Studies on temperature sensitive growth of phage ∅80

VIROLOGY

67, 168-178 (19%)

Studies

on Temperature

Sensitive

Growth

of Phage $80.

I. Prophage Excision

Department

of Microbial

SHIRO

AIZAWA

Genetics,

Research kami,

AIZO MATSUSHIRO

AND

Institute for Microbial Suita, Osaka, Japan

Accepted

April

Diseases,

Osaka Unioersity,

Yamada-

23, 1975

We have examined the effect of high temperature on the early stage of vegetative development of phage #SO. We have shown that the temperature sensitivity is mainly due to a failure of prophage excision. Furthermore, we have shown that the deficiency in phage DNA synthesis resulting from failure of prophage excision leads to a decrease in endolysin production and late mRNA synthesis at the high temperature. INTRODUCTION

There are indications that the regulatory system for the temperate phage $80 is generally analogous to that described for phage X (Matsushiro et al., 1964; Sato et al., 1968; Lozeron and Szybalski, 1969; Tomita, 1969), but that the two phages differ in several other respects (Szpirer and Brachet, 1970). In this series of experiments we have concentrated on one difference; at high temperature (42”), 480 is not able to produce a full yield of progeny particles after induction of the lysogen or after infection, whereas X is able to do so. The purpose of our experiment was essentially to exploit this temperature sensitivity to investigate vegetative development and morphogenetic assembly processes in phage $80. In addition, we expected to be able to use ts mutations as markers in the genetics of this phage. In this and the following paper it will be demonstrated that the wild type of phage 480 contains several temperature sensitive loci; (a) one or more temperature sensitive steps exist in the early stage of vegetative development; (b) there exists a temperature sensitive step during morphogenetic assembly in the late stage. In this paper we will concentrate on the former problem and present data showing that excision of the prophage is temperature sensitive. From

this fact we can explain the remarkable differences in DNA, mRNA, and endolysin synthesis shown by the induction and infection cycles. MATERIALS

(a) Bacteria Escherichia coli K12 strain 594(su-) and its lysogens were mainly used. 594trpEthy- was used as a thymineless strain for the labeling of phage DNA. Ymel su3+ was used as a permissive strain for phage carrying the Sam7 marker. The bacterial strain used for the physical study of prophage excision was AE-1 thy-l Ftrp, a strain which carries a chromosomal deletion for the attao region and harbors a sex fector, Ftrp, which contains the trp attao region. (b) Bacteriophages

Phage strains used in this study are listed in Table 1. The genetic constitution of all phage strains used is shown in Fig. 1. (c) Media

L broth (Lennox, 1955) and X-broth containing 1% w/v tryptone and 0.25% w/v NaCl were used for phage growth and cellular lysis experiments. Ca-M medium, minimal medium E (Vogel and Bonner, 1956) supplemented with 0.4% casamino

168 Copyright All rights

0 1975 by Academic Press, Inc. of reproduction in any form reserved.

AND METHODS

PROPHAGE

169

EXCISION

TABLE

1

PHAGE STRAINS Phage

Plaque

strain

formation at 42”

+

x

Growth at 42” after induction

Origin

i

xc1 XcIts857Sam7

Goldberg Matsushiro Thomas Nishimune Signer N. Franklin

480 680~ $80int2 hsoatt80i”(17,

18, 19)80

h”attsOiso h”atts0i”(17,

18, 19)80

h”att%” h”att”i”(l7, 18, 19)80 hsOattsOi” h*attW’(Q, S, R)”

+ (pin point) + + + +

“h”atteoi” was obtained by selecting recombinant hAatt’0i*(17, 18, 19)‘0withh~“att~0i”cIts857Sam7.

