE
MUTANTS
lli3
2-20
2 1-20
3-8 U21 U37
2-21 e47
c~~ ___ l( Ip
U,16
\I
(857 U46 3-1)
e71
1 \'1/r
U32 U,sl
I
uso
U9
I
~
0-1
%
recombination
FIG. 2. Sites of mutation in and Methods.
CI
resulting in heat inducibility. Procedure is given in Materials
TABLE
1
Recombination frequencies between C1 mutants
~2
~2
c47
x
3 (2)
14 (2)
33 (4)
35 (7)
51 (2)
40 (2)
64 (2)
e47
X
18 (3)
26 (3)
31 (3)
33 (3)
46 (1)
75 (3)
90 (1)
(1)
X
20 (3)
16 (4)
33 (1)
28 (1)
34 (2)
52 (1)
56 (1)
UI6
X
9 (6)
6 (2)
9 (1)
36 (2)
U21
X
3 (2)
12 (5)
25 (4)
(2)
x
8 (5)
33 (5)
37 (2)
54 (1)
U32
x
25 (5)
45 (4)
118 (2)
1
x
12 (4)
857
1·22
1-22 U16 U21 U37 U32
U37
1
857 U46
3·1
U9
U50 e7l
47 (1)
81 (1)
88
38 (1) 61
100 (1)
(1)
125 (1)
(2)
52 (1)
89 (2)
70 (1)
51 (1)
57 (1)
69 (3)
65 (3)
6 (3)
7 (3)
12 (2)
24 (4)
31 (3)
x
1 (1)
2 (1)
10 (1)
27 (1)
52 (1)
U46
x
0·2 (2)
6 (3)
(4)
X
8 (2)
16 (4)
t:"9
x
3·1
64
U50
74
15 43 (1)
17
35
(4)
(1)
X
10 (1)
The number of wild-type recombinants per 104 is listed above the number of crosses on which the calculation is based. All phages were irradiated with ultraviolet before infection (see Materials and Methods).
M. LIEB
154
markers (negative interference), so the map distances have been determined from crosses between adjacent markers. The map orders of tU46, tU3-1 and t857, and of tU37 and tU21, are not known with certainty. Three "hot spots" occur at the left end of the CI cistron at 2, 2-20 and U32. Reversion rates of the mutants have not been studied, but the frequency of spontaneous revertants in all stocks was less than 10- 4 • The mutants designated tl, t53, t59 and t64 in an earlier publication (Lieb, 1964a) had been isolated from- a single stock of ,\ containing 5BU. They do not recombine to give wild-type phage, and are probably members of a clone of spontaneous mutants. The slight differences in the ultravioletand heat-sensitivity of lysogenic cultures containing these mutants were apparently due to genetic differences in the host bacteria. To make sure that all of the heat-inducible mutants were in the same cistron, complementation tests were made. In spot tests (see Materials and Methods), turbidity was observed in some spots after incubation at 42°C. Some of the pairs of mutants that seemed to be complementing were tested further. A lysogen containing one mutant was superinfected with the second mutant, or with ,\ CO2 , which contains a mutation in the Crn gene. The rates of heat-killing of the superinfected cultures were compared. In every case studied, the increase in heat-resistance of a lysogen after superinfection with another CI mutant was much less than that observed when the superinfecting phage contained a mutation known to be in another cistron, Thus, all the heat-inducible mutants are in CI, but there may be some intragenic complementation. (c) Spontaneous induction
Cultures containing the prophages of mutants at the sites of t2, t2-20 and tU16 never reach stationary phase when grown at 25 or 35°C, but stop growing at the end of the logarithmic phase (3 to 5 X 108 baoteriajml.) and then lyse (Fig. 3). Induction occurs before there is any detectable change in the pH of the medium. It is not prevented by the addition of yeast extract or Mg2 + (10- 2 M). All three cultures are also
109
E
M3Wl)
'r
OD.
OJ
1:
M3W21 OD.
108
.2
c-, c;
0
0
u
30
PROPERTIES OF HEAT-INDUCIBLE A MUTANTS
155
induced when incubated in buffer plus 0·2% glucose at 35°0, or when transferred from TB into synthetic medium with or without Oasamino acids (step-down culture). Incubation in TB + 10 to 20 fLg/mJ . chloramphenicol at 35°0 for 90 to 120 minutes, which does not kill other lysogenic cultures, kills about 90% of the bacteria containing such growth-rate-sensitive mutants. The property of growth-rate-sensitivity has been helpful in the detection of recurrences of mutation at "hot spots". (d) Sensitive bacteria in lysogenic cuUures
AIl heat-induction (survival) curves have a "tail" (Fig. 4), indicating that a small fraction of the culture is resistant to heat induction. The heat-resistant bacteria are non-lysogenic; replica-plating of several thousand colonies from unheated stock
c
o
.~
u
o
.:::
0 ·0001l..----~---__,:,,;-----"""-
10
20
30
.......
Time (min) at 45° C FIG. 4. Induction at 45°C of lysogenic bacteria containing different A C1 mutants. Curve A, M3 (All), is representative of lysogenies containing A's tU32, tU3·1, tU46, tU50 and tU9. Curve B, M3 ('\tU37) and curve C, M3 ('\t857) are unique. Curve D, M3 ('\t2), represents lysogenies containing A's tl-22, tU16 and tU21.
cultures revealed 1/200 to 1/5000 non-lysogenic bacteria in strains carrying different mutant prophages. A lysogenic strain retains a characteristic frequency of nonlysogenies, even after re-isolation from a single colony of lysogenic bacteria. Only one non-lysogenic colony was found among 4100 colonies from a culture of M3 (,\+). Thus, there may be a higher rate of spontaneous "curing" in lysogenic bacteria containing heat-inducible ,\ mutants. When a culture lysogenic for a heat-inducible mutant is heated, the number of non-lysogenies per unit volume increases at the rate expected from the division of
156
M. LIEB
pre-existing sensitive bacteria. Curing of lysogenic bacteria by heat must occur rarely, if at all. When lysogenic cultures are grown continuously at an inducing temperature that permits division of non-lysogenic bacteria, and in the presence of anti-'\ serum to prevent infection, completely non-lysogenic cultures are obtained. (e) Heat-induction curves of various mutant prophages
At 45°C, the slopes of the heat-induction curves of lysogenic bacteria containing various prophages are quite similar (Fig. 4), suggesting that all the lysogens are equally heat-sensitive. However, at 43°C, the killing curves show a greater variety of slopes, and at lower temperatures, some lysogens are induced slowly whereas others are able to grow. Thus, mutations at different sites in C]: seem to result in different heat-labilities in the respective C]: products, although these differences cannot be detected when the lysogens are heated at 45°C. In some lysogenic cultures, no bacteria are induced until they have been heated for several minutes (Fig. 4, curve A). Sussman & Jacob (1962) found that the minimum heating period for t857 was five minutes at 40°C; in our experiments, we find a twominute delay in the killing curve at 45°C (curve C). A minimum heating period for induction is required for all prophages with mutations in the right side of Cr' When the prophage mutation is in the left side of cr , the minimal heating time is 30 seconds or less (curve D). One culture, M3 (tU32), gives a curve suggesting that induction starts immediately in some cells, but that the rate of induction is maximum only after five minutes at the high temperature. The tU32 mutation is in the left side of C]:, but is close to the middle of the C]: region (Fig. 2). (f) Induction after heating in chloramphenicol and puromycin
The absence of heat induction ofM3 (tl) in broth plus CAP orin saline was reported in an earlier publication (Lieb, 1964a). Puromycin (10- 3 M) also inhibits heat induction in M3 (t1). Both of these inhibitors of protein synthesis are reported preferentially to inhibit the synthesis of induced enzymes (Sypherd & Strauss, 1962). No induction of M3 (tl) takes place if starved cultures are incubated in buffer at 45°C. As additional mutants were studied, it was found that only six mutants were not induced when heated in media that inhibit protein synthesis. These prophages all have mutations in the right side of C]:. Lysogens the prophages of which have mutations in the left side of Cr are killed as the result of heating in media containing chloramphenicol or puromycin, or in buffer or saline (Fig. 5). Some of these lysogens have been tested for phage production after heating in CAP. After heating in CAP for 10 minutes, 90% of M3 (U16) or M3 (U37) cells produce phage. Longer heating in CAP results in additional killing and in a loss of plaque formers. It is not known whether the event that kills a heated lysogenic cell occurs during the heating, or later, on the plate. However, if a lysogen dies as the result of a treatment, I conclude that the treatment is responsible for initiating the lethal reaction. (g) UUraviolet inducibility of heat-inducible prophaqes
Three levels of ultraviolet inducibility were observed among the heat-inducible. mutants. Mutations in the right side of C]: result in prophages that are significantly more sensitive to ultraviolet induction than is wild-type ,\ (Lieb, 1964a). Mutations at three sites, all in the left side of Cr, cause the prophages to be extremely sensitive
PROPERTIES OF HEAT-INDUCIBLE 1",------=-::-::--:::-- - --
' ' ' " -D- ~O- -O . . D - - 0
-
-
~
MUTANTS
157
- -- - - - - ,
- - o - - - --o M3W ll CAP
tl\
,,
.~\ c 0 :;:;
0-\
u
\.\\~,. \
0
.t: c '"
'';
·E :> V)
\'6.
