The effect of phenethyl alcohol on Bacillus subtilis transformation

264

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96087

T H E E F F E C T OF P H E N E T H Y L ALCOHOL ON B A C I L L U S TRANSFORMATION

SUBTILIS

I. CHARACTERIZATION OF T H E E F F E C T

A R L A N G. R I C H A R D S O N AND F R A N K L I N R. LEACH

Department o/ Biochemistry, Oklahoma State University, Stillwater, Okla. (U.S.A.) (Received J u l y 26th, 1968)

SUMMARY

Phenethyl alcohol at a concentration of 0.05 % reduces neither the growth rate of Bacillus subtilis, as determined by Ae3o m~ measurements, nor the viable titer during a 6o-min exposure. When 0.05 % phenethyl alcohol is added concomitantly with DNA to competent B. subtilis, there is a 50-70 % inhibition of transformation without any effect on the viable titer of the cells. Treatment of isolated transforming DNA with 0.5 % phenethyl alcohol is without any effect on the specific biological activity, and no evidence for binding or interaction of [z~C~phenethyl alcohol and DNA was obtained by Sephadex chromatography, methylated albumin kieselguhr chromatography, or CsC1 density gradient centrifugation. Addition of phenethyl alcohol after the uptake of DNA was completed did not inhibit transformation, and the potential transformants became resistant to deoxyribonuclease and phenethyl alcohol inhibition at the same time. Incubation of competent cells with phenethyl alcohol prior to the addition of DNA resulted in a greater inhibition of transformation than when phenethyl alcohol was added simultaneously with DNA, and if this incubation was over an hour long, removal of the phenethyl alcohol by centrifugation and washing did not restore competence. Addition of more competent cells resulted in more transformants, but addition of an excess of DNA failed to change the number of transformants obtained from an inhibited mixture. Phenethyl alcohol inhibits the transport of radioactive DNA but not the initial attachment of DNA to competent cells.

INTRODUCTION

Phenethyl alcohol is bacteriostatic for Gram-negative bacteria I and reversibly inhibits the synthesis of DNA is Escherichia coli ~. TREICK AND KONETZKA8 demonstrated differential inhibitions of stationary and exponential phase cells. Addition of phenethyl alcohol (0.32 %) to stationary phase cells completely inhibits DNA synthesis while synthesis of RNA and protein continued. However, for exponential phase cells, DNA synthesis was inhibited only after a 1.4- to 1.6-fold increase in DNA as determined by chemical analysis (diphenylamine reaction). These results were Biochim. Biophys. Acta, 174 (1969) 264-275