-

Nishimune Prepared Prepared Prepared Prepared

+ Not tested

hAatt’0i*cIts857Sam7

in in in in

and our our our our

on Ymel s~3+/‘I’l

Matsushiro 1ab.O 1ab.O lab.< lab!

strain

in the cross of

bhhatthih(17,18,19)80 was selected on x-/T1 bacteria in the cross of h”0atta0iA(17,18,19)“0 with XcIts857Sam7. CW’attW was obtained by selecting recombinant hs0att~0i”cIts857Sam7 in the cross of h8°attsoi”(17, 18, 19)*O with hcIts857Sam7. d h”attW’(Q,S,R)” was selected on su- bacteria in the cross of h*a~t”is0(17,18,19)80 sus91 with XcIPam3. h: host range. att: attachment site. i: immunity region. cIts857: temperature sensitive mutation in immunity gene of X. Sam7: amber mutation in gene S of X. Pam3: amber mutation in gene P of X. sus91: amber mutation in gene 18 of 480. +: normal growth at 42” on permissive host; -: defective growth, lower than one-hundredth of growth at 35”

1

FIG. 1. Genetic

hA

OP

IA

ot+A

,mNl

I s I WA

constitution of X, $80, and $80-X hybrid phages. The genomes of X and $80 are represented as solid and empty lines, respectively. The position of selected genes on the h chromosome (A-J, att, int, N, cI, 0, P, Q, S, and R) is according to Fiandt et al. (1971). The position of genes on the 480 chromosome (l-19, art, int c1, 15, 14, and 16) is according to Sato (1970), Sato et al. (1968) and Matsushiro et al. (in preparation). The maps of 980-X hybrids employed in this study are shown in the lower part of the diagram. Solid lines indicate genetic regions derived from the h chromosome and empty lines those from the $80 chromosome. All locations for the junctions between 480 and X DNA are according to Fiandt et al. (1971).

170

AIZAWA AND MATSUSHIRO

acids and 100 pg/ml L-tryptophan was used [3H]uridine. [3H]RNA was prepared acfor the assay of mRNA synthesis and cording to the method of Imamoto (1969). endolysin synthesis. 2. DNA-RNA hybridization. The prepaCa-X broth containing 1% w/v casamino ration of DNA filter was carried out accordacids and 0.25% w/v NaCl was used for ing to Segawa and Imamoto (1974). 0.1 ml [3H]RNA sample suspended in 3x SSC phage DNA synthesis experiments. Tl buffer (Matsushiro et al., 1964) con- was annealed on a piece of Millipore filter taining 2 x 10m3M KCN (Tl-CN buffer) (type HA, 0.45 pm pore size) containing 5 was used for phage adsorption and UV pg of immobilized phage DNA, for 18 hr at irradiation for prophage induction. 66”. The filter was then removed, digested for 30 min at 37” with 5 pg/ml RNase, (d) Procedures for Infection and Induction washed three times with lx SSC, dried, Infection. Cells grown at 37” to a titer of and counted with 10 ml of scintillation 3 x 108/ml were centrifuged and resus- fluid in a Beckman LS250 scintillation counter. Total radioactivity of [3H]uridine pended at 1.5 x log/ml Tl-CN buffer. Phages were added at a multiplicity of incorporated into RNA was measured as about 4 and allowed to adsorb at 37” for 15 the trichloroacetic acid-precipitable material, min. Thermal induction. Lysogens for phages (g) Measurement of Phage DNA Synthesis carrying the CIts857 character were grown at 30” to 3 x 108/ml. They were induced by 1. Labeling and preparation of phage DNA. After infection or induction, the exposure to 45” for 10 min. Ultraviolet induction. Lysogens grown at cells were centrifuged, transferred into 37” to 3 x 108/ml were induced by exposure prewarmed Ca-X medium supplemented to ultraviolet irradiation in the Tl-CN with 2 pglml thymine, 200 pg/ml tryptophan, and 10 &i/ml [3H]thymidine. At buffer for 45 set (11 erg/mm’/sec). each time during phage development, 0.5 (e) Endolysin Assay ml samples were withdrawn and precipiAfter phage infection or induction, the tated by 5 ml cold 5% trichloroacetic acid cells were centrifuged and resuspended in (TCA). After 2 hr, they were twice washed with 5 ml cold 5% TCA and resuspended in Ca-M medium. They were then incubated for a suitable period, stopped by cooling in 0.5 ml of 0.5 N NaOH. They were then an ice bath and sonicated for 30 sec. The heat-denatured in boiling water for 15 min disrupted lysates were centrifuged to re- and neutralized with 0.33 M citric acid. 2. DNA-DNA hybridization. The DNAmove cell debris and the supernatants were DNA hybridization was carried out essenstored at 0” until use. Endolysin assay was carried out accord- tially according to the method of Denhardt ing to the method of Tsugita et al. (1968). (1966). The annealing was carried out for The endolysin activity of egg white lyso- 18 hr at 65” in a tightly sealed scintillation zyme was used as standard and the en- vial containing 0.7 ml of 6x SSC supplemented with 1 mM EDTA, 5 Fg of alkalidolysin activity of samples was calculated in terms of the corresponding amount of denatured DNA-carrying filter and 0.3 ml of sample. egg white lysozyme.