· 0.. .
0·01
\\ -
.<,'"'O'" \'~ "
. M3W ' ) " -oM 3( 2) CAP · - -· M3(.112)
. - ' -0",_
~
10
20 30 40 Time (min) at 43° C
FIG. 5. Effect of chloramphenicol on induction of M3 (Atl) and M3 (~t2)_ At time 0, chilled logph>tBe cultures were diluted 1 : 100 into TB ± 20,..g CAP/m!. Samples were assayed at the times indicated.
to ultraviolet induction. To prove that differences in levels of ultraviolet sensitivity are not due to mutations in the host bacterium, several independently isolated lysogens containing each mutant have been tested. I considered the possibility that increased ultraviolet sensitivity might reflect a different mechanism of ultraviolet induction. However, ultraviol et induction of all the heat-sensitive mutants is susceptible to both photo-repair and da rk repair (Lieb , 1964b). The latent period for ultraviolet induction of heat-inducible mutants is shorter than that of II'" (Fig. 7). One mutant is less susceptible to ultraviolet induction than is wild-type A. The ultraviolet killing curve of M3 (U32) is very similar to that of M3 (A ind-) , and few irradiated cells produce plaques. The site of the mutation that simultaneously makes a prophage inducible by heat and less inducible by ultraviolet is in the left side of Cr-
(h) Dominance relationships of heat-inducible mutants
When a bacterium contains both a prophage and a superinfecting phage, with different heat- or ultraviolet-sensitivities, the heat- or ultraviolet-sensitivity of the complex is determined by tho mutant which, as prophage, produces the lysogen that is more resistant to heat or ultraviolet (Fig . 6). Dominanco is not affected by the positions of the genomes, e.g., in prophage or superinfocting phage. Figure 7 also shows that superinfecting All protects a cell lysogenic for MU37 from induction in CAP. In all cases tested, mutants with alterations at sites on the right side of the Cr gene inhibited induction in CAP of mutants mapping on the left side. A+ is dominant to all the heat-inducible mutants. Additional results will be published elsewhere (Lieb , manuscript in preparation).
M3{U37)
0,\ c: 0 '';:;
u
0
.::: Ol
0,01
c: ' S; .~
::> (/)
0,001 " " 0
lB.CAP
20
10
60
30 Time (min) at 43° C
FIG. 6. M3 (U 37) superinfected with ,\ + or Atl and then heated at 43°C. Labels on the curves indicate the superinfecting phage. A culture of M3 ('\tU37) at 6 X 10s/ml. was diluted 1 : 1 with ,\+ or Atl at 5 X 109/mI . and incubated at 36°0 for 10 min. Nine ml. of TB was added, the super· infected cultures were bub bled at 36°0 for 20 min and then chille d . For heat induction, the cold bacteria were dil uted into TB or TB 20 p.g/mI. CAP pre-heated to 43°0, and sampled at intervals.
+
2-20 3-8 U21 U37
\-22
Ul6
+
+
+
+
+
50
60
U32 U51
8 57
U46 3-1
U9
U50
.l+
58
60
55
70
Surviving
Surviving fractio n IOminheat
043°C 145°C Heat induction in CAP Spo ntaneous lysis u .v, la tent period (min)
+ + 50
60
+
±
SO
ind-
60
58
FIo. 7. P roper t ies of heat-inducible prophages. T he mutants are arranged in probable map orde r.