PHENETHYL ALCOHOL AND TRANSFORMATION

265

interpreted as a completion of synthesis of DNA which is in the process of replication at the time of phenethyl alcohol addition and prevention of initiation of a second cycle of DNA replication. LARK AND LARK4 confirmed the above experiments on DNA replication and suggest that phenethyl alcohol inhibits the synthesis or modifies the structure of one of the two proteins required for the initiation of chromosome replication. ZAHN et al. 6 demonstrated an i n vitro inhibition of DNA nucleotidyltransferase (EC 2.7.7.7) (DNA polymerase) from calf thymus by 0. 5 ~o phenethyl alcohol and various analogues. ROSENKRANZ, CARR AND ROSEs found no effect of phenethyl alcohol on the physicochemical properties of isolated DNA, and DN'A isolated from cells treated with phenethyl alcohol has the same physicochemical properties as control DNA. MENDELSON AND FRASER~ found no effect of phenethyl alcohol on thermal denaturation, renaturation and viscosity of DNA. Additional effects of phenethyl alcohol on bacterial cells have been described, and the mode of action of phenethyl alcohol is currently disputed. ROSENKRANZ, CARR AND ROSEs found a 5 ° ~ inhibition of alkaline phosphatase induction with 0.07 % and 0.04 ~ phenethyl alcohol in resting and growing E. coli C6oo cells, resspectively. Since RNA synthesis as determined by a2p incorporation was more sensitive than E14Clamino acid incorporation, they concluded that messenger RNA synthesis is the point of phenethyl alcohol inhibition. PREVOST AND MOSES8 found that 0.3 % phenethyl alcohol inhibited RNA synthesis as measured by uracil incorporation as much as/~-galactosidase induction, and concluded that phenethyl alcohol was not specific for messenger RNA synthesis. A third site of action is indicated, since phenethyl alcohol alters the permeability of Neurospora crassa for ~-aminoisobutyric acid s. SILVER AND WENDT10 further investigated permeability effects by measuring acriflavine uptake and potassium leakage in E. coli B. There is a rapid and reversible breakdown in the permeability barrier when cells are treated with 0.25 ~o phenethyl alcohol or toluene. The authors suggest two mechanisms by which phenethyl alcohol may exert its inhibitory effect on the many different processes affected: (i) there may be direct coupling of the process to the bacterial cell membrane or (2) there may be leakage from the cytoplasm of essential small molecules. Several other processes are sensitive to phenethyl alcohol, e.g., phenethyl alcohol inhibits the development of the RNA bacteriophages,/5 and MS-2, whose replications do not involve DNA function 11. SLEPECKY12 found inhibition of sporulation and germination in Bacillus megaterium by phenethyl alcohol. Later studies 13 revealed that the inhibition of sporulation in Bacillus cereus was an effect on early forespore membrane formation. Bacterial transformation is a process which would allow study of the biological consequences of treatments of DNA with phenethyl alcohol in vitro; the measurement of transforming activity is more sensitive than physicochemical measurements. Expression during transformation requires the synthesis of messenger RNA and the protein corresponding to the newly introduced information. NESTER AND STOCKER14 found no tryptophan synthetase in B. subtilis cultures until 3-4 h following transformation; that is, expression was delayed. This allows exploitation of times of treatment or addition of phenethyl alcohol to differentiate between uptake and expression effects in transformation. Before the genetic information in the exogenous DNA can be used, the DNA must be taken up by the recipient cell. Thus, the completion of Biochim. Biophys. Acta, 174 (1969) 264-275

266

A. G. RICHARDSON, F. R. LEACH

transformation (as measured by colony formation) requires the proper functioning of the three processes described above. The results presented in this paper establish that phenethyl alcohol inhibits the uptake of radioactive DNA under conditions where there is no gross effect on cellular events such as growth; addition of phenethyl alcohol after uptake of transforming DNA is without effect on transformation. Phenethyl alcohol apparently does not react with DNA but exerts its inhibitory effect on the competent cells of the transformation mixture.

MATERIALS AND METHODS

Bacterial strains B. subtilis WT and 168 indole- were obtained from Dr. W. C. McDonald. B subtilis SB 25 (try2-his2-) was from Dr. F. E. Young, and thymine-requiring strains were from Dr. F. Rothman and Dr. I. C. Felkner (F H 2006 ind- thy-).

DNA preparation DNA was isolated by the procedure of SAITO AND MIURA1L The concentration was estimated by absorbance at 260 m# and the BURTON16 diphenylamine reaction.

Preparation o[ E3HlDNA B. subtilis strain F H 2oo6 (ind- thy-) was grown overnight on brain heart infusion agar plates and used to inoculate minimal medium 1~ supplemented with 0.05 °/o of acid-hydrolyzed casein, 5 °/~g/ml of tryptophan and 5 ° #g/ml of thymidine. These cells were grown at 37 ° with shaking until Ass0 m~ equals 0.64, and then centrifuged, washed twice and suspended at an A630 m# of o.I in 50 ml of minimal medium supplemented with 0.05 % of acid-hydrolyzed casein, 50/~g/ml of tryptophan and 2.5 mC of ESHlthymidine (io~g/ml). When A630 m# reached 0.8 (3 generations), the cells were harvested by centrifugation, washed twice and suspended in 2 ml of o.15 M NaC1, o.I M E D T A (pH 8.0). Lysozyme (12 rag) was added and the mixture was incubated until the solution became viscous. The mixture was frozen and thawed, and then 0.7 ml of a solution containing o.I M Tris, I °/o sodium dodecyl sulfate and o.I M NaC1 (pH 9.0) was added. The suspension was frozen and thawed two more times. Then pronase (8 mg/ml) was added, and the suspension was incubated for 7 h at 37 °. An equal volume of cold phenol saturated with o.I M Tris, I °/o sodium dodecyl sulfate and o.I M NaC1 (pH 9.0) was added, and the suspension was shaken in the cold for 20 min. The suspension was clarified by centrifugation at 18 o o o × g for 20 min. The aqueous layer was dialyzed against 3 changes (2 1 each) of o.I M NaC1 and o.oi M sodium citrate (pH 7.0) for 48 h. The specific activity of the DNA was i i i 800 counts/min per/~g.

