- Email: [email protected]
Inactivation of β-galactosidase induction by ultraviolet light
614
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 8337
INACTIVATION OF B-GALACTOSIDASE INDUCTION BY ULTRAVIOLET LIGHT ARTHUR B. PARDEE AND LOUISE S. PRESTIDGE
Biology Department, Princeton University, Princeton, N. J. (U.S.A.) (Received June Ioth, 1963)
SUMMARY
The ability of Escherichia coli to form /5-galactosidase (EC 3.2.1.23) is strongly inhibited (relative to total protein synthesis) by ultraviolet light if the bacteria are maintained in the same medium before and after irradiation. However, irradiation has a much smaller effect if the bacteria are transferred to a poorer medium after irradiation and before induction. High concentrations of inducer do not reverse the ultraviolet light inhibition. Ultraviolet light appears mainly to cause a "catabolite repression" of/%galactosidase formation, similar to action of asp decay or thymine starvation, and not to inactivate the structural gene for this enzyme. Ultraviolet light inactivation of/~-galactosidase induction is not an all or none phenomenon in each bacterium. Increasing doses of ultraviolet light progressively inactivate formation of the enzyme in an individual bacterium. In contrast, inactivation of galactoside permease induction is virtually complete in each damaged cell. A difference in ultraviolet light sensitivity before and after induction was noted. This is attributed to inactivation of galactoside permease which is initially essential for ~-galactosidase induction. After induction had commenced, inhibition of permease formation no longer reduced the ability of the bacteria to produce/~-galactosidase.
INTRODUCTION
Irradiation of Escherichia coli with ultraviolet light stops induced /~-galactosidase synthesis within a few minutes. The action spectrum suggests that the sensitive target is a high molecular weight nucleic acid 1. /5-Galactosidase synthesis is considerably more sensitive to ultraviolet light than is total protein synthesis 2. MCFALL AND MAGASANIK3 have proposed that inactivation of /~-galactosidase formation by 32p decay or thymine starvation results from a repression by catabolites accumulated in the damaged bacteria. As suggested by McFALL (personal communication) and demonstrated by BOWNE AND ROGERS4 this also seems to be the case ~or ultraviolet irradiation. The results reported here confirm this conclusion. Investigations presented here demonstrate that the individual bacteria gradually lose their ability to form 15-galactosidase, but galactoside permease is lost in an all or " Abbreviations: TMG, methyl-/~-D-thiogalactoside; ONPG, o-nitrophenyl-~-D-galactoside; IPTG, isopropyl-/~-D-thiogalactoside.
Biochim. Biophys. Acta, 76 (1963) 614~21
fl-GALACTOSIDASE INDUCTION AND ULTRAVIOLET LIGHT
615
none fashion. Also, the relative sensitivity of /~-galactosidase induction is greater before than after addition of inducer, owing to inactivation of permease induction. MATERIALS AND METHODS
E. cull B and various mutants of E. cull KI2 were used in these experiments. 2ooo~¢ is a wild-type F - organism, 4000 is a wild-type Hfr organism; C-6oo-I is an F-, galactoside-permease negative (Y-) mutant. An F - strain constitutive for alkaline phosphatase (EC 3.I.3.I), (ref. 5), (P+R2-) and inducible for/5-galactosidase was also used. The bacteria were usually grown aerobically by swirling at 37 ° in a synthetic medium (M63) containing per liter 2 g glycerol, 13.6 g KH2PO4, 2.0 g (NH4)2SO,, 0.2 g MgSO4-7H,O and 0.5 mg FeSO4.7H20, adjusted to pH 7.0 with KOH. In some experiments 0.o5 % acid hydrolyzed casein (Casamino Acids) was added. The TGSE medium of GAREN AND SIDDIQI5, with glycerol replacing glucose, was used when alkaline phosphatase was determined. Bacteria in the exponential phase of growth were irradiated with a I5-W Sylvania Germicidal-A Lamp having an intensity (mostly at 2537 A) of about 35 ergs/mm*/sec at the distance used. Protein was determined by the Folin method 6. fl-Galactosidase was induced with either 3" lO-3 M TMG or 5" lO-4 M IPTG. The enzyme was assayed b y the o-nitrophenyl-/5-D-galactoside procedure 1. Alkaline phosphatase was measured by determining the rate of hydrolysis of o-nitrophenyl phosphate s.
RESULTS AND DISCUSSION Nutritional e[/ects on ultraviolet light inactivation.
The medium on which the bacteria were grown did not strongly influence ultraviolet light inactivation of /5-galactosidase induction. Graphs of log residual enzyme forming ability versus ultraviolet dose showed a somewhat smaller shoulder or "hitness" if the bacteria were grown in rich rather than in synthetic medium, but the final slopes of these curves were similar. Quite a different result w~s obtained if bacteria grown in one medium were irradiated and then transferred into a different medium .(Fig. I). If the second medium permitted slower growth than the first, a dose of ultraviolet light was much less inhibitory than if the bacteria were kept in one medium or the other at all times. Thus, a shift from richer to poorer medium largely nullified the effect of ultraviolet light. Data of this sort were the basis for the catabolite repression hypothesis suggested by McFALL AND MAGASANIK3. If the medium into which the bacteria were transferred after irradiation was made even richer than that in which they were grown, ultraviolet light had a much greater effect. Not only was enzyme induction considerably reduced, but the lag before enzyme formation commenced was greatly extended. l"hese nutrient effects were readily reversible. When i~adiated bacteria in a glycerol-casein hydrolysate medium were shifted to a medium lacking glycerol, their rate of/5-galactosidase synthesis much less inhibited, as above. If this culture was again supplied with glycerol the rate of enzyme synthesis was once more conBiochira. Biophys. Acta, 76 (i963) 614-62i
616
A.P.
