Temperature-sensitive regulation system of prophage lambda induction

J. Mol. Biol. (1967) 23, 217-224,

Temperature-sensitive Regulation System of Prophage Lambda Induction TADAO H o m ~ o m t A~D HAu-,-O INOEUOHI

De2~artment of CAemi~ry, National In~itute of Health of Japa~ Shinagawaku, Tokyo, Ja~ar~ (Received 11 March 1966, and ~r, reviaezfform 21 June 1966) Mutants of the temperate bacteriophage A of Eacher$c/v~ col$ have been isolated, which have a temperature-sensitive repression system for prophage induction. These mutants can be classified into two types: At~ type I which is induced at 28°C in Penassay broth after treatment for ten minutes at 47-5°C in buffer, or at a high temperature during growth; and A~ type I I which is induced only when the culture is heated at a high temperature during growth. Type I and type IT mutations reside in the cistron cI controlling repressor substance (immunity substance) production. The correlation between ultraviolet- and temperature-sensitivity for the induction of A~s prophage has been examined. When irradiated with ultraviolet light, bacteria carrying some A~ prophage were induced at lower doses than the induction dose of wild-type prophage A. Others At~ prophage retained the ultraviolet sensitivity to induction of wild-type A. Mutants of phage Awhich are more sensitive to induction by ultraviolet irradiation than the wild-type phage yet as heat-stable as the wild type were isolated. These findings indicate that although many At~ prophage are induced by a small dose of ultraviolet tight, the ultraviolet sensitivity is not necessarily correlated with the temperature sensitivity. Bacterial mutants which suppress the temperature-sensitive characteristic of the system of prophage ~ were isolated. They also restored the ultraviolet sensitivity for the prophage induction to the level of the bacteria lysogenic for the wild-type A. A possible mechanism of heat inactivation of repressors produced by A~ prophage is discussed. 1. I n t r o d u c t i o n The regulatory systems for enzyme synthesis and induction of prophage have been shown to be v e r y siml]ar in the sense t h a t the initiation of both systems is repressed b y the mediation of repressor substance, a product of a regulator gene (Jacob & Monod, 1961,1963). Two types of mutations resulting in temperature-sensitive regulation of fl-galactosidase formation are known: one in which repression is released temporarily after heating either in a growing condition or in a non-growing condition (Horiuchi, Horiuchi & Novick, 1961); and the other in which repression is released only when grown at high temperatures (Novick, Lennox & Jacob, 1963). I t has been suggested that the repressor for fl-galactosidase synthesis produced by the former type is itself temperature sensitive (Horiuchi & Novick, 1965; Sadler & Novick, 1965). 1"Present address: Faculty of Pharmaceutical Sciences, Kyushu University, Fukuolm, Japan. 217

218

T. H O R I U C H I AND H. I I ~ O K U C H I

I n this paper, the isolation a n d characterization of various m u t a n t s with different t e m p e r a t u r e and]or ultraviolet sensitivities for prophage i n d u c t i o n are described. The experiments to t e s t t h e c o m p l e m e n t a t i o n between these m u t a n t s are presented. This work has been briefly r e p o r t e d elsewhere (Inokuchi & Horiuchi, 1964; Horiuchi & Inokuehi, 1966). 2. M a t e r i a l s a n d M e t h o d s (a) Bacteria and bacteriophage 8train~

