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The molecular nature of damage by oxygen to freeze-dried Escherichia coli
C1wOBIOLOGY
12, 15-25
(1975)
The Moleculur Nature of Damage by Oxygen Freeze-Dried Escherichia co/i ’ E. ISRAELI,
Israel Institute
A. KOHN,
for Biological
AND
JANINA
Research, Ness-Ziona
to
GITELMAN and Tel-Auiv University,
Medical School, Ramat-Aviv, Israel
Molecular oxygen is toxic to freeze-dried (FD) bacteria (14). Freeze-dried E. coli exposed to air lose the ability to form cob nies on nutrient media, but retain the ability to complete the ongoing DNA synthesis (16), and to serve as host for the production of infectious phage (8). It was suggested that the damage caused by oxygen might be connected with the comp1ex responsible for initiation of DNA synthesis ( 7). To support this hypothesis we present in this communication data, based on experiments with thymidine deficient mutants of E. coli. METHODS
1, Bacterial Strains a. E. co& K-12 auxotroph which is thy-, his-, prom, arg-, try-, thiamine-. b. E. coli K-12 120/6. Which is thy-, pro-, str* ts dna, received from Dr. Beyersman, Berlin ( 1) . c. E. coli B thy-.
CaCl,, 2 X 1O-4 M; FeC&, 2 X 1O-e M; Na$O,, 2.5 X 1O-3M; Tris, 0.12 M; glucose, 0.4%; KH2P04, 1.5 X 1O-3 M; (16) suppIemented with thymine, histidine, proline, and arginine (20 pg/ml of each), 30 pg/ml of tryptophan and 5 /*g/ml of thiamine. b. The temperature sensitive mutant was grown in TPG broth (Tris-Phosphate-Glucase) containing in 1 liter of water: NH&I 1.1 g, KC1 8 g, NaCl 0.5 g, MgCl, *6Hz0 0.2 g, KH2P04 0.023 g, sodium pyruvate 0.8 g, Tris (Z-amino 2 hydroxymethyl propan 1:3 dial) 12.1 g, CaC12 -1 ml of 0.1 N solution NazS04 -1 ml of 0.16 N sofution. Adjusted to pH 7 with 37% HCI. After autoclaving FeC& *6Hz0 1 ml of I pg/mI solution, and 0.2% gIucose were added. The medium was supplemented with proline, thymine, and thiamine 20 pg/ml of each. c. Tryptose agar served as plating medium. d. E. cola’ B was grown in L-broth (71, 3. Starvation for Amino Acids The AA auxotroph was grown in TGA broth with shaking at 37°C until logarithmic phase of growth was reached. The cells were then washed and half of the culture was resuspended in TGA lacking the required AA and shaked for 2 hr at 37°C. The other half continued growing in fully suppIemented TGA and served as control. The cells were then chilled to 4”C, washed
2. Growth Media a. The amino acids (AA) auxotroph was grown in TGA synthetic medium, the composition of which was: NaCl, 0.08 M; KC], 0.02 M; NH&I, 0.02 M; MgCl,, 10m3 M; Received
July 1, 1974.
1 This work is part of a Ph.D. thesis to be submitted
to the Tel-.4viv
University
by E. Israeli. 15
Copyright 6 1975 by Academic Press. Inc. All rights of reproductirrn in any form reserved.
16
ISRAELI,
KOHN
and freeze-dried, applying the foIlowing procedure. One-milliIiter amounts of a suspension containing 1011 cells/ml, washed and resuspended in distilled water, were pipetted into standard lo-ml ampoules. Each ampoule was spun around its axis at ca. 2000 rpm while dipped in a methanol-dry ice freezing mixture ( -78”). The frozen ampoules were then dried for 3 hr under vacuum, the fmal pressure in the system after complete dehydration being 5 e Hg. After completion of the drying process ampoules were sealed in vacua.
GITELMAN
6. 3H TdR Incorporation To 10 ml of bacterial suspensions (I2.108 cells/ml) 0.2 ml of 3H TdR was added (50 &i/ml lo-15 Ci/mM). Samples (0.1 ml) were drawn out at intervals to tubes containing 2 ml TCA ( 5% ), 0.2 ml sodium pyrophosphate 0.2 N and 200 pg/ml of non radioactive thymidine, at 0°C. After 18 hr at 4°C the samples were filtered through glass filters (Whatman GF/C), washed thrice with ice cold, water, once with ethanol, dried and counted in Packard’s Tricarb scintillator, using toluene based scintillation fiuid. 7. Formation of Filamentous Freeze-Dried Bacleria
4. Starvation for Thymine Escherichia coli K-12 was grown in TGA to a concentration of 10” cells/ml. The cuIture was divided in two: half continued growing, (control) and half was washed, and resuspended in the same medium lacking thymine, and incubated with shaking for 50 min. at 37°C. After another washing, the cultures were suspended in the same medium and grown in the presence of chloramphenicol (CAM 150-300 pug/ml) or in the absence of essential amino acids, and tritiated thymidine ( 3H TdR) incorporation was measured. Freeze-drying took place after the period of thymine starvation. 5. Growth at the Restrictive
AND
Temperature
Escherichia coli K-12 ts-dna was grown in TPG with shaking at 33”C, to a concentration of 2.107 cells/ml. The culture was divided in two: half was grown further at 33’C, the other at 42°C for 2.5 hr for the completion of rounds of replication (1). The cultures were then transferred to an ice bath, washed trite in water, and freezedried. In order to measure thymidine incorporation at different temperatures, the dried bacteria were resuspended in medium prewarmed to the desired temperature (42°C ar 33°C).
