WnOLOGY 26, 190-198 (1965)

On the Induction of Escherichia coli K(X) with High Hydrostatic Pressure

LARS R U T B E R G

Department of Bacteriology, Karolinska Instituter, Stockholm 60, Sweden

Accepted February 4, 1965

Escherichia coli K(X) has been treated with high hydrostatic pressure to induce about 10% of the bacteria. Incubation of the treated bacteria at 45°C or in the presence of chloramphenicol (CM) greatly reduces the number of bacterial survivors. The pressure-CM treated bacteria have lost their immunity to superinfection with homologous phage. A defectively lysogenic strain of E. coli K has similarly been found to have a greatly reduced immunity level after pressure treatment. The de- creased survival at 45 ° has been correlated with the production of phage endolysin in the bacteria. No effect of pressure on DNA synthesis in nonlysogenic E. coli K has been found. It is suggested that pressure induces phage formation in E. coli K(X) by affecting the immunity of the cells.

INTRODUCTION

It has recently been reported that short exposure of Escherichia coli K (k) to an in- creased hydrostatic pressure induces phage formation in a large fraction of the bac- teria (Rutberg, 1964a). The present paper concerns itself with some characteristics of this type of induction as compared with induction with ultraviolet light (UV).

MATERIALS AND METHODS

The following derivatives of E. coli K 12 have been used: K 38 = nonlysogenic E. coli K ; K 39 = K 38 carrying k prophage; K 48 = nonlysogenic E. coli K requiring thymidine, threonine, and leucine for growth; K 5I = a streptomycin-resistant derivative of K 38; P 22 = E. coli K carry- ing a defective X prophage. The m u t an t prophage present in this strain cannot initiate vegetat ive multiplication and does not synthesize endolysin or serum-blocking antigen (Jacob and Wollman, 1961). The strains were provided by Dr. G. Bertani except for strain P 22, which was obtained from Dr. F. Jacob.

Stocks of X were prepared from UV- induced K 39. Stocks of X sir ( = h - - 2,

Lederberg and Lederberg, 1953) was pre- pared on plates.

The bacteria were grown in Difco nutrient broth, p H 7.4, with 5 g NaC1 added per liter. In experiments concerning colorimet- ric analysis of nucleic acids 7 g of K2HPO4 and 3 g of KH2PO4 were added per liter (buffered broth).

Bacteria and phage were assayed by the top-layer agar method (Adams, 1959) on plates containing Difco t ryptone 10 g, NaC1 5 g, and Difco agar 12 g per liter distilled water. The top-layer agar con- tained 7 g of agar per liter.

The number of induced cells was assayed by the s treptomycin-spraying method of Six (1959) as described previously (Rut- berg, 1964a).

The pressure t rea tment employed was 4 minutes at 720 a t m at 37 ° unless otherwise mentioned in the text.

Chloramphenicol (CM) was obtained from Parke, Davis, & Co.

For colorimetric analysis of nucleic acids 5-ml samples were centrifuged in the cold, washed with phosphate buffer, and sus- pended in 5 ml of 5% TCA at 4 ° . After standing for about 10 minutes the samples

190

E S C H E R I C H I A COLI K(X) AND H Y D R O S T A T I C P R E S S U R E 191

were centrifuged and the pellets were sus- pended in 1/2 volume 5 % TCA at 90 ° and kept at this temperature for 30 minutes. The samples were then centrifuged, and the supernatants were assayed for nucleic acids. DNA was determined by the diphen- ylamine method and RNA by the orcinol method (Webb and Levy, 1958). Calf thymus DNA and yeast RNA, respectively, were used as standards. All reagents were of the purest commercially available quality.

