FAILURE OF PROTEIN SYNTHESIS AND NET RNA SYNTHESIS IN HEAT-KILLED ESCHERICHIA COIJ Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY ROGER GLEN DEAN 1976 ‘ This is to certify that the thesis entitled FAILURE OF PROTEIN SYNTHESIS AND NET RNA SYNTHESIS IN HEAT-KILLED ESCHERICHIA COLI presented by Roger Glen Dean has been accepted towards fulfillment of the requirements for DOCTOR or mumsnzm degree inflophysiw W274 654mg Date 171 / 41/74 0-7539 2 wide. .na um Er: m”. Aw”: (-— J {AD/£97 ) 0 ABSTRACT FAILURE OF PROTEIN SYNTHESIS AND NET RNA SYNTHESIS IN HEAT-KILLED ESCHERICHIA COLI by Roger Glen Dean High temperature treatment (480C and above) of Escherichia coli causes cell death as determined by the inability of the killed cells to form colonies upon plating. The kinetics of heat-killing, which is pseudo-first-order, lead to the conclusion that one or a small number of events or molecules are the cause of death. In addition to the first-order kinetics of killing, a number of molecular sites of thermal damage have been found in heated cells of various bacteria. These sites of damage include proteins, the membrane, rRNA, and DNA. There is evidence that much of the thermal damage produced by heating cells is not lethal. This non-lethal damage is expressed by the increased lag time necessary, after heating, for survivors to reinitiate growth. In order to separate more clearly thermal damage which causes death from thermal damage that is non-lethal, it is necessary to compare heat-killed cells and survivors which have been subjected to the same heating conditions. To accomplish this comparison, cells were heated until a mixed Roger Glen Dean population of heat-killed cells and survivors was achieved. Treat- ing this mixed population with penicillin causes lysis of the sur- viving cells. The heat-killed cells, or a class of heat-killed cells, is unaffected by this process and remain largely intact. Labeling the population of surviving and heat-killed cells immediately after heating with [3H]uracil or [140]1eucine provides for a comparison of protein or RNA synthesis after heat-killed and surviving cells are separated using the penicillin treatment. This technique has shown that protein synthesis and net RNA synthesis fails in heat-killed cells. Furthermore, this failure occurs in the earliest stages of recovery. These results indicate that the lethal damage occurring in heat-killed cells is directly coupled to protein and RNA synthe- sis and causes the immediate failure of these two synthetic processes. Several possible explanations of these results are considered. These explanations are based on the types of molecular damage known to occur in heated populations of bacteria. Most explanations are found to be inadequate because they are either inconsistent with the results of this thesis, they are inconsistent with the first-order kinetics of killing, or the types of damage have not been shown to be lethal. One alternative explanation does appear to have some merit and is proposed. This prOposal suggests that damage to the nucleoid RNA which holds the DNA together, is the rate-limiting step in heat-killing. This pr0posal agrees with the data of this thesis and with the first-order survival kinetics. FAILURE OF PROTEIN SYNTHESIS AND NET RNA SYNTHESIS IN HEAT-KILLED ESCHERICHIA COLI by Roger Glen Dean A DISSERTATION - Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biophysics 1976 ® Copyright by ROGER GLEN DEAN 1976 To my parents, wife, and children ii ACKNOWLEDGEMENTS The assistance of my advisor Dr. Estelle McGroarty with this research is very deeply appreciated. She had the difficult task of advising me in this work, which was outside her immediate area of research. Despite such an obstacle, her insight, patience, and con- siderable effort were instrumental in the deve10pment of this work. In addition I am most grateful for her assistance in the preparation of the text of this dissertation. I would also like to express my gratitude to the members of my dissertation committee: Dr. Barnett Rosenberg, Dr. Gabor Kemeny, and Dr. Harold Sadoff. Their continued support and assistance with this research was most helpful and is greatly appreciated. I must also thank my wife Barbara who typed all the rough drafts and the final draft of this dissertation. I was also supported in this work by the Michigan State Univer- sity College of Osteopathic Medicine and NIH Training Grant No GM 01422. 111 LIST OF TABLES . . . . LIST OF FIGURES . . . INTRODUCTION . . . . . Kinetics . . . . Molecular Sites of EXPERIMENTAL THEORY . MATERIALS AND METHODS Bacterial Strain TABLE OF Damage CONTENTS page C O O O O O O O O O O O O O C . Vii O O O O O O O O O O O O O O O O 16 Growth of Cells and Plate Counting . . . . . . . . . . . . 16 Assay Buffers and Media Heating . . . . . O O O I O O O O O O O O O O O O O O 17 O O O O O O O O O O O O O O O O 18 Centrifugation . . . . . . . . . . . . . . . . . . . . . . l9 Radioactive Labeling and Counting Techniques . . . . . . . 19 Separation Assay O O O O O O O O O O O O O O O O 21 RE SULTS O O O O O O O O O O O O O O O O O O O C O O O O O O O O 2 6 Kinetics of Heat-killing and Recovery . . . . . . . . . . . 26 [14C]leucine Contamination of Pellet Fraction . . . . . . . 32 Protein Synthesis in Heat-killed and Surviving Cells . . . 35 Contents of the Pellet Fraction . . . . . . . . . . . . . . 41 Pellet Fraction Losses of Incorporated [14C]leucine . . . . 45 Degradation of protein 0 O O O O O O O O O O O O O O O 46 iv Losses during heating--whole protein Whole cell losses during centrifugation . . . Net RNA Synthesis in Surviving and Heat-killed Cells [3H]uracil Contamination of the Pellet Fraction . . CONCLUSIONS DISCUSSION RECOMMENDATIONS APPENDIX A APPENDIX B BIBLIOGRAPHY page 49 54 6O 65 69 76 9O 93 94 96 LIST OF TABLES Loss of [14C]leucine from the heat-killed or pellet fraction during the "Separation Assay"a. . Amount of [14C]leucine found in cells before heating and in the total suspension after heatingaoooo00.00.000.00...o. Amount of [14C]leucine found in cells and extra- cellu13r1y after heatinga o e o o o o o o o o o 0 TCA precipitable [14C]leucine in filtrate from ce118 after heatinga. o o o o o o o o o o o o o 0 Cells remaining in the supernatant fraction after heating and centrifugation steps described in the "Separation Assay"a. . . . . . . . . . . . Living cells left in the supernatant fractions after centrifugation8 . . . . . . . . . . . . . . vi Page 43 51 52 53 55 57 LIST OF FIGURES Figure Page 1. Types of survivor curves observed during heating. N is the number of cells surviving at time t and No is the initial number of cells . . . . . . . . . . . . 