Trans[ormation procedure Transformation of recipient strains of B. subtilis SB 25 (try-~ his2- ) or 168 Biochim. Biophys. Acta, 174 (1969) 264-275

PHENETHYL ALCOHOL AND TRANSFORMATION

267

(try2-) was performed by a modification method of YOUNG AND SPIZIZEN18. The overnight growth of the recipient strain in tryptose blood broth was 14 h at 37 ° with shaking; the culture was diluted 3o-fold and incubated for 8 h at 37 ° with shaking. The culture was diluted Io-fold with minimal medium containing IO #g/ml of tryptophan and histidine (or tryptophan alone if strain 168 was being used) and o.oi °/o of acid-hydrolyzed casein, and grown for 3 h at 37 ° with shaking. Then DNA (5/zg/ml unless otherwise indicated) was incubated with competent cells for 30-45 min at 37 ° with shaking (either time allowed maximum transformation). Dilutions were then made at room temperature in minimal medium. Double transformants bearing the linked try2 + and his2+ markers (try + his +) were scored on minimal agar; total his + or try + transformants were scored on minimal agar supplemented with IO/zg/ml of tryptophan or histidine, respectively. The total viable cells were scored on minimal agar supplemented with IO #g/ml of both histidine and tryptophan. Frequencies of transformation of 0.2 to 2 % were obtained using this procedure.

Radioactive compounds and counting procedures x4C-Labeled phenethyl alcohol was prepared by LiA1H, reduction of the methyl ester of [I-14Clphenylacetic acid obtained from New England Nuclear Corporation. The product was 99.5 % radiochemically pure by gas chromatography and had a specific activity of 5°/~C/mmole. A Packard scintillation counter with BRAY'S19 scintillation fluid was used for counting.

Other materials Deoxyribonuclease (EC 3.1.4.5) I × crystallized was purchased from Worthington Biochemicals, and phenethyl alcohol was obtained from Eastman Organic Chemicals.

RESULTS

E//ect o/phenethyI alcohol on B. subtilis and on trans/ormation A phenethyl alcohol concentration of 0.05 % was selected because this concentration neither killed nor changed the exponential growth rate of B. subtilis. Table I shows a 74 ~o reduction in the number of transformants when phenethyl alcohol was present during a 45-min incubation of competent cells and transforming DNA. Part B shows that plating either the transformants or recipient population on plates containing 0.05 °/o phenethyl alcohol is without effect on either the number of transformants or the viability of the recipient population. Since phenethyl alcohol was present during the 3-4-h biosynthetic latent period x4, phenethyl alcohol does not inhibit expression. The transforming system contains two components which must interact for transformation to be accomplished: these are DNA and the competent cell. Experiments were devised to ascertain which of these two components of the transforming system, or the interaction of both, was being inhibited by phenethyl alcohol. Biochim. Biophys. Acta, I74 (1969) 264-275

268 TABLE EFFECT

A . G . RICHARDSON, F. R. LEACH I OF PHENETHYL

ALCOHOL

TREATMENT

ON

THE

B. subtilis T R A N S F O R M A T I O N

SYSTEM

Part A : C o m p e t e n t B. subtilis SB 25 cells were exposed to 5 p g / m l of D N A a n d / o r 0.05 % phenet h y l alcohol as indicated for 45 min. The cells were diluted a n d plated on the a p p r o p r i a t e media. Part B: C o m p e t e n t B. subtilis 25 cells were exposed to 5 F g / m l of D N A for 45 m i n and t h e n diluted and plated on the a p p r o p r i a t e media s u p p l e m e n t e d with and w i t h o u t p h e n e t h y l alcohol (o.o5 %). T r y p t o p h a n t r a n s f o r m a n t s are shown.