PARDEE, L. S. PRESTIDGE
tool
f
t
i
J
- -
50
20 ._1 > rr
i0
bo 5
\ I
I
I
I000
2000
ULTRAVIOLET-LIGFtT DOSE (er$:js/rnrn ~) Fig. I. E f f e c t of s h i f t i n g to a p o o r e r m e d i u m . A n a l k a l i n e p h o s p h a t a s e c o n s t i t u t i v e o r g a n i s m w a s g r o w n on a low p h o s p h a t e - g l y c e r o l - c a s e i n h y d r o l y s a t e m e d i u m . The b a c t e r i a w e re c e n t r i f u g e d , r e s u s p e n d e d in a m e d i u m l a c k i n g glycerol, a n d i r r a d i a t e d for v a r i o u s t i me s . G l y c e r o l w a s a d d e d b a c k to h a l f of e a c h c u l t u r e . V i a b i l i t y ([]---~) a n d f o r m a t i o n of p r o t e i n ( A - - ZX), a l k a l i n e p h o s p h a t a s e ( V - ~ 7 ) , a n d f i - g a l a c t o s i d a s e ( O - O ) w e r e d e t e r m i n e d i n t h e c u l t u r e s c o n t a i n i n g glycerol. f i - G a l a c t o s i d a s e w a s d e t e r m i n e d in t h e c u l t u r e l a c k i n g g l y c e r o l as w e l l ( G - C ) ) .
siderably reduced. Therefore, the richer and poorer media did not permanently damage and repair the irradiated bacteria. Lowering the nutrient supply brought ~-galactosidase inhibition in line with general inhibition of macromolecule synthesis. Protein synthesis, production of alkaline phosphatase (an enzyme not subject to catabolite repression), total RNA, and messenger RNA were approximately as sensitive to ultraviolet light as was fl-galactosidase after glycerol was removed (Table I). This result therefore suggests TABLE I INHIBITION BY ULTRAVIOLET IRRADIATION E . coli t3 g r o w i n g e x p o n e n t i a l l y a t 26 ° w e r e i r r a d i a t e d as i n d i c a t e d . S a m p l e s w e re t a k e n a t int e r v a l s up to 90 m i n for p r o t e i n , t o t a l R N A , a n d i n d u c e d ~ - g a l a c t o s i d a s e f o r m a t i o n . To d e t e r m i n e m e s s e n g e r R N A a t 28 m i n a f t e r i r r a d i a t i o n , 2/*C [3H]u ri di ne w a s a d d e d ; 2o sec l a t e r t h e b a c t e r i a were ch illed in a s o l u t i o n c o n t a i n i n g o.oi M N a N 3. A l i q u o t s (plus I m g s e r u m a l b u m i n ) w e re p r e c i p i t a t e d w i t h i o °/o t r i c h l o r o a c e t i c acid, t h e n e x t r a c t e d w i t h b o i l i n g 5 % t r i c h l o r o a c e t i c acid a n d t h e cold t r i c h l o r o a c e t i c a c i d - i n s o l u b l e , h o t t r i c h l o r o a c e t i c a c i d - s o l u b l e 3H w a s d e t e r m i n e d (20" ~ZNA). The b u l k of t h e c u l t u r e w a s d i s r u p t e d w i t h l y s o z y m e , t r e a t e d w i t h D N A a s e (EC 3.1.4.5), a n d s u b j e c t e d to sucrose g r a d i e n t (20- 3 %) c e n t r i f u g a t i o n a t 38 ooo r e v . / m i n for 135 m i n T. Dr ops w e r e c o l l e c t e d from a p i n h o l e m a d e in t h e b o t t o m of t h e c e n t r i f u g e t u b e . E a c h s a m p l e was c o u n t e d for cold t r i c h l o r o a c e t i c a c i d - i n s o l u b l e , h o t t r i c h l o r o a c t i c a c i d - s o l u b l e 3H. T h e s u m of c o u n t s fo und in t h e m e s s e n g e r - R N A a r e a is g i v e n as " m - R N A " i n t h e t a b l e . Synthesis, % o] control UV ergs/mm ~
fl-Galactosidase
Protein
20 sec RNA
m RNA
o iooo 15oo
ioo 3 0.2
IOO 5° 3°
ioo 38 24
ioo 4° 25
o 12oo
ioo 2
IOO 45
ioo 44
ioo (33)
o 12oo
ioo 5
ioo 45
Ioo 46 Biochim.
Biophys.
Acta,
76 (1963) 614-621
~-GALACTOSIDASE INDUCTION AND ULTRAVIOLET LIGHT
617
that excess nutrients create a specific inhibition of/~-galactosidase production in the ultraviolet irradiated bacteria.