6 2. Flow chart of the "Separation Assay". . . . . . . . . . . 24 3. Kinetics of heat-killing at 50.0°C (o), and 50.80C (0). Cells surviving heating (N) as a percentage of the orig- inal number (NC) at the end of 15 min of heating were 66% at 50.0°C and 56% at 50.8°C. The bars represent the two standard deviation interval for each measurement. . . 27 4. The kinetics of recovery after 15 min of heating was measured by viable cell counts. The temperature of killing in both cases was 49.900. This heating resulted in 35% survivors (o), and 83% survivors (0) immediately after heating when recovery was initiated . . . . . . . . 30 5. [140]leucine contamination of the pellet fraction. Cells were grown to an OD of 0.075, centrifuged and resuspended in 12 ml of heating gaffer with 4 ml of "recovery concentrate" added. [ C]leucine was added to a final concentration of 0.417 uCi/ml. Cells were not heated and the "Separation Assay" (Steps 4 through 10) was used to give a supernatant fraction (0), and pellet fraction (0) . . . . . . . . . . . . . . . . . 33 6. The measurement of [14C]leucine (0.417 uCi/ml) incorporated in surviving cells (0), and heat- killed cells (0) during recovery. The total num- ber of cells before heating was 5.16 x 107. After heating the number of survivors was 2.00 x 107, giving 39% survivors. The procedure followed was that of the "Separation Assay." . . . . . . . . . . . . . . . . . . . 36 vii Figure 7. 9. 10. 11. Page The rate of [14C]leucine incorporation calculated from the 10 min intervals of Figure 6. The rates were calculated by taking the difference in counts for each 10 min interval and plotting these values on the ordinate at the midpoint of the time interval on the abscissa. The dotted line is the extrapolation of the curve . . . . . . . . . . . . 38 Cells were labeled during growth with about 0.26 uCi/ml [14C]leucine for 55 min, then washed and heated for 30 min at 51°C which produced 22% survivors. Cells were cooled and "recovery concen- trate" supplemented with cold leucine (714 ug/ml) was added. TCA was added to 2 m1 samples at 1 hr intervals and cells were collected on membrane fil- ters (o). The sample taken at 5 hr (0) was col- lected on a membrane filter but was not treated with TCA. The filtrate of the 5 hr sample was also retained and counted (0). . . . . . . . . . . . . . . . . 47 The measurement of [3H]uracil (1.25 uCi/ml) incor- porated in surviving cells (0), and heat-killed cells (0) during recovery. The to al number of cells before heating was 4.90 x 10 . After heating, the number.of survivors was 1.84 x 107, giving 38% survivors. The procedure followed was that of the "Separation Assay". . . . . . . . . . . . . . . . . . . . 61 The rate of [3H]uracil incorporation calculated from the 10 min intervals of Figure 9. The rates were calculated by taking the difference in counts for each 10 min interval and plotting these values on the ordinate at the midpoint of the time interval on the abscissa. The dashed line is the extrapola- tion of the curve . . . . . . . . . . . . . . . . . . . . 63 [3H]uracil contamination of the pellet fraction. Cells were grown to an OD of 0.07, centrifuged and resuspended in 12 ml of heating buffer with 4 m1 of "recovery concentrate" added. [ H]uracil was added to a final concentration of 2.5 uCi/ml. Cells were not heated and the "Separation Assay" (Steps 4 through 10) was used with the addition of Triton x+100 (0.008% final concentration) in Step 5. The super- natant fraction (0) and the pellet fraction (0) is Show 0 O O C O O C O O O C O C C O O O O O O O O O O O O 66 viii INTRODUCTION The study of killing micro-organisms by means of heat has had a very long history. Initial efforts were directed toward developing methods of sterilizing and protecting food products. The early con- tributions of investigators were of tremendous practical importance to the development of microbiology, but very little was done toward understanding the mechanism of heat-killing. Many studies were car- ried out in which various organisms, primarily those organisms found in food, were heated in milk, fruit juice, and many other media related to food products. Studies of this kind were not directed towards elucidating the mechanism of heat-killing. When studies were directed towards finding the mechanism of heat-killing, no consistent method of growth, heating, or recovery was used, making it difficult to compare results between different laboratories. In addition, a number of inadequate methods of measuring the thermal resistance of micro-organisms were used in early experiments. Some of these diffi- culties persist in present experiments, particularly the variations in methods employed. Kinetics An important advance toward understanding the molecular mecha- nism of heat-killing was made when Chick (16) measured the number of surviving cells in bacterial cultures during heating as a function 2 of time. The number of surviving cells at various times of heating were determined by plating samples of the heated cells and counting the colonies which grew. These kinetic measurements resulted in the finding that the kinetics of heat-killing was exponential, typical of first40rder chemical kinetics. The functional relationship is found to be N/No - exp[-kt] (l) where N is the number of survivors, No is the number of cells in the initial papulation, and k is the rate constant. This relationship has been a central theme in most work concerned with heat-killing of micro-organisms. It has been interpreted, misinterpreted, and ques- tioned as to its general applicability, but it has always been con- sidered informative as to the molecular mechanism of heat-killing. This relationship is particularly important