Condition o/ incubation

Cells per rnl Trans/ormants

Viable

X 10 4

X 10 7

Trans/ormation (%)

Part A No D N A P h e n e t h y l alcohol, no D N A DNA D N A + p h e n e t h y l alcohol

o o 420 lO9

80 93 90 88

Trans/ormants

Viable

X IO 5

X Io 8

o o 0.46 o. 12

Part B None 0.05 % Pkenethyl alcohol

21 22

15 16

E//ect o~ lbhenethyl alcohol treatment o/ DNA on its irans/orrning ability The effect of treatment of transforming B. subtilis DNA with 0.5 % phenethyl alcohol was determined. Fig. I shows the dose-response curves for transformation o

0.4 z

o

< 0.3

o/

oo0o

,ii

~. 0.2 < I---

o

ot o

2:0

lol

: I0

l o0oo 20

FRACTION NUMBER

#g/ml DNA

Fig. i. Effect of t r e a t m e n t of D N A with p h e n e t h y l alcohol on its t r a n s f o r m i n g ability. One sample of D N A (O} w a s t r e a t e d for 2 h w i t h o. 5 % p h e n e t h y l alcohol, precipitated w i t h 95 ~o ethyl alcohol, w a s h e d twice and dissolved in o.15 M sodium chloride-o.oI 5 M sodium citrate. A n o t h e r sample of D N A ( O ) received equivalent handling except t h a t the p h e n e t h y l alcohol was omitted. The concentration of D N A was determined b y diphenylamine test. Recovery of t r e a t e d D N A was 87 °/o, and n o n t r e a t e d 9o 0/o• The effect of several concentrations of the two D N A p r e p a r a t i o n s on t r a n s f o r m a t i o n was determined using c o m p e t e n t B. subtilis cells (1.1o 9 cells/ml). Fig. 2. Sephadex c h r o m a t o g r a p h y of p h e n e t h y l alcohol and DNA. A sample (16o pg) ot D N A w a s incubated for 6o rain at 37 ° w i t h 33/~g of [x4C]phenethyl alcohol in i ml and then c h r o m a t o g r a p h e d on a S e p h a d e x G-25 c o l u m n 2 o m m × 15o ram. Elution was w i t h O.Ol 5 M sodium citrate, o.oool 5 IV[ sodium citrate and 5-ml fractions were collected. A 280 mu and the radioactivity were determined for each fraction. Note the b r e a k in the c o u n t s / m i n scale. O - O , A ~n0mr*; O - O , counts/rain.

Biochim. Biophys. Acta, 174 (1969) 264-275

P H E N E T H Y L ALCOHOL AND TRANSFORMATION

26 9

using control DNA and treated DNA. There was no apparent difference in the ability of the two DNA samples to transform either in regions where transformation was proportional to DNA concentration or at saturating concentrations. Thus, phenethyl alcohol treatment of DNA does not irreversibly modify indole transforming activity.

Studies on the interaction o/phenethyl alcohol and DNA Since the washing procedures used in the experiment described above do not eliminate a reversible interaction of the DNA and phenethyl alcohol, experiments were designed to test for an interaction using radioactive phenethyl alcohol. The DNA and [l~C]phenethyl alcohol were incubated together, and formation of a complex was sought as coincidence of radioactive and A260 m/z (DNA) peaks after the application of various procedures which would separate DNA from phenethyl alcohol on the basis of size, specificity or density. Fig. 2 shows the results of molecular sieve chromatography on Sephadex G-25 which separates on the basis of size. Only background radioactivity eluted with the DNA; thus there is no binding of phenethyl alcohol to DNA. Methylated albumin kieselguhr chromatography of incubated mixtures of radioactive phenethyl alcohol and DNA resulted in the elution of all the radioactivity prior to the elution of the DNA by increasing the ionic strength (results not shown). CsC1 density gradient centrifugation of incubated mixtures of phenethyl alcohol and DNA revealed only background radioactivity (determined from centrifuging phenethyl alcohol alone under the same conditions) at the position of the DNA peak. Three different experimental procedures failed to yield any evidence for complex formation between phenethyl alcohol and DNA. The second component of the transformation system is the competent cells; therefore, the effect of phenethyl alcohol on competent cells was examined and the inhibition was characterized.

