Gene photoinactivation in Escherichia coli which contain 5-bromodeoxyuridine-substituted DNA

112

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96414

G E N E PHOTOINACTIVATION IN E S C H E R I C H I A COLI WHICH CONTAIN 5-BROMODEOXYURIDINE-SUBSTITUTED DNA ROSELYN J. E I S E N B E R G * AND A R T H U R B. P A R D E E

Program in Biochemical Sciences, Mo]]ett Laboratory, Princeton University, Princeton, N.J. 08540 (U.S.A.) (Received September i2th, 1969)

SUMMARY

The DNA of Escherichia coli grown with 5-bromodeoxyuridine becomes a specific target for ultraviolet light at 313 nm. Bacteria not exposed to 5-bromodeoxyuridine have a low sensitivity at this wavelength. Sensitivity appears slowly and reaches a maximum in one generation, as measured by either inactivation of colony forming ability or capacity to induce fl-galactosidase. The bacteria are not sensitized when they are grown in the presence of nalidixic acid, an antibiotic that relatively specifically prevents DNA synthesis. When an F-lac episome is injected from a non5-bromodeoxyuridine containing donor into a recipient which had been grown in 5-bromodeoxyuridine medium, there is no loss of enzyme forming ability in the irradiated zygotes. This experiment argues against stable cytoplasmic targets of irradiation. After thymine auxotrophs are grown in 5-bromodeoxyuridine for one generation, irradiation kills or prevents enzyme formation by these bacteria by over 9 ° %. Since 5-bromodeoxyuridine can only be in one strand of the DNA after one round of replication, a lesion originating in either strand prevents both replication and transscription. Very possibly both strands are damaged. The photochemical damage can be localized in a single gene; inactivation of the gene for fl-galactoside permease without inactivation of the adjacent gene for fl-galactosidase was demonstrated. This method of labeling a gene with 5-bromodeoxyuridine during its replication and specifically inactivating it at another time can be applied to follow gene replication and transfer.

INTRODUCTION

Damage to DNA, as for example with ultraviolet light 1 or decay of incorporated 32p (ref. 2) or 3H incorporated in thymine, uracil or amino acids 3, can be used in the study of gene transcription. However, the ultraviolet light damage is not restricted to DNA (ref. i). The isotopes are built into macromolecules at the time they are Abbreviations: IPTG, isopropyl-fl-D-thiogalactoside; ONPG, o-nitrophenyl-fl-galactoside; TMG, methyl-thio-fl-galactoside. * Present address: Department of Microbiology, University of Pennsylvania, School of Dental Medicine, Philadelphia, Pa. 19104, U.S.A.

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made, a property that should be more specific for gene inactivation. But use of isotope decay for these studies has inconvenient drawbacks: the decay process takes a long time, and experiments are expensive and somewhat hazardous. In the hope of finding a more convenient but specific destructive agent, we turned our attention to the thymidine analog, 5-bromodeoxyuridine. G R E E R 4, among others, reported that substitution of the thymidine of DNA b y 5-bromodeoxyuridine makes bacteria much more susceptible to a photochemical loss of viability. SETLOW AND BOYCE5 showed that the m a x i m u m effect occurs at a wavelength about 31o nm where 5-bromodeoxyuridine absorbs fairly strongly. Normal DNA, containing thymine, absorbs very weakly at this wavelength and is little affected. However, the chemical basis for the increased sensitivity to longer wavelength ultraviolet light remains obscurO ,e. BONHOEFFER AND SCHALLER7 have used 5-bromodeoxyuridine sensitivity to select mutants conditionally defective in DNA synthesis. A process such as transcription or replication which depends on the integrity of DNA should be halted when 5-bromodeoxyuridine-DNA is irradiated, and could therefore be studied by this method. But reliable methods of incorporating 5-bromodeoxyuridine must first be developed, and it is important to be confident that the photochemical effect in a bacterium is confined to DNA, and is localized in its action. F o x AND MESELSON8 have used 5-bromodeoxyuridine incorporation and irradiation at 31o nm to demonstrate that different results are obtained b y damaging alternative strands of phage 2 DNA.