In the experiments on uptake of radio- active uracil or thymidine, the bacteria were grown in the following medium: NH4C1 2 g, Na2PO4 6 g, KH2PO4 3 g, Difco easamino acids 1 g, distilled water 1000 nfl (= CR-medium). After autoclav- ing, glucose and MgC12 were added to final concentrations of 2.5 g/1 and 0.001 M, respectively. Uracil-2-C ~4 and thymidine-2- C ~ were obtained from New England Nu- clear Corporation. The bacteria were diluted 1:200 from an overnight culture into fresh medium and grown at 37 ° with aeration. In experiments on the uptake of uracil, 20 ug/ml each of cold m-acil and cytosine was added (Kurland and Maal0e, 1962). In experiments on thymidine uptake 10 ug of thymidine and 20 ug of each of the requh~ed amino acids were added per nfilli- liter. When the culture had reached a den- sity of 5 to 10 X 107 bacteria per milliliter, it was centrifuged, the bacteria were washed once in cold medium and suspended in 1/10 volume of fresh medium without glucose or supplements. The bacteria were exposed to the various treatments and then diluted into fresh medium containing the appropriate isototopes at 37 ° with aeration. Two-milliliter samples were taken at inter- vals and diluted into an equal volume of 10 % TCA at 0 °. At the end of the experi- ment the samples were filtered through Millipore filters HA (Sekiguchi and Cohen, 1964). The filters were rinsed with cold 5 % TCA and mounted on planchets, which were dried and counted in a windowless flow counter. The counts were at least four times the background in all experiments.

The method for determination of endoly- sin activity was the one described by Gro- man and Suzuki (1963). The substrate em- ployed was acetone-powdered E. coli K

r FRACTION SUR- VIVING BACTERIA 1.o, ~ V

Pressure

O n i I , I i 20 40 60

MIN. OF EXPOSURE TO CM.

FIG. 1. E. coli K(X) was grown in b ro th to a densi ty of about 5 N 107 bac te r ia per milli l i ter. The bac te r ia were centr i fuged and suspended in phospha te buffer pH 7.0. One pa r t of the sus- pension was i r radia ted wi th UV for 15 seconds, the other pa r t was exposed to 720 a tm for 4 min- utes. The t rea ted samples, as well as an un t r ea t ed control sample, were di luted 1:10 into b ro th con- ta in ing 50 pg CM per milli l i ter. At in tervals al iquots were assayed for viable bacter ia . In- duct ion was about 10% in the pressure- t rea ted bacter ia and about 15% in the i r radia ted sample.

grown in a medium containing Difeo tryp- tone instead of trypticase soy extract. The bacteria were shaken with chloroform prior to assay for endolysin activity. The readings were made using a Unicam eolori- meter Sp. 1300 with filter no. 5. One unit of endolysin is defined as the amount giving a decrease in turbidity of one scale unit per minute.

E X P E R I M E N T A L RESULTS

Exposure of UV-induced E. coli K(X) to C5~ during the first 20-30 minutes follow- ing irradiation has been reported to cause a reduction of the induction and an increased survival of the bacteria (Whitfield, 1962). In Fig. 1 is shown an experiment where UV- or pressure-treated K(X) bacteria were incubated with C~/I immediately after treat- ment. The pressure-treated sample shows a substantial decrease in survival, not ob- served in the irradiated sample. This effect occurs only in the lysogenie strain and at concentrations of CM at which only protein synthesis (as opposed to nucleic

192 R U T B E R G

a c i d syn thes i s ) is s u b s t a n t i a l l y i n h i b i t e d ( T a b l e 1). T h e d e c r e a s e d s u r v i v a l is n o t a c c o m p a n i e d b y a s imi la r inc rease in t h e n u m b e r of p l a q u e - f o r m e r s ( T a b l e 2). P r e s - s u r e - t r e a t e d K(X) b e c o m e s r e f r a c t o r y to t h e l e t h a l e f fec t of C M 2 0 - 3 0 m i n u t e s a f t e r t r e a t m e n t (Fig . 2). T h e c a p a c i t y of p re s su re - a n d C M - t r e a t e d K(X) to p r o d u c e p h a g e u p o n s u p e r i n f e c t i o n is n o t lost , h o w e v e r , a s d e m o n s t r a t e d b y t h e r e su l t s p r e s e n t e d in T a b l e 3. T h e p r e s e n c e of a p r o p h a g e m a r k e r c a n be d e m o n s t r a t e d in t h e b u r s t s p r o d u c e d b y s u p e r i n f e c t e d b a c t e r i a , b u t t h e

TABLE 1

THE EFFECT OF CM ON PRESSURE-TREATED E. Coli K AND ~k a.