for the experiments dealt with in this thesis, since the survivor curve has been found to be exponential for Escherichia coli (16). Most investigators feel intuitively that the first-order rate of heat-killing is explained by events at the molecular level. Alone, first-order kinetics for a process does not imply that the process is the result of molecular events. For example, a popula- tion of ants on a well-traveled sidewalk will give an exponential survival curve, yet this killing can hardly be described as due to molecular events. A more important relation which implies the mole- cular nature of a process is the temperature dependence of the rate constant k. The temperature dependence of the rate constant for the survival of micro-organisms often follows quite closely an Arrhenius equation (28), which means there is an activation energy in the 3 process, and the events described by the rate constant are molecular. Thus, when applied to the case of the ants, this explanation is con- sistent with the conclusion that the death in ants is not a molecular event, since it does not show such temperature dependence. The molecular events which give rise to the logarithmic survival curve are better understood by deriving the relationship from proba- bility theory. The derivation of the exponential survivor curve de- mands three conditions. First, the process being described is a two- state process, going from state A to state B. In the case of heat- killing, the organism goes from living to dead. Secondly, the pro- cess is not reversible. Thirdly, the probability of a cell being killed in a unit of time dt is kdt, where k is the rate constant of the logarithmic survival curve. When these three conditions are met, the derived equation is identical to Equation 1 (see Appendix A). The meaning of the exponential survival curve is contained in the conditions set down which allowed its derivation. The most straight-forward, and perhaps the only interpretation of these conditions is that a single molecule in each cell makes the transition from state A to state B, which results in the death of the cell. This deceptively simple interpretation makes very good mathematical sense, but when applied to the complexities of the bac- terial cell, it is difficult to imagine how this could be true. A number of authors have argued against such interpretation (14), and some have made attempts at other interpretations (l9). Biologically, it is easier to think in terms of many molecules denaturing or for many steps to be included in the process.‘ Such possibilities, when examined carefully, are found to be inadmissible in the framework 4 of exponential killing. First, any process which is made up of several steps, each having a rate constant equal (or close to equal) to the others, requires a shoulder (an initial time when no death is observed) in the exponential survivor curve when plotted as the logarithm of survivors vs. time. This shoulder would occur if the steps are sequential or if there are a large number of molecules, each of which has to be destroyed before death can occur. The shoulder would appear, because the time taken to accomplish the steps prior to the last step is a period when the organisms are still alive. Therefore, a lag time would be necessary for the first steps to be accomplished before death would be due to the last event. This thinking is incorporated formally in the derivation of Equation 1 when we assume that the process of going from state A to state B has a constant probability, regardless of the organism's previous history of heating. If the death process in the organism had proceeded through several steps, then at any time t, some of these steps would have been accomplished, and the organism would have an increased probability of death in the next instant of time. This would not fit the theory. This line of reasoning does not imply that we have ruled out the possibility of many events. There can be any number of events, as long as the rate constants of these events are much greater than the rate constant for one of the events. Thus, the slowest event (the event with the smallest rate constant) becomes the rate-limiting step. Since all the steps before and after this step are rapid, the organism can be considered to live or die based on the rate-limiting step. Another possible molecular mechanism which would give an 5 exponential survivor curve is that there are several molecular sites, each of which can be lethal if it is destroyed. If there are n potentially lethal molecular sites in a cell any one of which can cause death when it is destroyed by heating, the relation for the fraction of surviving cells at any time during heating becomes N/No - exp[-nkt] (2) where N is the number of survivors, No is the initial number of cells, and k is the rate constant. This equation gives an exponential sur- vivor curve, but the slope of the curve is nk instead of k (29). The very significant finding that the order of death for many organisms is exponential has not been without exceptions. Numerous investigators have found, in addition to exponential rates of death, rates which deviate somewhat from a strict exponential decay. Fig- ure 1 shows some of the various kinds of curves obtained from studies of heat-killing. Extensive discussions of the curves have appeared in the literature (26,28,35,38,44). Curve B of Figure l is the typical exponential decline in popu- lation which we have discussed. Curve C of Figure 1 shows a popula- tion of cells which apparently increases their heat resistance with time. This type of curve is usually explained by either assuming that there is a heterogeneous papulation of cells (10,57), or that products leaked from the cells during heating have a protective effect on the cells remaining in the later stages of heating. Curve A shows a population which shows a shoulder before the exponential decline in survivors. In terms of the probability of death with time, this increasing sensitivity to heat can be explained in two ways. First, any sequential reaction which includes the 1.0 o o H I SURVIVING FRACTION N/No 0001 " TIME Figure 1. Types of survivor curves observed during heating. N is the number of cells surviving at time t and No is the initial number of cells. 