Rate of mutation to phage resistance in 2H2O medium

213

MUTATION RESEARCH

R A T E O F M U T A T I O N TO P H A G E

R E S I S T A N C E IN ~H20 M E D I U M

EI),NEST Pt)LLAI~,I)AND MARTHA LEMKE 13iophysics l)eDarlment, Pe~nsvlva~ia Slate U~iz,ersiO~, U~:iversitv Park, Pa. (U.N.d.) (l~eceived January I2th, ~O(G)

SUMMARY The rate of m u t a t i o n of Escherichia coli B to T I a n d T2 phage resistance in 2H~O m e d i u m was c o m p a r e d to the m u t a t i o n rate in H~O m e d i u m to d e t e r m i n e w h e t h e r the t u n n e l i n g of a p r o t o n from one base to a n o t h e r can account for m u t a t i o n s . The results suggest t h a t p r o t o n tunneling, if it causes m u t a n t s , is rare when c o m p a r e d with o t h e r m e c h a n i s m s which are responsible for forming m u t a n t s .

INTRODUCTION I t has been suggested, n o t a b l y b y FREESE 1 t h a t a change in a base p a i r in nuclei acid from an a d e n i n e - t h y m i n e pair, to a g u a n i n e - c y t o s i n e p a i r or vice-versa, c o n s t i t u t e s a process of m u t a t i o n . A f u r t h e r suggestion has been m a d e b y L6WDIN 2 t h a t this change could be m e d i a t e d b y t h e t u n n e l i n g of a p r o t o n from one base to another. The process of p r o t o n t u n n e l i n g involves the passage t h r o u g h a p o t e n t i a l barrier, a n d the p r o b a b i l i t y of passage P is given b y an expression of the form below

P = A e - f ~ { ~m(~'-E)}~d.~ where A is a c o n s t a n t , m is the mass of the t u n n e l i n g particle, h is P l a n c k ' s c o n s t a n t a n d E, V a n d x are as i n d i c a t e d in Fig. I. T h e t u n n e l i n g process involves the w a v e l e n g t h of the p a r t i c l e which does the tunneling, a n d this in t u r n involves the m a s s of the particle. If the p a r t i c l e is a p r o t o n t h e n it has one-half t h e mass of a deuteron, a n d for a given s i t u a t i o n t h e p r o b a b i l i t y of t u n n e l i n g will change b y i n t r o d u c i n g in t h e e x p o n e n t a factor of the square root of 2, which will m a t e r i a l l y change t h e a b s o l u t e m a g n i t u d e of the q u a n t i t y involved. If we suppose t h a t t h e p r o b a b i l i t y for m u t a t i o n due to this effect is a b o u t one in a million, a n d if this is due to the t u n n e l i n g of a proton, t h e n t h e presence of a d e u t e r o n w o u l d cause a change b y a factor from I0 -~ to a p p r o x . I 0 -~. T h u s if it should be t r u e t h a t the sole cause of m u t a t i o n was p r o t o n t u n n e l i n g in D N A , a n d it were possible to m a k e a full exchange of p r o t o n s for deuterons, t h e m u t a t i o n rate should d r a s t i c a l l y fall for b a c t e r i a which h a d been c u l t u r e d in ~H~O m e d i u m .

3/Iutation Research 2 (I905)

213--2

~7

214

1.;. P O L L A R D A N D M. L E M K E

I t is possible to phage. All challenged

r e a d i l y possible to grow f s c h e r i c l ~ i a coil cells in ~H~O m e d i u m , and it is m e a s u r e the process of m u t a t i o n to p h a g e resistance, b o t h for T1 a n d T~r t h a t is necessary is to p l a t e the b a c t e r i a on m e d i u m after t h e y have been b y phage, a n d the m u t a n t s a p p e a r as r e s i s t a n t colonies.