CM (pg/ml) Fraction surviving bacteria

K K(X)

0 1.2 1.1 1 1.0 1.0

10 1.0 0.22 25 1.1 0.18 50 0.99 0.11

100 O. 98 O. 09

E. coli K and E. coli K(X) bacteria were grown in broth to a density of about 5 X 107 cells per milli l i ter, centrifuged, and suspended in fresh broth. The bacteria were exposed to pressure and diluted in broth containing various amounts of CM as indicated in the table. Viable counts were performed at the t ime of dilution into CM-broth and after 30 minutes ' incubation in the presence of the drug at 37 ° .

e x p e r i m e n t s h a v e n o t p e r m i t t e d a n y q u a n - t i t a t i v e m e a s u r e m e n t s . N o i n c r e a s e d " c u r - i n g " of t h e p r o p h a g e cou ld be d e m o n - s t r a t e d in t h e b a c t e r i a t h a t s u r v i v e d pres- s u r e - C M t r e a t m e n t .

FF~ACTION SUR- VIVING BACTERIA

1.0'

0. ] I F I t 10 20 30 40 50

MIN. IN PLAIN BROTH BEFORE DIL.INTO CM

FIG. 2. Two cultures of E. coli K(X) were grown in broth to a density of about 5 X 107 bacteria per milliliter. The bacteria were centrifuged; one pellet was suspended in phosphate buffer pH 7.0, the other in fresh broth. The buffer suspension was UV-irradiated (induction about 50%), the broth culture was exposed to 720 arm for 4 minutes (induction about 10%). After t reatment the bac- teria were diluted 1:10 into broth at 37 °. From these suspensions the bacteria were further diluted into broth containing 25 pg CM per millil i ter at t imes indicated in the figure. Viable counts were performed at the t ime of dilution into CM-broth as well as after 30 minutes ' incubation in the presence of CM. © ©, pressure; A A, UV.

TABLE 2

COMPARISON BETWEEN VIABLE COUNT AND NUMBER OF PLAQUE FORMERS AFTER TREATMENT

WITH I°RESSURE AND C M a

Time of exposure to CM in minutes

Control bacteria Pressure-treated bacteria

Viable count Plaque formers Viable count Plaque formers

0 1.9 X 106 1.6 X 106 1.1 X 106 1.4 X 106 20 1.8 X 106 1.5 X 106 7.6 X 10 ~ 5.3 X 105 40 1.8 X 106 1.7 X 106 5.3 X 105 3.9 X 10 ~ 60 1.9 X 106 1.5 X 106 2.6 X 105 2.4 X 105

E. coli K(X) was grown in broth to a density of about 5 X 107 bacteria per milliliter. The bacteria were centrifuged and suspended in fresh broth to a density of about 1 X 10 s bacter iaper milliliter. Par t of the suspension was treated at 720 arm for 4 minutes (induction 10.5%). Treated and untreated bucteria were diluted into broth containing 30 g CM per milliliter. At intervMs viable counts were performed and dilutions of the bacteria were plated with indicator bacteria. After 2-3 hours' incubation at 37 ° these plates were l ight ly irradiated with UV and further incubated overnight.

E S C H E R I C H I A COLI K(X) AND HYDROSTATIC PRESSURE 193

TABLE 3

ThE EFFECT OF CM, CM ~- X, AND CM + Mi~ ON PRESSURE-TREATED BACTERIA a

Viable count Adsorbed phage Number of plaques Expt.

n o .

Treat - ment

I Pressure I

II None I Pressure I

No CM CM

1.6X 10 8 1 .5X 10 s 1.6 X l0 s 4.3 X 10 7 3.4 X 107 3.2 X 107 2.9 X 107 7.0 X 106