7 lethal damage can exhibit a shoulder if two or more steps have rate constants smaller than the other rate constants yet very close to each other in magnitude. Alternatively, there are two or more identical steps that must be accomplished (not necessarily in se- quence) in order for the organism to be killed. The familiar equation from target theory for this process is N/No - 1 - [1 - exp<-kt)1“ (3) where No is the number of cells in the original population, N is the number of survivors at time t, k is the rate constant for the event, and n is the number of events which must be accomplished. The variations in kinetics as illustrated in Figure 1 has given rise to a great deal of debate about the validity of the exponential order of death. This debate has not been very profitable for several reasons. First, the experimental methods employed may give spurious results as to the shape of the curve (44). Secondly, curves such as curves A and C are usually not very exaggerated. Curve A usually does not possess much of a shoulder, and when calcu- lations are made to find the number of events predicted by this curve, it is found that the number is quite small. Debate over whether the true state of affairs is l or 5 events has not been helpful in alert- ing the researcher as to what the lethal molecular damage might be. The only real question we might have is whether the organisms are somehow mimicking single-event kinetics with events of 1000 or more. The tail of curve C is usually found when the number of survivors is low. If it is true that the tail of curve C is due to a hetero- geneous population, this is more a question of the experimental design rather than an objection to the exponential order of death. 8 Molecular Sites gf_Damage The observation that the kinetics of heat-killing in bacteria is exponential or nearly exponential, gives the deceptive prospect that the molecular mechanism for heat-killing is simple, and there- fore, should be easy to detect. Indeed, it may be that the mole- cular mechanism of heat-killing is simple, but it is not at all easy to detect. At present, there is no adequate explanation of the mole- cular mechanism of heat-killing. A number of studies have been done on thermal inactivation of proteins and enzymes in_vi££o_(30). More important to the problem of heat-killing, but less well studied, is the inactivation of pro- teins in vivo. A number of enzymes involved with glycolysis and the tricarboxylic acid cycle have been shown to be inactivated in vivo in Salmonella typhimurium and Staphylococcus aureus (8,53) by heat- ing at lethal temperatures. An increase in extinction coefficient has been found at 500 nm in cultures of E. coli during heating (4,22). It was preposed that the increase in extinction coefficient may be due to changes in cytoplasmic proteins. Patterson and Gillespie (41) have shown that DNArdependent RNA polymerase is slightly inactivated when E. coli are heated to 44°C, a temperature at which they still grow. Presumably, RNA polymerase is inactivated to a greater degree at higher lethal temperatures. Some investigators have concluded that protein inactivation causes heat-killing in micro-organisms (52). The best evidence for this conclusion comes from thermodynamics. Absolute rate theory (29) describes the rate constant for first order kinetics by the equation T I kD = K'——— exp[AS /R] X exp[- AH h 1:IRT] (4) where AS and AH are the activation entropy and activation enthalpy respectively. It has been found that the activation entropy and enthalpy for various proteins follows a simple compensa- tion law (46). AS; - a.AH} + b (5) where a and b are constants. In addition, similar treatment of the data for various micro-organisms yields a compensation law relation with the same a and b constants as those for protein (46). The correlation of the a and b constants for both thermal death in micro- organisms and protein inactivation is quantitative evidence for protein denaturation as the rate-limiting step in thermal death in micro-organisms. Considering the extreme redundancy of most enzyme systems, it is difficult to justify protein inactivation as the cause of thermal death which gives exponential survivor curves (45). The contradiction between the demands of the exponential sur- vival curve and the redundancy found in most enzyme systems have lead researchers to interesting and novel explanations for how a single event of protein inactivation can cause the death of the cell. For example, if a repressor protein repressing a potentially lethal gene were denatured and allowed the gene to be expressed, this would kill the cell (Barnett Rosenberg, personal communication). Such models are quite hypothetical and very hard to demonstrate experimentally. It is safe to conclude that enzyme systems are inactivated at killing temperatures; what is not known is to what extent enzyme inactivation 10 may qualify as the rate-limiting lethal damage. There is considerable evidence that heating causes damage to the cytOplasmic membrane (14). It has been found that there is sig- nificant leakage of substances from the intracellular pool of Staph: ylococcus aureus. These substances were found to be made up primarily of RNAPlike material absorbing at 260 nm and having a positive orcinal test (25). In addition, amino acids have been found to leak from the intracellular pools (3,25). Russell and Harries showed a similar leakage of 260 nm absorbing material from heated suspensions of E. coli (49,50). In both Staph. aureus and §§_£21i, very little protein was found to be released (2,50). In addition, protein was found not to be degraded upon heating Staph. aureus (3). Tests have not been made for degradation of protein in E. coli. Total microscOpic cell counts of suspensions of E. coli before and after heating have been shown to remain constant (49), indicating that leakage was not due to lysis. Heated suspensions of E. coli spheroplast also showed no evidence of lysis (50). Therefore, it is evident that the damage to the membrane is not due to its rupture. This suggests that the leakage occurs uniformly in all