PROPERTIES OF HEAT.INDUCIBLE " MUTANTS
159
4. Discussion It is proposed that the initiation of any event dependent on the presence of the prophage be called "induction". In this study, "induction" refers to the initiation of an unknown event that causes the death of the lysogen. (a) The 'TfW,p of Cr
A diagrammatic representation of the properties of 12 heat-inducible mutants of ,\ is given in Fig. 7. The mutations in these phages map at 12 sites in the Cr region. In view of the different properties of the mutants, the Cr cistron seems to be divided into two regions: A, the left side of the Cr, contains mutant sites 2 to U32; B, the right side of Cr, contains sites I to U50. These regions together extend from the left of c47 to the vicinity of c71. (b) The nature of the Cr product The Cr gene is believed to contain the information for the synthesis of a product that prevents ,\ replication, cell killing and other events associated with phage production. Regions A and B in Cr are assumed to code for corresponding regions in the product of Cr' Mutations in the bacterium can suppress the effects of mutations in Cr (Jacob, Sussman & Monod, 1962; Thomas & Lambert, 1962). This suggests that the Cr product is a protein. Heat-inducibility may indicate that the protein produced by the mutant is heat-labile. Mutations at different sites would be expected to result in prophages with different sensitivities to heat. When various heat-inducible mutants were tested at temperatures from 38 to 43°0, marked differences in heat sensitivities were, indeed, observed. However, at 45°0 the killing curves of all lysogens are very similar. Perhaps the Cr product in all the mutants is inactivated very rapidly at 45°0, and the probability that a cellbe induced is determined by a subsequent process, the rate of which is not influenced by mutation in Cr. The possible nature of this process will be discussed later. (c) The "active site" for ultraviolet induction
Mutations in region A that result in heat-inducibility also affect the ultravioletinducibility of the prophage. Mutations at three sites result in greatly increased sensitivity to ultraviolet induction and a mutation at another site results in a prophage that is more resistant to ultraviolet induction than wild-type .\. Lysogens containing this mutant, or prophage .\t857, produce phage after ultraviolet only if they are also given a heat treatment sufficient to cause induction in the absence of ultraviolet. Goldthwaite & Jacob (1964) suggested that an inducer accumulates in the irradiated cell after ultraviolet, and that this substance combines with the Cr product, which they called the repressor. Lnd: mutants were postulated to have an alteration of the repressor that prevents effective combination with the inducer. The fact that there are Cr mutants with greater sensitivity to ultraviolet induction than wild-type A supports the notion that the Cr product combines with an inducer. Mutations affecting ultraviolet-inducibility map at several sites in the left side of Cr' The "active site" for interaction with the inducer may include a large portion of the Cr gene. (d) Unstable repressor
Some mutations in cr result in spontaneous induction, when the growth rate ofthe lysogenic culture decreases. Lysogenic cultures carrying these mutant prophages also
160
M. LIEB
contain large amounts of free phage. These phenomena can be attributed to the production of a very small amount of ex product, even at low temperatures, or to the instability of the ex product at low temperatures. (e) Protein synthesis and induction
Prophages with mutations in region A are induced when heated under conditions that prevent protein synthesis, indicating that induction (killing) is mediated by a pre-existing protein (enzyme) or does not require an enzyme. However, prophages with mutations in region B require protein synthesis for heat induction to occur. The question arises: if induction can occur in the absence of protein synthesis, what prevents certain lysogens from being induced when protein synthesis is blocked1 It could be that the synthesis of Cr product is blocked at high temperature in certain mutants, and that induction does not occur in the absence of protein synthesis because the Cr product is stable and persists to the end of the heating period. Several facts argue against this hypothesis. When a culture containing Atl is heated in a medium permitting growth, more than 90% of the bacteria are induced after ten minutes. At 45°C, the culture does not multiply, so that the Cr product is not diluted. If the ex product in these lysogens is stable, induction would not be expected to occur at such a high rate, unless additional assumptions are made. All the prophages that are not induced when protein synthesis is blocked have mutations at different sites in region B of ex, and it appears unlikely that mutation in one region of the gene would always result in a block to synthesis of the gene product at a high temperature. Finally, the experiments of Green (1966) demonstrate that when a lysogen containing Atl, mutated in region B, is heated in the presence of CAP, A mRNA rapidly increases in the bacteria, although they are not induced (killed). The ability of the Cr product to prevent the production of A mRNA is destroyed after two minutes at 45°C, indicating that this function of the ex product is heat-labile. However, the production of A mRNA is not sufficient, in these mutants, to produce cell killing or phage production unless protein synthesis accompanies heating. (f) Dominance of non-inactivated c/ product
A heat-inducible lysogen superinfeoted with wild-type ,\ before heating is not killed (Fig. 6), and does not produce ,\ mRNA (Green, 1966). A bacterium containing prophage.\t2 is induced (killed) when heated in a medium containing CAP. However, if the bacterium is infected with Atl shortly before heating, induction does not occur in CAP, or occurs at a much reduced rate. This "dominance" of active over inactivated Cr product indicates that the ex gene of the superinfecting phage is expressed in the lysogen. Additional information will be presented in a subsequent publication (Lieb, manuscript in preparation). (g) The activity of the c/ product: a model
The data presented here, together with those of Green (1966) show that the Cx product of ,\ has at least two inhibitory functions. One function is the inhibition of A mRNA production. The ex product also inhibits phage production and killing. The ex product of certain mutants can lose the first function but retain the capacity to prevent induction. I wish to propose a model in which a molecule of Cr product functions simultaneously as a repressor of ,\ mRNA synthesis and as an enzyme inhibitor (Fig. 8).
PROPERTIES OF HEAT·INDUCIBLE .\ MUTANTS
161
Region that codes / for'operator"
--,
t
DNA
After heating Mutation in region A
Active enzyme Mutation in region B
Inactive enzyme plus c r product
FIG. 8. Model of CI product in action. The product of CI is shown regulating the synthesis of the product of another gene. CI product could also regulate its own synthesis by aggregating with nascent CI protein.
The product of Cr is assumed to be synthesized constitutively. It combines with an enzyme, which is produced by another ,.\ gene. This enzyme is required for the initiation of the vegetative state; it might, for example, be a nuclease that releases A from the bacterial chromosome, and allows return of the ,.\ DNA to the vegetative form. As the messenger for this enzyme is made, it is translated by ribosomes that move along the messenger and separate it from the ,.\ DNA (Hartman, 1963). The studies of Byrne, Levin, Bladen & Nirenberg (1964) support the notion that ribosomes function in transcription and translation. Region A of the ex product combines with a part of the nascent enzyme; this part of the enzyme corresponds to the operator region. The enzyme cannot take its active configuration while it is combined with the Cr product. Region B of the attached ex product blocks the progression of the ribosome along the messenger. If the progression of this ribosome is required for mRNA release from the ,.\ DNA, the Cr product can block both transcription and translation; in non-nuclear ribosomes, the ex product blocks only translation, For reasons to be discussed elsewhere, I believe that molecules of Cr product aggregate, as shown in the diagram. Mutation anywhere in the Cr region can result in a Cr product that is unable to repress mRNA synthesis. When Cr product is altered in region A as the result of heating or ultraviolet irradiation, it releases the enzyme and simultaneously unblocks transcription and translation. The rate of ,.\ induction at high temperature could be limited by the rate of enzyme activation after release. The uncompleted molecule 11
162
M. LIEB
may be an active enzyme, or may be completed after the addition of a few amino acids. When C1 product with a mutation in region B is heated, region B no longer blocks the progression of the ribosome, and A mRNA is made. Region A of the C1 product remains attached to the enzyme and no irreversible event occurs until the concentration of enzyme molecules produced by the mRNA exceeds the coneentration of molecules of C1 product in a lysogen. Green has found that a lysogen containing At1 that has been heated in CAP continues to make large amounts of A mRNA after CAP is removed and cell growth has resumed. According to my model, translation of this RNA would result in enzyme synthesis and induction, unless the rate of synthesis of C1 product increased also. The model (Fig. 8) suggests that C1 product could regulate its own synthesis. Green proposes that the C1 product itself becomes an enzyme or "inducing protein", perhaps by disaggregation, when it is altered by heating. He suggests that when the C1 product has a mutation in region B, its enzymic activity is thermolabile and the synthesis of additional enzyme is necessary to cause induction. The site of action of the C1 product when it is a repressor is not specified. It may be possible to distinguish between the two models by studying A enzymes formed after inducing treatment. According to the model in Fig. 8, the operator is part of an enzyme made by a cistron other than C1• Certain changes in the enzyme (operator) might modify the rate of heat induction of particular C1 mutants. Double mutants containing mutations in C1 and cistrons N-R are being studied. Note added in proof: A double mutant A, containing CI mutations tl and t2, has been obtained. If mutation tl caused the CI "inducing protein" to be thermolabile (Green, 1966), the double-mutant prophage would be unable to kill the host when heated in CAP. However, lysogens containing A tlt2 are killed at 43°Cin CAP, so tl does not prevent heat induction in CAP when t2 is also present in the same genome.
