JOURNAL OF BACTERIOLOGY, Sept. 1969, p. 815-823Copyright © 1969 American Society for Microbiology

Vol. 99, No. 3Printed In U.S.A.

Effects of Ultraviolet Radiation on Respiration andGrowth in Radiation-resistant and Radiation-

sensitive Strains of Escherichia coli B1BARBARA A. HAMKALO2 AND P. A. SWENSON

Department of Zoology, the University of Massachusetts, Amherst, Massachusetts, and the Biology Division,Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830

Received for publication 2 June 1969

Ultraviolet (UV) irradiation at 254 nm causes different respiration and growthresponses in log-phase cultures of Escherichia coli B/r and B._1 . These differencesare correlated with the ability and inability, respectively, of these bacterial strainsto repair UV-induced lesions in deoxyribonucleic acid (DNA). After irradiation,B5_1 cells (radiation-sensitive) exhibit uncoupling of growth and respiration; growthand synthesis cease, whereas respiration continues. B/r cells (radiation-resistant)grown on glycerol exhibit severe temporary inhibition ofgrowth and respiration afterUV, and the coupling of these two processes is maintained, except at a very highUV dose. Inhibition begins at about the time DNA synthesis resumes and con-tinues for a period of time that is dependent upon dose. Glucose-grown cells do notexhibit severe respiratory, growth, and synthetic inhibitions; these processes remaincoupled in the cells during the postirradiation period. Photoreactivation treatmentdelays uncoupling of growth and respiration in B.-, and prevents inhibition of res-piration and growth in B/r. These results indicate that the postirradiation responsesresult from the presence of pyrimidine dimers in DNA. Ultraviolet irradiation ofB/r and B.-1 cells results in an accumulation of adenosine triphosphate by 30 minafter UV. This accumulation decreases with time and does not appear to be relatedto the inhibition of respiration in glycerol-grown B/r cells. The results on B/r areinterpreted in terms of a control mechanism for reestablishment of a balance amongmacromolecules in the irradiated cells so as to provide them with the potential tosurvive. The specific steps in such a reestablishment of balance appear to dependupon the substrate oxidized. In B.-, cells, which cannot repair UV-induced damagein DNA, some control mechanism that coordinates cellular processes may be in-activated.

Ultraviolet (UV) radiation produces intra-strand cyclobutane-type pyrimidine dimers indeoxyribonucleic acid (DNA) (28). These andother photoproducts (24) can be lethal, and theultimate fate of irradiated cells is a function ofthe numbers and types of such products (21)and the ability of cells to repair the UV-induceddamage (18). Some bacterial strains, such asEischerichia coli B/r, possess the ability to repair,in the dark, UV-induced lesions in DNA (4, 22),and consequently are more resistant to UV-

' Submitted by B. A. Hamkilo in partial fulfillment of the re-quirements for the Ph.D. degree.

2 Oak Ridge Graduate Fellow from the University of Massa-chusetts under appointment from the Oak Ridge Associated Uni-versities. Present address: Department of Biological Chemistry,Harvard Medical School, Boston, Mass. 02114.

killing than strains such as E. coli B.-1, whichlack the dark-repair mechanism (22).

After UV irradiation of B8.1 cells, inhibition ofDNA synthesis is permanent (23, 26) and DNAbreakdown follows (25). As a result of the dark-repair mechanism in B/r, DNA synthesis is in-hibited by UV, but resumes after a period oftime (23) if cells possess the potential to survive.However, not all of the cells are necessarilyviable in such a population. In fact, survival(colony-forming ability) may be quite low.

Physiological processes, such as respiration, arereportedly more resistant to UV irradiation thanis viability (8, 13). Thus, a population of cellsmay be inactivated, as indicated by low survival,but these "dead" cells may continue to carry out

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HAMKALO AND SWENSON

some cellular functions for a time after UVirradiation. The study of irradiated populationspresents difficulties in interpretation because wedo not know, for example, how the physiologyof an irradiated bacterium destined to form acolony differs from the physiology of an irradi-ated cell destined to die. Use of strains of bac-teria that represent extremes in radiation re-sistance and sensitivity facilitates interpretationsof the responses of cell populations to UV ir-radiation.

MATERIALS AND METHODS

Bacterial strains. Escherichia coli B/r (ORNL) andE. coil B.-1, characterized by extremes in radiationresistance and sensitivity, respectively, were used. Ob-tained from Ruth Hill, E. coli H/r, an irradiation-resistant strain which lacks the photoreactivatingenzyme and is phr- (10), was used in photoreactiva-tion (PR) experiments.

Media. M63 (17) contained (per liter): 2.0 g ofglycerol, 2.0 g of (NH4)2S04, 13.6 g of KH2PO4,0.2 g of MgSO4.7H20, 0.5 mg of FeSO4.7H20, and0.5 mg of thiamine. The pH was adjusted to 7.0 withconcentrated KOH. M9 (17) contained (per liter):4.0 g of glucose, 1.0 g of NH4Cl, 5.0 g of NaCl, 6.0 gof Na2HPO4, 3.0 g of KH2PO4, and 0.1 g of Mg.S047H20. Media were supplemented with a finalconcentration of 0.025% Casamino Acids (Difco);supplement is denoted as M63+ and M9+.