Forms by
Escherkhia coli B, dried or exposed to was resuspended in L-broth and wgen, grown for several hours at 37°C. Additional samples were inoculated onto microscope slides coated with a thin (l-mm) layer of agar (containing the same ingredients as the L-broth). Individual cells were photographed at different intervals in a Reichart microscope (magnification X 480). The slides were incubated at 37°C in a humid chamber. Samples from the liquid cuIture were fixed in 2.5% glutaraldehyde, postfrxed in 2% osmium tetroxide, and embedded in epon. Preparations were sectioned with Omu-2 Reichart ultramicrotome, stained with uranyl acetate and lead citrate, and observed in a JEM 100 B electron microscope. 8. Autoradiogrnphy (Modification Method for Animal C.&s (17)
of the
Samples of FD and oxygen exposed bacteria ( FDO ), were resuspended in full medium and allowed to incorporate “H TdR for 4-6 hr (specific activity in the medium = 0.1 Ci/mM, 5 &,/ml). The samples were then washed with growth medium and water, and fixed on microscope sbdes. The slides were covered with
OXYGEN
DAMAGE
TO LYOPHILIZED
E. cob
FIG. 1. *H TdR incorporation of FD and FDO E. coli K-12 after AA. starvation. E. coli K-12 wan grown in TGA to a concentration of 2.10’ cells/ml. The cells were washed and resuspended with AA (open circles) or without (closed circles). After FD (left panel) and resuspension, or FD and oxygen exposure (right panel) incorporation of *H TdR was measured. Oxygen exposure was at 28°C and 45% relative humidity (RH).
photographic film (Kodak scientific plates AR lo), dried and stored at 4°C for one and two weeks, then developed and fixed. Afterwards the preparations were stained with Giemsa-Felz stain for 2-3 hr. RESULTS
1. Incorporation
of “IS TdR in Escherichia
Coli (AA Deficient) This mutant, when grown in medium lacking the essential AA would complete the ongoing DNA synt!-esis, but is unable to reinitiate it (13). DNA synthesis begins anew onIy upon the addition of the missing AA, as measured by 3H TdR incorporation. When a fuIly supplemented culture was compared with one lacking AA, “H TdR incorporation proceeded at the same rate for 90 min but then, in the AA deficient me-
dium, it ceased. It was resumed only after 60 min after the addition of AA to the deficient culture (resuhs not shown), Having established this course of events, a culture of the mutant was prepared, so that it was in the quiescent stage, having completed the DNA cycle (about 120 min after withdrawal of AA). These bacteria were then washed and freeze-dried. After exposure in the dry state to air (oxygen) at 28°C and 45% relative humidity, the samples were resuspended in TGA and the incorporation of SH TdR into them was measured. In bacteria which were kept in vacua after lyophilization, the incorporation of “H TdR was similar both in those grown in the presence of AA, as in those held for 120 min in the absence of AA (Fig. I ) , whereas in the fully supplemented culture, the DNA synthesis started immediately upon resus-
ISRAELI,
18
KOHN AND GITELMAN
PER00 OF EXPOSLRE(HRS) FIG. 2. Survival curves of FD E. co8 K-12 upon exposure to oxygen. ( T = 28”C, RH = 45% ), Open circles-grown with AA; closed circles-grown without AA.
AA-deficient cells incorporated SH TdR exponentially even after 4 hr. These results indicate that when bacteria, that were not actively engaged in DNA synthesis, were freeze-dried and exposed to air their ability to synthetize DNA and to reinitiate it, was not impaired, while bacteria that were in the process of DNA synthesis when subjected to FD and oxygen, though able to complete the already initiated DNA synthesis could not reinitiate the synthesis of DNA. It thus seems that when the initiation site for DNA synthesis is not actively engaged at the time of FD and exposure to oxygen, it is not damaged by it. Consequently, it was not surprising to find that the survival curves of AA deficient bacteria indicated a considerable protection against the oxygen effect. (Fig. 2 and Table 1).
pension in full medium. In the AA deficient culture there was a delay of about 40 min before the DNA synthesis became evident. As for the FD bacteria exposed to oxygen, the situation was reversed. Reconstituted, fully supplemented bacteria ceased SH TdR incorporation after 2-3 hr of incubation, i.e., DNA synthesis ceased after the completion of one cycle. On the other hand, TABLE EFFECT OF
GROWTH
CONDITIUNS FREEZE-DRYINQ B wild
Mutant
In order to test the hypothesis that protection against the lethal action of oxygen involves an initiator protein or initiation complex we used bacteria in which protein synthesis is halted by chloramphenicol 3
ON SURVIVAL OF Escherichia coli NUT.%NTS AND EXPOSURE TO OXYOEN~ K-12 amino acid mquiring Amino mid atrmvation
Treatment
Decay constant of survival curyes
2. lncorporalion of 3H TdR in Escherichia coli K-12 after Thymine Sfaruation upon Blocking Protein Synthesti
CY T
1.9 1.1
Q-DNA temDerature aenaitive for DNA aynthesk
AFTER B log phase
GWKII with CAM
1.20 0.53
a Bacterial mutants were grown under conditions of optimal growth then transferred to restrictive conditions for l-Z.5 hr (see Methods). The bacteria were then frozen, freeze-dried and exposed to oxygen for varying periods of time in the dry state. The viability of reconstituted bacteria wae determined at these time intervals and from the results a decay curve WLI constructed. Decay rates (K) are shown in the table. Exposure to oxygen was at 2S°C, 45% relative humidity. b C = control bacteria (not treated). * Reference (7).
OXYGEN
DAMAGE
TO LYOPHILIZED
E. coli
19
FIG. 3. “HTdR incorporation in FD and FDO E. coli K-12, upon protein synthesis inhibition, after thymine starvation. Cells were starved for 50 min. After Iyophilization and oxygen exposure, the samples were resuspended in synthetic medium upon protein synthesis inhibition, Triangles-FD bacteria without protein synthesis inhibition; Closed circles-FD bacteria upon protein synthesis inhibition; Open circles-FDO bacteria (30 min exposure) upon protein synthesis inhibition.
(CAM ), or thymineless mutants which can synthetize DNA in the absence of protein synthesis (2). In wild-type bacteria, or in thymineless mutants growing in the presence of thymine, inhibition of protein synthesis by CAM causes cessation of DNA synthesis (13). On the other hand, in T- mutants deprived of thymine, there are produced stable initiator proteins so that even in the presence of CAM, DNA synthesis eontinues. It was expected that thymine starved mutants, which had accumulated initiation proteins should be less sensitive to the oxygen effect, than the same mutants growing in the presence of thymine, provided that the lethal effects of oxygen were indeed due to its direct interaction with such a protein( s ), If this be the case, the thymine-starved mutants, after exposure to oxygen, should incorporate more 3H TdR than controls, If, on the other hand, exposure of thymine-starved mutants to oxygen is associated with a decreased DNA
synthesis, one wouId have to assume that the oxygen did not directly interact with these proteins, but affected the initiation site or the membrane. Experimentally, when bacteria grown in the absence of AA and in the presence of CAM were lyophilized and held in vrzcuo before resuspension, the incorporation of 3H TdR proceeded for about 60-120 min, then ceased, having reached a level corresponding to doubling of DNA content (Fig. 3). The cessation of DNA synthesis after doubling was aIso observed in thyminestarved bacteria, grown in the absence of AA and with CAM, after lyophilization and exposure to oxygen. On the other hand, lyophilized thymine-starved bacteria, when reconstituted in medium permitting protein synthesis carried on 3H TdR incorporation exponentiahy for at least five hours. This result indicates that if after FD, repair is permitted, DNA will be synthetized. The damage due to FD per se is thus reversible, but becomes irreversible upon exposure to oriygen.