EHect o/ phenethyl alcohol concentration on inhibition o/ trans[ormation The effect of various phenethyl alcohol concentrations on inhibition of transformation is shown in Fig. 3. Concentrations below 0.oi % did not inhibit, while concentrations between 0.o2 °/o and o.06 % inhibited transformation without any effect on the titer of the recipients (not shown). Concentrations of phenethyl alcohol above o.15 % killed the recipient population as well as inhibited transformation.

E//ect o/ the time o/addition o~ phenethyl alcohol In the previous experiments, phenethyl alcohol was added to the transformation system simultaneously with DNA. Table II shows that maximum inhibition of transformation is obtained when phenethyl alcohol is incubated with competent cells 1-3 h prior to DNA addition. The addition of phenethyl alcohol after DNA addition becomes progressively less inhibitory with no inhibition resulting if phenethyl alcohol is added 20 min after DNA. Biochim. Biophys. Acta, I74 (I969) 264-275

270

A . G . RICHARDSON, F. R. LEACH

TABLE II ~FFECT OF TIME OF PHENETHYL ALCOHOL ADDITION P h e n e t h y l alcohol (o.o5 %) w a s added at the indicated times to the t r a n s f o r m a t i o n s y s t e m (SB25, t r y p t o p h a n z m a r k e r ) and incubated w i t h cells until dilution and plating. D N A was added at time zero and incubated for 30 rain at 37 ° w i t h shaking before dilution and plating.

Time o] phenethyl alcohol addition prior to DNA addition (h)

Trans]ormation (%)

3

o.14 o.13 o.32 o.44

1

0.5 o (same time)

Time of phenethyl alcohol addition a]ter DNA addition (h)

Trans/ormation (%)

o.17 o.25 o.33 o.5 o None

o.67 o.81 1.o6 o.93 I.IO

TABLE III EFFECT OF EXCESS COMPETENT CELLS AND D N A UPON ALLEVIATION OF PHEN1~THYL ALCOHOL INHIBITION OF TRANSFORMATION P h e n e t h y l alcohol (o.o 5 %) and D N A (5/,g/ml) were added as indicated, except in line 3 where io/~g/ml of D N A were added to c o m p e t e n t cells (7.1o 5 cells/ml) and incubated for 3 ° min at 37 ° w i t h shaking. Excess c o m p e t e n t cells were o b t a i n e d b y centrifugation, and this concentrated c o m p e t e n t cell suspension was added to the n o r m a l suspension s i m u l t a n e o u s l y w i t h D N A and p h e n e t h y l alcohol.

Additions

his + try + trans[ormants

Trans]ormation (%)

I. 2. 3. 4.

43 ° 230 238 254 °

0.7o 0.34 0.3o 0.58

None P h e n e t h y l alcohol 2 × D N A + p h e n e t h y l alcohol 7 × c e l l s + p h e n e t h y l alcohol

Alleviation o/phenethyl alcohol inhibition To gain further insight into the mechanism of the inhibition of transformation by phenethyl alcohol, factors which could reduce the inhibition were studied. Excess amounts of DNA and competent cells were added to a mixture of competent cells, DNA and phenethyl alcohol which had a partial inhibition; that is addition of more phenethyl alcohol would increase the inhibition observed. Table I I I demonstrates that the addition of IO #g/ml of DNA failed to increase the number of transformants obtained. However, when a 7-fold excess of competent cells was added, an increased number of transformants was observed. Since there was a defined level of inhibition, the addition of more of the substance with which phenethyl alcohol reacts would result in greater numbers of transformants and that substance was the competent cells. Biochim. Biophys. Acta, 174 (1968) 264-275

271

PHENETHYL ALCOHOL AND TRANSFORMATION T A B L E IV EFFECT

OF R E M O V A L

OF PHENETHYL

ALCOHOL BY CENTRIFUGATION

UPON

INHIBITION

OF

TRANSFORMATION

I n Part A, p h e n e t h y l alcohol (0.05 %) w a s a d d e d a t t h e i n d i c a t e d t i m e s d u r i n g a 3-h i n c u b a t i o n in t r a n s f o r m a t i o n m e d i u m . T h e p h e n e t h y l alcohol w a s r e m o v e d prior to t h e a d d i t i o n of D N A b y c e n t r i f u g a t i o n . T h e cells (also f r o m Part B) were s u s p e n d e d in f r e s h m i n i m a l m e d i u m s u p p l e m e n t ed w i t h i o / z g / m l each of t r y p t o p h a n a n d of histidine a n d o.oi % of acid casein h y d r o l y s a t e . D N A (5/~g/ml) was a d d e d a n d in Part B p h e n e t h y l alcohol w a s a d d e d as indicated, a n d a f t e r 3 ° rain i n c u b a t i o n t h e n u m b e r of t r y p t o p h a n t r a n s f o r m a n t s were d e t e r m i n e d b y a p p r o p r i a t e dilution a n d plating.