MATERIAL AND METHODS

Bacterial strains C S I o I - t h y - is an Hfr l a c + t h y - m e t - t h i - s t r s strain derived from CSIoI. 234o2 t h y - is an F - l a c I - l a c Z - t h y - p h e - t h i - s t r R derivative of 234o. 234o-2thy - (lac +) is a lac+ recombinant of CSIoI and 234o-2thy -. A327 is strS/F ' lac + obtained from Dr. S. LURIA. The thymine requiring mutants were isolated with the use of trimethoprim by the method of STACEY AND SIMSON9.

Bacterial growth The bacteria were grown aerobically by swirling at 37 ° in a New Brunswick gyrorotary shaker until they reached about 2-1o 8 cells/ml. A synthetic medium, M63 at p H 6.3 (ref. IO) plus 2 mg/ml glycerol and 2 mg/ml casamino acids (Difco) was used, supplemented with 5 #g/ml thiamin, 20 #g/ml amino acid and 40/zg/ml thymine as required. Generation times under these conditions were usually 50-60 rain. The bacteria were collected either b y centrifugation at 5000 rev./min for 15 rain or b y filtration on a membrane filter (Millipore type HA, 0.45 or 0.63 # pore size). The cells were then washed and resuspended in M63 salts to a density of 2. lO s cells/ ml.

5-Bromodeoxyuridine treatment Care was taken from this point on to protect the organisms from light. The cell suspension in red tinted flasks (Kimax Low Actinic glassware) was diluted with an equal volume of M63 salts containing 4 mg/ml casamino acids, 4 mg/ml glycerol, Biochim. Biophys. Acta, 204 (197 o) 112-119

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o.oi mg/ml thiamin, o.o4 mg/ml methionine or phenylalanine as needed, o.5 mg/ml adenosine and 0.2 mg/ml 5-bromodeoxyuridine or 0.08 mg/ml thymine. The bacteria were then grown for I h. They were filtered on a Millipore H A filter, were washed on the filter with M63 salts, and then the filter was placed in M63 salts at o°; the cells were suspended to give a density of 2. lO 7 to 5" lOT cells/ml.

Irradiation procedure Light from a high intensity mercury lamp (Philips, Type CS 5oo W) 11 was filtered through a 3 m m thick Corning filter No. 9863 taped to the cover of a Pyrex petri dish containing a 7.5 m m deep solution of 17.8 mM NiC12, 2.5 mM potassium biphthalate and 0. 5 mM potassium chromate 12. About 50 ~o of the light at 313 nm passed through the filter: most other light was filtered out. For irradiation, the bacterial suspension at a depth of 75-1oo m m in a beaker was placed in an ice bath on top of a magnetic stirrer 15 cm from the lamp. The suspension was stirred continuously with a small magnetic flea during irradiation.

fl-Galactosidase induction Portions of the cell suspension were placed in 25 m m diameter red tinted test tubes and warmed to 37 ° in a New Brunswick gyrorotary shaker, in a medium like the one used for growth but with no glycerol, to decrease catabolite repression 1°. fl-Galactosidase was usually induced for 30 rain with 5" I °-4 M isopropyl-fl-D-thiogalactoside. (Briefer inductions would have been desirable in order to avoid DNA synthesis in this period but the amount of enzyme formed would not give sufficient accuracy.) The enzyme was assayed by the O-nitrophenyl-/~-galactoside procedure 1°.