20
ISRAELI,
KOHN
AND
GITELMAN
AT 42’C
AT 33%
I
I
60pomBb300 TIME
60
ta
TIME
~MIN)
WIN)
FIG. 4. ‘HTdR incorporation in FD and FDO E. coli ts-dna at 33 and 42°C. Cells were grown in TPC, lyophilized and oxygen exposed (1.5 hr for bacteria grown at 33°C and 2.5 hr for those grown at 42°C). Resuspension was in TPG and the incorporation measured both at 33°C (a) and 42°C (b). Closed squares- Control bacteria grown at 33°C; Closed circlesControl bacteria grown at 42°C; Open trianglesFD control bacteria grown at 33°C; Closed triangles-FD control bacteria grown at 42°C; Open squares-FDO control bacteria grown at 33°C; Open circles-FDO control bacteria grown at 42°C.
3. Incorporation coIi K-12 ts-dna
of SH TdR in Escherichia
We have used a DNA ts-mutant of E. cola isolated by Beyersman ( 1) defective in the initiation of DNA synthesis (see aIso 2-4, 11, 12, 19). At the nonpermissive temperature (42°C) this mutant completes the DNA cycle, but is unable to reinitiate DNA synthesis unless it is brought back to the permissive temperature (33°C ) . One would expect that this mutant grown at the nonpermissive temperature would be in a physiological state similar to the thyminestarved, or AA-starved bacteria, and therefore after freezc-drying would be less sensitive to oxygen ( Table 1) . The incorporation of thymidine in FD &mutants was practically unaffected when they were frozen after growing at 33”C, but was significantly depressed when frozen from 42°C. Exposure to oxygen completely abohshed thymidine incorporation in FD bacteria grown at 42°C but permitted con-
siderable incorporation at the permissive temperature ( Fig. 4). The decay of these FD ts-mutant after exposure to oxygen was considerably smaller in bacteria held for 2.5 hr at nonpermissive temperature than in bacteria grown and held at 33°C (Fig. 5). In this respect, the results have the same character as in experiments with AA-starved cells, or with colicin-treated ones, namely that in the absence of initiation events, FD bacteria are less sensitive to oxygen. 4. Mimoscopical Electron)
Obsewations
( Optical and
While control bacteria upon seeding on nutrient agar start dividing within 30-60 min, and form microcolonies within 2-3 hr after seeding (Fig. 6a), FD bacteria produced at that time only long filaments (Fig. Bb) and only 10% of them produced colonies. Similar filamentous growth has been observed in E. coli deprived of magnesium (21) or thymine (18). When these fila-
OXYGEN
DAMAGE
TO LYOPHILIZED
E. di
mentous forms, observed in freeze-dried and reconstituted bacteria, were labeled cvith “H TdR and then autoradiographed, a diffuse pattern of grains (Fig. 7) indicated segregation of the DNA and multipoint initiations. No septa were seen in these filaments (Fig. 8), though pro&ction of septa per se is not SufFicient for ceILdivision: a ts-mutant of E. coli (MX174 ts27) at 41°C produces septa but does not divide (5). In FD bacteria exposed to oxygen (Fig. 6c), only about 1% of the cells grew to filaments, while the viability of the culture was as Iow as 10-5y0. DISCUSSION
When amino acids deficient
mutants
of
E. cc& that were not activeIy engaged in DNA synthesis, were freeze-dried and exposed to oxygen their ability to synthetizc DNA and to initiate it was not impaired, while similar treatment of bacteria that were in the process of DNA synthesis caused loss of reinitiation of DNA synthesis. Comparison of these results with those concerning the effect of colicin El (7) on oxygen exposed bacteria, indicate that in both cases the process of initiation of DNA synthesis seems to be affected, either by lack of synthesis of a necessary initiator protein, upon AA deprivation, or by binding of coIicin to initiator site, so as to protect the cells against the lethal action of oxygen (Table 1). In order to distinguish whether oxygen affects the initiation proteins or the initiation complex associated with the plasma membrane, we checked DNA synthesis in thymineless mutant after growth in medium lacking thymine. The results obtained with this mutant suggest, that the damage to the DNA synthesis mechanism is caused during freeze-drying. If after Iyophilization repair is permitted (protein synthesis), DNA is synthetized. The damage due to FD per se is thus reversible. It ceases to be reversible when the FD bacteria are exposed to oxygen, This finding is com-
PER100 OF EXPOSURE IHRS)
FIG. 5. Survival curves of FD E. cdi ts-dna after growth at different temperatures. Open circles-FD bacteria grown at 33°C; closed circlesFD bacteria grown at 42°C. (Exposure to oxygen was at 28°C and 45% RH.) patible with the results obtained from experiments on phage production in FD bacteria (8) and on membrane permeability (6). Since the initiator protein is stable and present in excess in the cells, it is more plausible that the damage occurring during FD would involve the initiation site rather than the initiator protein itself. Two conditions are required for the initiation of DNA synthesis: (a) there has to be RNA and protein synthesis ( 13, 15, ZO), and (b) the initiation complex has to be associated with the cytoplasmic membrane (9). By the use of inhibitory properties of CAM and phenethyl alcohol (PEA), two proteins were implicated to take part in initiation of DNA synthesis. One is inhibited by high concentration of CAM, but not by a low one. During growth in the presence of PEA, the CAM sensitive pro-
ISRAELI,
FIG, 6. Grawth of E. bacteria. c. FJX bacteria.