Time o/ incubation with phenethyl alcohol prior to centri/ugation (rain)

Frequency o/ trans/ormation

Part A 18o I2o 60 3°

0.04 0.04 o.I8 o.24 °.3°

5 Part B P h e n e t h y l alcohol a d d e d s i m u l t a n e o u s l y with DNA and not removed N o p h e n e t h y l alcohol a d d e d

o.12 0.32

0.4 o \o 0,2

\

O. L

TRY +

\

\ o\

o 0.05

,

o~5~

~

i

DgLUTE

a5_~

/\

o~

I

=

u_

O. OI

0,0 L o

\

\

0.005

i1 o

O.O 0 3 0

I

0.0 4

PER

CENT

i

0.08

0 ,I 2

PHENETHYL

I0

5

i

HOURS

0 .I 6

ALCOHOL

TIME 0.05% PHENETHYLALCOHOL pRESENT

Fig. 3. E f f e c t of c o n c e n t r a t i o n of p h e n e t h y l alcohol u p o n t r a n s f o r m a t i o n . P h e n e t h y l alcohol a t t h e i n d i c a t e d c o n c e n t r a t i o n s w a s a d d e d to c o m p e t e n t B. subtilis cells s i m u l t a n e o u s l y w i t h 5/*g[ ml of D N A a n d i n c u b a t e d w i t h t h e cells for 3 ° m i n a t 37 ° w i t h s h a k i n g before dilution a n d p l a t i n g . Fig. 4- R e g a i n of c o m p e t e n c e following c e n t r i f u g a t i o n after t r e a t m e n t w i t h p h e n e t h y l alcohol. H a l f of a c u l t u r e of B. subtilis cells, w h i c h were d e v e l o p i n g c o m p e t e n c e u n d e r t h e u s u a l regime, w a s t r e a t e d w i t h p h e n e t h y l alcohol (o.o 5 %) for 75 m i n (time period 1.74 to 3 h), a n d t h e n t h e p h e n e t h y l alcohol w a s r e m o v e d b y centrifugation. B o t h t r e a t e d ( 0 ) a n d u n t r e a t e d cells ( O ) were t a k e n u p in fresh m i n i m a l m e d i u m s u p p l e m e n t e d w i t h i o / ~ g / m l of t r y p t o p h a n a n d k i s t i d i n e a n d o.oi % casein h y d r o l y s a t e a n d i n c u b a t e d ~vith s h a k i n g a t 37 °. S a m p l e s were t a k e n e v e r y h o u r t e s t e d for t h e level of c o m p e t e n c e b y a d d i n g 5 / , g / m l of D N A a n d i n c u b a t i n g for 3o rain a t 37 ° prior to dilution a n d plating.

Biochim. Biophys. Acta, 174 (1969) 2 6 4 - 2 7 5

272

A . G . RICHARDSON, F. R. LEACH

Using E14CJphenethyl alcohol to follow the removal of phenethyl alcohol a single sedimentation of the cells b y centrifugation resulted in the removal of 99 % of the radioactivity. Table IV shows t h a t removal of phenethyl alcohol when less than 3o rain incubation had occurred completely reversed the inhibition, while in cases where incubation with the cells had been 2-3 h, no reversal was obtained.

Time o/ regain o/competence after centri/ugation The time course of regain of competence was studied by following the development of competence after removal of phenethyl alcohol b y centrifugation and dilution in fresh minimal medium. I n Fig. 4, the wave of competence developing after 3 h of incubation in transformation medium was inhibited in the treated culture, even though the cells were removed from the phenethyl alcohol-containing solution b y centrifugation prior to the onset of competence. The treated cells do not regain competence without going through the same physiological conditions as an ordinary culture (a similar magnitude and time of expression of the second periods of competence). This result suggests that phenethyl alcohol either prevents the development of competence or removes a factor essential for expression of competence.