Growth of cells. Cultures of bacteria were inocu-lated from nutrient agar plates into 10 ml of mediumin a 25-mi Erlenmeyer flask. An 0.2- to 0.3-ml amountof a cell suspension grown overnight was inoculatedinto 20 ml of the same medium, and cells were grownto log phase (2 X 108/mi, as determined by a Lu-metron colorimeter). For experiments in whichgrowth was observed after UV irradiation, cells wereincubated in 50-ml Erlenmeyer flasks to whichspectrophotometer cuvettes were attached as side-arms. Flasks were incubated by shaking at 37 C in arotatory, shaking water bath (New BrunswickScientific Co., New Brunswick, N.J.). Changes inabsorbance at 650 nm were measured in a DU spec-trophotometer (Beckman Instruments, Inc., Fuller-ton, Calif.).

Irradiations were performed by three methods. Alloperations during and after UV irradiation werecarried out in yellow light to avoid unwanted PR.

(i) Irradiation in Warburg vessels. A 2-ml amountof cells in growth medium at 2 X 108 cells per mlwere irradiated in quartz Warburg vessels (bottomdiameter, 3.3 cm) in a Warburg bath. Irradiationswere performed with a 15-w General Electric germi-cidal lamp (G15T8, principal emission at 254 nm)which rested at the bottom of the Warburg bath. Analuminum shield with a 0.32 cm slit was used tocover the lamp to obtain the desired incident doserate. The dose rate at the vessel bottom was 10ergs/mm2 per sec, as measured with a meter de-scribed by Jagger (11). Since the depth of the cellsuspension was about 2 mm, the absorption by the

suspension was small, and no corrections were madefor average intensity through the sample.

(ii) Irradiation at 265 nm. Log-phase cells (2 ml) at4 X 107 cells per ml were stirred and irradiated inquartz cuvettes with monochromatic light (265 nm)from a Hilger quartz-prism monochromator. Theincident intensity was 13 ergs/mm' per sec, as meas-ured with a calibrated photocell. The average intensitythrough the sample was calculated according to themethod of Morowitz (15).

(iii) Irradiation in open dishes. A 40-ml amount ofstirred bacterial suspension at 4 X 107 cells per mlwas irradiated in a crystallizing dish 100 mm indiameter. The depth of the suspension was 7 mm, andits surface was 35 cm below two 15-w General Electricgermicidal lamps (G15T8). The lamps were in adesk-lamp holder. The dose rate at the surface of thecell suspension was about 12.5 ergs/mm2 per sec, asmeasured with a meter described by Jagger (11). Nocorrections were made for the average intensitythrough the sample.The three methods for PR were comparable to the

three irradiation methods.(i) PR in the Warburg bath. PR treatment was

with a 15-w General Electric black lamp (F15T8-BLB,emission maximum 360 nm) that was located 1 cmfrom the sides of the shaking Warburg vessels. Il-lumination time was 10 min.

(ii) PR at 405 nm. A 2-ml amount of log phasecells at 4 X 107 cells per ml was stirred in glasscuvettes and illuminated with monochromatic light(405 nm) from a Hilger monochromator. The incidentintensity was measured with a calibrated thermopileand breaker amplifier. The 405-nm dose was 4.2 X 106ergs/mm2. Illumination time was 20 min.

(iii) PR in open dishes. A 40-ml amount of ir-radiated cells at 4 X 107/ml was stirred in a 100-mmdiameter crystallizing dish while being illuminated atroom temperature with light from two GeneralElectric black lamps (F15T8-BLB) mounted in adesk lamp 9 cm above the surface of the cell suspen-sion. Photoreactivation at 37 C was carried out in aWedco incubator. Output of the lamp is 300 to 400nm, with a maximum at 360 nm. Wavelengths below310 nm were filtered out by means of a plate ofwindow glass 0.64 cm thick. Black light dose was7.8 X 104 ergs/mm', as measured by a meter de-scribed by Jagger (11). Illumination time was 20 min.

Respiration measurements. Cells in Warburgvessels were equilibrated for 10 to 15 min at 37 C inthe Warburg bath. Irradiation followed equilibrationand was measured at 15-min intervals according tothe method of Umbreit et al. (27).

Survival curves. An 0.1-ml amount of irradiated(265 nm) cell suspension was removed after each doseof UV irradiation, and dilutions were made in theappropriate growth medium. Cells were plated eitheron nutrient agar plates or on synthetic medium plates,made by the addition of 1.5% agar to synthetic media(M63 or M9), plus Casamino Acids. Plates wereincubated overnight, or longer for the slower growingsynthetic medium plates, at 37 C in the dark.DNA synthesis. DNA synthesis was measured by

the incorporation of 3H-thymidine into cold tri-

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EFFECTS OF UV IRRADIATION ON E. COLI

chloroacetic acid-insoluble material by the filter-paperdisc method of Bollum (3). Each tube contained 5,liters of 3H-thymidine (6.7 c/mM; 1 mc/ml), 20,Aliters of adenosine (2.5 mg/ml), and 200 Mliters of acell suspension at 4 X 107 ml. The cell suspension wasadded to each tube at zero time; tubes were incubatedat 37 C; at intervals, 10-,uliter samples were withdrawnand spotted on Whatman no. 1 filter-paper discs. Theacid-soluble material was removed by washing thediscs in cold 5% trichloroacetic acid. Discs wererinsed with 95% ethyl alcohol, and were dried beforecounting in a Tri-Carb liquid scintillation spec-trometer (Packard Instrument Co., Inc., DownersGrove, Ill.). The scintillating fluid was 2,5-bis[2-5-(tert-butylbenzoxazolyl)] -thiophene (Packard In-strument Co.) in toluene. Isotopes were obtainedfrom New England Nuclear Corp., Boston, Mass.