coli
KORN AND GITELMAN
B m agar {3 hr growth) I 11.Control nontreated bacteria. b. FD
OXYGEN
DAMAGE
TO LYOPHILIZED
tein accumulates, but the synthesis of CAM-refractive protein (presumably a membrane protein), is inhibited (20). During thymine starvation, initiation protein (s > accumulate; this protein permits a number of initiations, while the initiation proteins produced in normally growing cells are destroyed after each initiation (IO). En the ts-mutant of E. coli we used, either the initiation protein per se is thermolabile, or the binding process to the membrane is thermolabile, perhaps because of change in phase transition properties of the membrane lipids. The results presented here tend to support the second alternative. The changes in membrane due to Oa are effective jn blocking initiation only if the initiation complex is bound to the membrane. If it is not, as in the case of adsorption of colicin, or because of phase transition in the &mutant at 42”C, the effect of oxygen is largely depressed (Table 1). The finding that FD and exposure to air produced the same effects in bacteria grown at :33”C (when initiation complex is
FIG. 8. Electron micrograph of 6lamentous suspended in L-broth and grown for 4 hr.
E. COG
23
FIG. 7. Autoradiography of FD E. coli B. Freezedried bacteria were resuspended in L-broth and grown for 4-6 hr in the presence of “HTdR (0.1 Ci/mhr, 5 ,&i/ml) _
operative) and those grown at 42°C (when it is not), has to be explained. It seems that the critical event is the formation of the initiation complex at 33°C and that this event is instantaneous. ts bacteria that are grown at 42°C have to be cooled to freez-
forms
of FD
E. c&
B. FD
bacteria
were
re-
24
ISRAELI,
KOBN
ing temperature. They pass through the permissive temperature (33°C ) , SD that initiation complex may be formed, and thus their sensitivity to oxygen would not be different from that of cells grown at 33°C. This result is incompatible with an assumption that a necessary protein is entirely missing in the ts-mutant grown at nonpermissive temperature, but is consistent with an alternative that the conformation of a protein necessary for initiaticn or binding to the membrane changes with temperature. An interesting point also to be explained is that in bacteria exposed to oxygen and reconstituted at 42”C, both initiation and DNA elongation are halted (Fig. 4a, open squares and circles ). One may perhaps postulate that there are at least three elements involved in DNA synthesis, all three required for initiation (15) but only two for elongation. One of the ekments (membranal) is affected by exposure to oxygen, another (an enzyme) by elevated temperature, so that at 42°C there is no DNA synthesis in oxygen-treated bacteria because two of the three elements are damaged. Damage to only one of the three factors (by oxygen treatment or high temperature), would affect initiation only. Our results indicate that the site sensitive to oxygen in the FD bacteria is a membranal one. Damage to this site stops initiation of DNA synthesis, the cells lose their ability to divide and form colonies and so they “die.” SUMMARY
The damage occurring in freeze-dried bacteria exposed to oxygen is mainly in the bacterial membrane and involves the DNAinitiation complex. This injury occurs in two stages: The primary damage is due to the freeze-drying itself, and is repaired upon reconstitution of bacteria and their subsequent incubation in nutrient broth. The repair process requires protein synthesis. In the next step, the exposure of freezedried bacteria to oxygen, the injury be-
AND
GITELMAK
comes irreversible and the bacteria ‘
J. Mol. Btid. 53,369387 ( 1970). 5. Inouye, M. Unlinking of cell division DNA
reolication
in a temoerature
from
sensitive
OXYGEN
DAMAGE
TO LYOPHILIZED
DNA synthesis mutant of Escherich~a coli. J. Bacterial.
99, 842-850
( 1969).
6. Israeli, E., Giberman, E., and Kohn, A. Cryobiology 11, 473-47-I (1974). 7. Israeli, E., and Kahn, A. Protection of lyophilized Escherich!u coli from oxygen by co!icin E, treatment. FEBS Letters 26, 323-326 ( 1972). 8. Israeli, E., and Shapira, A. Production of bacteriophage T4 by lyophilized and oxygen exposed Escherichia cc&. .l. Gela. Microbid. 79, 159-161
( 1973).
9. Jacob, F., Brenner, S., and C&n, F. On the regulation of DNA replication jn bacteria. Cold Spring Ha&. Symp. @era&. Biol. 28, 329-348 ( 1963 ) . 10. Kogoma, T., and Lark, K. G. DNA rephcation in Eschmichia coli: Replication in absence of protein synthesis after replication inhibition. 1. Mol. Biol. 52, 143-164 ( 1970). Il. Kuempel, I’. L. Temperature sensitive initiation of chromosome replication in a mutant of Escherichia coEi. J. Bacterid. 100, 13021310 (1969). 12. Lark, K. C. Regulation of chromosome replica-
tion and segregation in bacteria. Becteriol. Reo. 30, :3-32 ( 1966).
13. Lark, K. G., Repko, T., and Hoffman, E. J. The effect of amino acid deprivation on subsequent DNA repIication. Bbchim. Biophvs. A& 76, !3-24 ( 1963 ).
E. coU
25
14. Lion, M. B. Quantitative aspects of protection of freeze-dried Escherkhia coli against the toxic effect of oxygen. J. Gen Microbial. 32, 321329 (1963). 15. Messer, W. Initiation of deoxyribonucleic acid replication in Eschetichia coli B/r: chronology of events and transcriptiona contro1 of initiation. J. BucterioE. 112, 7-12 ( 1972). 16. Novick, O., Israeli, E., and Kahn, A. Nucleic acid and protein synthesis in reconstituted Iyophilized Eschetichia cc& exposed to air. J. AI& Bactera’ol. 35, 184-191 ( 1972), 17. Oren, R., and Kohn, A. Density dependent inhibition of cell growth in cultures of primary and established lines of cells. J. Cell. PhysioE. 74, 307-314 (1969). 18. Reeve, J, N., Groves, D. J,, and Clark, D. J. Regulation of cell division in Eschetichia co8: Characterization of temperature sensitive division mutants. J. Bacterial. 104, 1052-1064 (1970). 19. Schaubach, W. II., Whitmer, J. D., and Davem, C. I. Genetic control of DNA initiation in Eschwichia coli. J. Mol. Biol. 74, 205-221 ( 1973). 20. Smith, D. W. In “Progress in Biophysics and
Molecular Biology,” Vol-26, pp. 324-333, Pergamon Press, Oxford, 1973. 21. Webb, M. Effects of magnesium on celluIar division in bacteria. Science 118, 607-611 (1953).