Partial inactivation o/ enzyme [ormation in individual bacteria We may ask about the distribution of ability to synthesise fl-galactosidase in individual bacteria. Do some irradiated bacteria lose all ability to form fl-galactosidase while others remain unchanged, or does each bacterium lose a portion of its ability to be induced? If the former were the case, each of the still-inducible bacteria would produce as much enzyme as would a non-irradiated bacterium. In the latter case, each bacterium would produce less enzyme than does an unirradiated bacterium. One way of distinguishing between these possibilities is to compare the rates of hydrolysis of ONPG by extracts and by whole bacteria. The rate of ONPG hydrolysis by intact bacteria is limited by the rate of entry of ONPG and is relatively independent of the amount of enzyme in the cell. The latter, the actual amount of enzyme, is measured by assaying disrupted cells. The ratio of rate of hydrolysis by extracts to rate of hydrolysis by whole cells increases with increasing amount of enzyme per cell (Fig. 2), since the rate of entry remains the same independent of induction, in these bacteria which lack a permease, ,
L
,
I
I
I
. j
I: >
¢n.2 i-
z I
I0 20 30 40 UNITS ENZYME/ml (EXTRACTS)
Fig. 2. Hydrolysis rates of ONPG by intact vs. disrupted bacteria. Cultures of a permease-negative E. coli (C-6oo-i) were induced for intervals of up to 6o rain with 5" IO-* M IPTG. Samples were taken, at the same cell concentration, into 2 5 ~ug/ml chloramphenicol (to obtain intact cells), or into toluene and Sarkosyl (to obtain disrupted cells). The rates of ONIK} hydrolysis b y t h e s e preparations were determined. The curve indicates tS-galactosidase activities of bacteria versus extracts.
When ONPG hydrolysis by irradiated bacteria before and after disruption was compared, Fig. 3. was obtained. With low ultraviolet doses, the enzyme produced (measured in extracts) was much more strongly inhibited than the activity of the whole cell. If some cells had completely retained their enzyme-forming ability, the ratios of activities in extracts to those of whole cells would have remained constant. The actual result is predicted, from Fig. 2, if all individual bacteria lost a fraction of their enzyme-forming ability. Therefore, the ability of a bacterium to make fl-galactosidase is lost gradually and not in an all-or-none fashion. This partial inactivation of enzyme-forming ability might be attributed to the inactivation, one at a time, of the several nuclei contained in each bacterium. Each nuclear inactivation might reduce enzyme-forming ability to some extent. In order Bioc hi m . Bi ophy s . Acta, 76 (I963) 614-62I
618
A.B.
ULTRAVIOLET-LIGHT DOSE (er,cjs/mm=) 1000 2000
3000
I000
PARDEE, L. S. PRESTIDGE
,,,
-
""x cn30G -
N.%.
0 ).Z G. Z
I0O - -
30,
W
%.%
\
10
I
Fig. 3. O N P G hydrolysis b y intact and disrupted irradiated bacteria. E. coli C-6oo-i were irradiated for various times. The cultures were induced with 5" lO-4 M I P T G for 20 min. Abilities of broken ( O - G ) and whole ( 0 - 0 ) cells to hydrolyze ONPG were measured.
to test this idea the bacteria were starved of phosphate to make them uni-nucleate s. These bacteria were irradiated, induced, and assayed. The ratio of fl-galactosidase activities of intact and disrupted cells changed in the same way as it did with unstarved bacteria. Therefore, partial inactivation is not attributed to an inactivation of nuclei within a cell, one at a time, but is consistent with gradual inhibition.
Ultraviolet irradiation damage to galactoside permease production Ultraviolet irradiation inhibited induction of permease for accumulation of melibiose inside the bacteria '. The degree of inactivation was roughly the same as for inhibition of fl-galactosidase-forming ability. Activity of preformed permease and the permeability of the bacteria to galactosides were not altered b y irradiation. Another way of showing that permease formation is inhibited by ultraviolet light is based on the observation that a low concentration of inducer cannot induce fl-galactosidase unless a bacterium already possess galactoside permease 1°. This is seen in Fig. 4, Curve A: here uninduced bacteria are not induced by 1.5"Io-5M 1PTG. Curve B shows that bacteria which had previously been briefly exposed to 3" lO-4 M I P T G were readily induced b y 1. 5 • lO -5 M IPTG. These bacteria produced fl-galactosidase nearly as rapidly as did bacteria that were continually induced b y the higher I P T G concentration (Curve C). Therefore, presence of permease can be measured b y determining induction b y a low I P T G concentration. The inactivation of permease formation b y ultraviolet was measured in the same experiment. Bacteria were irradiated sufficiently to decrease fl-galactosidase induction with 3" lO-4 M I P T G by about 40 % (Curve D). After 12 min the inducer concentration was decreased to 1.5 "1o -5 M, whereupon the inhibition increased to Biochim. Biophys. Acta, 76 (1963) 614-62I
~-GALACTOSIDASE INDUCTION AND ULTRAVIOLET LIGHT
619
///J/ J~
"'
8
t$)
.-
of t~
oLld o
20
30
40
TIME OF INDUCTION (rain)
Fig. 4. Permease determination measured b y induction by dilute inducer. Sample A (non irradiated) of E. coli 2oooa was diluted 2o-fold and was induced with 1. 5. io -~ M IPTG. Samples ]3 and C (non-irradiated) and D and E (irradiated) were induced for 12 rain with 3' io-* M IPTG, and were then diluted 2o-fold into media (in which bacteria had previously been grown) lacking inducer (B and E) or to which 3" io-* M inducer was added (C and D). Assays for fl-galactosidase activity of disrupted cells were made at intervals.
about 75 % (Curve E). It is concluded the many (perhaps half) of the irradiated bacteria did not produce sufficient permease in the initial 12 rain to permit subsequent fl-galactosidase induction at the lower inducer concentration. Either a fraction of the irradiated bacteria lost all ability to produce permease, or most of the bacteria were able to produce permease at only a fraction of their original rate. To distinguish between these possibilities, the fl-galactosidase-forming ability attained after dilution of inducer was determined after different lengths of preinduction. The ability to form the enzyme did not increase as the preinduction period was lengthened. If many of the bacteria had retained ability to make some permease, the culture should have gradually gained ability to produce fl-galactosidase at low inducer concentration, as the time of pre-induction became longer. Therefore, permease-forming ability of individual bacteria seems to be reduced by ultraviolet light to a level too low to support fl-galactosidase induction, when it is reduced at all. Permease induction inactivation seems to be all-or-none, by this criterion. Ultraviolet irradiation sensitivity o[ fl-galactosidase /ormation be[ore vs. after induction
Penicillinase induction of Bacillus cereus n and fl-glucosidase induction of yeast ]~ are more sensitive to ultraviolet light before induction has started than after enzyme formation has commenced. The induction of fl-galactosidase by TMG in BiocMm. Biophys. Acta, 76 (1963) 614-621
620
A.B.