POTENTIAL ENERGY

(v)

DISTANCE (~)

F i g . ~. l ) i a g r a m of a h y p o t h e t i c a l

potcnLial barrier.

The question arises as to w h e t h e r cells grown on m e d i u m in which the components are of n o r m a l h y d r o g e n while the w a t e r is h e a v y water, do have a p p r e c i a b l e s u b s t i t u t i o n of d e u t e r i u m for p r o t i u m . In our own l a b o r a t o r y we can offer the following evidence. T1 phage p r o d u c e d b y infection of cells grown on "H~O m e d i u m (normal n u t r i e n t b r o t h m a d e in ~H~()) shows m a r k e d l y different t h e r m a l i n a c t i v a t i o n from p h a g e grown on H~O grown cells. This indicates a real change has been m a d e in a phage which has to be formed from the m e t a b o l i c o p e r a t i o n s of the cell. Also / # g a l a c t o s i d a s e from cells grown in "H~O m e d i u m has different t h e r m a l i n a c t i v a t i o n constants. W e conclude, as is reasonable, t h a t the biochemical processes which involve w a t e r cause the i n c o r p o r a t i o n of d e u t e r i u m in place of n o r m a l hydrogen. A certain a m o u n t of (:are m u s t be exercised in this k i n d of s t u d y because m u t a tion to p h a g e resistance m a y not i m m e d i a t e l y a l t e r the surface of a b a c t e r i u m in such a w a y as to p r e v e n t an a t t a c h m e n t of phage, which is the basis for the blocking of the infective process. Thus om~ m u s t allow sufficient time for the m u t a t i o n to become expressed as phage resistance. PROCE1)URES

Cells of E . coli, strain B (ATCC 113o1 ) were grown in n u t r i e n t b r o t h , with a g i t a t i o n overnight. In the m o r n i n g IOO ml of fresh n u t r i e n t b r o t h were i n o c u l a t e d ~ i t h I ml of the o v e r n i g h t culture a n d allowed to grow for a b o u t an hour before .~ampling. Ever)." half hour 7 ml were taken, a n d used to measure (a) t u r b i d i t y , (b) dilution a n d p l a t i n g for cell colony count a n d (c) challenge for m u t a n t s . "[his last was done b y a d d i n g 2 ml of p h a g e at lO '~ per ml to 2 ml of cells, allowit~g to s t a y m i x e d for two hours, diluting a n d p l a t i n g b y the a g a r o v e r l a y m e t h o d with an a d d i t i o n a l o. 5 ml of phage a d d e d to the t u b e a g a r before i~ouring on tt~e plate. The plates were i n c u b a t e d overnight at 37 ° and m u t a n t colonies counted. The same p r o c e d u r e xYas used for ~H.aO cells w'hich were grown in n u t r i e n t b r o t h dissolved in ~H~O, first o v e r n i g h t and t h e n transferred. 3~u/a/hm l~'scar~/~ ~ (~965t ~ 3 ~ ~7

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Fig. 2. The n u m b e r of m u t a n t s for T I and T2 scored as cells m u l t i p l y in H~O b r o t h at 37 °. Fig. 3. The d e v e l o p m e n t of T I and T2 l n u t a n t s in eH~O b r o t h at 37 °. The scale is changed for T2 m u t a n t s .

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Fig. 4. The n u m b e r of E. coli B m u t a n t s to T I resistance in relation to the n u m b e r of cells in the culture ai; 37 °. Fig. 5. The n u m b e r of E. coli m u t a n t s to T2 resistance in relation to the n u m b e r of cells in the culture. 2~l,dation Research 2 (1965) 213 2i 7