heated cells, not only in cells that are being killed. Furthermore, correlations between the amount of leakage and the number of cells being killed cannot be made, and it may be that the reason for heat-killing is not leakage of essential intermediates (3). Degradation of ribosomes and rRNA has been found in several different organisms. In suspensions of Staph. aureus which were heated at sub-lethal temperatures, degradation occurred primarily in the 308 ribosomal subunit and the associated l6S RNA (48). The ll degradation of the 303 ribosomal subunit under these conditions varies from 85% to 100% (47). Similar degradation of ribosomes and rRNA has been shown to occur in Salmonella typhimurium (54). In Bacillus subtilis, both the 168 RNA and 23S RNA have been found to be degraded (37). The mechanism of this degradation is not known (48); however, evidence indicates that degradation of the 168 rRNA may be the result of a ribonuclease (47). It has also been shown - that degraded rRNA can be lost through the cytoplasmic membrane during heating, and these materials must be resynthesized for sur- vival (25). Bridges, Smith, and Munson (12) have noted in E. coli strains a close correlation between the sensitivity of cells to ionizing radiation and their sensitivity to heating. These authors suggest that thermal damage and damage due to ionizing radiation act simi- larly in their lethal effect on micro-organisms. Woodcock and Grigg (58) have found that single- and double-strand breaks occur in the DNA of E. coli that have been heated at lethal temperatures. The damage appears to be restricted to single- and double-strand breaks, since no degradation of the DNA takes place (3,43,58). Furthermore, death due to heating was not dependent upon the strand breaks as much as it was on the repair of these breaks (58). It is apparent that a number of molecular sites suffer damage at temperatures at or near the killing temperatures. Heat affects every site in the cell (14). Therefore, it is not surprising that a number of molecular sites show damage. Furthermore, not all mole- cular damage is lethal. Cells that survive heating have no lethal damage, yet they do reflect the fact that there is a great deal of 12 non-lethal damage due to heating. Cells that survive heating show a significant lag time before they reinitiate growth (27,32,37,55). This lag can only be interpreted as the time necessary for the cells to repair extensive non-lethal damage which has occurred due to heating. All experiments to date can only show that some particular dam- age has been doen to heated populations of cells. They have not been able to show that the observed damage is the critical or lethal dam- age. This failure to single out the lethal damage is explicit or implicit in the way the experiments were done. In these experiments, damage due to heating was observed as a deviation from the behavior of unheated cells. This is a poor comparison. Since there is wide- spread damage in the cell, the investigator, after observing a par- ticular damage, must rely on ad hog_explanations as to the critical nature of the damage in order to justify killing. Such explanations are hazardous and require great caution. EXPERIMENTAL THEORY The biggest single difficulty in detecting the lethal damage which causes heat-killing is separating the lethal damage done to the cell from all the other non-lethal damage which also occurs. Comparisons based on differences in damage done to heated papula- tions and unheated populations cannot conclusively show if the dam-. age observed is lethal. The only valid comparison that can be made which has a chance of singling out the lethal damage is that of come paring heat-killed cells with survivors of heating. If a comparison of heat-killed cells and survivors can be made, the only difference between them should be the lethal damage which occurred in the heat- killed cells and did not occur in the survivors. Furthermore, the exponential survival curve demands that the lethal damage occurs in a single rate-limiting step. The lethal damage does not accumulate, so survivors should have no record of the lethal damage. All non- lethal damage should appear equally in both survivors and heat- killed cells immediately after heating. A comparison between heat-killed and surviving cells requires a separation of the two populations so that measurements can be done on each set of cells and then compared. In the experiments that follow, this separation has been accomplished. Before separating heat-killed from surviving bacteria for comparative purposes, we must decide what molecular events are to be compared. Of course it 13 14 is not possible to directly isolate and compare the lethal damage, since it is not known what it is or where it is to be found. It is possible to compare specific functions in heat-killed cells to those in the survivors. Furthermore, the lethal damage appearing in the heat-killed cells must express itself as a loss of some function. This same function in the survivors should not be lost at all. Some caution is necessary before differential loss of function between heat-killed and surviving cells can be interpreted as a direct result of lethal damage. It is obvious that the lethal damage in the heat-killed cells must eventually be expressed broadly in the loss of all functions. Nevertheless, those functions whose loss are expressed first are the functions that are most directly coupled to the lethal damage. Thus, in determining the correlation between the function ob- served and lethal damage, we have two criteria. First, the function must be lost in the heat-killed cells and not lost in the survivors. Secondly, the loss of function must be expressed immediately after heating as a difference between heat-killed and surviving cells. Another question which comes to mind in connection with func- tional loss in heat-killed cells is whether or not any metabolic activities can be expected to be found in heat-killed cells. It could be said that if a cell is killed, then it should not function at all. It must be remembered that the definition of death that is used in all experiments of this kind is the inability of a cell to