Mating procedure Exponential phase F' and F - bacteria (I-IO 8 cells/ml) were suspended in M63 medium supplemented with growth requirements but lacking glycerol. Thymine or 5-bromodeoxyuridine was added to these cultures at various times prior to or after mating. Asparagine (final conc. 1.35 mg/ml) was added to the F - cells just prior to mating. The cells mixed in a ratio of I F' : 2 F - were incubated in a shallow layer with slow shaking usually for IO rain. Then further mating was prevented by adding Duponol (sodium dodecyl sulfate) at a final concentration of 30 #g/ml and b y vigorous shaking. Streptomycin (final conc. 250#g/ml) was added to prevent enzyme synthesis b y the F' cells. The cells were placed in an ice bath during irradiation, and then returned to the 37 ° shaker for induction of fl-galactosidase.

Plating to determine viability Portions of the irradiated cells along with appropriate controls were plated on Penassay Agar (Difco) supplemented with 0. 5 ~tg/ml thiamin and 4 °/zg/ml thymine and incubated overnight at 37 ° in the dark.

Sources o/chemicals Nalidixic acid was a gift from Mr. C. E. Carl of Winthrop Laboratories.

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RESULTS

Dosage response o/ 5-bromodeoxyuridine-containing bacteria to 3z3-nm light S t r a i n 2 3 4 o - 2 t h y - was grown in 5 - b r o m o d e o x y u r i d i n e m e d i u m for one generation time (6o rain) so t h a t only one s t r a n d of the double s t r a n d e d D N A helix would be labeled. Growth was e x p o n e n t i a l for a b o u t 7 ° m i n a n d t h e n stopped. W h e n this p o p u l a t i o n was i r r a d i a t e d (Fig. I), the decline in colony forming ability was a function of the r a d i a t i o n dose, declining to 1 % of the u n i r r a d i a t e d controls. I n d u c t i o n of fl-galactosidase dropped to less t h a n IO % of the u n i r r a d i a t e d control. Briefer 5 - b r o m o d e o x y u r i d i n e i n c o r p o r a t i o n gave a biphasic curve for e n z y m e f o r m a t i o n vs. ultraviolet light dose. F o r example, 30 m i n growth plus 5 - b r o m o d e o x y u r i d i n e resulted in a m a x i m u m loss of 30 %, reached with a b o u t 15 m i n irradiation. More t h a n 9 ° % of the i r r a d i a t e d cells were killed. This suggests t h a t n e a r l y all cells took u p 5 - b r o m o d e o x y u r i d i n e b u t only 30 % of t h e m were labeled in the lac region. The result argues against a general i n h i b i t i o n such as could be caused b y catabolite repression. T h y m i n e grown cells were i n a c t i v a t e d at only a b o u t IO % the rate of those grown in 5 - b r o m o d e o x y u r i d i n e for I h. Similar losses of e n z y m e f o r m a t i o n a n d v i a b i l i t y were observed with all of the strains t h a t required a high (4 ° # g / m l ) c o n c e n t r a t i o n of t h y m i n e . There was less effect on one s t r a i n t h a t required only 2/~g/ml. A slight shoulder in the i n a c t i v a t i o n curve for e n z y m e i n d u c t i o n was sometimes seen b u t n o t always.

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Fig. i. Inactivation of 234o-2thy- by 313-nm light. Cells were grown in 5-bromodeoxyuridine (O, A) or thymine (O, A) medium for i h, washed and resuspended in M63 salts. 5-ml portions were irradiated for various times. Part of each sample was diluted and plated on Penassaythymine (&, A) and the rest was induced for fl-galactosidase with 5' lO-4 IPTG (S, O)- The number of cells or amount of enzyme in the irradiated samples is expressed as percent of the value for the unirradiated samples. Fig. 2. Sensitization by growth of CSIoI thy- in 5-bromodeoxyuridine medium. Samples of a culture growing in 5-bromodeoxyuridine medium were removed at intervals. These samples were then quickly filtered, washed and resuspended in cold M63 salts. For each time interval, one portion was irradiated for io rain and another was not (unirradiated control); then both samples were induced for fl-galactosidase with 5" lO-4 M IPTG. The enzyme formed in the irradiated sample is expressed as % of its unirradiated control.