I wish to thank Dr J. J. Weigle for his continued interest and help, and Dr M. Green for stimulating discussions. Mrs Ernestine Pruiet and Mr Percy Sanderman gave valuable technical assistance. This work was supported by grant no. G-24165 from the National Science Foundation, grant no. AI-05367 from the National Institutes of Health, and a Career Development Award (5-K3-GM-1546) from the National Institutes of Health. REFERENCES Bertani, G. (1958). Advanc. Virus Res. 5, 151. Byrne, R., Levin, J. G., Bladen, H. A. & Nirenberg, M. W. (1964). Proc. Nat. Acad. s«; Wash. 52, 140. Campbell, A. (1962). Advanc. Genetics, 11, 101. Goldthwaite, D. & Jacob, F. (1964). C.R. Acad. Sci. Paris, 259, 661. Gray, C. H. & Tatum, E. L. (1944). Proc. Nat. Acad. Sci., Wash. 30, 404. Green, M. (1966). J. Mol. Biol. 16, 134. Hartman. P. E. (1963). Proc, 11th Congr. Genetics, 2, 123. Oxford: Pergamon Press. Jacob, F. & Campbell, A. (1959). C.R. Acad. Sci. Paris, 248, 3219. Jacob, F. & Monod, J. (1961). Cold Spr. Harb. Symp. Compo Biol. 26, 193. Jacob, F., Sussman, R. & Monod, J. (1962). C.R. Acad. Sci. Paris, 254, 4214. Kaiser, A. D. (1957). Virology, 3, 42. Kaiser, A. D. & Jacob, F. (1957). Virology, 4, 509. Kaudewitz, F. (1959). Z. Naiur], 14b, 528. Lieb, M. (1953). J. Bact. 65, 642. Lieb, M. (1964a). Science, 145, 175. Lieb, M. (1964b). Virology, 23, 381. Lwoff, A. (1953). Bact. Rev. 17, 269.
PROPERTIES OF HEAT-INDUCIBLE A MUTANTS Okada., T., Yanagisawa, K. & Ryan, F. S. (1961). Z. Vererbunqslehre, 92, 403. Radding, C. (1964). Biochem. Biophys. Res. Comm. 15, 8. Sussman, R. & Jacob, F. (1962). C.R. Acad. Sci. Paris, 254, 1517. Sypherd, P. S. & Strauss, N. (1962). Proc, Nat. Acad. Sci., Wash. 49,400. Thomas, R. & Lambert, L. (1962). Bull. Acad. roy. Belg. 48, 668. Weigle, J. J. (1953). Proc, Nat. Acad. Sci., Wash. 39,628.
163
Studies of Heat-inducible Lambda Bacteriophage I. Order of Genetic Sites and Properties of Mutant Prophages MARGARET LIEB
Department of Microbiology, University of Southern Oalifornia School of Medicine and Los Angeles Oounty General Hospital Los Angeles, Oalif., U.s.A. (Received 8 July 1965, and in revised form 1 November 1965) When a lysogen containing a heat-inducible A mutant is subjected to an appro. priate heat treatment, phage is usually produced and the bacterium dies. Under certain conditions, the heated bacterium dies although phage production does not take place. Non-lysogenic cells, and lysogens containing wild-type A, do not die when heated. Therefore, death after heating must be due to an activity of the mutant prophage. In this study, killing of the lysogen is called induction, whether or not phage is produced. Mutations resulting in heat-inducible prophage have been mapped at 12 different sites in the A CI cistron, Some of the heat-inducible prophages with mutations in the left side of the cistron are extremely sensitive to ultraviolet induction. A heat-inducible mutant that is simultaneously resistant to ultraviolet induction maps on the left side of CI, as does A isui: , Prophages with mutations in the right side of CI are not induced to kill the lysogen when heated in media containing chloramphenicol or puromycin, or in the absence of a carbon or nitrogen source. Protein synthesis during the heating period appears to be required for induction of these mutants. However, when the mutation resulting in heat-inducibility is in the left part of CI, induction occurs under conditions that block protein synthesis. In a bacterium containing phage genomes of both types, heat induction requires protein synthesis. It is proposed that the CI product is a protein molecule with two functions. The part of the molecule corresponding to the right side of CI represses transcription of A DNA. The part of the cI product corresponding to the left side of the CI is assumed to inhibit a phage-specified enzyme(s) present in the lysogenic bacterium.
1. Introduction Mutations that prevent bacteriophage>. from becoming a stable prophage are located in the cI region of the phage (Kaiser, 1957; Kaiser & Jacob, 1957). The ex product, or "immunity substance" (Bertani, 1958) is necessary for the maintenance of the prophage, and also prevents the replication of superlnfeoting phage in a lysogenic bacterium. It has been suggested that thec! product is a repressor that combines with an operator and thereby prevents transcription of>. genes the products of which are required for>. replication (Jacob & Monod, 1961). As I shall try to show, the product of the ex gene may have more than one function, so I shall refer to it as the "ex product" rather than "repressor". Induction of a lysogenic bacterium was defined by Lwoff (1953) as the "action of provoking the development of bacteriophage ... or the initiation of the vegetative 149
150
M. LIEB
state". In practice, phage production by the lysogen has often been used to measure induction. However, the "initiation of the vegetative state" may not always lead to phage production. In this study I observed that under certain conditions, heatinducible lysogens were killed by heating to 43 to 45°C, and yet did not produce phage. In a non-permissive host, sus mutations can prevent a prophage from producing completed progeny, but the lysogen is still killed by a dose ~f ultraviolet radiation that does not kill non-lysogenic bacteria (Campbell, 1962): A new exonuclease accumulates after ultraviolet radiation in bacteria containing defective ,\ mutants (Radding, 1964). Thus one can detect the induction of some phage functions in a lysogen even when no phage is produced. I propose to define "induction" of a lysogen as the initiation of any event dependent on the presence of the prophage. In the following report, I study the induction of killing of the lysogen. The induction of ,\ mRNA synthesis (Green, 1966) is also discussed. In addition to non-lysogenizing mutants, two other types of Cr mutants are known. Bacteria containing'\ ind- prophages, unlike lysogens containing wild-type A, are not sensitive to ultraviolet killing, and they do not produce phage after ultraviolet irradiation (Jacob & Campbell, 1959). Other mutations in 01 result in the incapacity to maintain the prophage state at high temperatures. For example, At857 prophage is induced when the lysogen is heated at 40 to 45°C in a growth medium (Sussman & Jacob, 1962). Sussman & Jacob observed that there was no induction (killing of the lysogen) when an auxotroph containing At857 was deprived of a growth factor requirement during heating. They suggested that the mutant repressor was not thermolabile, but that the synthesis of repressor was prevented at the high temperature. Presumably, growth of the culture was required for the dilution of repressor and subsequent induction. In this report, I describe the properties of At857 and of a series of new heat-inducible ,\ mutants. Mutations have been located at 12 sites in the 01 gene. Five of the mutants resemble At857, but others result in heat induction of the lysogen in the presence of chloramphenicol or under other non-growing conditions. Such mutants contain alterations mapping in the left side of Cr, while t857 and similar mutations map in the right side. I suggest that the Cr product in all the heat-inducible mutants is heatlabile, and propose a model in which two different parts of the 01 product act, respectively, as an enzyme inhibitor and a repressor of'\ mRNA synthesis.