12, 15-25
(1975)
The Moleculur Nature of Damage by Oxygen Freeze-Dried Escherichia co/i ’ E. ISRAELI,
Israel Institute
A. KOHN,
for Biological
AND
JANINA
Research, Ness-Ziona
to
GITELMAN and Tel-Auiv University,
Medical School, Ramat-Aviv, Israel
Molecular oxygen is toxic to freeze-dried (FD) bacteria (14). Freeze-dried E. coli exposed to air lose the ability to form cob nies on nutrient media, but retain the ability to complete the ongoing DNA synthesis (16), and to serve as host for the production of infectious phage (8). It was suggested that the damage caused by oxygen might be connected with the comp1ex responsible for initiation of DNA synthesis ( 7). To support this hypothesis we present in this communication data, based on experiments with thymidine deficient mutants of E. coli. METHODS
1, Bacterial Strains a. E. co& K-12 auxotroph which is thy-, his-, prom, arg-, try-, thiamine-. b. E. coli K-12 120/6. Which is thy-, pro-, str* ts dna, received from Dr. Beyersman, Berlin ( 1) . c. E. coli B thy-.
CaCl,, 2 X 1O-4 M; FeC&, 2 X 1O-e M; Na$O,, 2.5 X 1O-3M; Tris, 0.12 M; glucose, 0.4%; KH2P04, 1.5 X 1O-3 M; (16) suppIemented with thymine, histidine, proline, and arginine (20 pg/ml of each), 30 pg/ml of tryptophan and 5 /*g/ml of thiamine. b. The temperature sensitive mutant was grown in TPG broth (Tris-Phosphate-Glucase) containing in 1 liter of water: NH&I 1.1 g, KC1 8 g, NaCl 0.5 g, MgCl, *6Hz0 0.2 g, KH2P04 0.023 g, sodium pyruvate 0.8 g, Tris (Z-amino 2 hydroxymethyl propan 1:3 dial) 12.1 g, CaC12 -1 ml of 0.1 N solution NazS04 -1 ml of 0.16 N sofution. Adjusted to pH 7 with 37% HCI. After autoclaving FeC& *6Hz0 1 ml of I pg/mI solution, and 0.2% gIucose were added. The medium was supplemented with proline, thymine, and thiamine 20 pg/ml of each. c. Tryptose agar served as plating medium. d. E. cola’ B was grown in L-broth (71, 3. Starvation for Amino Acids The AA auxotroph was grown in TGA broth with shaking at 37°C until logarithmic phase of growth was reached. The cells were then washed and half of the culture was resuspended in TGA lacking the required AA and shaked for 2 hr at 37°C. The other half continued growing in fully suppIemented TGA and served as control. The cells were then chilled to 4”C, washed
2. Growth Media a. The amino acids (AA) auxotroph was grown in TGA synthetic medium, the composition of which was: NaCl, 0.08 M; KC], 0.02 M; NH&I, 0.02 M; MgCl,, 10m3 M; Received
July 1, 1974.
1 This work is part of a Ph.D. thesis to be submitted
to the Tel-.4viv
University
by E. Israeli. 15
Copyright 6 1975 by Academic Press. Inc. All rights of reproductirrn in any form reserved.
16
ISRAELI,
KOHN
and freeze-dried, applying the foIlowing procedure. One-milliIiter amounts of a suspension containing 1011 cells/ml, washed and resuspended in distilled water, were pipetted into standard lo-ml ampoules. Each ampoule was spun around its axis at ca. 2000 rpm while dipped in a methanol-dry ice freezing mixture ( -78”). The frozen ampoules were then dried for 3 hr under vacuum, the fmal pressure in the system after complete dehydration being 5 e Hg. After completion of the drying process ampoules were sealed in vacua.
GITELMAN
6. 3H TdR Incorporation To 10 ml of bacterial suspensions (I2.108 cells/ml) 0.2 ml of 3H TdR was added (50 &i/ml lo-15 Ci/mM). Samples (0.1 ml) were drawn out at intervals to tubes containing 2 ml TCA ( 5% ), 0.2 ml sodium pyrophosphate 0.2 N and 200 pg/ml of non radioactive thymidine, at 0°C. After 18 hr at 4°C the samples were filtered through glass filters (Whatman GF/C), washed thrice with ice cold, water, once with ethanol, dried and counted in Packard’s Tricarb scintillator, using toluene based scintillation fiuid. 7. Formation of Filamentous Freeze-Dried Bacleria
4. Starvation for Thymine Escherichia coli K-12 was grown in TGA to a concentration of 10” cells/ml. The cuIture was divided in two: half continued growing, (control) and half was washed, and resuspended in the same medium lacking thymine, and incubated with shaking for 50 min. at 37°C. After another washing, the cultures were suspended in the same medium and grown in the presence of chloramphenicol (CAM 150-300 pug/ml) or in the absence of essential amino acids, and tritiated thymidine ( 3H TdR) incorporation was measured. Freeze-drying took place after the period of thymine starvation. 5. Growth at the Restrictive
AND
Temperature
Escherichia coli K-12 ts-dna was grown in TPG with shaking at 33”C, to a concentration of 2.107 cells/ml. The culture was divided in two: half was grown further at 33’C, the other at 42°C for 2.5 hr for the completion of rounds of replication (1). The cultures were then transferred to an ice bath, washed trite in water, and freezedried. In order to measure thymidine incorporation at different temperatures, the dried bacteria were resuspended in medium prewarmed to the desired temperature (42°C ar 33°C).