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Acquisition o! photosensitivity The number of cells sensitive to IO min irradiation increased approximately linearly during growth in medium containing 5-bromodeoxyuridine, for 60 min (Fig. 2).

Prevention by nalidixic acid o/the 5-bromodeoxyuridine inactivation Nalidixic acid which is known to block DNA synthesis in E. coli (ref. 13) should prevent 5-bromodeoxyuridine incorporation into DNA and its consequences. Table I shows that for two thymineless strains of E. coli exposed to both 5-bromodeoxyuridine and nalidixic acid the enzyme forming ability and colony forming ability of the irradiated samples were nearly the same as the unirradiated controls. TABLE I EFFECT

OF NALIDIXIC

ACID ON 5-BROMODEOXYURIDINE

EFFECTS

Cells were g r o w n in m e d i u m containing t h y m i n e or 5 - b r o m o d e o x y u r i d i n e and nalidixic acid (2o/~g/ml) for i h, w a s h e d and resuspended in M63 salts. A 5-ml p o r t i o n was irradiated for IO min while a n o t h e r 5-ml portion was not. P a r t of each sample was diluted and plated on P e n a s s a y t h y m i n e agar and the rest was induced for fl-galactosidase.

fl-Galactosidase induction Viability alter irradiation (% non-irradiated control) after irradiation (% non-irradiated control)

Growth condition

Strain : Thymine T h y m i n e + nalidixic acid 5-]3romodeoxyuridine 5 - B r o m o d e o x y u r i d i n e + nalidixic acid

234o-2thy-

CSIoI-thy-

234o-2thy-

CSIoi-thy-

94 92 15 iio

i io lO4 15 lO 5

I io 14o 0.9 57

113 13 ° o. 8 200

Unexpectedly, nalidixic acid itself was extremely toxic to these strains. At a concentration of 20 #g/ml the antibiotic reduced both viability and enzyme forming ability of strain C S I o I - t h y - b y over 9 ° %. Even at very low nalidixic acid concentrations (2 #g/ml) there was a considerable loss of enzyme forming ability. In strain 2340-2 thy-: the effect was less severe but still substantial. These results were especiaUy surprising since the inhibitor was only present for I h and was removed before induction or plating.

Conjugation o/ an F' lac with a 5-bromodeoxyuridine containing recipient To determine whether 5-bromodeoxyuridine acts in the cytoplasm, the recipient strain F - 2340-2 t h y - (lac-) was preloaded b y growing it plus 5-bromodeoxyuridine for I h. Then it was mated for 2o min with a t h y + donor that was not exposed to 5-bromodeoxyuridine, in the presence of thymine. The zygotes were irradiated with a strong dose of 313-nm light./3-Galactosidase forming ability in the irradiated bacteria was not reduced (Table II). Under these conditions recipient cells which contain 5-bromodeoxyuridine are sensitive to irradiation as shown by a drop in viability. It is concluded that the sensitive target does not reside in stable cytoplasmic molecules contributed b y the recipient. Other mating experiments plus 5-bromodeoxyuridine support this conclusion 14. Biochirn. Biophys. dcta, 204 (197 o) t12-119

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TABLE II E X P R E S S I O N OF AN

F ' LAC E P I S O M E IN A R E C I P I E N T C O N T A I N I N G 5 - B R O M O D E O X Y U R I D I N E

Strains A327 and 2340-2 t h y - were g r o w n to a b o u t I- Io*/ml. Half of t h e F - s t r a i n was g r o w n plus o.i m g / m l 5 - b r o m o d e o x y u r i d i n e (final conc.) d u r i n g the final hour; at the time m a t i n g w a s started, o.I m g / m l t h y m i n e (final conc.) w a s added. Mating was t e r m i n a t e d at 2o rain, s t r e p t o m y c i n w a s added and p o r t i o n s of the cultures were irradiated for io rain. Following irradiation fl-galactosidase w a s induced with I P T G (5' lO-4 M). Viability was m e a s u r e d b y plating s a m p l e s of F - cells which were carried t h r o u g h the same procedure except t h e y were n o t mated.