Adenosine triphosphate assays. A 2.0-ml amountof cells at 2 X 108/ml were collected by centrifugationat 8,000 X g for 5 min. The supernatant fluid waskept on ice for assay, and the pellet was suspended byvortex mixing in 2 ml of medium. Cells were brokenopen by using a Bronson Sonifier (scale setting, 1) for15 sec. ATP was assayed by the procedure of Seligerand McElroy (19), with crude extracts of fireflylanterns (Sigma Chemical Co., St. Louis, Mo., andWorthington Biochemical Corp., Freehold, N.J.)used as a source of luciferin and luciferase. In thepresence of oxygen, excess luciferase reacts withluciferin and ATP to produce a light flash. Over arange of ATP concentrations from 10 mg/ml to 5Jg/ml, the intensity of the flash is linearly propor-tional to the ATP concentration.The reaction mixture of 2.1 ml of glycylglycine

(0.025 M, pH 7.5), 0.1 mg of MgSO4 (0.1 M), and 10to 50 ,uliters of firefly extract was placed in a glasscuvette. Four cuvettes were mounted in the turretof a light-tight sample holder (Schoeffel Instruments,Westwood, N.J.). The cells could be successivelyrotated into a position directly in front of an end-window photomultiplier.To elicit the light flash, 0.2 ml of the sample was

injected through a syringe cap into the cuvette directlyin front of the photomultiplier. The current output ofthe photomultiplier developed a voltage across aresistor; this voltage was applied to a voltage-to-frequency converter (Hewlett-Packard Co., PaloAlto, Calif.) that produced a chain of pulses (104/secper v). The rate of these pulses is directly proportionalto the applied voltage, and, hence, to the intensity ofthe light flash, and also to the concentration of ATPin the sample. The pulses were counted by electroniccounters (Hewlett-Packard) for 10 sec, 2 min, or 4min. The 10-sec counting was controlled electroni-cally by a separate preset counter with an internalclock (Hewlett-Packard).

RESULTSRespiration after irradiation in B/r and B.-1

cells grown on glycerol. Fig. 1 shows the effectsof different UV doses on the respiration of log-phase cultures of E. coil B/r and B9.1 cells,grown on glycerol. Respiration in B,,- (Fig. la)

is only slightly affected by UV irradiation. InB/r (Fig. lb), severe respiratory inhibition isobserved after doses in the range of 375 to 700ergs/mm2. These observations were not expectedon the basis of the abilities of cells to repair UVdamage, nor on the basis of the l /e doses (re-quired to reduce survival to 37%) for the twostrains (200 ergs/mm2 for B/r, and 1 erg/mm2for B8.1).The postirradiation respiratory responses of

B/r cells shown in Fig. lb are not simple dose-dependent decreases in respiratory rate. A lowdose of UV irradiation (125 ergs/mm2) decreasesthe initial rate of oxygen consumption for 1 hr,after which respiration resumes at a rate closeto that of unirradiated controls. After higherUV doses, respiration is relatively unaffected for1 hr. After this time, respiration is severely in-hibited. A decrease in the number of survivorsdoes not appear to be the reason for the lack ofoxygen consumption, since respiratory inhibitionbegins at the same time, regardless of dose, andafter the same amount of oxygen consumption.

Unirradiated B.-1 cells grow more slowly andrespire at a lower rate compared to B/r cells(Fig. 1). The division times are 60 and 50 min,respectively, in M63+ medium. The rate of res-

a UNIRRAD.

500-E5cC 125

/Lo/o 250 ergs/mm2*0 t

300- 500J

X10012

0 40 80120160200

Q b UNIRRAD//O 1

0 20 40 60 80 100 120 140TIME AFTER UV (min)

FIG. 1. Respiration in glycerol-grown, UV-irradi-ated, log-phase cultures of E. coli B,1 (a) and B/r (b).Irradiation method (i) was used (see Materials andMethods).

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piration of B8.1 cells after three different dosesof UV irradiation is the same as that of the con-trols for about 60 min after UV. After this time,the rate of oxygen consumption decreases withdose. Inhibition of respiration is exaggeratedbecause these cells are not growing or dividing,as are unirradiated cells (see Fig. 2c).

Respiration and growth after UV. Growth andrespiration are normally coupled in log-phasecultures of aerobic microorganisms. In B/r cellsgrowth and respiration remain coupled afterdoses of 375 and 500 ergs/mm2 (Fig. 2a). Whenrespiration is severely inhibited, the growth rateis reduced concomitantly. Ribonucleic acid andprotein synthesis are also severely inhibited dur-ing respiratory inhibition (B. A. Hamkalo,Ph.D. thesis, Univ. of Massachusetts, 1968).The maintenance of coupling of growth andrespiration in irradiated B/r cells is shown for atypical experiment in Table 1. The values ofAMliter 02/A optical density (OD) remain closeto control values. Although Fig. 2a representsa typical experiment, some runs do show oxygenconsumption of from 1 to 2 uliters of 02/minduring the period of severe respiratory andgrowth inhibition. In Fig. 2a, it is also seen thatthe duration of inhibition of these processes inB/r is dose dependent, although the time atwhich inhibition sets in is independent of dose.