X Xvir

1 .5X 10 7 2.1 X 10 7 1.4 X 10 7 2.1 X 10 7 3.8 X 10 5 4 . 5 X 106 3 . 6 X 106 4 .6X 10¢

No phage or CM

1.3 X 10 ~ 1.6 X 1@ 3.8 X 10 ~ 3.2 X 10 ~

CM CM q- X

1 .1X 10~ 1 .6X 106 2.0 X 10 ~ 1.3 X 107 2.9 X 10 ~ 3.1 X 10a 4 . 5 X 105 3 .6X 106

CY[ + Xvir

2.0 X 107 1.8 X 107 4.6 X 106 4.2 X 106

E. coli K(X) bacteria were grown in broth, centrifuged, and resuspended in fresh broth. Part of the suspension was exposed to pressure. Treated and untreated bacteria were incubated 30 minutes in broth containing 35 #g CM per milliliter. Viable counts were made at the time of dilution into CM-broth and at the end of the incubation. The bacteria were then centrifuged, suspended in 0.01 M MgSO4, and superinfected with X or Xvir. After proper time for adsorption the bacteria were centrifuged and the supernatant was assayed for free phage. Adsorption was 65-95%. The bacteria were washed once with MgSO4 and dilutions were plated with streptomycin-resistant indicator bacteria. After 3 hours' incuba- tion at 370 the plates were sprayed with streptomycin, placed at 4 ° for 1 hour, then incubated overnight at 37 ° .

I t should be noted t h a t in the above ex- per iments , as well as in the fol lowing ones, the pressure dose appl ied will induce abou t 10 % of the bac te r ia ; the kil l ing effect a t this dose is slight. Never the less , the major - i ty of the bac te r ia respond to the post- pressure t r e a t m e n t s employed. This sug- gests t h a t the s tabi l i ty of the lysogenic s ta te has been upse t in the m a j o r i t y of the cells even though mos t of t h e m would r eve r t to s table lysogeny under o rd inary condit ions.

T h e next t y p e of exper iment consisted in incuba t ing p ressure - t rea ted K(X) a t 45 ° and measur ing su rv iva l of the bacter ia . T h e resul ts of one such exper iment are presented in Tab l e 4. I n c u b a t i o n a t the e leva ted tern- pe ra tu re leads to a large decrease in surv iva l of the lysogenic bacter ia , a decrease accom- panied by some increase in the number of p laque-formers . A t least 50 % of the bac- teria, however , cannot be accoun ted for ei ther as colony-formers or as p laque-for-

mers. UV- i r r ad i a t ed K(X) is no t affected

by the same t r e a tmen t , nor is the nonlyso-

genie strain. Microscopic observa t ions of the

bac ter ia suggested t h a t lysis of a large

f rac t ion of the cells a l ready occurred af ter

30-40 minu te s ' i ncuba t ion at the elevated

t empera tu re . P ressure - t rea ted K(X) be-

comes re f rac to ry to the le thal effect of in-

TABLE 4

TRE EFFECT OF INCUBATION OF E. coli K(X) AT 4 5 ° A F T E R V A R I O U S T R E A T M E N T S a

Treatment

Min- None Pressure UV utes

at 45 ° Strain

K(X) K 105 It(X) _ _ _•(x)

0 7.3 x 105 2.0 x 7.0 x 105 ] 1.2 x 106 20 - - 1.0 × 105] - - 40 - - 8 . 8 X 1 0 4 - -

60 7.5 X 105[ 2.1 X 10 ~ 3.4 X 104 1.3 X 106 I

a The bacteria were grown in broth to about 5 X 107 cells per milliliter, centrifuged, and sus- pended into fresh broth, or, in the case of UV irradiation, in phosphate buffer. The bacteria were then treated as indicated in the table, di- luted into fresh broth at 45 °, and viable counts were performed. The UV dose employed is suffi- cient to induce about 20% of the bacteria. The values for K(k), control and pressure, are from the same experiment, the other values are from separate experiments.

cubation at the elevated temperature 20-30 minutes after treatment.

These results suggested the action of some lyric substance, more specifically, the action of phage-induced endolysin. The

194 R U T B E R G

T A B L E 5

CALCULATION OF THE ~UMBER OF ~NDOLYSIN PRODUCING BACTERIA AT 45 ° AFTER

PRESSURE TREATMENT a

Expt. Induced Endolysin units Calculated No. of endolysin- Bacteria "lost" at producing bacteria 45 ° after pressure Treatment

no. bacteria 37oC 45oc 37oc 45oc b treatment

I 1.3 X 108 19.7 4.3 - - - - - - UV II 1.0 X lO s 26.1 7.1 - - - - - - UV

I I I 1.3 X 10 s 19.6 4.6 - - - - - - UV IV 3.5 X 107 10.8 13.2 3.5 X 107 1.8 X 10 s 1.9 X 108 Pressure