produce a colony upon plating. This definition in no way implies that all functions are lost immediately after heating, even though the metabolic action will step eventually. The immediate functional 15 loss in heat-killed cells should be coupled to the lethal damage. Indeed, there are cases in which a cell will not produce a colony when plated, but continues to function in a number of way after receiving lethal damage (6,33). In the experiments to be described, the functions of protein synthesis and RNA synthesis will be measured and compared between heat-killed and surviving cells. It is quite possible that the effects of heating on two such broad and important activities as protein and RNA synthesis will provide some information about the lethal damage which occurs in heat-killed cells. MATERIALS AND METHODS Bacterial Strain Escherichia coli K-12, CR63 was obtained from Dr. Loren Snyder and was used throughout the following experiments. This organism is prototrOphic, contains a sup D60 mutation, and is lambda sensi- tive (7). Growth g£_Cells and Plate Counting Cells were kept on slants of nutrient agar (Difco), 23 g per liter of water, supplemented with (per liter of water): yeast ex- tract (Difco), 2 g; glucose, 1 g; and NaCl, 1 g. These slants were stored at 5°C until used. Cultures were started from these slants by inoculating a starter broth composed of (per liter of water): nutrient broth (Difco), 6 g; yeast extract (Difco), 2 g; glucose, 1 g; and NaCl, 1 g. This culture was grown for about 3 hours, with aera- tion, and then transferred by diluting (about 1/20) into minimal media, and grown for an additional 6 hours. Minimal media used for growth had the following composition (per liter of water): NaZHPO4, 6 g; KH2P04. 3 g; MgSO4'7HZ), 0.15 g; NaCl, 1 g; CaClz, 0.01 g; NHACI, 2 g; and glucose, 3 g. Glass double-distilled water was used in all preparations. This culture was again transferred to fresh minimal media and grown overnight (10 hours), yielding a very turbid culture. The 16 17 final step in growth was the transfer of this overnight culture by dilution (about 1/100) into fresh minimal media. The cells were then incubated at 37°C until they reached an optical density of 0.15 at 560 nm. The time necessary to reach this Optical density represented about four doubling times, so that all cells were in logarithmic phase before experiments were carried out. The concentration of cells at the Optical density of 0.15 was about 1.5 x 108 cells per ml. Estimates of growth were made in each experiment by measuring the time required for the culture to double in Optical density. Dilutions for plate counts were done in buffer which was of the same composition as the buffer used in heating. Plate counts were done by the pour plate method, using tubes of liquid agar medium at 41°C. The composition of these materials is given below. Assay Buffers and Media Heating buffer and dilution buffer was made up of (per liter of water): NaZHPO4, 6 g; KH2P04, 3 g; Mg804-7H20, 0.15 g; and NaCl, 1 3. Recovery media was made by adding a concentrated 5X solution of amino acids, glucose, N34c1, and CaCl2 (recovery concentrate) to the heat- ing buffer. The final composition of the recovery media was (per liter of water): NaZHPOA, 2 0.01 g; NH4CI, 2 g; and glucose, 3 g. In addition, 6 g; KH P04, 3 g; MgSOA-7H20, 0.15 3; NaCl, 1 g; CaClz, the recovery media had the following concentrations of amino acids: alanine, glycine, lysine, serine, valine, and glutamic acid--20 ug per ml; threonine, proline, isoleucine, araginine, glutamine, methi- onine, asparagine, and phenylalanine-10 ug per ml; tyrosine and leu- cine-2 ug per ml; aspartic acid-16 ug per ml; histadine-5 ug per 18 ml; tryptOphan-l ug per ml, and cysteine--0.4 ug per ml. Lysis media was made by adding a concentrated solution (lysis concentrate) of beef extract, yeast extract, peptone (Difco), Mg804°7H20, NaCl, and sucrose to the recovery media containing the cells. The final concentration of the added materials (per liter of water) was: beef extract, 0.5 g; yeast extract, 0.5 g; peptone, 1.5 g; NaCl, 2 g; and MgSO4'7H20,-10 mM. Sucrose was present at either 8% or 5%, depending on the experiment. The composition of the recovery media, except for NaCl, was diluted by 2/3 when the lysis media was made. Potassium penicillin G (E. R. Squibb and Sons) was added to the lysis media at a final concentration of 1111 units per ml or 0.66 mg per ml when lysis was required. Plating agar was made with minimal media supplemented with the 20 amino acids at the same concentrations found in the recovery media, and 6.5 g per liter of water of Bacto agar (Difco). Except for the agar, the plating agar was identical in every respect to the recovery media. Resuspension buffer, which was used to resuspend the first pellet after lysis, was 50 mM KH P04 and made to pH 6.6 2 using KOH. Heating Heating was carried out in two steps. The first step was to preheat the cells at 44.7°C for 5 minutes. This was a temperature at which no killing took place, and it served to bring the cell sus- pension up to a temperature which would prevent heat shock. Heat shock may have taken place if cells at room temperature were suddenly subjected to killing temperatures. This step also reduced the time l9 necessary for the suspension to come up to the killing temperature when the cells were added to the predwarmed buffer at killing tem- perature. The time for the cell suspension to reach the set killing temperature was approximately 45 seconds. Cells were heated at killing temperatures of 49.900 to 51°C in water baths with bath control model 33 (Fisher) with an accuracy of 0.05°C and a varia- tion of i 0.0100. Cell suspensions were aerated during heating. Centrifugation All centrifugation steps were carried out in a model HN-S cen- trifuge with swing buckets (International Equipment Company) at a range of temperatures from room temperature to approximately 35°C. The increase in temperature was due to moderate heating of the cen- trifuge at high speeds and long times. Room temperature was used to prevent killing due to low temperature shock (25). Graduated coni- cal centrifuge tubes (15 ml) were used in all centrifugation