Forms by
Escherkhia coli B, dried or exposed to was resuspended in L-broth and wgen, grown for several hours at 37°C. Additional samples were inoculated onto microscope slides coated with a thin (l-mm) layer of agar (containing the same ingredients as the L-broth). Individual cells were photographed at different intervals in a Reichart microscope (magnification X 480). The slides were incubated at 37°C in a humid chamber. Samples from the liquid cuIture were fixed in 2.5% glutaraldehyde, postfrxed in 2% osmium tetroxide, and embedded in epon. Preparations were sectioned with Omu-2 Reichart ultramicrotome, stained with uranyl acetate and lead citrate, and observed in a JEM 100 B electron microscope. 8. Autoradiogrnphy (Modification Method for Animal C.&s (17)
of the
Samples of FD and oxygen exposed bacteria ( FDO ), were resuspended in full medium and allowed to incorporate “H TdR for 4-6 hr (specific activity in the medium = 0.1 Ci/mM, 5 &,/ml). The samples were then washed with growth medium and water, and fixed on microscope sbdes. The slides were covered with
OXYGEN
DAMAGE
TO LYOPHILIZED
E. cob
FIG. 1. *H TdR incorporation of FD and FDO E. coli K-12 after AA. starvation. E. coli K-12 wan grown in TGA to a concentration of 2.10’ cells/ml. The cells were washed and resuspended with AA (open circles) or without (closed circles). After FD (left panel) and resuspension, or FD and oxygen exposure (right panel) incorporation of *H TdR was measured. Oxygen exposure was at 28°C and 45% relative humidity (RH).
photographic film (Kodak scientific plates AR lo), dried and stored at 4°C for one and two weeks, then developed and fixed. Afterwards the preparations were stained with Giemsa-Felz stain for 2-3 hr. RESULTS
1. Incorporation
of “IS TdR in Escherichia
Coli (AA Deficient) This mutant, when grown in medium lacking the essential AA would complete the ongoing DNA synt!-esis, but is unable to reinitiate it (13). DNA synthesis begins anew onIy upon the addition of the missing AA, as measured by 3H TdR incorporation. When a fuIly supplemented culture was compared with one lacking AA, “H TdR incorporation proceeded at the same rate for 90 min but then, in the AA deficient me-
dium, it ceased. It was resumed only after 60 min after the addition of AA to the deficient culture (resuhs not shown), Having established this course of events, a culture of the mutant was prepared, so that it was in the quiescent stage, having completed the DNA cycle (about 120 min after withdrawal of AA). These bacteria were then washed and freeze-dried. After exposure in the dry state to air (oxygen) at 28°C and 45% relative humidity, the samples were resuspended in TGA and the incorporation of SH TdR into them was measured. In bacteria which were kept in vacua after lyophilization, the incorporation of “H TdR was similar both in those grown in the presence of AA, as in those held for 120 min in the absence of AA (Fig. I ) , whereas in the fully supplemented culture, the DNA synthesis started immediately upon resus-
ISRAELI,
18
KOHN AND GITELMAN
PER00 OF EXPOSLRE(HRS) FIG. 2. Survival curves of FD E. co8 K-12 upon exposure to oxygen. ( T = 28”C, RH = 45% ), Open circles-grown with AA; closed circles-grown without AA.
AA-deficient cells incorporated SH TdR exponentially even after 4 hr. These results indicate that when bacteria, that were not actively engaged in DNA synthesis, were freeze-dried and exposed to air their ability to synthetize DNA and to reinitiate it, was not impaired, while bacteria that were in the process of DNA synthesis when subjected to FD and oxygen, though able to complete the already initiated DNA synthesis could not reinitiate the synthesis of DNA. It thus seems that when the initiation site for DNA synthesis is not actively engaged at the time of FD and exposure to oxygen, it is not damaged by it. Consequently, it was not surprising to find that the survival curves of AA deficient bacteria indicated a considerable protection against the oxygen effect. (Fig. 2 and Table 1).
pension in full medium. In the AA deficient culture there was a delay of about 40 min before the DNA synthesis became evident. As for the FD bacteria exposed to oxygen, the situation was reversed. Reconstituted, fully supplemented bacteria ceased SH TdR incorporation after 2-3 hr of incubation, i.e., DNA synthesis ceased after the completion of one cycle. On the other hand, TABLE EFFECT OF
GROWTH
CONDITIUNS FREEZE-DRYINQ B wild
Mutant
In order to test the hypothesis that protection against the lethal action of oxygen involves an initiator protein or initiation complex we used bacteria in which protein synthesis is halted by chloramphenicol 3
ON SURVIVAL OF Escherichia coli NUT.%NTS AND EXPOSURE TO OXYOEN~ K-12 amino acid mquiring Amino mid atrmvation
Treatment
Decay constant of survival curyes
2. lncorporalion of 3H TdR in Escherichia coli K-12 after Thymine Sfaruation upon Blocking Protein Synthesti
CY T
1.9 1.1
Q-DNA temDerature aenaitive for DNA aynthesk
AFTER B log phase
GWKII with CAM
1.20 0.53
a Bacterial mutants were grown under conditions of optimal growth then transferred to restrictive conditions for l-Z.5 hr (see Methods). The bacteria were then frozen, freeze-dried and exposed to oxygen for varying periods of time in the dry state. The viability of reconstituted bacteria wae determined at these time intervals and from the results a decay curve WLI constructed. Decay rates (K) are shown in the table. Exposure to oxygen was at 2S°C, 45% relative humidity. b C = control bacteria (not treated). * Reference (7).
OXYGEN
DAMAGE
TO LYOPHILIZED
E. coli
19
FIG. 3. “HTdR incorporation in FD and FDO E. coli K-12, upon protein synthesis inhibition, after thymine starvation. Cells were starved for 50 min. After Iyophilization and oxygen exposure, the samples were resuspended in synthetic medium upon protein synthesis inhibition, Triangles-FD bacteria without protein synthesis inhibition; Closed circles-FD bacteria upon protein synthesis inhibition; Open circles-FDO bacteria (30 min exposure) upon protein synthesis inhibition.
(CAM ), or thymineless mutants which can synthetize DNA in the absence of protein synthesis (2). In wild-type bacteria, or in thymineless mutants growing in the presence of thymine, inhibition of protein synthesis by CAM causes cessation of DNA synthesis (13). On the other hand, in T- mutants deprived of thymine, there are produced stable initiator proteins so that even in the presence of CAM, DNA synthesis eontinues. It was expected that thymine starved mutants, which had accumulated initiation proteins should be less sensitive to the oxygen effect, than the same mutants growing in the presence of thymine, provided that the lethal effects of oxygen were indeed due to its direct interaction with such a protein( s ), If this be the case, the thymine-starved mutants, after exposure to oxygen, should incorporate more 3H TdR than controls, If, on the other hand, exposure of thymine-starved mutants to oxygen is associated with a decreased DNA
synthesis, one wouId have to assume that the oxygen did not directly interact with these proteins, but affected the initiation site or the membrane. Experimentally, when bacteria grown in the absence of AA and in the presence of CAM were lyophilized and held in vrzcuo before resuspension, the incorporation of 3H TdR proceeded for about 60-120 min, then ceased, having reached a level corresponding to doubling of DNA content (Fig. 3). The cessation of DNA synthesis after doubling was aIso observed in thyminestarved bacteria, grown in the absence of AA and with CAM, after lyophilization and exposure to oxygen. On the other hand, lyophilized thymine-starved bacteria, when reconstituted in medium permitting protein synthesis carried on 3H TdR incorporation exponentiahy for at least five hours. This result indicates that if after FD, repair is permitted, DNA will be synthetized. The damage due to FD per se is thus reversible, but becomes irreversible upon exposure to oriygen.