2. Materials and Methods (a) Media Tryptone broth (TB): 1% Bacto tryptone, 0·5% NaOI; Hershey broth (HB): 0'5% Bacto peptone, 0'8% Bacto nutrient broth, 0'5% NaOI, 0·1 % glucose. Medium A: Gray & Tatum's synthetic medium (1944) with 0·2% glucose and 0'2% casein hydrolysate; K buffer (Kaudewitz, 1959). Plates (TA) were poured with Tryptone agar (TB + 1·2% Bacto agar); HB plus 0'2% sodium citrate and 0'6% Bacto agar was used as atop layer for phage assays. Cultures were grown in TB on a reciprocating shaker at 34 to 35°0 to a concentration of 2 to 4 X 10 s/ml. (b) Bacteria and bacteriophage etocke Escherichia coli strain M3 is a histidine-requiring derivative of K12S (Lieb, 1953). OR 34P-, a. thymine-requiring strain of K12, was isolated by Okada, Yanagisawa & Ryan (1961). Lysogenic strains were prepared by plating the desired phage on M3 at 33°0
PROPERTIES OF HEAT-INDUCIBLE A MUTANTS
151
and later streaking out from growth in a plaque. The streak was replicated to M3 to determine whether all colonies were lysogenic; if sensitive colonies were also present, one or more lysogenic colonies were restreaked until a pure clone was obtained. Each lysogenic stock is derived from a single lysogenic colony. To obtain phage stocks, lysogenic cultures were grown in broth to 4 X 10 8 bacteriajml., transferred to 45°C for 12 min, and then grown at 36°C until lysis. Stocks were sterilized by the addition of chloroform. A C1 mutants c47 and e7l are described by Kaiser (1957). The heat-inducible CI mutant t857 is described by Sussman & Jacob (1962) and was obtained from S. Silver. This is a double mutant and also contains the ind: mutation in eI which makes the prophage noninducible by ultraviolet (Jacob & Campbell, 1959). A new heat-inducible A mutant and a stock of A containing 5-bromouracil, from which this mutant had been isolated, were furnished by J. J. Weigle. Weigle's mutant was designated as A crt! (Lieb, 1964a), but for brevity will be referred to as tl. One additional mutant, t2; was found in this 5BUt A stock. (c) Isolation of heat-inducible mutants
Four new stocks of 5BU A were prepared, starting with small inocula of CR 34T- (,\+). Ten-ml. cultures were grown in synthetic medium A (0,2 mgjml. thymidine) to about 4 X 10B/mI., and resuspended in medium A without thymidine. After 1 hr at 35°C, 20 JLg/ml. of 5BU was added, and the cultures allowed to lyse. The identifying numbers for mutants obtained from these stocks are preceded by the stock number, e.g., 1.20, 2.22, etc. In both the original stock from Weigle, and in stock 3, large "jackpots" of identical mutants were found. The total numbers of identical mutants in the cultures were 105 or more. It appears unlikely that such jackpots could have arisen as the result of the incorporation of 5BU; therefore, mutants tl and t3·1 are probably spontaneous. Ultraviolet-induced mutants (designated by "U" preceding the identifying number) were obtained from ,\ + irradiated to give a survival of 3 X 10- 3 and then plated on ultraviolet-treated M3S to 50% survival (Weigle, 1953). Phages grown in 5BU were plated on unirradiated bacteria. The plates were incubated at 41 to 43°C; prospective mutants (clear plaques) were replated at the high temperature and at 33 or 37°C. Phages that gave turbid plaques at the low temperature but clear plaques at the high temperatures were selected for further study, and lysogenic stocks were prepared as described above. (d) Induction curves of lysogenic cultures
Lysogenic bacteria. were grown in TB on a reciprocating shaker to about 2 X 108/m I. and then chilled to O°C. Heat induction was initiated by diluting (usually 1 : 100) into TB at the desired temperature. Samples were removed from the warmed medium at intervals and either first chilled to O°C or assayed immediately at room temperature. For ultraviolet induction, bacteria were diluted or resuspended in cold 0'9% saline or K buffer at O°C. Small volumes were irradiated in Petri dishes with constant shaking. The ultraviolet source was 2 Westinghouse Sterilamps each delivering 4 ergs/mm 2jsec to the target area, After irradiation, the bacteria were kept chilled, and plating for eolony-formera and plaque-formers was done under yellow lights. For photoreactivation, bacteria. were placed in covered Petri dishes under a bank of G.E. Blacklight lamps. (e) Mapping of
mutants To increase recombination frequencies, phage stocks were irradiated with an ultraviolet dose allowing about 30% survival. An M3S culture was infected with 10 phages from each of two mutant phages, treated with antiserum and then allowed to lyse. Lysates were plated with M3S at 43°C; wild-type ,\ forms turbid plaques at this temperature, while the heat-inducible mutants give clear plaques. Since there is considerable variation in reo combination frequency observed for the same markers in repeated crosses, the map distances are only approximate. In complementation studies, about 2 X 10 7 bacteria containing a mutant prophage were spread on a TA plate. After drying, drops of phages to be tested (about 10 7jdrop) were spotted on the lawn of lysogenic bacteria, and the plates were incubated at 41 to
t
C1
Abbreviations used: 5BU, 5-bromouracil; CAP, chloramphenicol.
1152
Y. LIEB
43°C. After 12 to 18 hr, the background had lysed almost completely, and the areas corresponding to the phage spots were examined for bacterial growth. When only a few isolated colonies appeared in tho spots, these were considered to be lysogenic bacteria containing wild-type recombinant phage. A continuous film of growth was considered to indicate complementation.
3. Results (a) Heat-induction and cell killing
Sensitive bacteria, or bacteria lysogenic for wild-type A, are not killed when heated in nutrient medium at 45°C for more than an hour; at 37 to 44°C, they divide about every 35 minutes. When bacteria containing a heat-inducible>. mutant are heated in B nutrient medium, the cells are killed and the fraction of survivors at any time depends on the temperature and the genotype of the mutant. Cells of M3 (tl) heated to 43°C start to die after a five-minute lag (Fig. 1). If the lysogens are plated for plaque formers, these are found to increase, as shown. At least 99% of the lysogenic bacteria can become plaque formers.
0-001'---~--~----;c!;:------:;;--~ 10 20 30 40 Time (min) at 43° C
FIG. 1. Induction of M3 ("tIl at 43°C. Procedure given in Materials and Methods. Colonies; --0--0--, plaques.
-e-e-,
When lysogens carrying a heat-inducible prophage are heated at 45°C instead of 43°C, the number of plaque formers first increases, and then decreases after about 10 minutes. The number of surviving bacteria continues to decrease with time. Since lysogens with wild-type prophage are not killed at 45°C, I conclude that the presence of heat-inducible prophage is the cause of the death of the cells at high temperature, even when they produce no phage. I shall call this killing "induction" . (b) Mapping and "lwt spots"
In Fig. 2, the location of 12 heat-inducible A mutations and mutants c47 and e7l is given. The data are in Table 1. The recombination frequency of two distant markers was usually less than the sum of recombination distances between the intervening
~
PROPERTIES OF HEAT-INDUCIBLE
MUTANTS
lli3
2-20
2 1-20
3-8 U21 U37
2-21 e47
c~~ ___ l( Ip
U,16
\I
(857 U46 3-1)
e71
1 \'1/r
U32 U,sl
I
uso
U9
I
~
0-1
%
recombination
FIG. 2. Sites of mutation in and Methods.