Escherichia coli K12 C600 (Appleyard, 1954) was used for the plaque assay and for the preparation of phage stocks. Selective indicator CR63 (Appleyaxd, McGregor & Baird, 1956) is resistant to the wild type of h but sensitive to the h mutant. Another sensitive derivative of K12, W3623, F-gal-try-str ~ isolated by J. Lederberg, was used as a host in lysogenization of At~ phage. W3350 carylng F'3, with the chromosomal segment including the gal and A locus (Jacob, Sehaeffer & Wollman, 1960), was kindly supplied by Dr F. Jacob. The phage strains used are the wild-type A and its derivatives, Acl, Acol and Ace2 (Kaiser, 1957). Various mutants which produce an altered represser substance upon lysogenization were isolated from the wild-type phage as described later. Spontaneous phage mutants of various types which can infect CR63 were isolated and designated h mutants. Ai~34 is a hybrid of phage A and 434 (Kaiser & Jacob, 1957). (b) Media Ponassay broth contains 17"5 g of Dffco Bacto-Penassay broth in 1 1. water. A-broth contains 10.0 g of Polypeptone (Daigo-Eiyo Chemicals) and 2.5 g NaCI/1. of water. For plating, A-broth was solidified with 0.6% agar for the top layer and 1% agar for the bottom layer. M9 buffer was used for heat induction of Ats lysogenic bacteria. This buffer is identical to M9 medium (Anderson, 1946) except t h a t the carbon source is omitted. (o) Induction by heating or ultravi~Zet irradiation Bacteria carrying prophage ht~ in the exponential phase of growth were centrifuged, washed with M9 buffer and resuspended in one-tenth of the original volume of buffer or Penassay broth. The suspension was heated at 47.5°C for 10 rain. The heated bacteria were plated immediately or cultured in Penassay broth at a low temperature. Plate~ were incubated either at 28 or 43°C. For ultraviolet irradiation, 2 to 5 ml. of M9 buffer containing 2 × 107 to 2 × 10a ceils ml. were shal~en in a Petri dish exposed to a 15-w germicidal lamp (Toshiba) at a distance of 90 cm. The dose rate was 1.9 erg/mm2/sec as determined b y a Toshiba germicidal ultraviolet meter. (d) Other methods Bacterial density was measured as optical density at 660 m~ using a Beckman model DB spectrophotometer. A culture with an optical density 0.1 contains approximately 0.5 × 103 bacteria/ml. Bacteria carrying two types of prophage were prepared in two ways. One method was by superinfection of bacteria carrying one t y p e of prophage with the other. The other method was by mating of W3350 carrying F ' gal harboring one type of prophage and W3623 carrying another type of prophage. The mating was carried out at 28°C. Merozygotes were selected on eosin-methylene blue-glaetose medium supplemented with streptomycin. Complementation for lysogenization between various mutants was tested as described by Kaiser (1957).

3. R e s u l t s (a) Isolation and classification of temTerature-sensitive mutants of ?t phage I n order to o b t a i n temperature-sensitive m u t a n t s for t h e repression s y s t e m of prophage ~, the phage was i r r a d i a t e d b y u l t r a v i o l e t light to a survival of a b o u t 10 -8 • a n d p l a t e d on ~-sensitive bacteria a n d i n c u b a t e d a t 43°C. Clear plaques were picked

T H E R M O S E N S I T I V E I I ~ D U C T I O ~ OF P R O P H A G E A

219

and spotted on two plates containing the same bacteria. The plates were incubated separately at 28 and 43°C. Plaques whichwere clear at 43°0 and turbid a t 28°0 were saved. Temperature-sensitive m u t a n t s of ;~ phage were also obtained from the ethyl methane sulfonate-treated lysogenic bacteria which lyse at 43°C and can grow a t 28°0. W3623 was lysogenized b y several temperature-sensitive mutants. These bacterial cultures were heated under various conditions. I t was found t h a t the m u t a n t s fall into two classes according to the response of the bacteria lysogenized b y the mutants. Bacteria lysogenic for M~I-1 lysed completely when subsequently grown a t 28°0 after heat t r e a t m e n t for a short time in either buffer or a complete medium, while bacteria lysogenic for ~ I I . 1 lysed only after heating in a complete m e d i u m t. The response of lysogenic ceils heated in a complete m e d i u m with chloramphenicol (100 ~g/ml.) is slml]ar to t h a t in buffer for b o t h types. The lysis of bacteria containing t8 prophage after heat t r e a t m e n t was caused b y production of ;~8 phage. The burst size was between 20 and 100. -----r----n

l Broth 37°C_

1, ~

-

0.01 ~ : 5 °

I

I

Buffer 37°C 47-S °C

-

C

0-001 . _ _ . . J _ _ _ _ L _ _ 0 I0 7.0 (a)

47'5~C~~

_

_

I0 20 (b)

0

I

I

I0

20 (c)

_ _

0

I0

20 30 (d)

Time (rain) l~Io. 1. Survival of the lysogenic bacteria carrying ;~ prophage as a function of the time of heating at various temperatures. W3623 lysogenized for (a) AtaI-lh, or (b) ;ttaII-1 were cultured" at 28°C. The cultures were diluted 100-fold at time zero into Penassay broth warmed to various temperatures as shown on the curves. At a given time, they were sampled and plated for the viable bacterial count. Experiments (c) and (d) were performed as (a) and (b), with the exception~that the heating was carried out in buffer at various temperatures. Time courses of the change in the surviving fraction of bacteria contalnln~ /8 prophage after heat t r e a t m e n t a t various temperatures are shown in Fig. 1. Here it can be seen clearly t h a t t y p e I a n d t y p e I I belong to the different groups. T y p e I prophage can be induced to effect lysis of the lysogenic bacteria b y heating in buffer, whereas t y p e I I prophage cannot in the same conditions (Fig. l(c) and (d)). The induction of W3623(~8I-1) when heated during growth is more efficient t h a n when it is heated in a resting condition. 1"Because of the operational similarity of the thermoseusitive regulatory systems of ~-galactosidase synthesis and ;~ induction, the heat-inducible mutants of;t are classified as type I and I I as described in the text. They may correspond to class TT and I, respectively, of Green (J. MoL Biol. 1966, 16, 134).