V 1.0 X 10 s 21.6 24.6 1.0 X 108 4.9 X 108 4.9 X 108 Pressure

Logar i thmicMly growing E. coli K(X) bac te r ia in b ro th were cent r i fuged and suspended in (a) phos pha t e buffer for i r rad ia t ion or (b) f resh b ro th for pressure t r e a t m e n t . After t r e a t m e n t the bacter ia were d i lu ted into b ro th at 37 ° or 45 ° and heavi ly aerated. Samples were t aken and p ipe t t ed into tubes a t room t e m p e r a t u r e con ta in ing a smal l vo lume of chloroform. These tubes were v igorous ly shaken , left a t room t empera tu re for 15 minu tes , and t hen s tored at 4 ° unt i l assayed. All assays were per formed wi th in 24 hours of sampl ing . Dur ing this t ime no decrease in ac t iv i ty was found. Induc t ion was 30-60% in the i r rad ia ted samples and 10-17% in the p ressu re - t r ea ted samples . In the i r radia ted samples the m a x i m u m level of endolys in was found af ter abou t 150 minu te s a t 37 ° and 60-80 minu t e s at 45 °. The corresponding figures for p ressu re - t r ea ted bac ter ia were 120 minu t e s and 40-60 minu tes , respect ively .

b Calcu la ted wi th the correct ion factor 4,3 as de te rmined f rom exper iments I - I I I .

presence of endolysin was tested for in these bacteria. As less endolysin is produced per bacterium at the higher temperature (Groman and Suzuki, 1963), a correction factor had to be determined. This factor was taken as the ratio of the amounts of endoly- sin produced by UV-induced bacteria at 37 ° and 45 ° , respectively. Taking this fac- tor into account, all the bacteria lost as a consequence of the pressure-temperature treatment could be accounted for as endoly- sin-producing bacteria (Table 5).

The survival of the defective lysogenic strain P22 was not affected by incubation at 45 ° following pressure treatment. When this strain was superinfected with immune- sensitive X phage in connection with pressure treatment, however, its survival was low- ered by incubation at 45 °. Superinfection of this strain with X after pressure treat- ment led to the production of viable phage in one-third to one-half of the bacteria, as measured by the streptomycin spraying technique.

I t is characteristic of most inducing agents to interfere in some way with the synthesis of DNA in the bacterium (Jacob, 1954). Experiments were performed to study the effects of pressure on nucleic

N- ~ VIABLE BACTERIA TOTAL pq DNA TOTAL/v 9 RNA

3×10 -- -- 300

-- A NAc°ntr°[ -- 200

f 2 a C L c o n t r o L

lo'

5x10

~.. . .~NA cant rot

,0

I I l [ 15 15 30 45 60 NIN.

Fie . 3. E. coli K was grown in 400 ml of buffered b ro th on a shaker wi th aera t ion to a dens i ty of 1 to 2 X 108 bac ter ia per milli l i ter. The bac te r ia were centr i fuged and suspended in 3-4 ml of f resh medium. Pa r t of the bacter ia were exposed to pressure . T rea t ed as well as u n t r e a t e d bac ter ia were inocula ted into 100 ml of f resh NBB, re- spectively. Samples were t aken for determi- na t ions of viable counts and nucleic acids as described in Mater ia ls and Methods .

ESCHERICHIA COL[ K(X) AND HYDROSTATIC PRESSURE t95

[ CPM PER 107 BACT, 1800

1500

1000 1 o

F //

10 20 30 MIN Fro. 4. E. coli K thy- was grown in CR~medium

to a density of 1 to 2 X 10 s bacteria per milliliter. The bacteria were centrifuged and suspended in the same medium without added glucose. The bacteria were exposed to pressure, and pressure- treated as well as untreated bacteria were inoeu- lated into fresh medium containing radioactive thymidine. Samples were taken at intervals as described and used to determine acid-insoluble radioactivity. In the figure are included data from another experiment in which the bacteria were suspended in phosphate buffer, pH 7.0, and ir- radiated with a UV dose sufficient to induce 10-15% of the bacteria. • • , control bacteria; © O, pressure-treated bacteria; v7 v1, UV- treated bacteria.

acid synthesis in K and K(X) in the ab- sence and presence of CM.