Kinetics o/stopping o/trans/ormation by phenethyl alcohol If phenethyl alcohol prevents the transport of D N A into competent cells, the transformation process should become refractive to phenethyl alcohol inhibition at the same time as transformants are no longer sensitive to deoxyribonuclease; t h a t is, neither inhibitor would be effective after the D N A has been taken up b y the competent cells. Experiments in which phenethyl alcohol and deoxyribonuclease were added at various times after the addition of D N A to competent cells revealed t h a t the n u m b e r of transformants was not reduced if deoxyribonuclease was added 16 min after the D N A or if phenethyl alcohol was added 15. 5 min after the DNA. This observation is consistent with phenethyl alcohol inhibiting D N A transport.

E//ect o/ phenethyl alcohol on DNA uptake Since the inhibition was not upon the potential transformants and was maxiTABLE V EFFECT OF PHENI~THYL ALCOHOL UPON THE UPTAKE OF

[3H]DNA

C o m p e t e n t B. subtilis S B 2 5 c e l l s (3 m l ) w e r e t r e a t e d for 45 m i n w i t h [ 3 H ] D N A ( o . i / 2 g / 3 m l of

cells) and/or phenethyl alcohol (0.05 %). At the end of the transformation period, the cells were treated with deoxyribonuclease (50 #g/ml) for IO rain (magnesium concentration i raM) and collected by centrifogation. The cells were washed twice with minimal medium and finally taken up in i ml of minimal medium and counted using BRAY'S19 scintillation fluid. No radioactivity was incorporated when the DNA was incubated with deoxyribonuclease prior to the addition of competent cells.

Addition

counts/rain

Trans/ormants (his+) (IoS/ml)

i. None 2. 0.05 % phenethyl alcohol

317 34

300 36

Biochim. Biophys. Acta, I74 (I969) 264-275

273

PHENETHYL ALCOHOL AND TRANSFORMATION

mal when phenethyl alcohol was added prior to DNA addition, the effect of phenethyl alcohol on the uptake of [6H]DNA into the cells was measured as radioactivity associated with the cells after removal of exogenous DNA by deoxyribonuclease treatment. Table V demonstrates that phenethyl alcohol decreased both the uptake of E6H]DNA and the frequency of transformation to the same extent.

E[]ect o] phenethyl alcohol upon the reversible attachment o/ [aH]DNA to competent cells Since the transport of DNA is presumably preceded by a reversible binding of the DNA to the exterior of the cell~°, the effect of phenethyl alcohol upon the reversible (deoxyribonuclease sensitive) binding of E6H]DNA was investigated. Table VI shows that the [6H]DNA associated with the cells after one washing was the same for treated and untreated cells. Treatment with deoxyribonuclease removed 95 % of the radioactivity associated with the cells, demonstrating that the E6H]DNA was reversibly associated with cells. Thus, phenethyl alcohol (o.o5 %) is not inhibiting the initial attachment stage of transformation.

TABLE VI EFFECT OF PHENETHYL ALCOHOL UPON THE REVERSIBLE BINDING OF D N A TO B. subtilis C o m p e t e n t B. subtilis SB25 cells (6 ml) were obtained as described in MATERIALS AND METHODS and t h e n were chilled in an ice b a t h for IO miD. P h e n e t h y l alcohol (0.05 %) w a s added at this time. [3H]DNA (o.i/zg/3 ml of cells) w a s t h e n added and i n c u b a t e d w i t h the chilled cells for 6o miD. The cells were centrifuged down, w a s h e d w i t h cold minimal medium, and centrifuged again. The cells were t h e n suspended in 2 ml of m i n i m a l m e d i u m and a i-ml sample w a s t a k e n to determine the radioactivity associated w i t h the cells after one washing. The other sample w a s diluted to 3 ml with w a r m e d minimal m e d i u m s u p p l e m e n t e d w i t h o. I m g ] m l of deoxyribonuclease ( m a g n e s i u m c o n c e n t r a t i o n i mM). The cells were incubated w i t h the deoxyribonuclease for 20 miD at 37 °, and t h e n r e m o v e d b y centrifugation and suspended in I ml of m i n i m a l medium, a n d the radioactivity was determined. The results given are an average of 2 experiments.