220

T. HORIUCHI

AND

H. INOKUCHI

Since Ms prophage can be induced and produce clear plaques at a high temperature, it was presumed t h a t the ~ mutations were located in the c / r e g i o n . To t e s t this assumption, complementation experiments for lysogenization between ts phage (MsI-1, MsI-2, MslI.1, MslI.2 and Ms/I-4) and various clear mutants were carried out. None of the t~ mutants of either t y p e showed complementation with )lcl at 43°C, whereas they did complement with ~col (mutated in the region c/I), ~lco2 (mutated in the region o H / ) . These results indicated t h a t these ts mutations are located in the region cI. Since the repression system of prophage M s / i s inactivated b y heating in buffer, the simplest explanation would be t h a t the represser itself is thermolabfle. I t is also reasonable to assume t h a t M s / / p r o p h a g e also produces an altered represser. (b) TemTerature-sensitivity of the bacteria carrying both 2tsI and )ttslI Trophage I t is of interest to know which type of the ts characters is dominant in bacteria carrying MsI and tslI and whether or not the repression systems of M s / a n d tsII are complementary. To answer these questions, we utilized the fact t h a t the bacteria carrying MsI-1 and those carrying MslI.1, in addition to the different response upon heating in buffer, differ in the following respect. The bacteria carrying MsII-1 are induced within one hour by heating at 37°C; but those carrying Ms/-1 are not induced even in complete medium at this temperature (see Fig. 1). i~Ierodiploid bacteria, carrying MsI- lh on the chromosome (gal-) and Ms//- lh + on the F-gal + episome and vice versa, were selected and examlned (Fig. 2 and Table 1). Results are summarized as follows: (1) No lysis of the culture was observed after transfer to 37°C (curve A). (2) Heating in buffer at 47.5°C for ten m~nutes does not cause phage induction (curve C). (3) Lysis was observed when the bacteria were transferred to a complete medium at 43°C (curve B). (4) The bacteria grown at 28°C were transferred to 37°C for one hour; then the bacteria were centrifuged, washed and heated in buffer at 47.5°C. No lysis

50

A

I0

A

C

C

05

0'1

O'OS 1

i

o1½; (Q)

I

I

I

I

0 1 2 3 (b)

Time(hr) l~m. 2. Effect of heating on the growth of bacteria containing Ms/and MslI prophage. W3623(MaI-lh)/F'(MaII-lh+) (a) and W3623(MsI/-lh+)/F'(M~I-lh) (b) were grown at 28°C and divided into three portions. Cultures A and B were transferred at time zero from 28 to 87 and 43°C, respectively. Culture C was centrifuged, washed and resuspended in buffer, then heated at 47.5°C for 10 v,~n and finally grown at 28°C in the broth.

THERMOSEI~SITIVE II~DUCTION OF PROPHAGE h

221

T~BLE 1

Effect of heat trcatmen~ on bacteria carrying htsI and )ttsII prophage Fraction (%) of surviving bacteria after heating at 47.5°C for 10 mln in buffer in broth

Strains

W3623(~t~I-lh, ;(talI-1) W3623(~I-1h)/F'(~H-1)

99.4 90.3 95.3

2.9 9.0 4.7

wa623(~!-1h)

1.7

1.0

W3623()~t~II-1)

86.5

1.0

W3623(~telI-1)/F'(~4~I-1h)

Bacteria grown for 1 hr at 37°C in Penassay broth were heated, except W3623 (~tslI-1), which was grown at 28°0.

was observed (Table 1). (5) W3623 doubly-lysogenized for ~tsI-lh and k~II-1 behaves slml]arly. The result (3) indicates that the repression systems of 2tsI-1 and of ~slI.1 do not complement significantly at 43°C. I f the complementation is not significant at 37°G in broth or 47.5°0 in buffer, the results (1), (2) and (4) can be explained by rapid regain of activity or rapid synthesis of tslI-1 repressor after the return to 28°0. Complementation tests between various type I and type II phages in various conditions are now under way.