CI
resulting in heat inducibility. Procedure is given in Materials
TABLE
1
Recombination frequencies between C1 mutants
~2
~2
c47
x
3 (2)
14 (2)
33 (4)
35 (7)
51 (2)
40 (2)
64 (2)
e47
X
18 (3)
26 (3)
31 (3)
33 (3)
46 (1)
75 (3)
90 (1)
(1)
X
20 (3)
16 (4)
33 (1)
28 (1)
34 (2)
52 (1)
56 (1)
UI6
X
9 (6)
6 (2)
9 (1)
36 (2)
U21
X
3 (2)
12 (5)
25 (4)
(2)
x
8 (5)
33 (5)
37 (2)
54 (1)
U32
x
25 (5)
45 (4)
118 (2)
1
x
12 (4)
857
1·22
1-22 U16 U21 U37 U32
U37
1
857 U46
3·1
U9
U50 e7l
47 (1)
81 (1)
88
38 (1) 61
100 (1)
(1)
125 (1)
(2)
52 (1)
89 (2)
70 (1)
51 (1)
57 (1)
69 (3)
65 (3)
6 (3)
7 (3)
12 (2)
24 (4)
31 (3)
x
1 (1)
2 (1)
10 (1)
27 (1)
52 (1)
U46
x
0·2 (2)
6 (3)
(4)
X
8 (2)
16 (4)
t:"9
x
3·1
64
U50
74
15 43 (1)
17
35
(4)
(1)
X
10 (1)
The number of wild-type recombinants per 104 is listed above the number of crosses on which the calculation is based. All phages were irradiated with ultraviolet before infection (see Materials and Methods).
M. LIEB
154
markers (negative interference), so the map distances have been determined from crosses between adjacent markers. The map orders of tU46, tU3-1 and t857, and of tU37 and tU21, are not known with certainty. Three "hot spots" occur at the left end of the CI cistron at 2, 2-20 and U32. Reversion rates of the mutants have not been studied, but the frequency of spontaneous revertants in all stocks was less than 10- 4 • The mutants designated tl, t53, t59 and t64 in an earlier publication (Lieb, 1964a) had been isolated from- a single stock of ,\ containing 5BU. They do not recombine to give wild-type phage, and are probably members of a clone of spontaneous mutants. The slight differences in the ultravioletand heat-sensitivity of lysogenic cultures containing these mutants were apparently due to genetic differences in the host bacteria. To make sure that all of the heat-inducible mutants were in the same cistron, complementation tests were made. In spot tests (see Materials and Methods), turbidity was observed in some spots after incubation at 42°C. Some of the pairs of mutants that seemed to be complementing were tested further. A lysogen containing one mutant was superinfected with the second mutant, or with ,\ CO2 , which contains a mutation in the Crn gene. The rates of heat-killing of the superinfected cultures were compared. In every case studied, the increase in heat-resistance of a lysogen after superinfection with another CI mutant was much less than that observed when the superinfecting phage contained a mutation known to be in another cistron, Thus, all the heat-inducible mutants are in CI, but there may be some intragenic complementation. (c) Spontaneous induction
Cultures containing the prophages of mutants at the sites of t2, t2-20 and tU16 never reach stationary phase when grown at 25 or 35°C, but stop growing at the end of the logarithmic phase (3 to 5 X 108 baoteriajml.) and then lyse (Fig. 3). Induction occurs before there is any detectable change in the pH of the medium. It is not prevented by the addition of yeast extract or Mg2 + (10- 2 M). All three cultures are also
109
E
M3Wl)
'r
OD.
OJ
1:
M3W21 OD.
108
.2
c-, c;
0
0
u
30
PROPERTIES OF HEAT-INDUCIBLE A MUTANTS
155
induced when incubated in buffer plus 0·2% glucose at 35°0, or when transferred from TB into synthetic medium with or without Oasamino acids (step-down culture). Incubation in TB + 10 to 20 fLg/mJ . chloramphenicol at 35°0 for 90 to 120 minutes, which does not kill other lysogenic cultures, kills about 90% of the bacteria containing such growth-rate-sensitive mutants. The property of growth-rate-sensitivity has been helpful in the detection of recurrences of mutation at "hot spots". (d) Sensitive bacteria in lysogenic cuUures
AIl heat-induction (survival) curves have a "tail" (Fig. 4), indicating that a small fraction of the culture is resistant to heat induction. The heat-resistant bacteria are non-lysogenic; replica-plating of several thousand colonies from unheated stock
c
o
.~
u
o
.:::
0 ·0001l..----~---__,:,,;-----"""-
10
20
30
.......
Time (min) at 45° C FIG. 4. Induction at 45°C of lysogenic bacteria containing different A C1 mutants. Curve A, M3 (All), is representative of lysogenies containing A's tU32, tU3·1, tU46, tU50 and tU9. Curve B, M3 ('\tU37) and curve C, M3 ('\t857) are unique. Curve D, M3 ('\t2), represents lysogenies containing A's tl-22, tU16 and tU21.
cultures revealed 1/200 to 1/5000 non-lysogenic bacteria in strains carrying different mutant prophages. A lysogenic strain retains a characteristic frequency of nonlysogenies, even after re-isolation from a single colony of lysogenic bacteria. Only one non-lysogenic colony was found among 4100 colonies from a culture of M3 (,\+). Thus, there may be a higher rate of spontaneous "curing" in lysogenic bacteria containing heat-inducible ,\ mutants. When a culture lysogenic for a heat-inducible mutant is heated, the number of non-lysogenies per unit volume increases at the rate expected from the division of
156
M. LIEB
pre-existing sensitive bacteria. Curing of lysogenic bacteria by heat must occur rarely, if at all. When lysogenic cultures are grown continuously at an inducing temperature that permits division of non-lysogenic bacteria, and in the presence of anti-'\ serum to prevent infection, completely non-lysogenic cultures are obtained. (e) Heat-induction curves of various mutant prophages
At 45°C, the slopes of the heat-induction curves of lysogenic bacteria containing various prophages are quite similar (Fig. 4), suggesting that all the lysogens are equally heat-sensitive. However, at 43°C, the killing curves show a greater variety of slopes, and at lower temperatures, some lysogens are induced slowly whereas others are able to grow. Thus, mutations at different sites in C]: seem to result in different heat-labilities in the respective C]: products, although these differences cannot be detected when the lysogens are heated at 45°C. In some lysogenic cultures, no bacteria are induced until they have been heated for several minutes (Fig. 4, curve A). Sussman & Jacob (1962) found that the minimum heating period for t857 was five minutes at 40°C; in our experiments, we find a twominute delay in the killing curve at 45°C (curve C). A minimum heating period for induction is required for all prophages with mutations in the right side of Cr' When the prophage mutation is in the left side of cr , the minimal heating time is 30 seconds or less (curve D). One culture, M3 (tU32), gives a curve suggesting that induction starts immediately in some cells, but that the rate of induction is maximum only after five minutes at the high temperature. The tU32 mutation is in the left side of C]:, but is close to the middle of the C]: region (Fig. 2). (f) Induction after heating in chloramphenicol and puromycin
The absence of heat induction ofM3 (tl) in broth plus CAP orin saline was reported in an earlier publication (Lieb, 1964a). Puromycin (10- 3 M) also inhibits heat induction in M3 (t1). Both of these inhibitors of protein synthesis are reported preferentially to inhibit the synthesis of induced enzymes (Sypherd & Strauss, 1962). No induction of M3 (tl) takes place if starved cultures are incubated in buffer at 45°C. As additional mutants were studied, it was found that only six mutants were not induced when heated in media that inhibit protein synthesis. These prophages all have mutations in the right side of C]:. Lysogens the prophages of which have mutations in the left side of Cr are killed as the result of heating in media containing chloramphenicol or puromycin, or in buffer or saline (Fig. 5). Some of these lysogens have been tested for phage production after heating in CAP. After heating in CAP for 10 minutes, 90% of M3 (U16) or M3 (U37) cells produce phage. Longer heating in CAP results in additional killing and in a loss of plaque formers. It is not known whether the event that kills a heated lysogenic cell occurs during the heating, or later, on the plate. However, if a lysogen dies as the result of a treatment, I conclude that the treatment is responsible for initiating the lethal reaction. (g) UUraviolet inducibility of heat-inducible prophaqes
Three levels of ultraviolet inducibility were observed among the heat-inducible. mutants. Mutations in the right side of C]: result in prophages that are significantly more sensitive to ultraviolet induction than is wild-type ,\ (Lieb, 1964a). Mutations at three sites, all in the left side of Cr, cause the prophages to be extremely sensitive
PROPERTIES OF HEAT-INDUCIBLE 1",------=-::-::--:::-- - --
' ' ' " -D- ~O- -O . . D - - 0
-
-
~
MUTANTS
157
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20 30 40 Time (min) at 43° C
FIG. 5. Effect of chloramphenicol on induction of M3 (Atl) and M3 (~t2)_ At time 0, chilled logph>tBe cultures were diluted 1 : 100 into TB ± 20,..g CAP/m!. Samples were assayed at the times indicated.