Nucleoside present during growth of F prior to mating

Add thymine at time of mating

Irradiated after mating

Arbitrary units ot fl-galactosidase formed in zygotes

Viable F - cells/ml, × zo -~

Thymine Thymine 5-Bromodeoxyuridine 5-Bromodeoxyuridine

yes yes yes yes

no yes no yes

324 320 IOO 87

5.9 6.9 12.5 o.o6

Di]/erent e//ects on induction by two inducers

o/ fl-galactosidase /ormation

An experiment was next performed to learn if the photochemical lesion extends over more than one gene. The principle is the same as one used previously with light at 260 nm (ref. IO). If a Y gene which controls fl-galactoside permease formation could be inactivated without inactivation of the adjacent Z gene which determines the structure of /~-galactosidase, this cell would be able to make the enzyme but not the transport system. I t should be induced by I mM I P T G which can induce Y mutants maximally and does not require the permease, but not by I mM methylthio-fl-galactoside which needs the permease to achieve a maximal rate of induction within about 20 min (seen with the unirradiated sample of Fig. 3)-

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Fig. 3- I r r a d i a t i o n of 5 - b r o m o d e o x y u r i d i n e labeled cells: induction w i t h t w o inducers. Strain C S I o I t h y - w a s g r o w n for 6o m i n in 5 - b r o m o d e o x y u r i d i n e medium. D u r i n g t h e last 15 m i n one p a r t of t h e culture was induced w i t h io -s M I P T G (open symbols). The r e m a i n d e r of the culture was n o t induced (solid symbols). B o t h cultures were filtered, w a s h e d and r e s u s p e n d e d in cold M6 3 salts. One p a r t of each culture was irradiated for 4 m i n ( A, A) a n d a n o t h e r was n o t ( O , O ) . These cultures were divided again and fl-galactosidase was induced b y lO -a M TMG or b y lO -3 M IPTG.

t~iochim. Biophys. Acta, 2o 4 (i97o) 112-119

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R.j.

EISENBERG, A. B. PARDEE

After irradiation, I P T G induced enzyme much more rapidly than did TMG (Fig. 3). When I P T G was the inducer, the rate of enzyme formation after irradiation b y a low dose of 313 nm light was 33 °//o of the unirradiated control; but when TMG was the inducer the rate of enzyme formation was only IO °/o of its control. The final rate was reached b y 20 rain in all cases. However, if the culture was preinduced for 15 min prior to irradiation, to form permease, the rate of induction in the irradiated samples were similar for the two inducers. This preinduction had no effect on enzyme formation after irradiation when I P T G was the inducer. The results are consistent with inactivation of the Y gene without inactivation of the Z gene in m a n y cells.