If B/r cells are irradiated with a massive doseof UV (5,000 ergs/mm2), some cell lysis occurs;further, growth and respiration no longer showparallel responses (Fig. 2b). The fact that such ahigh dose destroys the coordination betweengrowth and respiration may indicate the lack ofrepair of UV-irradiation damage, and damage toorganelles in heavily irradiated cells. This situa-tion is similar to the one seen in less heavilyirradiated B8.1 cells (Fig. 2c and Table 1), in whichrespiration continues for several hours after 500ergs/mm2 of UV irradiation, but in which thereis little net increase in cell mass. The A,uliters

02/AOD value of irradiated B81 cells is 10-foldhigher than the value in unirradiated controls.Such a deviation in this value from that of controlcells is an indication of uncoupling of growthfrom respiration.

Photoreactivation (PR). PR treatment after UVirradiation was used to establish the relationshipbetween pyrimidine dimers in DNA and theobserved physiological changes. In Fig. 3a, it isobserved that the severe temporary respiratoryinhibition in B/r is photoreactivable by eithernear-UV irradiation (black light), or mono-chromatic illumination (405 nm). The facts that405-nm treatment reverses the UV irradiationeffects and that PR of respiratory inhibition doesnot take place in E. coli H/r (Fig. 3b), a strainwhich is deficient in the PR enzyme, almostcertainly implicate DNA pyrimidine dimers in

TABLE 1. Coupling of growth and respiration afterUV irradiationa

Glycrol-rown Time intervalG rrwns after UV Asliters 02/AOD6b0strains irradiation

Escherichia coliB/r

Unirradiated 15-30 min 103/.078 = 1,400105-120 min 124/.08 = 1,600

500 ergs/mm' 15-30 min 122/.062 = 2,000105-120min 12/.009= 1,300

5,000 ergs/ 15-30 min 40/0 = Xmm2

E. coli B.1Unirradiated 15-30 min 25/.018 = 1,400

105-120 min 40/.03 = 1,300500 ergs/mm' 15-30 min 30/.01 = 3,000

105-120 min 25/.0025 = 10,000500 ergs/mm2 15-30 min 35/.018 = 2,000

105-120 min 18/.008 = 2,300

a The data for B/r are taken from Fig. lb and2b; those for B.-, are from Fig. la and 4.

a 375 rs /mmIE. coli 8/r /

I~~~~~~~~~~~~~~~~~~~~~~~/-////°/^0.|FF t Z / Sz / /~~~~~d'/ _.~~~~~~~I-

-055

045

035

025

0.15

-005

bE col/ i/r5000 ergs/I,2

- 004

w --0.-4-0.04

40, 80 120 20024.

280 3203600 40 80 120 160 200 240 280 320 360 0 20 40 60 80 100

- cEcoll BS_

UNIRRA / - 0.35

. 0.25 o

ayo- 0.150 5000 ergs/mmI i ~~~~0.050 40 80 120 i60 200

TIME AFTER UiV (min)

FiG. 2. Respiration and growth, as measured by changes in turbidity at 6S0 nm, in unirradiated and UV-ir-radiated, glycerol-grown, log-phase cultures ofE. coli Blr (a) and (b), and E. coli B.-1 (c). Circles indicate oxygen

consumption; triangles, turbidity changes. irradiation method (i) was used.

500

0

0

A.

aw

300-

100*

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EFFECIS OF UV IRRADIATION ON E. COLI

the temporary inhibition of respiration (20).Figure 4 shows the effect of PR treatment onrespiration and growth of UV-irradiated culturesof B.-1, and it can be seen that PR treatmentpermits growth to continue for several hours(Fig. 4). Thus, PR reestablishes the coordinationbetween growth and respiration, and also reducesthe AMliter 02/AOD value of irradiated B/r cellsfrom 10,000 to 2,000 (Table 1).

Effects of UV irradiation on the oxidation ofglucose by B/r. Figure 5 shows that respirationof UV-irradiated, glucose-grown cultures of B/ris not severely inhibited after a dose of 500 ergs/mM2 of UV irradiation, although there are slightinflections in the curve at 60 and 100 min. Therespiratory rate is only slightly lower than thatof unirradiated cells, in agreement with Kelner(14).

Survival after UV irradiation. The respirationdata for glycerol- and glucose-grown B/r cellsshow a difference in metabolic response to UVirradiation that is dependent upon the carbonsource oxidized by the cells. Differences in sur-

vival of cells after UV irradiation could be cor-

related with differences in respiration after irradi-ation. To test this idea, cells were irradiated withmonochromatic light at 265 nm, and survivalwas determined by plate counts on nutrient-agarplates and synthetic medium plates. Althoughsurvival differs with the plating medium, thenumber of survivors, after a given UV dose, are

nearly the same for glucose- and glycerol-growncells (Fig. 6). Survival at times after UV irradia-tion was also measured in cultures incubated inliquid medium at 37 C. For the first 3 to 4 hr afterUV irradiation, there was little increase in thenumber of surviving cells. Therefore, the differ-ences in oxygen consumption after UV irradia-tion of glycerol- and glucose-grown cells is not areflection of differences in the numbers of sur-viving cells. In fact, physiological responses afterUV appear to bear little relationship to survival.