PARDEE, L. S. PRESTIDGE
wild type E. coli was also more sensitive to ultraviolet irradiation prior to induction than after enzyme formation has commenced (Fig. 5A). An explanation of this changed sensitivity is based on the fact that induction of galactoside permease is required in order to induce fl-galactosidase with TMG. Irradiation prior to induction damages both permease- and fl-galactosidase-forming abilities, and therefore inhibits 3
A
I
I
I
I
I
I
I
I
I>tr.
g (.3
3 <1
o,
20
40 O 20 TIME OF" INDUCTION (rnin)
4.0
Fig. 5. I r r a d i a t i o n before ( O - O ) a n d a f t e r ( A - A ) i n d u c t i o n ; ( O - O ) n o t irradiated. W i l d - t y p e E. coli 4ooo were i n d u c e d for io rain w i t h 5" IO-4 M T M G (A), or for 5 m i n w i t h i o -8 M I P T G (]3). A n o t h e r c u l t u r e w a s n o t i n d u c e d . O n e p a r t of e a c h c u l t u r e w a s t h e n irradiated, a n d a n o t h e r w a s not. fl-Galactosidase i n d u c t i o n w a s t h e n d e t e r m i n e d in e a c h of t h e s e cultures. A, p e r m e a s e r e q u i r e d ; B, p e r m e a s e n o t required.
induction by TNIG in two separate ways. But after induction has commenced and the permease is present, only the sensitivity of fl-galactosidase formation itself remains. Thus, after induction has started the number of ultraviolet light-sensitive targets is reduced from two to one. This hypothesis is supported by the fact tha~ induction with IPTG, an inducer which does not require a permease, is equally sensitive to ultraviolet light before or after induction (Fig. 5B). This suggests that the extra sensitivity of induction by Ti~G is related to formation of a permease. Another experiment which favors this conclusion is that when E. coli B were pre-induced with galactinol s so that they contained permease but not fl-galactosidase, their ultraviolet light sensitivity was then the same with either TMG or IPTG, and equal to the sensitivity of IPTG-induced bacteria. It is concluded that the specially ultraviolet light-sensitive step required to start fl-galactosidase induction is simply the induction of permease. CONCLUSIONS
Ultraviolet light inactivation of fl-galactosidase induction is complex. Ultraviolet light does not simply inactivate the structural gene for the enzyme. Rather, the general synthetic ability of the cell is inhibited slightly, through nucleic acid-containing targets. The great effect on fl-galactosidase synthesis seems at present best explained by a repression of this enzyme by catabolites accumulated by the damaged bacteria. A paradox is seen in that the inhibition of permease induction in an individual Biochim. Biophys. Acta, 76 (x963) 6 1 4 - 6 2 I
fl-GALACTOSIDASE INDUCTION AND ULTRAVIOLET LIGHT
621
bacterium can be virtually complete at doses of ultraviolet light which only partly inhibit/~-galactosidase induction. Ultraviolet light sensitivity of fl-galactosidase induction is greater prior to induction than afterwards. This is explained by the requirement for formation of permease before but not after induction has commenced. Changes in ultraviolet light sensitivity of other inductions might similarly be attributed to the existence of twostep inductions, one step of which (not necessarily permease) is eliminated shortly after induction has commenced. ACKNOWLEDGEMENTS
are indebted to D r . R . D o I for help with the technique of sucrose gradient c e n T h i s w o r k was aided by a Grant AI-o44o 9 from the United States Public Health Service. We
trifugation.
REFERENCES i G. RUSHIZKY, IV[. RILEY, L. S. PRESTIDGE AND A. B. PARDEE, Biochim. Biophys. Acta, 45 (196o) 7° . M. MASTERS AND A. B. PARDEE, Biochim. Biophys. Acta, 56 (I962) 6o9. a E. MCFALL AND B. 1V[AGASANIK, Biochim. Biophys. Acta, 55 (1962) 9o0. 4 S. Vq. BOWNE, Jr. AND P. ROGERS, J. Mol. Biol., 5 (1962) 90. A. G'AREN AND O. SIDDIQI, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1121. e O. H. LOWRY, N. J. ROSEBROUGVI, A. L. FARR, AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265 . ? M. NOMURA, B. D. HALL AND S. SPIEGELMAN, J. Mol. Biol., 2 (196o) 3o6. E. 1VicFALL, A. ]3. PARDEE AND G'. S. STENT, Biochim. Biophys. Acta, 27 (1958) 282. A. B. PARDEE, J. Bacteriol., 73 (1957) 376. :0 A. N o v l c K AND M. WEINER, Proc. Natl. Acad. Sci. U.S., 43 (1957) 553. n A. M. TORRIANI, Biochim. Biophys. Acta, 19 (1956) 224. 1~ H. HALVORSON AND L. JACKSON, J. Gen. Microbiol., 14 (1956) 26.