DISCUSSION

The results presented here indicate that after 5-bromodeoxyuridine is incorporated into either strand of E. coli DNA, irradiation with 313-nm light kills the cells and also inactivates enzyme formation. F o x AND MESELSON8 showed that photochemical damage to one but not the other 5-bromodeoxyuridine-containing strand of phage 2 DNA was lethal. This result is the opposite of ours. The difference might depend on replication of the undamaged strand of the phage prior to its function, as they suggest, or it might be because they prevented secondary effects of free radicals with the agent AET. In E. coli the damage appears to be localized in a single gene, since some cells after irradiation seem to be able to induce fl-galactosidase but not galactoside permease which is coded for by an adjacent gene. This result could alternatively be explained if both inductions were partly inactivated in each cell, as for example by catabolite repression. This could create a long lag (greater than 8o min to explain Fig. 3) before maximal rate is reached in irradiated cells induced by TMG. This hypothesis is not supported b y results obtained with ultraviolet light at 254 nm, which show the effects are all-or-none for enzyme and permease formation and that catabolite repression is not serious under these conditions 1°. It is noted that the gene inactivation rate is surprisingly high relative to lethality, about 1/7 as rapid. Similar ratios have been noted previously with other inactivation techniques 1-3, which might be in part attributed to gradual reactivation after plating and to some residual effects of catabolite repression in reducing enzyme forming ability. The gradual appearance over i h of sensitivity as measured by/3-galactosidase inducibility suggests that 5-bromodeoxyuridine incorporation commences at random points on the chromosome of asynchronously growing cells. ABE AND TOMIZAWA 1~ reported that when E. coli K-I2 are placed in 5-bromodeoxyuridine medium, replication ceases at the growing point of the chromosome, and is reinitiated at an origin located between the lysine and histidine genes, with replication proceeding in a clockwise direction. Replication then would require 20-30 rain to reach the lac operon, which is located more than halfway around the chromosome from this origin. Little or no/5-galactosidase inducibility should be lost until the entire population replicates the lac operon, whereupon a steep decline in induction should occur. Instead, as seen in Fig. 2, we found an approximately linear decrease in/~-galactosidase inducibility for about I h. Probably in our system 5-bromodeoxyuridine is Biochim. Biophys. Acta, 204 (197 o) i I 2 - i t 9

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incorporated into DNA strands at the existing growing point of the K-I2 chromosome, not at an induced origin. The reason for the disparity between our conclusion and that of ABA AND TOMIZAWAmay be a technical one. WOLF et al. 1~ reported that traces of thymine in the 5-bromodeoxyuridine medium prevent reinitiation at the origin. The problem may be related to our use of strains with a high (4° pg/ml) thymine requirement; ABE AND TOMIZAWA used low-thymine requiring mutants. These results indicate that 5-bromodeoxyuridine can be used to study growth of the chromosome.

ACKNOWLEDGEMENTS

This work was aided by Grant AI-o44o9 from the U.S. Public Health Service. Preliminary experiments were performed by L. S. Prestidge AND Dr. Y. Takagi. R.J.E. is a Postdoctoral Research Fellow of the National Institutes of Health. REFERENCES i 2 3 4 5 6 7 8 9 io II 12 13 14 15 16

A.. B. PARDEE AND L. S. PRESTIDGE, J. Bacteriol., 93 (1967) 121o. E. McFALL, A. B. PARDEE AND G. S. STENT Bioehim. Biophys. Acta, 27 (1958) 282. M. RACHMELER AND A. B. PARDEE, Biochim. Biophys. Acta, 68 (1963) 62. S. GREER, J. Gen. Microbiol., 22 (196o) 618. R. B. SETLOW AND R. BOYCE, Bioehim. Biophys. Acta, 68 (1963) 455, M. B. LION, Biochim. Biophys. Acta, 155 (1968) 505 . F. BONHOEFFER AND H. SCHALLER, Biochem. Biophys. Res. Commun., 2o (1965) 93. E. F o x AND M. MESELSON, J. Mol. Biol., 7 (1963) 583 • K. A. STACEY AND E. SIMSON, J. Bacteriol., 90 (1965) 554. A. ~B. PARDEE AND L. S. PRESTIDGE, Biochim. Biophys. Aeta, 76 (1963) 614. J. R. WALKER AND A. B. PARDEE, J. Bacteriol., 95 (1968) 123. A. D. McLAREN AND D. SHUGAR, Photochemistry o] Nucleic Acids and Proteins, P e r g a m o n , N.Y., 1964, p. 372. W. A. Goss, W. H. DEITZ AND T. M. COOK, J. Bacteriol., 89 (1965) lO68. R. EISENBERG AND A. B. PARDEE, J. Mol. Biol., 46 (i969) 355. 1V[. ABE AND J. TOMIZAWA, Proc. Natl. Acad. Sei. U.S., 58 (1967) 1911. B. WOLF, A. NEWMAN AND D. A. GLASER, J. Mol. Biol., 32 (1968) 611.

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