Effects of change in the carbon source before or

after UV irradiation of B/r cells. Since glucose-grown cells do not exhibit severe inhibition ofrespiration after UV, we attempted to preventthe inhibition of respiration in glycerol-growncells by the removal of the cells from glycerolmedium and the addition of glucose either im-mediately before, or immediately after UV. Insuch experiments, respiration follows a patternsimilar to glycerol-grown cells, i.e., respirationfor about 60 min after UV irradiation, followedby severe temporary respiratory inhibition (Fig.7). The same result is obtained if glucose isadded immediately before UV irradiation. Theseexperiments indicate that glycerol-grown cells are

not capable of oxidizing glucoseradiation, a situation which doesglucose-grown cells.

700-

_-

a 500-

z

o 300-

100-

B/r PR

UNIRRAD>/ 405nm

UV

&- -NO0 BL

b

after UV ir-not exist for

H/ra

UNIRRAD/

/UV

//11- NO BL

y&oL,-- l lll0 20 40 60 80 1 120 14 00 20 4 60 80 iiO 120 140

TIME AFTER PR TREATMENT (min)

FIG. 3. Effects of PR treatment on the respirationof UV-irradiated (500 ergs/mm2) cultures of (a) B/rand (b) H/r cells. Irradiation was by method (iii) andPR treatment by method (ii) for 405 nm and by method(iii) for BL (black lamp).

300- co/iBs

PRO

itoo2 VRESPIRATIONw ~~~0 0.175

Cn 0/,g0NO PR

PR0PR. J0.125 O100

o,0 GROWTH -0.075 <

NO PR -0.025

0 40 80 120 160TIME AFTER PR (min)

FIG. 4. Effects of PR treatment on respiration andgrowth of a UV-irradiated B.-1 culture. The UV dosewas 500 ergs/mm'. Irradiation was by method (i) andPR treatment by method (i).

E co/i B/r {

500 UNIRRAD.

400 A A"

wAX/ A'IRRAD.Z 300 /

O 200 /

100-

0 40 80 120 160 200TIME AFTER UV (min)

FIG. 5. Respiration ofunirradiated E. coli B/r grownon glucose and irradiated with 500 ergs/mm1. Irradia-tion was by method (iii).

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HAMKALO AND SWENSON

)0 800 0 200 400 600 800DOSE (ergs/mm2 at 265 nm)

FIG. 6. Dose response curves of survival of E. coliB/r grown on glycerol (M63+) or glucose (M9+)medium. The cell suspensions were irradiated withincremental doses of265 nm and plated on (a) nutrient-agar or (b) synthetic medium plates. The latter con-tained the same carbon source as did the medium onwhich the cells were grown. Irradiation was by method(ii).

400-

400 - olUNIRRAD./

~300- IRRAD.C

2:200- / D

0 40 80 120 160 200TIME AFTER UV (min)

FIG. 7. Respiration ofE. coli Blr grown on glycerol,washed with glycerol-free medium, and given glucoseimmediately after irradiation (500 ergs/mm2). Irradi-ation was by method (i).

ATP measurements. Since ATP pool levelsexert control over respiration (1), we measuredATP pool levels in irradiated B/r cells. Theamount of ATP per unit of cell mass, normalizedto a value of 100 for unirradiated cells at zerotime, remains fairly constant with the growth ofthese cells (Fig. 8a). However, by 30 min after500 ergs/mm2 of UV irradiation, there is an ac-cumulation of ATP to about 2.5 times the controlvalues. The ATP level then falls rapidly, andcontrol values are approached by 1 hr after UV,the time at which respiratory inhibition sets in.

Although PR at room temperature preventsrespiratory inhibition, it does not alter ATPlevels; such treatment can reduce the ATP levelonly if given at 37 C (Table 2). These data, inaddition to the fact that ATP is accumulated incells grown on glucose (Fig. 8b), lend support tothe argument that respiratory inhibition is not aresult of excessively high ATP pool levels. Thereduction in the pool size of irradiated cells seemsto be a result of the balance between the utiliza-tion of ATP for synthesis and the phosphoryla-tion of adenosine diphosphate to form ATP.DNA synthesis after UV irradiation. Although

irradiated populations of glucose- and glycerol-grown cells show about the same survival afterUV, and because PR studies indicate that respira-tory inhibition is a result of pyrimidine dimersin DNA, it was of interest to measure DNA syn-thesis for both cultural conditions after UVirradiation (500 ergs/mm2). DNA synthesis re-sumes about the same time (50 min) in both cul-tures (Fig. 9a, 9b). This time coincides with thetime of the onset of respiratory inhibition inglycerol-grown cells. Upon recovery, however,glucose-grown cells synthesize DNA at a ratenearly parallel to unirradiated cultures, whereas

4001 b

3001- IRRAD.~200j ~RAD,

ffis°°TUNIRRAD.+ ~~UNIRRADA

0 40 80 20 160 200 240 280 0 40 80 120 160TIME AFTER UV (min)

FIG. 8. Time course of ATP pool levels in unir-radiated and UV-irradiated glycerol-grown (a) andglucose-grown (b) E. coli Blr. Irradiation was bymethod (i).