steps. These tubes had the tap lips cut off so that they could be fitted with Bacti-capall covers. These covers prevented dust from entering the tubes. Radioactive Labeling_and Counting Techniques Cells were labeled with [14CJleucine, uniformly labeled with a specific activity of 325 mCi per m mol in the protein synthesis ex- periments. In the RNA synthesis experiments, [6-3H1uracil at a Specific activity of 26.2 C1 per m mol was used. Both radiochemi- cals were obtained from New England Nuclear. In most experiments, except where otherwise noted, carrier levels for non-radioactive 20 leucine of 2 ug per ml, or for noneradioactive uracil of 0.1 or 0.3 ug per ml were used. Two variations of liquid scintillation counting techniques were employed. Mbst counts were taken from samples collected on membrane filters. Liquid scintillation fluid for counting these filters was 2,5-diphenyloxazole, 6 g; and l,4-bis[2-(4~methyl-5—phenyl-oxazolyl)]- benzene, 0.25 g in 1 liter of toluene. In experiments where only samples collected on filters were counted, the results are given in terms of Cpm. Some experiments required counting of liquid samples containing water in addition to counting samples collected on fil- ters. In these cases, the sample was mixed with PCStm cocktail (Amersham/Searle Corporation) so that the mixture had a water con- tent of 12% or lower. In the experiments where samples containing water were compared with samples collected on filters, results are given in dpm, so that comparisons can be made between the samples. Disintegrations per minute were calculated on selected samples by the internal standardization method using [1401toluene which had an activity of 3.000 x 105 dpm. This carbon-l4 standard was obtained from New England Nuclear. It was necessary to standardize only in the case of experiments utilizing [14C11eucine. All samples were counted in a Packard Tri-carb Liquid Scintil- latiOn spectrometer Model 3320. For carbon-l4 samples, the gain was set at 8%, and the discriminators were set at 50 and 1000 respec- tively. For the tritium samples, the gain was set at 70%, and the discriminators were set at 50 and 1000. All samples were counted in glass vials with foil-lined tops. 21 Separation Assay The experimental procedure used throughout these experiments was designed to separate heat-killed from surviving organisms. In addi- tion, radioactive labels were added immediately after heating to mea- sure and compare the biosynthetic processes of heat-killed and sur- viving cells. The procedure, as outlined below, was modified in var- ious experiments to provide control experiments or to obtain specific information pertinent to the investigation. The methodology for the penicillin and lysis steps is a modification of the Kaback (31) method for isolating membranes. Figure 2 shows a flow chart of the "Separation Assay." Step 1. During the final growth before heating, an estimate Of the growth rate was made by measuring the optical density of the cul- ture using a Beckman model B spectrOphotometer. When an optical den- sity of 0.15 was reached, 6.3 ml of the culture was removed from the bubbler tube and centrifuged at 3400 rpm (1400 x g-l900 x g) for 15 minutes. After centrifugation, the supernatant fluid was removed, leaving only the pelleted cells. These cells were resuspended in 4.3 ml of heating buffer, transferred to a heating tube, and pre- heated as described previously. Step 2. Four ml of the preheated culture was poured into 14 ml of heating buffer which had been preheated to the killing tempera- ture. The cells were mixed, and a 1 ml sample was withdrawn for plate counting immediately. Heating was continued with aeration for 15 minutes, at which time the culture was quickly cooled and a 1 ml sample was again taken for plate counting so that survivors could 22 be counted. Step 3. Immediately after cooling, a l*ml sample was taken for plate counting, 4 ml of recovery charge was added to the culture, and [14C]1eucine was added so that the final concentration was 0.417 uCi per m1. For RNA synthesis studies, the concentration of [3H]uracil was 1.25 uCi per ml. Exceptions to these amounts are noted. Step 4. As soon as possible, 2 ml aliquots of the cell sus- pension were dispensed into each of nine test tubes (25 mm diameter) and incubated at 37°C. At 10 minute intervals for 90 minutes, cold leucine was added to one of the 9 tubes so that the final concentra- tion of cold leucine was 714 ug per ml. This addition of leucine severely reduced the incorporation of the labeled leucine by the cells. All the tubes were incubated at 37°C for a total of 2.5 hours. Step 5. At the end of the recovery period, 1 ml of the lysis concentrate and 1111 units of penicillin C were added to each sample. Incubation was continued for 1.5 hours. In some experiments, Triton Xr100 was added at 0.008% to 0.01% 1 hour after the addition of the lysis concentrate. After the addition of Triton X-100, incu- bation was continued for 30 minutes. This step lyses or produces spherOplast in the surviving population. Step 6. Cells from the incubation tubes were poured into cen- trifuge tubes and centrifuged at 3200 rpm (1200 x g-l600 x g) for 30 minutes. After centrifugation, the supernatant fluid was care- fully removed, leaving a 0.3 ml pellet volume. The supernatant fluid was put in numbered tubes on ice. Step 7. The pellet was partially resuspended in the 0.3 ml 23 pellet volume. Then 3 ml of resuspension buffer was added and mixed vigorously to resuspend the cells. The resuspended cells were incu- bated for 15 minutes at 37°C. One-tenth ml of 100 mM ethylenedi- aminetetraacetic acid (EDTA) was added to give a 3 mM final concen- tration to each sample. The samples were incubated an additional 15 minutes; then magnesium sulfate was added to give a final concentra- tion of Mg++of about 8 mM. Step 8. The cell suspension was centrifuged at 2600 rpm (800 x g-1100 x g) for 30 minutes. After centrifugation, the supernatant fluid was removed, leaving a 0.2 ml pellet volume. This supernatant fraction was put on ice with the first supernatant fraction. The pellet was resuspended in 3 ml of resuspension buffer and put on ice. Step 9. To