20
ISRAELI,
KOHN
AND
GITELMAN
AT 42’C
AT 33%
I
I
60pomBb300 TIME
60
ta
TIME
~MIN)
WIN)
FIG. 4. ‘HTdR incorporation in FD and FDO E. coli ts-dna at 33 and 42°C. Cells were grown in TPC, lyophilized and oxygen exposed (1.5 hr for bacteria grown at 33°C and 2.5 hr for those grown at 42°C). Resuspension was in TPG and the incorporation measured both at 33°C (a) and 42°C (b). Closed squares- Control bacteria grown at 33°C; Closed circlesControl bacteria grown at 42°C; Open trianglesFD control bacteria grown at 33°C; Closed triangles-FD control bacteria grown at 42°C; Open squares-FDO control bacteria grown at 33°C; Open circles-FDO control bacteria grown at 42°C.
3. Incorporation coIi K-12 ts-dna
of SH TdR in Escherichia
We have used a DNA ts-mutant of E. cola isolated by Beyersman ( 1) defective in the initiation of DNA synthesis (see aIso 2-4, 11, 12, 19). At the nonpermissive temperature (42°C) this mutant completes the DNA cycle, but is unable to reinitiate DNA synthesis unless it is brought back to the permissive temperature (33°C ) . One would expect that this mutant grown at the nonpermissive temperature would be in a physiological state similar to the thyminestarved, or AA-starved bacteria, and therefore after freezc-drying would be less sensitive to oxygen ( Table 1) . The incorporation of thymidine in FD &mutants was practically unaffected when they were frozen after growing at 33”C, but was significantly depressed when frozen from 42°C. Exposure to oxygen completely abohshed thymidine incorporation in FD bacteria grown at 42°C but permitted con-
siderable incorporation at the permissive temperature ( Fig. 4). The decay of these FD ts-mutant after exposure to oxygen was considerably smaller in bacteria held for 2.5 hr at nonpermissive temperature than in bacteria grown and held at 33°C (Fig. 5). In this respect, the results have the same character as in experiments with AA-starved cells, or with colicin-treated ones, namely that in the absence of initiation events, FD bacteria are less sensitive to oxygen. 4. Mimoscopical Electron)
Obsewations
( Optical and
While control bacteria upon seeding on nutrient agar start dividing within 30-60 min, and form microcolonies within 2-3 hr after seeding (Fig. 6a), FD bacteria produced at that time only long filaments (Fig. Bb) and only 10% of them produced colonies. Similar filamentous growth has been observed in E. coli deprived of magnesium (21) or thymine (18). When these fila-
OXYGEN
DAMAGE
TO LYOPHILIZED
E. di
mentous forms, observed in freeze-dried and reconstituted bacteria, were labeled cvith “H TdR and then autoradiographed, a diffuse pattern of grains (Fig. 7) indicated segregation of the DNA and multipoint initiations. No septa were seen in these filaments (Fig. 8), though pro&ction of septa per se is not SufFicient for ceILdivision: a ts-mutant of E. coli (MX174 ts27) at 41°C produces septa but does not divide (5). In FD bacteria exposed to oxygen (Fig. 6c), only about 1% of the cells grew to filaments, while the viability of the culture was as Iow as 10-5y0. DISCUSSION
When amino acids deficient
mutants
of
E. cc& that were not activeIy engaged in DNA synthesis, were freeze-dried and exposed to oxygen their ability to synthetizc DNA and to initiate it was not impaired, while similar treatment of bacteria that were in the process of DNA synthesis caused loss of reinitiation of DNA synthesis. Comparison of these results with those concerning the effect of colicin El (7) on oxygen exposed bacteria, indicate that in both cases the process of initiation of DNA synthesis seems to be affected, either by lack of synthesis of a necessary initiator protein, upon AA deprivation, or by binding of coIicin to initiator site, so as to protect the cells against the lethal action of oxygen (Table 1). In order to distinguish whether oxygen affects the initiation proteins or the initiation complex associated with the plasma membrane, we checked DNA synthesis in thymineless mutant after growth in medium lacking thymine. The results obtained with this mutant suggest, that the damage to the DNA synthesis mechanism is caused during freeze-drying. If after Iyophilization repair is permitted (protein synthesis), DNA is synthetized. The damage due to FD per se is thus reversible. It ceases to be reversible when the FD bacteria are exposed to oxygen, This finding is com-
PER100 OF EXPOSURE IHRS)
FIG. 5. Survival curves of FD E. cdi ts-dna after growth at different temperatures. Open circles-FD bacteria grown at 33°C; closed circlesFD bacteria grown at 42°C. (Exposure to oxygen was at 28°C and 45% RH.) patible with the results obtained from experiments on phage production in FD bacteria (8) and on membrane permeability (6). Since the initiator protein is stable and present in excess in the cells, it is more plausible that the damage occurring during FD would involve the initiation site rather than the initiator protein itself. Two conditions are required for the initiation of DNA synthesis: (a) there has to be RNA and protein synthesis ( 13, 15, ZO), and (b) the initiation complex has to be associated with the cytoplasmic membrane (9). By the use of inhibitory properties of CAM and phenethyl alcohol (PEA), two proteins were implicated to take part in initiation of DNA synthesis. One is inhibited by high concentration of CAM, but not by a low one. During growth in the presence of PEA, the CAM sensitive pro-
ISRAELI,
FIG, 6. Grawth of E. bacteria. c. FJX bacteria.
coli
KORN AND GITELMAN
B m agar {3 hr growth) I 11.Control nontreated bacteria. b. FD
OXYGEN
DAMAGE
TO LYOPHILIZED
tein accumulates, but the synthesis of CAM-refractive protein (presumably a membrane protein), is inhibited (20). During thymine starvation, initiation protein (s > accumulate; this protein permits a number of initiations, while the initiation proteins produced in normally growing cells are destroyed after each initiation (IO). En the ts-mutant of E. coli we used, either the initiation protein per se is thermolabile, or the binding process to the membrane is thermolabile, perhaps because of change in phase transition properties of the membrane lipids. The results presented here tend to support the second alternative. The changes in membrane due to Oa are effective jn blocking initiation only if the initiation complex is bound to the membrane. If it is not, as in the case of adsorption of colicin, or because of phase transition in the &mutant at 42”C, the effect of oxygen is largely depressed (Table 1). The finding that FD and exposure to air produced the same effects in bacteria grown at :33”C (when initiation complex is
FIG. 8. Electron micrograph of 6lamentous suspended in L-broth and grown for 4 hr.
