JOURNAL OF BACTERIOLOGY, Feb. 1976, p. 643-654 Copyright 0 1976 American Society for Microbiology
Vol. 125, No. 2 Printed in U.S.A.
Lethal Effect of Mitomycin C on Haemophilus influenzae GARY D. SMALL,I JANE K. SETLOW,* JAN KOOISTRA, AND ROSLYN SHAPANKA Biology Department, Brookhaven National Laboratory, Upton, New York 11973
Received for publication 10 November 1975
The sensitivity of ultraviolet-sensitive strains to inactivation by mitomycin C (MC) is at the most only a factor of two greater than that of the wild type. The presence of inducible prophage has very little effect on the sensitivity. Genes which control excision of ultraviolet-induced pyrimidine dimers also control repair of MC-induced cross-links, as measured by resistance of denatured deoxyribonucleic acid (DNA) from treated cells to S1 nuclease digestion. However, endonucleolytic breaks in MC-damaged DNA, as judged by decreased single-strand molecular weight upon incubation of treated cells, are independent of these genes and probably are caused by monoadducts. After long periods of incubation there is a return to the molecular weight of untreated DNA. DNA degradation after MC treatment of various strains is not correlated with sensitivity to inactivation. Stationary-phase cells of all strains are more than twice as sensitive to MC as exponentially growing cells, and the sensitivity difference agrees with the measured difference in the number of cross-links after MC treatment of cells in the two growth stages. Evidence has been obtained that these phenomena result from differences in uptake of MC, which can be influenced by cyclic adenosine monophosphate. Small deviations in MC sensitivity from that of the wild type observed in mutants lacking the adenosine 5'-triphosphate-dependent nuclease are postulated to result from differences in MC uptake. These mutants, although no more ultraviolet sensitive than the wild type, are more sensitive to streptomycin, which also must be taken up by the cell to be effective.
Mitomycin C (MC) has lethal and mutagenic effects on bacteria (10) and induces phage in lysogenic strains (21). After undergoing enzymatic reduction inside the cell, or chemical reduction in vitro, MC can induce cross-links in deoxyribonucleic acid (DNA) (11, 12). Strains of Escherichia coli or Bacillus subtilis which are able to excise pyrimidine dimers from their DNA are also able to remove MC-induced cross-links (18, 26). Excision-defective strains are much more sensitive to killing by MC, suggesting that cross-links are important as lethal lesions. However, there are differences in sensitivity in strains of E. coli that cannot be explained by differences in the ability to excise cross-links (26). Sensitivity to MC has been used for screening in the isolation of mutants of B. subtilis (20) and Haemophilus influenzae (13-15), some of which are not excision defective. In an effort to clarify the molecular basis of MC sensitivity, we determined the MC sensitivity of the wild type and mutant strains of H. influenzae and determined cross-links as a func' Present
address: Department of Biochemistry, University of South Dakota, Vermillion, S. D. 57069.
tion of post-treatment incubation. We found that the disappearance of cross-links requires a functional excision mechanism. However, the absence of such a repair system makes relatively little difference to the survival, indicating that cross-links are not the only biologically important MC damage. Endonucleolytic nicks are made and sealed after MC treatment even in strains defective in dimer excision, suggesting that MC damage other than cross-links may be repaired by an enzyme system that does not work on ultraviolet (UV)-induced pyrimidine dimers. We have also obtained evidence that differences in uptake of MC can account for some of the differences in sensitivity, and that cyclic adenosine monophosphate (cAMP) may play a role in enhanced uptake and sensitivity. We postulate that mutants defective in adenosine 5'-triphosphate (ATP)-dependent nuclease are MC sensitive because they are more permeable to the compound. MATERIALS AND METHODS Microorganisms. Table 1 gives a list of strains used, with their relevant phenotypes. Two of the