the combined supernatant fractions, 0.7 ml of cold 50% trichloroacetic acid (TCA) was added, and to the pellet fraction, 0.35 ml. of 50% TCA was added. This addition of TCA gives a final concentration of about 5%. Both the pellet and the supernatant frac- tions were placed on ice, and the protein or RNA precipitate was allowed to form for 30 minutes. Step 10. The precipitates or cells were collected on Metriceltm membrane filters, type GAr8 with pore size of 0.2 um (Gelman Instru- ment Company). The pellet fraction collected was washed with 12 ml of cold 5% TCA, and the supernatant fraction was washed with 24 ml of cold 5% TCA. The filters were dried and counted. The above "Separation Assay" describes the method of separating dead from living cells. This separation is achieved because cells that recovered and lived were susceptible to penicillin lysis, while 24 cats EIOM lOGAIIIHMIC "MS! 030er AEE WAsreo ONCE AHo nEIeArEo IO «'c. 4M1.0F rHEsE CELL: I AEE tHEH AOOEO to 14m. OE some A! We. HEA: 15 Mel. c001, Aoo LAEEL IlEucmE-c"-u on mAca-s-H’) AND RECOVEEV MEOAIM. 2m. SAMPlES mcusArE 37'C A! to MIHmE INTERVALS Aoo HOHEAOIO- g , ActIVE LEUCIHE (714th) on UIACII. 5 C (300 mm). INCUIATE 2 V2 HE. no is see-- 3 sees-eso- o so 40 so so so 90 1 1 1 1 1 1 J 1 l I I I I I I I : 5 5 g 5 - Aoo EHEICHEO MEDIA, PENICILIJN (1m 5 mus/ML), mrou x-Ioo (noon)- ; C omouAL. IchsAtE w: HA. L 1 1 1 4 1 1 1 1 f /l I I I I I I I / LIVE 9 '° CEHmEOOE EACH SAME """"""">. 3° 1 2 AI 3200 m (1200-16000) A .30 FOR 30 MIN. . 40 Ann com «A (5:); COOL OH . so . ICE so mu. COLLEcr EEE- . cmrAtEs AHo WHOLE CEllS 6° WWW n >4 OH 0.2.. MEMsaAHE' nuns. . 7° "'"'"' WASH WITH 4 vowMEs or 5s '0 cow TCA; on AHO coum. _ 90 EEsuerHo IH 3M]. 11,», some ,H 6.6. IncusArE 15 MIN. 37'c. mm mm 90 1--..-.» Ass EorA (311M); IchsAtE W ,0 15 MIA. Aoo MAGNESIUM (MAM); 7° CENIIIFUGE A: zooo am (soo- ‘ so “000) Eon so Mm. EEsuerHo IH am new. EUEEEE so ’ N pH 6.6 ‘0 mm H > . O a m J---------->Ozo DEAD O ,0 Figure 2. Flow chart of the "Separation Assay." 25 heat-killed cells could not be lysed by penicillin. During the dif- ferential centrifugation steps of this experiment, the debris of sur— viving cells which lyse should be found in the supernatant, while the heat-killed cells which do not lyse but remain as whole cells would be found in the pellet. _Thus, the separation of surviving from heat- killed cells can be viewed as a separation of supernatant fraction from pellet fraction. The terms will be used interchangably through- out this text. RESULTS Kinetics of_Heat-killing_and Recovery Before the method of separating heat-killed from living cells can be employed, it is necessary to inquire about some basic char- acteristics of the cells regarding their kinetics of death and re- covery. In separating surviving from heat-killed bacteria, it is neces- sary to be able to generate a mixed population of living and heat- killed cells. The only requirement is that significant numbers appear in both pOpulations so that measurements of biosynthesis can be done. Measurements of the kinetics of death also can give infor- mation concerning the number of molecular events involved. The kinetics of heat-killing are shown in Figure 3. Death was approxi- mately exponential, with possibly a slight shoulder at both tempera- tures used. The 15-minute heating time shows that a significant num- ber of heat-killed cells have been produced, so this time was chosen as the time to be used in further experiments. Figure 3 does not give an accurate picture of the reproduc- ibility of the death kinetics. At any particular temperature, sep- arate experiments showed rather wide variation in the percent of cells surviving after 15 minutes of heating. The variation ranged from about 35% to 70% survivors. I was unable to eliminate this 26 27 Figure 3. Kinetics of heat-killing at 50.0°C (o), and 50.800 (0). Cells surviving heating (N) as a percentage of the original number (N ) at the end of 15 min of heating were 66% at 50.0°C and 56% at 509800. The bars represent the two standard deviation interval for each measurement. 28 .II 45 \ x \ \ \ ) \ n TI\.|.. IOKIII imam w. I \ T \ m \ n \ x m \ x n rqdbn. In \ \\ x \\ C \ \ \x _ .—LTL~L P b p P _. _ oz 1. 11 m Figure 3. 29 variation, but in most cases, it was acceptable. This variation would interfere with experiments only if there were too few survivors or too few heat-killed cells to measure their incorporation of radio- label. Since the percent of cells surviving heating is a better mea- sure of the severity of heating than either temperature or time of heating, the percent of cells surviving heating (percent survivors) will be given in all data. The kinetics of recovery are shown in Figure 4. Cells were heated, recovery concentrate was added, and the cells were then incu- bated at 37°C with aeration. The difference in the percent of cells surviving in these two cases illustrates the problem of reproduc- ibility mentioned above. The important quantity that was determined in recovery experiments was the time taken for the cells to recover and grow at the rate at which they would have grown in recovery media had they not been heated. The doubling time of normal cells in the recovery media is about 40 minutes as determined by optical density measurements. In Figure 4, the cells with 83% survival reach this growth rate in approximately 1.5 hours. The cells with 35% sur- vival reach this growth rate at approximately 2.5 hours. The dif- ference in time taken to reach maximum growth rate is apparently related to the percent of cells surviving, which is a measure of the severity of heat-killing. This relation between the percent of cells surviving and recovery time was found to be a common feature in heat- killed cells. It was found that when a culture of E. coli B is recovering from heating, it is not lysed by ampicillin (a drug similar to 30 Figure 4. The kinetics of recovery after 15 min of heating was mea- sured by viable cell counts. The temperature of killing in both cases was 49.9°C. This heating resulted in 35% survivors (o), and 83% sur- vivors (0) immediately after heating when recovery was initiated. 31 _ p — IL 7 L .6 5 manmu mo mmmZDz A mo OHH