(f) Measurement

of Phage Specific mRNA

Synthesis 1. Labeling of phage mRNA with [3H]uridine. After infection or induction, the cells were centrifuged and transferred into prewarmed Ca-M medium. After incubation for a suitable period, they were pulse labeled for 30 set with 10 &i/ml

(h) Detection of Excised Phage Genome In principle, excised covalent circular form of phage DNA should sediment far ahead of the linear host chromosomal fragments in an alkaline sucrose gradient (Freifelder, et al., 1971). It is suggested that the efficiency of detection of excised covalent circular form of phage DNA is

PROPHAGE

remarkable in the case of the use of O- or P- prophage. We used a prophage marked Pam3. On the basis of above mentioned principles, the experimental protocol was as follows; AE-1 thy-/Ftrp(su-) cells lysogenized with hs0atts0iAcIts857Pam3 hybrid phage were radioactively labeled by growth at 32” in Ca-X medium supplemented with 100 pug/ml L-tryptophan, 2 pg/ml thymine, 50 &X/3 ml [3H]thymidine, and 200 pg/ml uridine for 3 hr, washed and resuspended in 6 ml chilled, nonradioactive medium containing 20 pglml thymine and 200 &ml uridine. For the zero time control, a 2-ml sample was collected by centrifugation, resuspended in lysis buffer, and stored at 0”. The remainder was induced by exposure to 45” for 5 min. A 2-ml sample was then incubated at each temperature (37”, 42”) for 25 min, chilled at O”, collected by centrifugation, and resuspended in lysis buffer. All procedures for lysis, sample preparation, centrifugation in alkaline sucrose gradients, and radioactive sample preparation and counting were carried out essentially according to the method of Freifelder et al. (1971). The control experiment was carried out by using bacteria containing F’450 (XcIts8570am29 PamSO) kindly provided by Dr. D. Freifelder. (i) Measurement of Tryptophan tase (TSase) Activity

EXCISION

171

Hybrid phage h80attsoi”cIts857Sam7 (Fig. 1) cannot form plaques at 42”, although XcIts857Sam7 can form plaques at 42” on Ymel su3+ strain. This hybrid phage is composed of the left arm of 480 and the right arm of X. This indicates that $80 has one or more temperature sensitive steps at the late stage of phage development. On the other hand, hybrid phage h”att*Oi*O forms pinpoint plaques at 42” (Fig. 1 and Table 1). This hybrid phage is composed of the left arm of X and the right arm of 480. This indicates that the right arm region also contains another weak temperature sensitive gene(s). The experiments in this section were designed to investigate the temperature sensitivity of the right arm region of 480. During the investigation of this point, we discovered a remarkable difference in phage development between infection and induction. 1. Endolysin synthesis. UV-induced 480 lysogens do not lyse and not produce active phage particles at high temperature (42”) (see Fig. 2). This is in clear contrast to X lysogens. The levels of endolysin produced at high (42”) and low (35”) temperatures were determined (Table 2). The endolysin level in the lysate prepared at high temperature was only a few percent of that ob-