Addition

counts~rain associated with chilled cells

counts ]min associated with cells after deoxyribonuclease treatment

None o.o 5 % p h e n e t h y l alcohol

129o 126o

8o 89

DISCUSSION

Three major sites of action for phenethyl alcohol in bacteria have been described in the work that was reviewed in the introduction: (I) inhibition of the initiation of DNA replication at the chromosome level; (2) RNA synthesis was inhibited with low concentrations; (3) there is a change in the permeability and transport properties of cell membranes. The physicochemical methods for detection, of the interaction of a small molecule, such as phenethyl alcohol, and DNA involve the use of the smaller molecule at approx. I. lO -8 M (refs. 6, 7). Using radioactive phenethyl alcohol interactions with DNA involving phenethyl alcohol concentrations of lO -8 M would be detectBiochim. Biophys. Acta, 174 (1969) 264-275

274

A.G. RICHARDSON, F. R. LEACH

able; that is, a IOOO-foldincrease over the sensitivity of the methods used previously6,7, Using the values for molecular weight and gene size for B. subtilis DNA obtained b y NESTER, SCHAFER AND LEDERBERG~1 the level of detection of complex formation is of the order of IO phenethyl alcohol sites per gene. Fig. 2 shows no evidence for interaction of DNA and phenethyl alcohol at the level of lO-6 M phenethyl alcohol, which is the concentration of smaller ions used for complex formation with DNA in the cases of acridine orange 22 and actinomycin D (refs. 23, 24). Treatment of transforming DNA with phenethyl alcohol is without effect on its biological activity. After uptake of DNA has occurred, the donated piece of DNA is recombined with the resident genome. BODMER~'~ found that this process in B. subtilis involves little, if any, new DNA synthesis. NESTER AND STOCKERI¢ demonstrated a biosynthetic latency (3-4 h) in the synthesis of the enzyme for which the genetic information has been introduced. Addition of phenethyl alcohol after the uptake of DNA, but before expression has occurred, results in no inhibition of bacterial transformation. Thus, phenethyl alcohol does not inhibit expression. Inhibition by phenethyl alcohol in the transforming system is on the competent cells since (I) greater inhibition resulted when competent cells were treated with phenethyl alcohol prior to the addition of DNA (see Tables I and II); (2)phenethyl alcohol inhibited transformation even though it had been removed from the competent cells by centrifugation and washing prior to the addition of DNA, if the treatment of competent cells with phenethyl alcohol has been longer than an hour (see Fig. 4); (3) the inhibition of the transformation system was ameliorated by the addition of excess competent cells but not by the addition of excess DNA (see Table III). SILVER AND WENDT10 demonstrated an effect of 0.25 % phenethyl alcohol on permeability in E. coli which differs in several ways from the observations made in this paper. Their effect was transitory, with healing occurring within IO min, and was also obtained with an equivalent concentration of toluene; neither situation is observed with B. s~tbtilis transformation. The initial attachment of DNA to competent cells was not influenced by phenethyl alcohol, whereas the transport of radioactive DNA into the cell was inhibited to the same extent as transformation.

ACKNOWLEDGMENTS

This investigation was supported by American Cancer Society Grant E-299, Oklahoma Agricultural Experiment Station Project lO96, an NDEA Fellowship to A. G. R., National Institutes of Health grants 3TI GM 665 and CA-o7488 and National Institutes of Health Research Career Program Award CA-K3-6487 to F.R.L. It was taken in part from the P h . D . thesis of A.G.R. submitted to the Oklahoma State University Graduate College, 1968. We wish to acknowledge the free exchange of results with J. URBAN and O.WYss after discovery that two groups were studying these effects. Dr. I. C. FELKNER provided labeled DNA and thymine requiring strains. Dr. F. ROTHMAN and Dr. W. R. RO~tlG also gave stocks of thymine-requiring cells. The recipient strain was obtained from Dr. F. YOUNG who also gave helpful suggestions concerning development of competence. Dr. F. REGNIER aided in the gas chromatography analysis of E14CJphenethyl alcohol. Biochim. Biophys. Acta, 174 (1969) 264-275

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275

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