Biochim. Biophys. Acta, 76 (1963) 614-621
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 8337
INACTIVATION OF B-GALACTOSIDASE INDUCTION BY ULTRAVIOLET LIGHT ARTHUR B. PARDEE AND LOUISE S. PRESTIDGE
Biology Department, Princeton University, Princeton, N. J. (U.S.A.) (Received June Ioth, 1963)
SUMMARY
The ability of Escherichia coli to form /5-galactosidase (EC 3.2.1.23) is strongly inhibited (relative to total protein synthesis) by ultraviolet light if the bacteria are maintained in the same medium before and after irradiation. However, irradiation has a much smaller effect if the bacteria are transferred to a poorer medium after irradiation and before induction. High concentrations of inducer do not reverse the ultraviolet light inhibition. Ultraviolet light appears mainly to cause a "catabolite repression" of/%galactosidase formation, similar to action of asp decay or thymine starvation, and not to inactivate the structural gene for this enzyme. Ultraviolet light inactivation of/~-galactosidase induction is not an all or none phenomenon in each bacterium. Increasing doses of ultraviolet light progressively inactivate formation of the enzyme in an individual bacterium. In contrast, inactivation of galactoside permease induction is virtually complete in each damaged cell. A difference in ultraviolet light sensitivity before and after induction was noted. This is attributed to inactivation of galactoside permease which is initially essential for ~-galactosidase induction. After induction had commenced, inhibition of permease formation no longer reduced the ability of the bacteria to produce/~-galactosidase.
INTRODUCTION
Irradiation of Escherichia coli with ultraviolet light stops induced /~-galactosidase synthesis within a few minutes. The action spectrum suggests that the sensitive target is a high molecular weight nucleic acid 1. /5-Galactosidase synthesis is considerably more sensitive to ultraviolet light than is total protein synthesis 2. MCFALL AND MAGASANIK3 have proposed that inactivation of /~-galactosidase formation by 32p decay or thymine starvation results from a repression by catabolites accumulated in the damaged bacteria. As suggested by McFALL (personal communication) and demonstrated by BOWNE AND ROGERS4 this also seems to be the case ~or ultraviolet irradiation. The results reported here confirm this conclusion. Investigations presented here demonstrate that the individual bacteria gradually lose their ability to form 15-galactosidase, but galactoside permease is lost in an all or " Abbreviations: TMG, methyl-/~-D-thiogalactoside; ONPG, o-nitrophenyl-~-D-galactoside; IPTG, isopropyl-/~-D-thiogalactoside.
Biochim. Biophys. Acta, 76 (1963) 614~21
fl-GALACTOSIDASE INDUCTION AND ULTRAVIOLET LIGHT
615
none fashion. Also, the relative sensitivity of /~-galactosidase induction is greater before than after addition of inducer, owing to inactivation of permease induction. MATERIALS AND METHODS
E. cull B and various mutants of E. cull KI2 were used in these experiments. 2ooo~¢ is a wild-type F - organism, 4000 is a wild-type Hfr organism; C-6oo-I is an F-, galactoside-permease negative (Y-) mutant. An F - strain constitutive for alkaline phosphatase (EC 3.I.3.I), (ref. 5), (P+R2-) and inducible for/5-galactosidase was also used. The bacteria were usually grown aerobically by swirling at 37 ° in a synthetic medium (M63) containing per liter 2 g glycerol, 13.6 g KH2PO4, 2.0 g (NH4)2SO,, 0.2 g MgSO4-7H,O and 0.5 mg FeSO4.7H20, adjusted to pH 7.0 with KOH. In some experiments 0.o5 % acid hydrolyzed casein (Casamino Acids) was added. The TGSE medium of GAREN AND SIDDIQI5, with glycerol replacing glucose, was used when alkaline phosphatase was determined. Bacteria in the exponential phase of growth were irradiated with a I5-W Sylvania Germicidal-A Lamp having an intensity (mostly at 2537 A) of about 35 ergs/mm*/sec at the distance used. Protein was determined by the Folin method 6. fl-Galactosidase was induced with either 3" lO-3 M TMG or 5" lO-4 M IPTG. The enzyme was assayed b y the o-nitrophenyl-/5-D-galactoside procedure 1. Alkaline phosphatase was measured by determining the rate of hydrolysis of o-nitrophenyl phosphate s.
RESULTS AND DISCUSSION Nutritional e[/ects on ultraviolet light inactivation.
The medium on which the bacteria were grown did not strongly influence ultraviolet light inactivation of /5-galactosidase induction. Graphs of log residual enzyme forming ability versus ultraviolet dose showed a somewhat smaller shoulder or "hitness" if the bacteria were grown in rich rather than in synthetic medium, but the final slopes of these curves were similar. Quite a different result w~s obtained if bacteria grown in one medium were irradiated and then transferred into a different medium .(Fig. I). If the second medium permitted slower growth than the first, a dose of ultraviolet light was much less inhibitory than if the bacteria were kept in one medium or the other at all times. Thus, a shift from richer to poorer medium largely nullified the effect of ultraviolet light. Data of this sort were the basis for the catabolite repression hypothesis suggested by McFALL AND MAGASANIK3. If the medium into which the bacteria were transferred after irradiation was made even richer than that in which they were grown, ultraviolet light had a much greater effect. Not only was enzyme induction considerably reduced, but the lag before enzyme formation commenced was greatly extended. l"hese nutrient effects were readily reversible. When i~adiated bacteria in a glycerol-casein hydrolysate medium were shifted to a medium lacking glycerol, their rate of/5-galactosidase synthesis much less inhibited, as above. If this culture was again supplied with glycerol the rate of enzyme synthesis was once more conBiochira. Biophys. Acta, 76 (i963) 614-62i
616
A.P.