(c) Sensitivity of the, repression system of bacteria carrying various mutant~ of Thage ~t to ultraviolet light IAeb (1964) reported that, upon appropriate ultraviolet irradiation, the survival of bacteria presumably containing a ~tsII prophage is decreased as compared with that of bacteria lysogenic for wild-type h. We compared the sensitivity to induction by heating to that by ultraviolet irradiation. As shown in Fig. 3, bacteria carrying various 2ts prophage of either type I or I I show different sensitivities to ultraviolet irradiation and some bacteria show a response similar to bacteria carrying wild-type prophage. I t should be mentioned that the W3623 carrying both )~I-1 and ~slI.1 has similar ultraviolet sensitivity to W3623(~tslI.1). I t is clear that the ultraviolet sensitivity to prophage induction is not necessarily correlated with temperature sensitivity. From this it follows that a mutant may be found in which prophage induction is heat-stable and ultraviolet-sensitive. To select such a type of mutant, phage irradiated with ultraviolet light were plated with 0.11zg of mitomycin C per plate. In this condition, ~ I - 1 makes a clear plaque while the wild-type h makes a turbid plaque. Sixty clear plaques on the mitomycin C plate incubated at 37°C were picked and tested. Two mutants of )~ phage made turbid plaques on the normal plate at 43°C. As shown in Fig. 3 (c) and (d), the bacteria lysogenic for these mutants (designated as )~ind') are not induced at high temperature whereas they are induced by a small dose of ultraviolet light. On a plate with 0.1 pg of mitomyein G, )~inds complements to form a turbid spot with )~col or hco2 but not with hcl.

T. HORIUCHI

222

i

i

i

AI~D H. Ilg0KUCHI

i

(~Sl-3)

(Cl +)

(~SE-4)

k

(C~+)

(~81-3)

\ \ V , s.- ,

I-I)_ (indS-1)~ (a) Heat

0"001

c) Heat

(b) u.v.

I

i

,o

2o

Time(min)

!

o

2o

/

4o

Time(sec)

6oo

(d) u.v.

i

i

io

2o

Time(mln)

0

20 40 60 Time(sec)

Fro. 3. Survival of t h e lysogenic strains carrying various kt~, XCnd"a n d wild t y p e as a function of t h e time of heating a t 43°C ((a) a n d (c)) a n d as a flmction of ultraviolet dose ((b) a n d (d)).

(d) SuTpressor mutation of bacteria of the, temperature.sensitive character of kts It has been reported that the formation of represser substance of phage )t is susceptible to the action of some bacterial suppressor mutations (Jacob, Sussman & 1VIonod, 1962). An attempt was made to isolate suppressor mutants of bacteria for a temperature-sensitive repression system of Mz prophage. Several cycles of low- to high-temperature shift were applied to the culture of W3623(~tsI-lh) and of W3623 (~tsII-1) and heat-resistant survivors were isolated and tested. Among 100 colonies tested in each case, a few colonies showed the expected character. The phage produced by ultraviolet irradiation of the lysogenio heat-resistant strains showed the original ts character when they lysogenized W3623 or C600. The bacterial suppressor mutation will be designated as sull for htsI-1 and su21 for that of ~ts//-1. The nonlysogenic sull strain were re-lysogenized by ~sI.lh or by )¢zlI-1 and tested for their heat resistance. W3623 sull(ktsI-lh ) was shown to be heat resistant like W3623()J~), but W3623 su~(htsII-1) was shown to be heat-sensitive like W3623(~ts//-1). The sensitivity to ultraviolet irradiation of W3623 sul~(ktsI.lh ) and W3623 su2~(~ts//-1 ) is almost restored to the level of bacteria lysogenic for the wild-type h. A presumed single suppressor mutation in bacteria restores to the prophage the sensitivity to heating and ultraviolet irradiation of the wild-type prophage. It may therefore be concluded that a temperature-sensitive mutant prophage which is simultaneously ultraviolet-sensitive is produced by a single mutation. However, an increase in the ultraviolet sensitivity to induction by a mutation in a prophage is not necessarily accompanied by an increase in temperature sensitivity, l~urther, the increase of temperature sensitivity is not necessarily accompanied by an increase of ultraviolet sensitivity. Furthermore, the temperature-sensitive mutation which simultaneously increases the ultraviolet sensitivity is not correlated with the types of the temperature-sensitive mutations.