to ultraviolet induction. To prove that differences in levels of ultraviolet sensitivity are not due to mutations in the host bacterium, several independently isolated lysogens containing each mutant have been tested. I considered the possibility that increased ultraviolet sensitivity might reflect a different mechanism of ultraviolet induction. However, ultraviol et induction of all the heat-sensitive mutants is susceptible to both photo-repair and da rk repair (Lieb , 1964b). The latent period for ultraviolet induction of heat-inducible mutants is shorter than that of II'" (Fig. 7). One mutant is less susceptible to ultraviolet induction than is wild-type A. The ultraviolet killing curve of M3 (U32) is very similar to that of M3 (A ind-) , and few irradiated cells produce plaques. The site of the mutation that simultaneously makes a prophage inducible by heat and less inducible by ultraviolet is in the left side of Cr-
(h) Dominance relationships of heat-inducible mutants
When a bacterium contains both a prophage and a superinfecting phage, with different heat- or ultraviolet-sensitivities, the heat- or ultraviolet-sensitivity of the complex is determined by tho mutant which, as prophage, produces the lysogen that is more resistant to heat or ultraviolet (Fig . 6). Dominanco is not affected by the positions of the genomes, e.g., in prophage or superinfocting phage. Figure 7 also shows that superinfecting All protects a cell lysogenic for MU37 from induction in CAP. In all cases tested, mutants with alterations at sites on the right side of the Cr gene inhibited induction in CAP of mutants mapping on the left side. A+ is dominant to all the heat-inducible mutants. Additional results will be published elsewhere (Lieb , manuscript in preparation).
M3{U37)
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FIG. 6. M3 (U 37) superinfected with ,\ + or Atl and then heated at 43°C. Labels on the curves indicate the superinfecting phage. A culture of M3 ('\tU37) at 6 X 10s/ml. was diluted 1 : 1 with ,\+ or Atl at 5 X 109/mI . and incubated at 36°0 for 10 min. Nine ml. of TB was added, the super· infected cultures were bub bled at 36°0 for 20 min and then chille d . For heat induction, the cold bacteria were dil uted into TB or TB 20 p.g/mI. CAP pre-heated to 43°0, and sampled at intervals.
+
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58
FIo. 7. P roper t ies of heat-inducible prophages. T he mutants are arranged in probable map orde r.
PROPERTIES OF HEAT.INDUCIBLE " MUTANTS
159
4. Discussion It is proposed that the initiation of any event dependent on the presence of the prophage be called "induction". In this study, "induction" refers to the initiation of an unknown event that causes the death of the lysogen. (a) The 'TfW,p of Cr
A diagrammatic representation of the properties of 12 heat-inducible mutants of ,\ is given in Fig. 7. The mutations in these phages map at 12 sites in the Cr region. In view of the different properties of the mutants, the Cr cistron seems to be divided into two regions: A, the left side of the Cr, contains mutant sites 2 to U32; B, the right side of Cr, contains sites I to U50. These regions together extend from the left of c47 to the vicinity of c71. (b) The nature of the Cr product The Cr gene is believed to contain the information for the synthesis of a product that prevents ,\ replication, cell killing and other events associated with phage production. Regions A and B in Cr are assumed to code for corresponding regions in the product of Cr' Mutations in the bacterium can suppress the effects of mutations in Cr (Jacob, Sussman & Monod, 1962; Thomas & Lambert, 1962). This suggests that the Cr product is a protein. Heat-inducibility may indicate that the protein produced by the mutant is heat-labile. Mutations at different sites would be expected to result in prophages with different sensitivities to heat. When various heat-inducible mutants were tested at temperatures from 38 to 43°0, marked differences in heat sensitivities were, indeed, observed. However, at 45°0 the killing curves of all lysogens are very similar. Perhaps the Cr product in all the mutants is inactivated very rapidly at 45°0, and the probability that a cellbe induced is determined by a subsequent process, the rate of which is not influenced by mutation in Cr. The possible nature of this process will be discussed later. (c) The "active site" for ultraviolet induction
Mutations in region A that result in heat-inducibility also affect the ultravioletinducibility of the prophage. Mutations at three sites result in greatly increased sensitivity to ultraviolet induction and a mutation at another site results in a prophage that is more resistant to ultraviolet induction than wild-type .\. Lysogens containing this mutant, or prophage .\t857, produce phage after ultraviolet only if they are also given a heat treatment sufficient to cause induction in the absence of ultraviolet. Goldthwaite & Jacob (1964) suggested that an inducer accumulates in the irradiated cell after ultraviolet, and that this substance combines with the Cr product, which they called the repressor. Lnd: mutants were postulated to have an alteration of the repressor that prevents effective combination with the inducer. The fact that there are Cr mutants with greater sensitivity to ultraviolet induction than wild-type A supports the notion that the Cr product combines with an inducer. Mutations affecting ultraviolet-inducibility map at several sites in the left side of Cr' The "active site" for interaction with the inducer may include a large portion of the Cr gene. (d) Unstable repressor
Some mutations in cr result in spontaneous induction, when the growth rate ofthe lysogenic culture decreases. Lysogenic cultures carrying these mutant prophages also
160
M. LIEB
contain large amounts of free phage. These phenomena can be attributed to the production of a very small amount of ex product, even at low temperatures, or to the instability of the ex product at low temperatures. (e) Protein synthesis and induction
Prophages with mutations in region A are induced when heated under conditions that prevent protein synthesis, indicating that induction (killing) is mediated by a pre-existing protein (enzyme) or does not require an enzyme. However, prophages with mutations in region B require protein synthesis for heat induction to occur. The question arises: if induction can occur in the absence of protein synthesis, what prevents certain lysogens from being induced when protein synthesis is blocked1 It could be that the synthesis of Cr product is blocked at high temperature in certain mutants, and that induction does not occur in the absence of protein synthesis because the Cr product is stable and persists to the end of the heating period. Several facts argue against this hypothesis. When a culture containing Atl is heated in a medium permitting growth, more than 90% of the bacteria are induced after ten minutes. At 45°C, the culture does not multiply, so that the Cr product is not diluted. If the ex product in these lysogens is stable, induction would not be expected to occur at such a high rate, unless additional assumptions are made. All the prophages that are not induced when protein synthesis is blocked have mutations at different sites in region B of ex, and it appears unlikely that mutation in one region of the gene would always result in a block to synthesis of the gene product at a high temperature. Finally, the experiments of Green (1966) demonstrate that when a lysogen containing Atl, mutated in region B, is heated in the presence of CAP, A mRNA rapidly increases in the bacteria, although they are not induced (killed). The ability of the Cr product to prevent the production of A mRNA is destroyed after two minutes at 45°C, indicating that this function of the ex product is heat-labile. However, the production of A mRNA is not sufficient, in these mutants, to produce cell killing or phage production unless protein synthesis accompanies heating. (f) Dominance of non-inactivated c/ product
A heat-inducible lysogen superinfeoted with wild-type ,\ before heating is not killed (Fig. 6), and does not produce ,\ mRNA (Green, 1966). A bacterium containing prophage.\t2 is induced (killed) when heated in a medium containing CAP. However, if the bacterium is infected with Atl shortly before heating, induction does not occur in CAP, or occurs at a much reduced rate. This "dominance" of active over inactivated Cr product indicates that the ex gene of the superinfecting phage is expressed in the lysogen. Additional information will be presented in a subsequent publication (Lieb, manuscript in preparation). (g) The activity of the c/ product: a model
The data presented here, together with those of Green (1966) show that the Cx product of ,\ has at least two inhibitory functions. One function is the inhibition of A mRNA production. The ex product also inhibits phage production and killing. The ex product of certain mutants can lose the first function but retain the capacity to prevent induction. I wish to propose a model in which a molecule of Cr product functions simultaneously as a repressor of ,\ mRNA synthesis and as an enzyme inhibitor (Fig. 8).