PARDEE, L. S. PRESTIDGE
tool
f
t
i
J
- -
50
20 ._1 > rr
i0
bo 5
\ I
I
I
I000
2000
ULTRAVIOLET-LIGFtT DOSE (er$:js/rnrn ~) Fig. I. E f f e c t of s h i f t i n g to a p o o r e r m e d i u m . A n a l k a l i n e p h o s p h a t a s e c o n s t i t u t i v e o r g a n i s m w a s g r o w n on a low p h o s p h a t e - g l y c e r o l - c a s e i n h y d r o l y s a t e m e d i u m . The b a c t e r i a w e re c e n t r i f u g e d , r e s u s p e n d e d in a m e d i u m l a c k i n g glycerol, a n d i r r a d i a t e d for v a r i o u s t i me s . G l y c e r o l w a s a d d e d b a c k to h a l f of e a c h c u l t u r e . V i a b i l i t y ([]---~) a n d f o r m a t i o n of p r o t e i n ( A - - ZX), a l k a l i n e p h o s p h a t a s e ( V - ~ 7 ) , a n d f i - g a l a c t o s i d a s e ( O - O ) w e r e d e t e r m i n e d i n t h e c u l t u r e s c o n t a i n i n g glycerol. f i - G a l a c t o s i d a s e w a s d e t e r m i n e d in t h e c u l t u r e l a c k i n g g l y c e r o l as w e l l ( G - C ) ) .
siderably reduced. Therefore, the richer and poorer media did not permanently damage and repair the irradiated bacteria. Lowering the nutrient supply brought ~-galactosidase inhibition in line with general inhibition of macromolecule synthesis. Protein synthesis, production of alkaline phosphatase (an enzyme not subject to catabolite repression), total RNA, and messenger RNA were approximately as sensitive to ultraviolet light as was fl-galactosidase after glycerol was removed (Table I). This result therefore suggests TABLE I INHIBITION BY ULTRAVIOLET IRRADIATION E . coli t3 g r o w i n g e x p o n e n t i a l l y a t 26 ° w e r e i r r a d i a t e d as i n d i c a t e d . S a m p l e s w e re t a k e n a t int e r v a l s up to 90 m i n for p r o t e i n , t o t a l R N A , a n d i n d u c e d ~ - g a l a c t o s i d a s e f o r m a t i o n . To d e t e r m i n e m e s s e n g e r R N A a t 28 m i n a f t e r i r r a d i a t i o n , 2/*C [3H]u ri di ne w a s a d d e d ; 2o sec l a t e r t h e b a c t e r i a were ch illed in a s o l u t i o n c o n t a i n i n g o.oi M N a N 3. A l i q u o t s (plus I m g s e r u m a l b u m i n ) w e re p r e c i p i t a t e d w i t h i o °/o t r i c h l o r o a c e t i c acid, t h e n e x t r a c t e d w i t h b o i l i n g 5 % t r i c h l o r o a c e t i c acid a n d t h e cold t r i c h l o r o a c e t i c a c i d - i n s o l u b l e , h o t t r i c h l o r o a c e t i c a c i d - s o l u b l e 3H w a s d e t e r m i n e d (20" ~ZNA). The b u l k of t h e c u l t u r e w a s d i s r u p t e d w i t h l y s o z y m e , t r e a t e d w i t h D N A a s e (EC 3.1.4.5), a n d s u b j e c t e d to sucrose g r a d i e n t (20- 3 %) c e n t r i f u g a t i o n a t 38 ooo r e v . / m i n for 135 m i n T. Dr ops w e r e c o l l e c t e d from a p i n h o l e m a d e in t h e b o t t o m of t h e c e n t r i f u g e t u b e . E a c h s a m p l e was c o u n t e d for cold t r i c h l o r o a c e t i c a c i d - i n s o l u b l e , h o t t r i c h l o r o a c t i c a c i d - s o l u b l e 3H. T h e s u m of c o u n t s fo und in t h e m e s s e n g e r - R N A a r e a is g i v e n as " m - R N A " i n t h e t a b l e . Synthesis, % o] control UV ergs/mm ~
fl-Galactosidase
Protein
20 sec RNA
m RNA
o iooo 15oo
ioo 3 0.2
IOO 5° 3°
ioo 38 24
ioo 4° 25
o 12oo
ioo 2
IOO 45
ioo 44
ioo (33)
o 12oo
ioo 5
ioo 45
Ioo 46 Biochim.
Biophys.
Acta,
76 (1963) 614-621
~-GALACTOSIDASE INDUCTION AND ULTRAVIOLET LIGHT
617
that excess nutrients create a specific inhibition of/~-galactosidase production in the ultraviolet irradiated bacteria.