643
644
J. BACTERIOL.
SMALL ET AL. TABLE 1. Strains of H. influenzae Mutation
Strain
transformed into strain Rd
Phenotype
KW31 (add-i) KW17 (add-6)
Wild type Defective in excision of pyrimidine dimers Defective in excision of' primidine dimers Recombination defective Recombination defective Recombination and excision def'ective Lacks ATP-dependent nuclease Lacks ATP-dependent nuclease
JK30 (add-6) JK57 (add-7) JK43 (add-8) BC200 BC200 (HP1C1) KW31 (HPlc1) Rd (HP1c1)
Lacks ATP-dependent nuclease Lacks ATP-dependent nuclease Lacks ATP-dependent nuclease Lacks inducible prophage BC200 lysogen of phage HP1cl KW31 lysogen of phage HP1c1 Rd lysogen of'phage HPlc1
Rd uvr-I uvr-2
rec-1 rec-2
rec-1 uvr-2
mutants defective in ATP-dependent nuclease (add) the gift of Kent W. Wilcox and had been originally selected on the basis of enzyme deficiency (29). Since one of these (KW31) was already a wild-type transformant, it was used as is. A lysate of' the other (KW17) was made, and the enzyme defect was transformed into strain Rd with the use of MC (3 X 10-2 ug /ml in agar plates) as a screening agent. Two other Add- strains were originally selected as MCwere
sensitive strains and subsequently were found lacking in the enzyme. Both of these mutations were transformed into strain Rd, selected for MC sensitivity and assayed for enzyme (JK57 and JK43). The lysogen of KW31 was made and tested as described before (22). Growth of cells. Media have been described (24). Growth stage was monitored by turbidity read at 675 nm in a Bausch and Lomb spectrophotometer. Exponentially growing cells were taken at a turbidity of 0.4, and stationary-phase cells were taken at a turbidity of 1.0. Cells were diluted in 3% Eugonbroth (Difco). ATP-dependent nuclease assay. Extracts were prepared by sonic treatment followed by centrifugation to remove cell debris. The enzyme was assayed essentially by the method of Friedman and Smith (8), except that the pH of the tris(hydroxymethyl)aminomethane buffer was 8.3 and the mercaptoethanol was omitted. Mitomycin C treatment and UV irradiation. Cells were treated at 37 C with MC for various times in the brain heart infusion growth medium in which they were grown, unless otherwise stated, and immediately diluted in the cold by a factor of at least 100 before they were plated. UV irradiation was performed as described previously (24). Because we were attempting to measure small differences in sensitivity, it was convenient to eliminate many of the experimental errors in the comparison of different
Reference
+
17
+
17
+
23 23 17
+ +
+ 4
+
+
29 K. W. Wilcox, unpublished data This paper This paper This paper 2, 22 22 This paper 4
strains by treating three or four strains with MC or UV irradiation in the same container. This method was made possible when each of' the strains was made resistant to a different antibiotic by transformation. One set of dilutions was made for each time point. The cells were plated in agar without antibiotic and then layered with the appropriate antibiotic-containing agar. By individual treatment of strains with and without markers conferring resistant to streptomycin (250 ,g/ml), novobiocin (2.5 gg/ml), erythromycin (10 gg/ml), and nalidixic acid (2 4g/ml), it was determined that the sensitivity was not altered by the antibiotic markers or the plating with antibiotics. A big advantage of the simultaneous treatment method is that it is labor saving. Measurement of DNA cross-links. Reaction of MC with DNA yields interstrand cross-links that prevent the DNA f'rom becoming irreversibly denatured (11). The amount of cross-linked DNA was determined by measuring the amount of DNA resistant to S1 nuclease (1), a single-strand-specific nuclease, after heat denaturation of the DNA. The DNA was labeled by growing cells in medium containing 5 MCi of [3H]thymidine per ml (20 Ci/mmol) for two or three generations to a final turbidity of around 0.4 or 1.0. Cells were centrifuged and suspended in fresh medium at the same concentration unless otherwise stated. MC was added, and the mixture was incubated at 37 C for varying times. The MC was removed by centrifuging and washing the cells in fresh medium. After the appropriate post-treatment incubation, portions were centrifuged and washed with 0.01 M tris(hydroxymethyl)aminomethane-0.01 M ethylenediaminetetraacetate, pH 7.4. The cells were suspended at a concentration of 109 to 5 x 109/ml in 0.2 ml of' the same buffer plus 0.09 ml of lysozyme (10 mg/ml). After 10 min on ice, 0.06 ml of' 5Nc