Synthe-

After phage infection, the cells were centrifuged and transferred into prewarmed Ca-M medium supplemented with 50 pg/ml L-tryptophan. They were incubated for a suitable period and stopped by the addition of chloramphenicol (100 pg/ ml) and cooling in ice. The preparation and the TSase assay of extracts were carried out according to the method of Ito and Imamoto (1968). RESULTS

(A) Difference Between Induction fection Cycles

and In-

80-X hybrid phages should be very useful for the determination of temperature sensitive region(s) in the 480 chromosome, for it is known that X is far less temperature sensitive than $80.

FIG. 2. Lytic gens, incubated Turbidity at 550 Spectrophotometer. 594(@80) at 37”; 37”; A = 594(h)

curves for 594(@0) and 594(X) lysoafter UV-induction at 37” and 42”. nm was followed in a Coleman Junior Symbols are as follows; 0 = l = 594(680) at 42”; A = 594(X) at at 42”.

172

AIZAWA

AND

tained at low temperature. Thus, poor lysis at high temperature might be ascribed to the low endolysin level. The possibility that $80 endolysin itself is temperature sensitive was ruled out by the fact that the lysogen harbouring hybrid phage h”att”i” (17, 18, 19)80 (Fig. 1) can lyse at high temperature, though it has the structural gene for $30 endolysin. It seems likely, therefore, that poor lysis at high temperature is due to the temperature sensitivity of endolysin production but not of its activity. The endolysin level present in the lysate prepared after infection with 480 was examined. The results obtained are presented in Table 3. In the case of infection, the endolysin level of the lysate obtained at TABLE ENDOLYSIN

LEVEL

Temp.b

Expt

I

Expt

II

2

IN UV-INDUCED

Endolysin level’

35” 42” 35” 42”

LYSATES”

Ratio (42”/35”)

23.6 0.5 19.6 2.3

0.02 0.12

“594(@0) cells were cultured for 180 min at each temperature (35”, 42”) after UV-induction and the endolysin levels in their lysates were determined by following the reduction of the optical density at 450 nm at room temperature. Endolysin level is calculated from the rate of reduction of optical density. A standard curve was constructed by measuring the absorbance given by a series of different concentrations of egg white lysozyme. bTemp.: incubation temperature. ‘Endolysin level was expressed as micrograms equivalent to egg white lysozyme. TABLE ENDOLYSIN

Temp.

Expt

I

Expt

II

3

LEVEL IN INFECTED WITH f#18oC”

35” 42” 35” 42”

Em$lysi

13.8 8.9 11.4 4.4

LYSATES

Ratio (42”/35’)

0.64 0.39

D 594 Cells infected at a multiplicity of infection about 4, were cultured for 90 min at each temperature (35”, 42’) and the endolysin level was determined described in Table 2 and Materials and Methods.

of as

MATSUSHIRO

high temperature was 40-60s of that obtained at low temperature. Therefore, the difference was less pronounced after infection as compared with induction. 2. Transcription of the late region. In other experiments, it was shown that phage 480 produced poor serum blocking power at high temperature, especially in induced lysates (data not shown). The temperature sensitivity of the synthesis of endolysin and serum blocking power suggests that the late region of phage $80 might be poorly transcribed at high temperature. Hence the transcription of the late region in induced lysogens was examined. The rate of late mRNA synthesis was determined by the DNA-RNA hybridization method (Tomita, 1969; Lozeron and Szybalski, 1969; Szybalski et al., 1970). Messenger-RNA corresponding to the region of genes 17, 18, and 19 was determined as the difference between values of [3H]RNA hybridizable with h”att”i” (17,18, 19)*‘-DNA and X-DNA (Fig. 1). Messenger-RNA corresponding to the region of genes 1-13 (left arm) was measured as the difference between values of [3H]RNA hybridizable with hsoattsoi”-DNA and h”attsoi”-DNA (Fig. 1). The results obtained after UV-induction are presented in Fig. 3a and b. It is apparent that the rate of synthesis of late mRNA at high temperature (42”) is only 10-20s or less of that at low temperature (35”). The low level of endolysin obtained at high temperature could be explained by a decrease in the rate of mRNA synthesis from the gene 17-19 region, because gene 19 is the structural gene for endolysin (Sato, 1970). The decrease of serum blocking power at high temperature can be similarly explained. The rate of late mRNA synthesis after infection was examined, and the results obtained are presented in Fig. 4. The effect of high temperature is not so remarkable as after induction. The extent of decrease in endolysin synthesis at high temperature is reflected by the decrease in late mRNA. 3. DNA synthesis. A decrease of the rate of late mRNA synthesis could be explained from either of the following two possibilities.