TABLE 2. Effects of UV irradiation and PR treat-ment with black light (BL) on ATP levels in

Escherichia coli B/r (glycerol-grown)aATP/OD

Sequence of experimental manipulation before assay (arbitraryunits)

No UV, noBL............................. 100BL (20 min) at 37C ........................ 125UV irradiation followed by:

30 min at 37 C............................ 25620 min ofBL at 37C ..................... 13030 min at 37 C, 20 min of BL at 37 C........ 14520 min of BL at room temp............... 20020 min of BL at room temp, 30 min at 37 C. 215

aThe UV dose was 500 ergs/mm2. Irradiationand PR treatment were by method (iii), as de-scribed in Materials and Methods.

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EFFECTS OF UV IRRADIATION ON E. COLI

glycerol-grown cells synthesize DNA at a muchlower rate. The fact that division of unirradiatedcells has occurred by the time DNA synthesisresumes after UV irradiation indicates that whenDNA synthesis resumes in irradiated, glucose-grown cells, the rate, per cell, is greater than fornormal cells.

DISCUSSION

In 1953, Kelner (14) proposed a model to ex-plain the effects of UV irradiation and subse-quent PR treatment on nucleic acid synthesis,growth, and respiration in E. coil B/r. He viewedthe sequence of events as follows. Inactivation byUV irradiation of the bacterial nucleus inhibitsDNA synthesis; cytoplasmic reactions, such asrespiration, RNA, and protein synthesis, con-tinue after UV, but those processes under thecontrol of the bacterial nucleus are eventuallyinhibited, since DNA is not functioning. Photo-reactivating treatment had been shown by Kelnerto reverse UV-induced lethal effects (13), and torelease the inhibition ofDNA synthesis (14). He,therefore, assumed that if processes which are in-hibited by UV irradiation could be photoreac-tivated, they are controlled by the bacterialgenome. Kelner demonstrated PR of a slightinhibition of respiration by massive UV doses tostationary phase B/r cultures and PR of a slightinhibition of growth in log-phase B/r cells (14),thus showing that both respiration and growthare under control at the bacterial nucleus.B/r cells possess a dark-repair mechanism not

found in B.-, cells, but both strains have theability to photoreactivate. As a basis for dis-cussion of our results, we shall update Kelner'smodel as follows. The primary UV-irradiationdamage (pyrimidine dimers) causes inhibition ofDNA synthesis, and also causes secondary effects,such as inhibition of growth and respiration. Therepair of the dimers by a dark-repair process or byPR results in at least a partial reversal of theseinhibitory effects. We have shown that growthand respiration are uncoupled after UV irradia-tion of glycerol-grown cultures of E. coli B8-1.This uncoupling appears to reflect the destructionof cellular control of coordinated activities.Photoreactivability of the uncoupling indicatesthat the disappearance of pyrimidine dimers fromDNA by photoenzymatic monomerization caneffect sustained coordination among cellularprocesses. When B/r cells are irradiated with amassive UV dose (5,000 ergs/mm'), uncouplingof growth from respiration occurs, as in the B.-,cells at lower doses. We feel that the loss of co-ordination is the result of excessive damage toDNA, but the high doses may also cause damage

81

6-

4-

2-

to0

4-

2-

B/r (GLUCOSE)a

/UNIRRAD.

/,? IRRAD./

10-

A1

b B/r (GLYCEROL)o

/UNIRRAD.

0 0

I~~~~IR0' 0

O 40 80 120 160 200TIME AFTER UV (min)

FIG. 9. DNA synthesis in E. coli B/r grown onglucose (a) or glycerol (b). DNA was measured bythe incorporation of 3H-labeled thymidine into coldtrichloroacetic acid-insoluble material. The UV doseswere 500 ergs/mm2. Irradiation was by method (iii).

to such cellular components as ribosomes andtransfer RNA.

If glycerol-grown B/r cells are irradiated withlower UV doses (300 to 600 ergs/mm2), one ob-serves sustained coordination of respiration andgrowth, even during severe inhibition of respira-tion. This inhibition in B/r is prevented by PRtreatment; inhibition with no reversal by PR in aphr- strain indicates that the inhibition is a sec-ondary response to the presence of pyrimidinedimers in DNA. As would be expected from therespiration and growth studies, RNA and proteinsynthesis are severely inhibited at the same time(B. A. Hamkalo, Ph.D. Thesis, Univ. of Massa-chusetts, 1968). The fact that the inhibition of allthese processes persists for a period of time whichincreases with increasing dose and sets in at thetime that DNA synthesis resumes suggests acorrelation between the respiratory inhibitionwith the dark-repair process in B/r and thephysiological changes which occur in the cellsduring repair.