In Fig. 3 are presented the results of an experiment in which D N A and R N A syn- thesis was measured in pressure-treated and untreated K in the absence of CM. The nucleic acids were determined colori- metrically as described under Materials and Methods. No difference was found between the treated and untreated bacteria with respect to growth or synthesis of nu- cleic acids. The effect of pressure on the rate of synthesis of D N A was next followed in K thy- by measuring incorporation of radio-

active thymidine into acid-insoluble prod- ucts (Fig. 4). No delayed uptake of label was observed in the pressure-treated bacteria. In the experiments were included K thy- bacteria irradiated with a UV dose giving about the same induction as the pressure employed. A lag in the uptake of label was observed in these bacteria. F rom these experiments it is concluded tha t the pres- sure employed in the current experiments does not affect D N A synthesis in the non- lysogenic E. coli K.

I~NA synthesis was next followed in K(X) by measuring incorporation of radioactive uracil into acid-insoluble products. After pressure t rea tment an initially reduced incorporation of label was found both in the absence and presence of Cdl. In the pres- ence of CM there is very little uptake of label after about 60 minutes ' incubation (Fig. 5). There is no turnover of any sub- stantial fraction (more than 10 %) of R N A in pressure-treated K(X) with or without CM. In Fig. 5 is also show11 the incorpora- tion of radioactive uracil in the presence of CM by K(X) irradiated with a UV dose giving either the same induction as the pressure employed or 95 % induction. At the latter dose a reduced uptake of label is seen, similar to tha t observed in the pres- sure-treated bacteria. Similar experiments performed with the nonlysogenic strain, K, are complicated by the fact tha t R N A syn- thesis in this strain is more susceptible to CM than in K(X). I t has been reported tha t a reduced R N A synthesis occurs in E. coli K following irradiation with UV, ir- respective of the presence or absence of lambda prophage (Waites and Fry, 1964).

Pressure-treated K(X) was grown with radioactive uracil in the presence of CM. After 5, 10, and 15 minutes ' incubation aliquots were chilled and mixed with an ex- cess of unlabeled, untreated K(X) bacteria. R N A was extracted from the bacteria with phenol and centrifuged in linear sucrose gradients (Kurland and MaaIOe, 1962). The tubes were then punctured in the bot- tom and 3-drop fractions were collected. The adsorbancy at 2600 A and the radio- act ivi ty of each fraction were measured. Three peaks were identified, and adsorb- ancy and radioactivity followed each other

196 RUTBERG

CPM

1500

tOO0

50O

I I [ I ~ I 30 60 90 120 I 0 180

MIN

FIG. 5. E. coli K(X) was grown in CR-medium in the presence of nonradioactive uracil as de- scribed. The bacteria were centrifuged. One part was suspended in fresh medium without glucose, one part into phosphate buffer. Part of the bac- teria suspended in fresh medium was exposed to pressure. The bacteria in buffer were irradiated with U¥ at two doses, one which gives about 10% induction (10 seconds) and one which gives 95% induction (50 seconds). After respective treatments the bacteria were diluted into fresh medium containing radioactive uracil, and sam- ples were taken as described for determination of acid-insoluble radioactivity. • • , control bac- teria; 0 - - 0 , pressure-treated bacteria; [] [~, UV 10 seconds; I I II, UV 50 seconds.

closely. These experiments suggest tha t the R N A made by K(X) during the first 15 minutes following pressure t reatment is not different from that made by the untreated bacteria.

Attempts to induce P2 prophage in E. coli strains B, C, and K did not meet with any success. :Repeated experiments also failed to demonstrate an inducing effect of pressure on E. coli C(X) (Rutberg, 1964b). Thus the specificity of pressure induction seems to be determined by the constitu- tion both of the prophage and of the host. A similar situation has been described for UV in a B. megaterium phage system (Io- nesco, 1956).

DISCUSSION

In the present experiments E. coli K(X) has been exposed to a pressure dose suffi-

cient to induce about 10 % of the bacteria; the killing effect at this dose is slight (Rut- berg, 1964a). Upon exposure of the pressure- treated bacteria to CM or to an elevated temperature, a large decrease in bacterial survival has been observed. This effect of CM and temperature is found only in the lysogenie strain; the presence of the pro- phage acts as a lethal factor under the above conditions.