THERMOSE1RSITIVE

INDUCTION

OF P R O P H A G E

~

223

4. Discussion Two types of mutation resulting in temperature-sensitive regulation of prophage induction have been described in this report. The mutations of ~ s I and ~ s I I are present in the same cistron, cI, and are recessive to the ci + allele. The response of the cells carrying ~ s I to heating can be interpreted as inactivation of the repressor. There are several possibilities which may account for the character of the response of cells carrying prophage AtsII: (i) the repressor is inactivated at high temperatures even in buffer, but the inactivated repressor regains its activity at a low temperature; (ii) the repressor is inactivated at high temperatures, but new repressor is synthesized rapidly when the bacteria are returned to a low temperature; (iii) the repressor cannot be formed at high temperatures during growth, but the ~n~Rhed repressor is stable at high temperatures. In addition, because prophage induction occurs quickly when the bacteria are grown at high temperatures, the finished repressor should be metabolically unstable, as suggested in the system of alkaline phosphatase formation (Gallant & Stapleton, 1963) and fl-galactosidase formation (Sadler & Novick, 1965). Using immune non-lysogenic segregants from cells infected with ~b2 tsII-1, 0gawa & Tomizawa (1967) have shown that the heat-inactivated repressor of AtsI-1 was reactivated very slowly and the heat-inactivated repressor of of AtsII-1 was reactivated very rapidly at low temperatures after heating at 47.5°0 in buffer. These results support possibility (i), namely, that the repressor produced by AtsII is inactivated at high temperatures but the inactivated repressor regains its activity when the bacteria are returned to a low temperature. The different responses to heating between the repression system of type I and that of type II may be caused by the difference of the rate of reactivation of the inactivated repressor. It is possible that the presence of two types of mutants represents two modes of inactivation of the repressor. If the repressor is an allosteric protein polymer, as suggested by Jacob & l~Ionod (1963) and Sadler & Novick (1965) for the repressor of fl-galactosidase formation, heating may cause destruction of the subunits in the case of type I, and dissociation of subunits in the case of type II. Recently, a hypothesis on the dual function of a repressor was proposed by Stent (1964), in which the repressor is supposed to possess the ability to initiate as well as ~nhibit formation of an enzyme. I f the repressor substance produced by the prophage A~I has a thermolabfle ~nhlbition site and a thermostable initiation site, and if both the sites of the repressor substance produced by the prophage ~ s I I are thermolabfle and the initiation site can be synthesized at a sufficient rate to function only in the growing condition, then the characters of both the mutants would also be explained. If this is the case, type I mutation should be dominant over type I I mutation at a high temperature unless no complementation is present. As shown in the results, type I mutation is not dominant over type I I mutation. This possibility is excluded, assuming that there is no significant complementation of the cl gene. We acknowledge the encouragement and sthnulating discussions of Dr J. Torn~.awa and members of the Department of Chemistry. This investigation has been aided by a grant from The Jane CoffinChilds Memorial Fund for Medical Research (project no. 165). REFERENCES Anderson, E. H. (1946). Proc. -hro~. Aco~. Sc~., Waah. 32, 120. Appleyard, R. K. (1954). Genetica, 39, 440.

224

T. H O R I U C H I

AND H. INOKUCHI

Appleyard, R. K., McGregor, J. F. & Baird, K. M. (1956). Virology, 2, 565. Gallant, J. & S~apleton, R. (1963). Proc. Nat. Acad. •ci., Wash. 50, 348. Horiuchi, T., Horiuchi, S. & Novick, A. (196i). g. Mol. BIOL 8, 703. Horiuohi, T. & Inokuohi, H. (1966). J. MoL Biol. 15, 674. Horiuohl, T. & l~ovick, A. (1965). Biochim. 5iophys. Acta, 108, 687. Inokuchi, H. & Horiuchi, T. (1964). Viru~ (in Japanese), 14, 299. Jacob, F. & Monod, J. (1961). Cold ,Spr. Har5. ~ymp. Quant. Biol. 26, 193. Jacob, F. & Monod, J. (1963). In Cytvdifferentiation and Macromolecular ~ynthe~, ed. by M. Locke, p.30. New York: Academic Press. Jacob, 1~., Schaeffer, P. & WoUman, E. L. (1960). In Microbial Genetics, ed. by W. Hayes & R. C. Clowes, p.67. Cambridge: The University Press. Jacob, 1~., Sussman, R. & Monod, J. (1962). C.R. Acad. Sci. Par~, 254, 4214. Kaiser, A. D. (1957). Virology, 3, 42. Kaiser, A. D. & Jacob, F. (1957). Virology, 4, 509. Lieb, M. (1964). Science, 145, 175. Novick, A., Lennox, E., & Jacob, F. (1963). Cold Spr. Harb. Syrup. Quant. Biol. 28, 397. 0gawa, T. & Tomlzawa, J-I. (1967). J. Mol. Biol. 23, 225. Sadler, J. R. & Novick, A. (1965). J. MoL Biol. 12, 305. Stcnt, G. S. (1964). ~c/ence, 144, 816.