E. COG
23
FIG. 7. Autoradiography of FD E. coli B. Freezedried bacteria were resuspended in L-broth and grown for 4-6 hr in the presence of “HTdR (0.1 Ci/mhr, 5 ,&i/ml) _
operative) and those grown at 42°C (when it is not), has to be explained. It seems that the critical event is the formation of the initiation complex at 33°C and that this event is instantaneous. ts bacteria that are grown at 42°C have to be cooled to freez-
forms
of FD
E. c&
B. FD
bacteria
were
re-
24
ISRAELI,
KOBN
ing temperature. They pass through the permissive temperature (33°C ) , SD that initiation complex may be formed, and thus their sensitivity to oxygen would not be different from that of cells grown at 33°C. This result is incompatible with an assumption that a necessary protein is entirely missing in the ts-mutant grown at nonpermissive temperature, but is consistent with an alternative that the conformation of a protein necessary for initiaticn or binding to the membrane changes with temperature. An interesting point also to be explained is that in bacteria exposed to oxygen and reconstituted at 42”C, both initiation and DNA elongation are halted (Fig. 4a, open squares and circles ). One may perhaps postulate that there are at least three elements involved in DNA synthesis, all three required for initiation (15) but only two for elongation. One of the ekments (membranal) is affected by exposure to oxygen, another (an enzyme) by elevated temperature, so that at 42°C there is no DNA synthesis in oxygen-treated bacteria because two of the three elements are damaged. Damage to only one of the three factors (by oxygen treatment or high temperature), would affect initiation only. Our results indicate that the site sensitive to oxygen in the FD bacteria is a membranal one. Damage to this site stops initiation of DNA synthesis, the cells lose their ability to divide and form colonies and so they “die.” SUMMARY
The damage occurring in freeze-dried bacteria exposed to oxygen is mainly in the bacterial membrane and involves the DNAinitiation complex. This injury occurs in two stages: The primary damage is due to the freeze-drying itself, and is repaired upon reconstitution of bacteria and their subsequent incubation in nutrient broth. The repair process requires protein synthesis. In the next step, the exposure of freezedried bacteria to oxygen, the injury be-
AND
GITELMAK
comes irreversible and the bacteria ‘
J. Mol. Btid. 53,369387 ( 1970). 5. Inouye, M. Unlinking of cell division DNA
reolication
in a temoerature
from
sensitive
OXYGEN
DAMAGE
TO LYOPHILIZED
DNA synthesis mutant of Escherich~a coli. J. Bacterial.
99, 842-850
( 1969).
6. Israeli, E., Giberman, E., and Kohn, A. Cryobiology 11, 473-47-I (1974). 7. Israeli, E., and Kahn, A. Protection of lyophilized Escherich!u coli from oxygen by co!icin E, treatment. FEBS Letters 26, 323-326 ( 1972). 8. Israeli, E., and Shapira, A. Production of bacteriophage T4 by lyophilized and oxygen exposed Escherichia cc&. .l. Gela. Microbid. 79, 159-161
( 1973).
9. Jacob, F., Brenner, S., and C&n, F. On the regulation of DNA replication jn bacteria. Cold Spring Ha&. Symp. @era&. Biol. 28, 329-348 ( 1963 ) . 10. Kogoma, T., and Lark, K. G. DNA rephcation in Eschmichia coli: Replication in absence of protein synthesis after replication inhibition. 1. Mol. Biol. 52, 143-164 ( 1970). Il. Kuempel, I’. L. Temperature sensitive initiation of chromosome replication in a mutant of Escherichia coEi. J. Bacterid. 100, 13021310 (1969). 12. Lark, K. C. Regulation of chromosome replica-
tion and segregation in bacteria. Becteriol. Reo. 30, :3-32 ( 1966).
13. Lark, K. G., Repko, T., and Hoffman, E. J. The effect of amino acid deprivation on subsequent DNA repIication. Bbchim. Biophvs. A& 76, !3-24 ( 1963 ).
E. coU
25
14. Lion, M. B. Quantitative aspects of protection of freeze-dried Escherkhia coli against the toxic effect of oxygen. J. Gen Microbial. 32, 321329 (1963). 15. Messer, W. Initiation of deoxyribonucleic acid replication in Eschetichia coli B/r: chronology of events and transcriptiona contro1 of initiation. J. BucterioE. 112, 7-12 ( 1972). 16. Novick, O., Israeli, E., and Kahn, A. Nucleic acid and protein synthesis in reconstituted Iyophilized Eschetichia cc& exposed to air. J. AI& Bactera’ol. 35, 184-191 ( 1972), 17. Oren, R., and Kohn, A. Density dependent inhibition of cell growth in cultures of primary and established lines of cells. J. Cell. PhysioE. 74, 307-314 (1969). 18. Reeve, J, N., Groves, D. J,, and Clark, D. J. Regulation of cell division in Eschetichia co8: Characterization of temperature sensitive division mutants. J. Bacterial. 104, 1052-1064 (1970). 19. Schaubach, W. II., Whitmer, J. D., and Davem, C. I. Genetic control of DNA initiation in Eschwichia coli. J. Mol. Biol. 74, 205-221 ( 1973). 20. Smith, D. W. In “Progress in Biophysics and
Molecular Biology,” Vol-26, pp. 324-333, Pergamon Press, Oxford, 1973. 21. Webb, M. Effects of magnesium on celluIar division in bacteria. Science 118, 607-611 (1953).