Partial inactivation o/ enzyme [ormation in individual bacteria We may ask about the distribution of ability to synthesise fl-galactosidase in individual bacteria. Do some irradiated bacteria lose all ability to form fl-galactosidase while others remain unchanged, or does each bacterium lose a portion of its ability to be induced? If the former were the case, each of the still-inducible bacteria would produce as much enzyme as would a non-irradiated bacterium. In the latter case, each bacterium would produce less enzyme than does an unirradiated bacterium. One way of distinguishing between these possibilities is to compare the rates of hydrolysis of ONPG by extracts and by whole bacteria. The rate of ONPG hydrolysis by intact bacteria is limited by the rate of entry of ONPG and is relatively independent of the amount of enzyme in the cell. The latter, the actual amount of enzyme, is measured by assaying disrupted cells. The ratio of rate of hydrolysis by extracts to rate of hydrolysis by whole cells increases with increasing amount of enzyme per cell (Fig. 2), since the rate of entry remains the same independent of induction, in these bacteria which lack a permease, ,
L
,
I
I
I
. j
I: >
¢n.2 i-
z I
I0 20 30 40 UNITS ENZYME/ml (EXTRACTS)
Fig. 2. Hydrolysis rates of ONPG by intact vs. disrupted bacteria. Cultures of a permease-negative E. coli (C-6oo-i) were induced for intervals of up to 6o rain with 5" IO-* M IPTG. Samples were taken, at the same cell concentration, into 2 5 ~ug/ml chloramphenicol (to obtain intact cells), or into toluene and Sarkosyl (to obtain disrupted cells). The rates of ONIK} hydrolysis b y t h e s e preparations were determined. The curve indicates tS-galactosidase activities of bacteria versus extracts.
When ONPG hydrolysis by irradiated bacteria before and after disruption was compared, Fig. 3. was obtained. With low ultraviolet doses, the enzyme produced (measured in extracts) was much more strongly inhibited than the activity of the whole cell. If some cells had completely retained their enzyme-forming ability, the ratios of activities in extracts to those of whole cells would have remained constant. The actual result is predicted, from Fig. 2, if all individual bacteria lost a fraction of their enzyme-forming ability. Therefore, the ability of a bacterium to make fl-galactosidase is lost gradually and not in an all-or-none fashion. This partial inactivation of enzyme-forming ability might be attributed to the inactivation, one at a time, of the several nuclei contained in each bacterium. Each nuclear inactivation might reduce enzyme-forming ability to some extent. In order Bioc hi m . Bi ophy s . Acta, 76 (I963) 614-62I
618
A.B.
ULTRAVIOLET-LIGHT DOSE (er,cjs/mm=) 1000 2000
3000
I000
PARDEE, L. S. PRESTIDGE
,,,
-
""x cn30G -
N.%.
0 ).Z G. Z
I0O - -
30,
W
%.%
\
10
I
Fig. 3. O N P G hydrolysis b y intact and disrupted irradiated bacteria. E. coli C-6oo-i were irradiated for various times. The cultures were induced with 5" lO-4 M I P T G for 20 min. Abilities of broken ( O - G ) and whole ( 0 - 0 ) cells to hydrolyze ONPG were measured.
to test this idea the bacteria were starved of phosphate to make them uni-nucleate s. These bacteria were irradiated, induced, and assayed. The ratio of fl-galactosidase activities of intact and disrupted cells changed in the same way as it did with unstarved bacteria. Therefore, partial inactivation is not attributed to an inactivation of nuclei within a cell, one at a time, but is consistent with gradual inhibition.
Ultraviolet irradiation damage to galactoside permease production Ultraviolet irradiation inhibited induction of permease for accumulation of melibiose inside the bacteria '. The degree of inactivation was roughly the same as for inhibition of fl-galactosidase-forming ability. Activity of preformed permease and the permeability of the bacteria to galactosides were not altered b y irradiation. Another way of showing that permease formation is inhibited by ultraviolet light is based on the observation that a low concentration of inducer cannot induce fl-galactosidase unless a bacterium already possess galactoside permease 1°. This is seen in Fig. 4, Curve A: here uninduced bacteria are not induced by 1.5"Io-5M 1PTG. Curve B shows that bacteria which had previously been briefly exposed to 3" lO-4 M I P T G were readily induced b y 1. 5 • lO -5 M IPTG. These bacteria produced fl-galactosidase nearly as rapidly as did bacteria that were continually induced b y the higher I P T G concentration (Curve C). Therefore, presence of permease can be measured b y determining induction b y a low I P T G concentration. The inactivation of permease formation b y ultraviolet was measured in the same experiment. Bacteria were irradiated sufficiently to decrease fl-galactosidase induction with 3" lO-4 M I P T G by about 40 % (Curve D). After 12 min the inducer concentration was decreased to 1.5 "1o -5 M, whereupon the inhibition increased to Biochim. Biophys. Acta, 76 (1963) 614-62I
~-GALACTOSIDASE INDUCTION AND ULTRAVIOLET LIGHT
619
///J/ J~
"'
8
t$)
.-
of t~
oLld o
20
30
40
TIME OF INDUCTION (rain)
Fig. 4. Permease determination measured b y induction by dilute inducer. Sample A (non irradiated) of E. coli 2oooa was diluted 2o-fold and was induced with 1. 5. io -~ M IPTG. Samples ]3 and C (non-irradiated) and D and E (irradiated) were induced for 12 rain with 3' io-* M IPTG, and were then diluted 2o-fold into media (in which bacteria had previously been grown) lacking inducer (B and E) or to which 3" io-* M inducer was added (C and D). Assays for fl-galactosidase activity of disrupted cells were made at intervals.
about 75 % (Curve E). It is concluded the many (perhaps half) of the irradiated bacteria did not produce sufficient permease in the initial 12 rain to permit subsequent fl-galactosidase induction at the lower inducer concentration. Either a fraction of the irradiated bacteria lost all ability to produce permease, or most of the bacteria were able to produce permease at only a fraction of their original rate. To distinguish between these possibilities, the fl-galactosidase-forming ability attained after dilution of inducer was determined after different lengths of preinduction. The ability to form the enzyme did not increase as the preinduction period was lengthened. If many of the bacteria had retained ability to make some permease, the culture should have gradually gained ability to produce fl-galactosidase at low inducer concentration, as the time of pre-induction became longer. Therefore, permease-forming ability of individual bacteria seems to be reduced by ultraviolet light to a level too low to support fl-galactosidase induction, when it is reduced at all. Permease induction inactivation seems to be all-or-none, by this criterion. Ultraviolet irradiation sensitivity o[ fl-galactosidase /ormation be[ore vs. after induction
Penicillinase induction of Bacillus cereus n and fl-glucosidase induction of yeast ]~ are more sensitive to ultraviolet light before induction has started than after enzyme formation has commenced. The induction of fl-galactosidase by TMG in BiocMm. Biophys. Acta, 76 (1963) 614-621
620
A.B.