PROPERTIES OF HEAT·INDUCIBLE .\ MUTANTS
161
Region that codes / for'operator"
--,
t
DNA
After heating Mutation in region A
Active enzyme Mutation in region B
Inactive enzyme plus c r product
FIG. 8. Model of CI product in action. The product of CI is shown regulating the synthesis of the product of another gene. CI product could also regulate its own synthesis by aggregating with nascent CI protein.
The product of Cr is assumed to be synthesized constitutively. It combines with an enzyme, which is produced by another ,.\ gene. This enzyme is required for the initiation of the vegetative state; it might, for example, be a nuclease that releases A from the bacterial chromosome, and allows return of the ,.\ DNA to the vegetative form. As the messenger for this enzyme is made, it is translated by ribosomes that move along the messenger and separate it from the ,.\ DNA (Hartman, 1963). The studies of Byrne, Levin, Bladen & Nirenberg (1964) support the notion that ribosomes function in transcription and translation. Region A of the ex product combines with a part of the nascent enzyme; this part of the enzyme corresponds to the operator region. The enzyme cannot take its active configuration while it is combined with the Cr product. Region B of the attached ex product blocks the progression of the ribosome along the messenger. If the progression of this ribosome is required for mRNA release from the ,.\ DNA, the Cr product can block both transcription and translation; in non-nuclear ribosomes, the ex product blocks only translation, For reasons to be discussed elsewhere, I believe that molecules of Cr product aggregate, as shown in the diagram. Mutation anywhere in the Cr region can result in a Cr product that is unable to repress mRNA synthesis. When Cr product is altered in region A as the result of heating or ultraviolet irradiation, it releases the enzyme and simultaneously unblocks transcription and translation. The rate of ,.\ induction at high temperature could be limited by the rate of enzyme activation after release. The uncompleted molecule 11
162
M. LIEB
may be an active enzyme, or may be completed after the addition of a few amino acids. When C1 product with a mutation in region B is heated, region B no longer blocks the progression of the ribosome, and A mRNA is made. Region A of the C1 product remains attached to the enzyme and no irreversible event occurs until the concentration of enzyme molecules produced by the mRNA exceeds the coneentration of molecules of C1 product in a lysogen. Green has found that a lysogen containing At1 that has been heated in CAP continues to make large amounts of A mRNA after CAP is removed and cell growth has resumed. According to my model, translation of this RNA would result in enzyme synthesis and induction, unless the rate of synthesis of C1 product increased also. The model (Fig. 8) suggests that C1 product could regulate its own synthesis. Green proposes that the C1 product itself becomes an enzyme or "inducing protein", perhaps by disaggregation, when it is altered by heating. He suggests that when the C1 product has a mutation in region B, its enzymic activity is thermolabile and the synthesis of additional enzyme is necessary to cause induction. The site of action of the C1 product when it is a repressor is not specified. It may be possible to distinguish between the two models by studying A enzymes formed after inducing treatment. According to the model in Fig. 8, the operator is part of an enzyme made by a cistron other than C1• Certain changes in the enzyme (operator) might modify the rate of heat induction of particular C1 mutants. Double mutants containing mutations in C1 and cistrons N-R are being studied. Note added in proof: A double mutant A, containing CI mutations tl and t2, has been obtained. If mutation tl caused the CI "inducing protein" to be thermolabile (Green, 1966), the double-mutant prophage would be unable to kill the host when heated in CAP. However, lysogens containing A tlt2 are killed at 43°Cin CAP, so tl does not prevent heat induction in CAP when t2 is also present in the same genome.
I wish to thank Dr J. J. Weigle for his continued interest and help, and Dr M. Green for stimulating discussions. Mrs Ernestine Pruiet and Mr Percy Sanderman gave valuable technical assistance. This work was supported by grant no. G-24165 from the National Science Foundation, grant no. AI-05367 from the National Institutes of Health, and a Career Development Award (5-K3-GM-1546) from the National Institutes of Health. REFERENCES Bertani, G. (1958). Advanc. Virus Res. 5, 151. Byrne, R., Levin, J. G., Bladen, H. A. & Nirenberg, M. W. (1964). Proc. Nat. Acad. s«; Wash. 52, 140. Campbell, A. (1962). Advanc. Genetics, 11, 101. Goldthwaite, D. & Jacob, F. (1964). C.R. Acad. Sci. Paris, 259, 661. Gray, C. H. & Tatum, E. L. (1944). Proc. Nat. Acad. Sci., Wash. 30, 404. Green, M. (1966). J. Mol. Biol. 16, 134. Hartman. P. E. (1963). Proc, 11th Congr. Genetics, 2, 123. Oxford: Pergamon Press. Jacob, F. & Campbell, A. (1959). C.R. Acad. Sci. Paris, 248, 3219. Jacob, F. & Monod, J. (1961). Cold Spr. Harb. Symp. Compo Biol. 26, 193. Jacob, F., Sussman, R. & Monod, J. (1962). C.R. Acad. Sci. Paris, 254, 4214. Kaiser, A. D. (1957). Virology, 3, 42. Kaiser, A. D. & Jacob, F. (1957). Virology, 4, 509. Kaudewitz, F. (1959). Z. Naiur], 14b, 528. Lieb, M. (1953). J. Bact. 65, 642. Lieb, M. (1964a). Science, 145, 175. Lieb, M. (1964b). Virology, 23, 381. Lwoff, A. (1953). Bact. Rev. 17, 269.
PROPERTIES OF HEAT-INDUCIBLE A MUTANTS Okada., T., Yanagisawa, K. & Ryan, F. S. (1961). Z. Vererbunqslehre, 92, 403. Radding, C. (1964). Biochem. Biophys. Res. Comm. 15, 8. Sussman, R. & Jacob, F. (1962). C.R. Acad. Sci. Paris, 254, 1517. Sypherd, P. S. & Strauss, N. (1962). Proc, Nat. Acad. Sci., Wash. 49,400. Thomas, R. & Lambert, L. (1962). Bull. Acad. roy. Belg. 48, 668. Weigle, J. J. (1953). Proc, Nat. Acad. Sci., Wash. 39,628.
163