PROPHAGE

(b)g.ne

l-13

*

Time after

induction

/

173

EXCISION

the case of induction, the fact that the DNA synthesis is remarkably affected by high temperature suggests a temperature sensitivity of prophage excision, a function restricted to the induction cycle. We will present evidence on this point in the next section.

(min.)

FIG. 3a and b. Transcription of the late region after induction; gene 17-19 region (a) and gene 1-13 region (b). Induced cells of 594(@0) were aerated at 35” (O--O) and 42” (O--O), and pulse-labeled for 30 set with [aH]uridine at the indicated times. [3H]RNA prepared from pulse-labeled cultures was assayed as described in Materials and Methods. The ordinate indicates the percentage of the total counts which is hybridizable with the phage DNA.

Time

after

induction(min.1

FIG. 5. DNA synthesis after induction. Induced cells of 594trpE-&y-(&30) were aerated at 35” (O--O) and 42” (O---O), and labeled with [3H]thymidine for the indicated times. [3H]DNA prepared from labeled cultures was assayed as described in Materials and Methods. The ordinate indicates the counts hybridizable with phage DNA. x109

FIG. 4a and b. Transcription of the late region after infection; gene 17-19 region (a) and gene 1-13 region (b). Cells infected with q%Oc were aerated at 35” (O--O) and 42” (O--O), and pulse-labeled for 30 set with [3H]uridine at the indicated times. For details see Materials and Methods and Fig. 3.

1. Decrease in the number

of gene copies due to decreased DNA synthesis. 2. Decrease in the transcription rate. The rate of phage DNA synthesis was examined by the DNA-DNA hybridization assay, which revealed that possibility 1. is more likely. The result obtained after induction is presented in Fig. 5. In this case there is a remarkable difference in the amount of DNA synthesis between low and high temperature. As shown in Fig. 6, in the case of infection there is some difference in the amount of DNA synthesis. In

‘?i

50 -

P 0 ;

40 u N 2 30& % = 204: z

’

lo-

“= .o

20

40

Time

after (Min.

80

80

infection 1

FIG. 6. Phage DNA synthesis after infection. The 594trpEthycells infected with @3Oc were aerated at 35O (O--O) and 42” (O--O), and labeled with [3H]thymidine for the indicated times. For details see Materials and Methods and Fig. 4.