210

E. POLLAllD AND M. LEMKE

A c o m p a r a b l e set of experiments were done in Roberts' C m i n i m a l m e d i u m (NH~C1, ~ g / l ; Na~HPO~, 6 g / l ; KHePO~, 3 g / l ; NaC1 3 g/l; MgCI=, o.o~ g/i; Na~S(),, o.o26 g / l ; glucose 6 g/l). Typical data are shown in Fig. ~ a n d Iqg. 3. I n these experiments the colony count of the growing cell culture is shown as one line, the n u m b e r of m u t a n t s to "f~ resistance is shown as a second line, a n d the n u m b e r of m u t a n t s to "1"~ resistance is shown as a t h i r d line. Similar d a t a taken in ~H~O m e d i u m a r e s h o u n in lqg..3. It can be seen at once t h a t there is no striking difference in the n u m b e r of m u t a n t s . I n Fig. 4 we show the n u m b e r of m u t a n t s per bacterial cell as the cultnre develops. The overall progress of the d e v e l o p m e n t of m u t a n t s can be seen in each case here shown for T I . In Fig. 5 similar d a t a for T2 arc shown. Table I shoxxs the n u m b e r of m u t a n t s per cell at each time for each kind of mediuln.

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E x p e r i m e n t s done in C m i n i m a l m e d i u m showed a higher overall rate of m u t a t i o n , b u t no systematic difference between -~H~O a n d H2(). Only T I resistance was studied. Cultures ~vhich had been stored overnight in '-'H20 a n d H20 did not show a n y appreciable difference in the u u m b e r of m u t a n t s . DISCUSSION We conclude from this p r e l i m i n a r y e x p e r i m e n t t h a t it is not safe to assume t h a t the o,dy basis for the formation of m u t a n t s is the process of proton t u n n e l i n g . We do n o t claim t h a t in these experiments we have e l i m i n a t e d it as a means of causing m u t a t i o n s , b u t merely suggest t h a t the discovery of m u t a n t s due to proton t u u n e l i n g m i g h t be done b y this means. We also suggest t h a t it will be found to be rare compared with other m e c h a n i s m s which are responsible for the formation of m u t a n t s . Our d a t a could be i n t e r p r e t e d as showing a smaller m u t a t i o n rate in ~H20 medium. It is likely t h a t this reduced rate is due to other factors which are k n o w n to influence m u t a t i o n expression, b u t if the claim were made t h a t this reduction is due to reduced proton t u n n e l i n g , t h e n the ratio of o.6 j - o.3 for T I between 2H20 m e d i m n m u t a n t s a n d H20 m e d i u m m u t a n t s should be compared with theoretical t u n n e l i n g ratios of 1.4 " Io ~. If we suppose t h a t a fraction of m u t a n t s is due to t u n n e l i n g , a n d I - - f is not d e p e n d e n t on 2H~O in a n y other way, t h e n the observed ratios can be used to calculate f , using the theoretically expected ratio as given above. We get for the calculated ratio ~lfulali
MUTATION RATE TO PHAGE RESISTANCE IN 2I-[20 MEDIUM

'~H~O/H,~O --- 1"4 " I o a f @ I - - f I

for T2 and

217

6.5" IO a f @ I - - f for TI I

Thus f is 40% in the first case and 3 °/o o/ in the second. Since it is very likely that the assumption that I - - f does not depend on ~H,O is not valid, the value o f f calculated above is probably too high. For this reason we feel it is safe to conclude that other mechanisms of mutation exist. ACKNOWLE1) GEMENTS

We wish to thank Dr. S. PERSON for advice and criticism. This work was supported by research grant NsG 324 of the National Aeronautics and Space Agency. REFERENCES i FREESE, E., T h e difference b e t w e e n s p o n t a n e o u s a n d b a s e - a n a l o g u e i n t r o d u c e d m u t a t i o n s of p h a g e T 4. Pro& Natl. Acad. Sci. U.S., 45 (1959) 622-63. 2 1,6WDIN, P.-O., P r o t o n t u n n e l i n g in D N A a n d its biological implications. Rev. Mod. Phys., 35 (3) (1963) 724-732. Mulalion Research 2 (t965) 213 217