In UV-irradiated B/r and B.- cells, RNA andprotein are synthesized during the time that DNAsynthesis is inhibited. Upon resumption of DNAsynthesis, the amount of RNA and protein rela-tive to the amount ofDNA in the irradiated cellswould be higher than that of the control cells.At any given time in the bacterial cell cycle, thereare fairly constant macromolecular ratios (5).Donachie et al. (6) suggested that the ratio of

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HAMKALO AND SWENSON

DNA to protein is a controlling factor in theinitiation of cell division. Macromolecular syn-thesis data after UV irradiation (26) show thatthis ratio is indeed disturbed in these cells, andthe subsequently observed physiological changescould be directed toward the reestablishment ofnormal macromolecular ratios. Severe respiratoryand synthetic inhibition, exclusive of DNA syn-thesis, in the postirradiation period would permitcells to accumulate an amount of DNA criticalto cell division. In fact, irradiated E. coli cellsdivide once or twice after UV irradiation, evenif these cells do not give rise to colonies (9).

UV-irradiated, glucose-grown B/r cells exhibitonly a slight transitory inhibition of respirationcompared to glycerol-grown cells, but both cul-tures have nearly the same survival curves. Thus,glucose-grown cells lose nothing by failing to turnoff their respiratory system for a period of time.There might be a relation between respirationrates after UV irradiation and the rates of DNAsynthesis upon resumption of synthesis. After thesame UV dose (500 ergs/mm2), glucose-growncells resume synthesis at a rate close to that of theunirradiated control, whereas glycerol-grown cellsresume DNA synthesis at a very low rate. Themacromolecular imbalances which result fromthe inhibition of DNA synthesis may be morerapidly adjusted in glucose-grown cells as a resultof rapid DNA synthesis. Therefore, severe inhibi-tion of the oxidation of glucose may not be neces-sary.The experiments which showed that the shift

of glycerol-grown cells to glucose after UV ir-radiation results in severe temporary inhibition ofrespiration, could be interpreted as the oxidationof glucose by a pathway which is not present, oris inactive in glycerol-grown cells. If glucose isnecessary to induce or activate such a pathway,then cells given glucose immediately after UVirradiation would not possess this pathway in anactive form.

B. A. Hamkalo (Ph.D. Thesis, Univ. of Massa-chusetts, 1968) showed that succinate- and acetate-grown cells exhibit severe temporary respiratoryinhibition after UV irradiation and also resumeDNA synthesis at a very low rate after irradiation.It is possible that only the Krebs cycle is availablefor the oxidation of these carbon sources, andthis cycle could be selectively inhibited as a conse-quence of the presence of pyrimidine dimers inDNA. Since inhibition is delayed for 60 min afterUV irradiation, the degree of repair during thefirst hour after UV irradiation may influence themetabolic responses of cells. The correlation thatexists between the rate of DNA synthesis uponresumption and the rate of respiratory response

implicates the DNA content of cells as a criticalcontrolling factor in cellular physiology.The observation by Doudney (7), that above a

certain UV dose DNA synthesis resumes at afixed time but at lower rates as the dose increases,indicates that at the time of resumption the DNAtemplate still contains photoproducts. Billen (2)noted that semiconservative replication is re-initiated upon such a template, and proposed thatthe reinitiation of synthesis is dependent upon thecellular capacity to repair UV-induced lesions. Alower rate ofDNA synthesis uponresumption mayindicate that synthesis past a photoproduct isslower than normal. The DNA that is made mayhave a base sequence altered from that of theparent molecule, and the messenger RNA trans-cribed may code for biologically inactive proteins.Such events could lead to the death of cells whichhave divided after the repair of some of the UV-induced damage.Our approach to the localization of the im-

mediate result of the inhibition of DNA synthesisafter UV irradiation that causes severe temporaryinhibition of respiration and growth was themeasurement of ATP pool levels in irradiatedcells. Contrary to the results of Kanazir andErrera (12), we found a marked difference inATP/unit of cell mass after UV irradiation. Al-though high ATP concentration can inhibitglycolysis and the Krebs cycle (1), it is not afactor in the respiratory inhibition of glycerol-grown B/r cells, because this inhibition sets inabout the time ATP per unit of cell mass hasfallen to control values. Also, glucose-grown cellsshow similar variations in ATP pool levels, al-though no respiratory inhibition is seen. The pho-toreactivability of respiratory inhibition in glyc-erol-grown B/rcells at room temperature, withouta concomitant decrease in the ATP pool levels,also supports the conclusion that the control ofrespiration effecting inhibition after UV irradia-tion is not the ATP level.

In summary, UV irradiation causes changes incellular physiology which are not necessarily cor-related with survival after UV irradiation. Thephysiological responses depend upon the bac-terium utilized and the cultural conditions before,during, and after irradiation. Cells which possessthe ability to repair UV-induced damage to DNAappear to maintain coordination among normallycoupled processes and may exhibit control overthe relative amounts of macromolecules in orderto prepare cells for division. Cells which are un-able to repair UV-induced damage to DNA can-not effect control over cellular processes shortlyafter UV irradiation, and cell death is certain.

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EFFECTS OF UV IRRADIATION ON E. COLI

ACKNOWLEDGMENTS

We thank R. B. Setlow and J. S. Cook for valuable discussionsand criticism of the manuscript. We also thank J. W. Longworthfor advice and for the use of his light-detection equipment forthe ATP assays.

This research was sponsored by the U.S. Atomic Energy Com-mission under contract with the Union Carbide Corporation.

LITERATURE CITED

1. Atkinson, D. E. 1965. Biological feedback at the molecularlevel. Science 150:851-857.

2. Billen, D. 1968. Comparison of the rate and extent of repairand semiconservative deoxyribonucleic acid syntheses inbacteria exposed to ultraviolet light. Biophys. J. 8:A52.