174

AIZAWA

AND

MATSUSHIRO

In summary, the temperature sensitive decrease in endolysin synthesis can be explained from the decrease in late mRNA synthesis which in turn is dependent on a decrease in the number of phage DNA copies. In addition, the reasons why hybrid phage h”utP’P’ forms pinpoint plaques at 42” as already mentioned will be interpreted from two additive effects;,( 1) the genetic constitution of this phage which forms essentially rather small plaques at 35” and (2) the partial inhibition of DNA synthesis after infection of $80 at high temperature as shown in Fig. 6. (B) Temperature Sensitivity of the Prophage Excision Process 1. DNA synthesis in an integration defective, int, mutant. To explain the difference in phage DNA synthesis between infection and induction, it could be assumed that the process of prophage excision from the host chromosome is temperature sensitive and that the difficulty of excision at high temperature affects the extent of DNA replication. In the case of phage X, when a lysogen harbouring a intprophage is induced, the extent of X-DNA synthesis after induction appears to be two to four times lower than in an induced int+ lysogen of XcIts857 (Stevens et al., 1971). We examined whether a similar result is obtained with phage 480. Bacteria lysogenie for a @Ointm mutant were used for this purpose. As shown in Fig. 7, the extent of DNA synthesis with the 480 int- mutant after induction is much less than that of $80 wild type. DNA replication of the 480 wild type increases logarithmically, but that of 480 int- is linear. The pattern of DNA synthesis with the $80 int- mutant is comparable to that of 480 wild type induced at 42’. These results support the validity of the proposal that the temperature sensitivity resides in the process of 480 prophage excision. 2. Physical study of prophage excision. In order to get more direct evidence for the temperature sensitivity of the process of prophage excision, a physical study of prophage excision was carried out. We have used the method of Freifelder et al.

a

20

40

60

Time

after

induction

80

too (min.)

FIG. 7. Phage DNA synthesis in an integration defective lysogen after UV-induction. UV induced cells of 594trpE-thy-(&l) (O--O) and 594trpEthy-(q#Oint2) (e--O) were aerated at 35’ and labeled with [3H]thymidine for the indicated times.

(1971) to determine whether prophage excision occurs during incubation at 42” after induction in the case of 480. Freifelder et al. (1973) have recently shown that when lysogens containing replication defective prophage h are incubated at 42” after heat induction, excised DNA circles can be efficiently detected. We have confirmed his result after heat induction of su- bacteria containing F’4.50 (XcIts857Oam29Pam80) and observed excised X DNA circles at both 37 and 42”. The hs0atts0i”cIts857Pam3 hybrid phage used in this experiment (Fig. 1) contains the left arm of phage 480, including genes intso and xisBo, and the right arm of phage X with a mutation in gene P. Figure 8 shows alkaline sucrose gradient sedimentation profiles for cultures prelabeled with [3H]thymidine at 32” and incubated at each temperature (37 or 42”) after heat induction. As shown in Fig. 8, during incubation at 37” after heat induction of lysogen, the circles of the hybrid phage DNA appeared efficiently, but, scarcely any were found at 42”. The fraction of phage DNA circles was also identified by DNA-DNA hybridization (Fig. 9). These

PROPHAGE

results clearly indicate that prophage excision proceeds normally at 37” but fails at 42” when this prophage excision is carried out by the inP and xiP gene products. The expression of genes intso and xi.P in this hybrid phage is regulated under the control of N”-operon (see Fig. 1). From the fact that the excision of phage h is carried out efficiently at high temperature, the transcription of genes intao and xisao under the control of N” in the hybrid phage must not be affected at high temperature. Accordingly, the temperature sensitivity of prophage excision of the hybrid phage must be due to temperature sensitivity of intBo or riSBo gene product itself. This is also supported by the following experiments. 3. Expression of int-xis region after infection. Transcription of early region after infection of phage $80 was measured and the results obtained are presented in Fig. 10a and b. Messenger-RNA corresponding

L

_I 5

10 Fraction

15 No.

20

FIG. 8. Alkaline sucrose gradient sedimentation profiles for lysates of AE-1 thy-/Ftrp(su-) cells containing F’trp (h*0attBoi”~Its857Pam3). Cells were heat-induced at 45” for 5 min and then incubated for 25 min at each temperature: A, control without heat-induction. 0, 37”; 0, 42”. 0.15 ml of lysate was layered on a 5 ml 5-20s alkaline sucrose gradient. The centrifugation time was 42 min at 32,000 rpm at 22” in a Spinco SW50.1 rotor. Sedimentation is from right to left. Each gradient had 33 fractions but only the first 20 fractions are shown.

175

EXCISION

5

10 Fraction

15

20

No.