The results suggest that pressure affects the expression of immunity in the cells. This suggestion is based on the following findings: (1) After pressure t reatment of a defective lysogenie strain a large fraction of the bacteria give plaques when super- infected with an otherwise immunity- sensitive phage (an enhancing effect on the induction is also observed when E. coli K(X) is superinfeeted with co-immune phage in connection with pressure treatment) . (2) After pressure-CM treatment of E. coli K(X) most of the bacteria produce phage if superinfected with co-immune phage. CM treatment by itself neither affects the immunity of the cells nor in- creases their inducibility (Levine and Cox, 1961). (3) Incubation of pressure treated E. coli K(X) at 45 ° leads to the production of a vegetative phage product, endolysin, in the vast majori ty of the cells.

The number of ceils induced at constant pressure is proportional to the time of ex- posure (Rutberg, 1964a). Assuming values of 20-30 molecules of immunity substance per cell (Bertani, 1961; Jacob et al., 1962), direct inactivation of immunity substance seems unlikely. The basis for these cal- culations is very uncertain, however. Inter- ferenee of pressure with the synthesis of immunity substance as the cause of pressure induction seems unlikely, considering the apparent stability of immunity substance (Luria et al., 1958).

CM treatment of bacteria infected with temperate phages has a variety of effects (Bertani, 1957; Ting, 1960; Zichichi and Kellenberger, 1963). A general feature seems to be tha t the effect is very much dependent on the time of addition of the drug (Bertani, 1957; Fry, 1963). RN A synthesis will proceed at a somewhat re- duced rate in pressure-treated E. coli KX)

ESCHERICHIA COLI K(X) AND HYDROSTATIC PRESSURE 197

in the presence of CM; after 60-90 minutes there is very little net increase or turnover of R N A in these bacteria. No evidence has been found for the synthesis of any par- titular type of R N A in these bacteria. The lethal effect of CM on pressure-treated E. coli K(X) remains obscure. The results suggest, however, that protein synthesis is not required for the initial phase of induc- tion (Melechen, 1964).

The mechanism of action of incubating pressure-treated E. coli K(X) at an elevated temperature seems n'tore easily explainable. Incubation at an elevated temperature will speed up the various reactions occurring in the cell. In the phage-infected cell, as well as in the induced lysogenic one, lysis is known to oeem ~ earlier at higher tempera- tures, and the burst size is severely reduced (Groman and Suzuki, 1962, 1963). This effect is probably due to a higher rate of synthesis of endolysin as compared to other phage- directed synthesis or to some step(s) lead- ing to lysogeny (Zichiehi and Kellenberger, 1963), as well as to an increased rate of action of the lytic enzyme on its substrate. If the pressure-treated population has a strongly reduced immunity, incubation of the same population at an elevated tempera- ture could lead to the preferential expres- sion of a vegetative phage function, endoly- sin synthesis.

No effect of pressure treatment on the rate of synthesis of D N A has been found in the nonlysogenic bacteria. Synthesis of a qualitatively altered D N A (Seno and Melechen, 1964) seems unlikely consider- ing the fact that the bacteria resume growth immediately after treatment.

The effects of pressure on bacteria and bacteriophage are quite different from those of UV (Rutberg, 1964b). A likely hypothesis seems to be that pressure interferes pri- marily with the expression of immunity in the lysogenie bacteria, whereas the induc- ing effect of UV is associated with some effect on the D N A synthesis of the bac- terium.

ACKNOWLEDGMENTS

My sincere thanks are due to Dr. G. Bertani for his kind and generous help throughout this investigation. My thanks are also due to Miss

Kerstin Nilsson for skilful technical assistance. This work was supported by U.S. Public Health Service research grant C-5799 from the National Cancer Institute, Public Health Service, Bethesda, Maryland, and a grant from Stiftelsen Therese och Johan Anderssons Minne.

REFERENCES

ADAMS, M. H. (1959). "Bacteriophages." Wiley (Interseienee), New York.

BEaTANI, L. E. (1957). The effect of inhibition of protein synthesis on the establishment of lysogeny. Virology 4, 53-71.

B~RTANI, L. E. (1961). Levels of immunity to superinfection in lysogenic bacteria as affected by prophage genotype. Virology I3,378--380.

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