3. -Bollum, F. J. 1959. Thernral conversion of nonpriming de-oxyribonucleic acid to primer. J. Biol. Chem. 234:2733-2734.

4. Boyce, R. P., and P. Howard-Flanders. 1964. Release ofultraviolet light-induced thymine dimers from DNA inE. coil K-12. Proc. Nat. Acad. Sci. U.S.A. 51:293-300.

5. Dean, A. C. R., and C. Hinshelwood. 1966. Growth, functionand regulation in bacterial cells. Clarendon Press, Oxford.

6. Donachie, W. D., D. G. Hobbs, and M. Masters. 1968.Chromosome replication and cell division in Escherichiacoli 15T after growth in the absence of DNA synthesis.Nature 219:1079-1080.

7. Doudney, C. 0. 1965. Ultraviolet light effects on deoxyribo-nucleic acid replication, p. 120-141. In Cellular radiationbiology, 18th symposium on fundamental cancer research.Williams & Wilkins Co., Baltimore, Md.

8. Giese, A. C. 1942. Stimulation of yeast respiration by ultra-violet radiations. J. Cell. Comp. Physiol. 20:35-46.

9. Haefner, K., and U. Striebeck. 1967. Radiation-inducedlethal sectoring in Escherichia coil B/r and B8_. . Mutat.Res. 4:399-407.

10. Harm, W., and B. Hillebrandt. 1962. A non-photoreactivablemutant of E. coil B. Photochem. Photobiol. 1:271-272.

11. Jagger, J. 1961. A small and inexpensive ultraviolet dose-ratemeter useful in biological experiments. Radiat. Res. 14:394-403.

12. Kanazir, D., and M. Errera. 1953. Teneur en adenosine tri-phosphate (ATP) de E. coli apres irradiation U.V. Bio-chim. Biophys. Acta 11:451-452.

13. Kelner, A. 1949. Effect of visible light on the recovery ofStreptomyces griseus conidia from ultraviolet irradiationinjury. Proc. Nat. Acad. Sci. U.S.A. 35:73-79.

14. Kelner, A. 1953. Growth, respiration and nucleic acid syn-

thesis in ultraviolet-irradiated and in photoreactivatedEscherichia coli. J. Bacteriol. 65:252-262.

15. Morowitz, H. J. 1950. Absorption effects in volume irradia-tion of microorganisms. Science 111:229-230.

16. Pardee, A. B., F. Jacob, and J. Monod. 1959. The geneticcontrol and cytoplasmic expression of "inducibility" in thesynthesis of ,l-galactosidase in E. coli. J. Mol. Biol. 1:165-178.

17. Roberts, R. B., P. H. Abelson, D. B. Cowie, E. T. Bolton,and R. J. Britten. 1955. Studies of biosynthesis in Escher-ichia coli. Carnegie Institution of Washington, Washing-ton, D.C.

18. Rupert, C. S. 1964. Photoreactivation of ultraviolet damage,p. 283-327. In A. Giese (ed.), Photophysiology, vol. II.

Action of light on animals and microorganisms; photobio-chemical mechanisms; bioluminescence. Academic PressInc., New York.

19. Seliger, H. H., and W. D. McElroy. 1965. Light: physical andbiological action. Academic Press Inc., New York.

20. Setlow, J. K. 1967. The effects of ultraviolet radiation andphotoreactivation, p. 157-208. In M. Florkin and E. Stotz(ed.), Comprehensive biochemistry, vol. 27. Elsevier Pub-lishing Co., Amsterdam.

21. Setlow, R. B. 1964. Molecular changes responsible for ultra-violet inactivation of the biological activity of DNA,p. 291-307. In C. Pavan, C. Chagas, 0. Frota-Pessoa, andL. R. Caldas, (ed.), Mammalian cytogenetic and relatedproblems in radiobiology. Pergamon Press, New York.

22. Setlow, R. B., and W. L. Carrier. 1964. The disappearanceof thymine dimers from DNA: an error-correcting mecha-nism. Proc. Nat. Acad. Sci. U.S.A. 51:226-231.

23. Setlow, R. B., P. A. Swenson, and W. L. Carrier. 1963. Thy-mine dimers and inhibition of DNA synthesis by ultra-violet irradiation of cells. Science 142:1464-1466.

24. Smith, K. C. 1966. Physical and chemical changes inducedin nucleic acids by ultraviolet light. Radiat. Res., (Suppl.)6:54-76.

25. Suzuki, K., E. Moriguchi, and Z. Horii. 1966. Stability ofDNA in Escherichia coli B/r and B8_1 irradiated with ultra-violet light. Nature 212:1265-1267.

26. Swenson, P. A., and R. B. Setlow. 1966. Effects of ultravioletradiation on macromolecular synthesis in Escherichia coil.J. Mol. Biol. 15:201-219.

27. Umbreit, W. W., R. H. Burris, and J. F. Stauffer. 1957.Manometric Technique. Burgess Publishing Co., Min-neapolis, Minn.

28. Wacker, A., H. Dellweg, and D. Weinblum. 1960. Strah-lenchemische veranderung der bakterien-deoxyribonu-cleinsaure in vivo. Naturwissenschaften 47:477.

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