FIG. 9. Hybridization assay of each fraction from the 37” lysate shown in Fig. 8. (O--O) The radioactivity of each fraction. (O--O) Percentage of input labeled DNA of each fraction hybridized with hsoattsoih phage DNA.

to early region was determined as the difference between values of 3H-labeled RNA hybridizable with h”att”i8”(Q,S,R,)“DNA and X-DNA. Messenger-RNA corresponding to att-int region was determined as the difference between values of 3Hlabeled RNA hybridizable with h”attsoiADNA and X-DNA. It is apparent that the rate of synthesis of early mRNA at high temperature (42”) is not less than that at low temperature (35”) (Fig. 10a and b), whereas that of late mRNA at high temperature is less than that at low temperature (Fig. 1Oc and Fig. 4). Messenger RNA synthesis of the early region rapidly turns off at both temperatures in the same manner as 1mRNA synthesis of phage X (Kumar et al., 1971). Several lines of evidence (Tomita, 1969; Lozeron and Szybalski 1969; Szpirer, 1972) show that the pattern of transcription of $80 is very similar to that of X and that the transcription of early region is directed by positive regulatory gene, Nso, corresponding to N”. Therefore, one may conclude that the early positive regulatory gene product of ~$80 is not temperature sensitive because the

176

AIZAWA

AND

(b)

c-3 10

,.--,

5

,'-‘.

5

-,*.’ L

i 0

,*

. . . . . . 4’ 10

20

30 40 Time after

0 10 20 infection(min.)

30

40

MATSUSHIRO

late genes which code for structural proteins of the phage head and tail are localized in the left arm region of the chromosome, and genes to specify the functions of lysogenization, DNA replication, and cellular lysis map in the right arm region. However, since native phage $80, in contrast to phage X, cannot form plaques at high temperature, some gene or genes of $30 must be naturally temperature sensitive. Since a hybrid phage whose right arm region consists of 480 chromosome, i.e., h”uttsoiso hybrid phage, does not lyse at

FIG. 10. Transcription of the early region after infection with +8Oc; (a) early region, (b) att-int region, (cl gene 17-19 region. The procedures were the same as those indicated in the legend of Fig. 4, except that cells infected were pulse-labeled for 30 set with 20 &i/ml [‘Hjuridine at 35” (O--O) and 42” (O--O).

transcription of early region of ~$30 occurs normally at high temperature. The result in Fig. 11 has confirmed the fact that the transcription of att-int region is not temperature sensitive (Fig. lob). In @Opt used here the att-int region of 480 is replaced with the genes of the tryptophan (trp) operon of E. coli (see Fig. 12). As seen in Fig. 11, the synthesis of tryptophan synthetase (TSase) at high temperature (42”) after infection with $SOpt is rather higher than that at low temperature (35 “) . The expression of tryptophan operon integrated into this @Opt is specially controlled by the phage immunity substance. The trp gene should be expressed by readthrough of leftward transcription which initiates at the phage promoter site, P, (Sate and Matsushiro, 1965; Franklin, 1971; Imamoto and Tani, 1972; Inoko et al., 1974). As indicated in Fig. 12, the regulation of expression of the gene int and/or xis in 480 should be the same as that of the trp operon in qb80pt. It is concluded that the temperature sensitivity of excision process must be due to that of int or xis gene product. DISCUSSION

It has been reported that the arrangement of the genes on the ~$80 chromosome is similar to that of h (Sato et al., 1969); the

He

1 0

10 20 after infection (min.)

Time

30

FIG. 11. Formation of TSase in trpBrecipient after infection with #8OptABI in the presence of L-tryptophan. TrpBm(B-5797) cells infected with @OptABI were aerated at 35” (LO) and 42” (60) for the indicated times in Ca-M medium supplemented with 50 rg/ml L-trp. head

att

tail

DNA synthesis

cl

c%

lysis

080 +-

transcription 0fmptA8~

&A

8

N8’

cl

trp

FIG. 12. Schematic representation of the genes in 680ptABI. This @Opt contains the A, B, and a part of C cistrons but not the promoter and operator of tryptophan operon. Furthermore, 1$80ptABI should contain the action site (t,) of NE’ because it grows on recA- bacteria (Zissler, Signer, and Schaefer, 1971; Franklin, 1971). Xis gene has not yet been identified in 480.