PARDEE, L. S. PRESTIDGE
wild type E. coli was also more sensitive to ultraviolet irradiation prior to induction than after enzyme formation has commenced (Fig. 5A). An explanation of this changed sensitivity is based on the fact that induction of galactoside permease is required in order to induce fl-galactosidase with TMG. Irradiation prior to induction damages both permease- and fl-galactosidase-forming abilities, and therefore inhibits 3
A
I
I
I
I
I
I
I
I
I>tr.
g (.3
3 <1
o,
20
40 O 20 TIME OF" INDUCTION (rnin)
4.0
Fig. 5. I r r a d i a t i o n before ( O - O ) a n d a f t e r ( A - A ) i n d u c t i o n ; ( O - O ) n o t irradiated. W i l d - t y p e E. coli 4ooo were i n d u c e d for io rain w i t h 5" IO-4 M T M G (A), or for 5 m i n w i t h i o -8 M I P T G (]3). A n o t h e r c u l t u r e w a s n o t i n d u c e d . O n e p a r t of e a c h c u l t u r e w a s t h e n irradiated, a n d a n o t h e r w a s not. fl-Galactosidase i n d u c t i o n w a s t h e n d e t e r m i n e d in e a c h of t h e s e cultures. A, p e r m e a s e r e q u i r e d ; B, p e r m e a s e n o t required.
induction by TNIG in two separate ways. But after induction has commenced and the permease is present, only the sensitivity of fl-galactosidase formation itself remains. Thus, after induction has started the number of ultraviolet light-sensitive targets is reduced from two to one. This hypothesis is supported by the fact tha~ induction with IPTG, an inducer which does not require a permease, is equally sensitive to ultraviolet light before or after induction (Fig. 5B). This suggests that the extra sensitivity of induction by Ti~G is related to formation of a permease. Another experiment which favors this conclusion is that when E. coli B were pre-induced with galactinol s so that they contained permease but not fl-galactosidase, their ultraviolet light sensitivity was then the same with either TMG or IPTG, and equal to the sensitivity of IPTG-induced bacteria. It is concluded that the specially ultraviolet light-sensitive step required to start fl-galactosidase induction is simply the induction of permease. CONCLUSIONS
Ultraviolet light inactivation of fl-galactosidase induction is complex. Ultraviolet light does not simply inactivate the structural gene for the enzyme. Rather, the general synthetic ability of the cell is inhibited slightly, through nucleic acid-containing targets. The great effect on fl-galactosidase synthesis seems at present best explained by a repression of this enzyme by catabolites accumulated by the damaged bacteria. A paradox is seen in that the inhibition of permease induction in an individual Biochim. Biophys. Acta, 76 (x963) 6 1 4 - 6 2 I
fl-GALACTOSIDASE INDUCTION AND ULTRAVIOLET LIGHT
621
bacterium can be virtually complete at doses of ultraviolet light which only partly inhibit/~-galactosidase induction. Ultraviolet light sensitivity of fl-galactosidase induction is greater prior to induction than afterwards. This is explained by the requirement for formation of permease before but not after induction has commenced. Changes in ultraviolet light sensitivity of other inductions might similarly be attributed to the existence of twostep inductions, one step of which (not necessarily permease) is eliminated shortly after induction has commenced. ACKNOWLEDGEMENTS
are indebted to D r . R . D o I for help with the technique of sucrose gradient c e n T h i s w o r k was aided by a Grant AI-o44o 9 from the United States Public Health Service. We
trifugation.
REFERENCES i G. RUSHIZKY, IV[. RILEY, L. S. PRESTIDGE AND A. B. PARDEE, Biochim. Biophys. Acta, 45 (196o) 7° . M. MASTERS AND A. B. PARDEE, Biochim. Biophys. Acta, 56 (I962) 6o9. a E. MCFALL AND B. 1V[AGASANIK, Biochim. Biophys. Acta, 55 (1962) 9o0. 4 S. Vq. BOWNE, Jr. AND P. ROGERS, J. Mol. Biol., 5 (1962) 90. A. G'AREN AND O. SIDDIQI, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1121. e O. H. LOWRY, N. J. ROSEBROUGVI, A. L. FARR, AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265 . ? M. NOMURA, B. D. HALL AND S. SPIEGELMAN, J. Mol. Biol., 2 (196o) 3o6. E. 1VicFALL, A. ]3. PARDEE AND G'. S. STENT, Biochim. Biophys. Acta, 27 (1958) 282. A. B. PARDEE, J. Bacteriol., 73 (1957) 376. :0 A. N o v l c K AND M. WEINER, Proc. Natl. Acad. Sci. U.S., 43 (1957) 553. n A. M. TORRIANI, Biochim. Biophys. Acta, 19 (1956) 224. 1~ H. HALVORSON AND L. JACKSON, J. Gen. Microbiol., 14 (1956) 26.
Biochim. Biophys. Acta, 76 (1963) 614-621