PROPHAGE

high temperature, it is clear that at least one of the ts genes is included in the right arm region of the 480 chromosome. First, the poor lysis was shown at high temperature. Subsequently, the reason for poor endolysin production at high temperature was examined. It was revealed that the decrease in mRNA synthesis from the endolysin gene (gene 19) was mainly coincident with the decrease in phage DNA synthesis at high temperature (Figs. 3 and 5). Therefore, the reduced amount of endolysin production most likely results from a limitation in the number of gene copies because of the low level of phage DNA synthesis. A similar correlation was also observed in the case of infection, but the reduction at high temperature was to a lesser degree (Figs. 4 and 6). However, at present it is still rather difficult to discuss precisely the quantitative relationships of endolysin production, mRNA synthesis, and DNA copies. The reason why phage DNA synthesis is decreased at high temperature was subsequently examined. The remarkable decrease in DNA synthesis following induction as compared to an infection cycle (section A 3) and the decreased phage DNA synthesis in an excision defective lysogen (section B 1) suggest that the process of prophage excision is temperature sensitive. This was clearly detected by the physical study; the excised phage DNA was detectable at 37” but not at 42” (Fig. 8). The detection of circular F’trp DNA with the prophage genome not excised at 42” would be desirable, but we could not detect this kind of DNA. The reason why excised phage DNA circles are clearly detectable but F’trp DNA circles are not, is not clear. As mentioned above, even though it is clear that the process of prophage excision is the main point of temperature sensitivity in the early stage of development of this phage, there must be other temperature sensitive process, because upon infection without the excision process the amount of phage DNA synthesis also decreases at high temperature. Therefore, there are two temperature sensitive processes in the early stage of the development of 480. One is the prophage

177

EXCISION

excision process and the other, although partial, is concerned with the process of DNA synthesis. If the positive regulator gene product of phage 480 corresponding to the N gene of phage X were temperature sensitive, prophage excision and the initiation of DNA synthesis would be suppressed at high temperature and the transcription of the early region must be also suppressed. The fact shown is that the transcription of the early region is not temperature sensitive. As seen in Fig. lOa, the transcription of the early region of 480 is turned off in the same pattern as X (Pero, 1970; Kumar, Calef, and Szybalski, 1971; Inoko et al., 1974). But the turn off of the transcription of att-int region is not detectable. The utilization of strand-separated phage DNA may be necessary for the exact measurement of the kinetics of the early transcription. The temperature resistance of the early transcription is sufficiently concluded from the result in Fig. 10 a and b. As the early transcription of phage $80 after UV-induction is not temperature sensitive (data not shown), the derepression of prophage 480 is efficiently carried out at high temperature. As mentioned in the introductory section, there exists an absolute temperature sensitive step during morphogenetic assembly in the late stage. This is shown in Table 1, in which the hybrid phage hsOuttsoiA cannot form plaques at high temperature. We will describe in detail the temperature sensitivity during the tail morphogenetic process of this phage elsewhere (Haga and Matsushiro, in preparation). ACKNOWLEDGMENTS We would like to express our appreciation to Dr. I. P. Crawford for his kindly aid in the preparation of the manuscript. We are grateful to Miss Mariko Haga for capable technical assistance and to Drs. F. Imamoto, K. Sato, Y. Nishimune, and T. Segawa for helpful discussion. This work was supported by Grant from the Ministry of Education of Japan. REFERENCES D. T. (1966). A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23, 641-646. EISEN, H., BRACHET, P., DA SILVA, PEREZ and JACOB, DENHARDT,

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