VOL. 125, 1976
EFFECT OF MITOMYCIN C ON H. INFLUENZAE
645
Sarkosyl was added, and the DNA was denatured by ied. Table 2 gives a summary of some of the heating at 100 C for 6 min followed by cooling on survival data. MC sensitivity is arbitrarily deice. To the cooled samples was added 0.6 ml of a solution containing 50 mM sodium acetate, pH 4.6, fined as the reciprocal of the number of minutes 8.3 mM ZnSO4, 0.23 M NaCl and 33 Mg of sonically of treatment required to inactivate the culture treated denatured calf thymus DNA per ml. Dupli- down to 10-3. It is seen that mutations in the cate 0.1-ml aliquots were removed to determine the uvr-1, uvr-2, and rec-1 loci increase the MC total amount of DNA present. To the remainder was added 5 ul of Si nuclease prepared as previously described (25), and the mixture was incubated at 50 C. Samples (0.1 ml) were removed after 10, 15, 20 and 25 min and absorbed on 2.3-cm Whatman 3MM filter disks. The disks were washed twice in 5% trichloroacetic acid and twice in 95% ethanol, dried, and counted with a toluene-based scintillation fluid. DNA synthesis and degradation. Synthesis was measured as uptake of [3H ]thymidine (19). For degradation the DNA was labeled by growing the cells in medium containing 5 MCi of [3H ]thymidine per ml for 90 min to a turbidity of around 0.4. The cells were washed with growth medium and divided into two portions. One portion was treated with 5Sug of MC per ml for 10 min at 37 C. The treated cells were washed to remove the excess MC and then suspended in fresh growth medium. Both the MC-treated and untreated control samples were incubated at 37 C, and 0.05-ml portions were removed at various times, placed on 2.3-cm Whatman 3MM filter disks, and submersed in cold 5% trichloroacetic acid. The disks were processed as above. Sedimentation of DNA. The single-strand molecular weight of cellular DNA after reaction with MC was determined by sedimentation in alkaline sucrose gradients. The DNA was labeled by growing the cells in medium containing 10 iCi of [3H ]thymidine per ml for 90 min to a turbidity of approximately 0.4. The cells were then washed, suspended in fresh growth medium, treated with 0.5 Mg of MC per ml for 5 min at 37 C, washed, and suspended in fresh medium at the same density. Portions (0.3-ml) were removed at various times of further incubation at 37 C, centrifuged, and suspended in 0.15 ml of 0.01 M tris(hydroxymethyl)aminomethane-0.01 M ethylenediamine6 8 10 tetraacetate, pH 7.4. A 0.1-ml amount of the cell M%itomycin C (min) suspension was lysed in 0.1 ml of 1 N NaOH on top of 5 to 20% sucrose gradients containing 0.5 M NaCl, FIG. 1. Inactivation of various strains of H. in0.2 N NaOH and 1 mM ethylenediaminetetra- fluenzae in stationary phase by MC at 0.5 Mg/ml. acetate. Centrifugation was for 90 min at 30,000 rpm in an SW50.1 rotor. Approximately 30 fractions were collected and processed as described previously TABLE 2. Relative MC sensitivities of various strains (5). Molecular weights were estimated with the use of of H. influenzae in stationary phase T2 as a reference. The single-strand size of DNA synthesized after Relative Strain MC treatment was determined as previously desensitivity scribed (16). 1.0 Rd 1.0 rec-2 RESULTS rec-1 1.5 1.5 uvr-2 Influence of excision and Rec genes on MC 1.6 uvr-1 sensitivity. Figure 1 shows typical MC dose2.1 rec-1 uvr-2 effect curves for various mutants and the wild 0.8 BC200 type (strain Rd), in which the time of treatment 1.0 BC200 (HP1C1) with a single MC concentration had been var-
646
SMALL ET AL.
sensitivity, whereas the rec-2 mutant has the same sensitivity as the wild type. The double mutant rec-1 uvr-2 is somewhat more sensitive than those of the corresponding single mutants. The rec-1 and uvr-2 mutations affect sensitivity to the same extent. Synthesis of DNA after MC treatment. Figure 2 shows DNA synthesis in three strains after MC treatment (0.5 ,g/ml) for various times (0, 3, and 6 min). The responses of the three strains are not very different. There is no evidence of a resumption of synthesis after delay. DNA cross-links induced by MC. The amount of cross-linking increases linearly with time of reaction with MC in uvr-2 (Fig. 3). The number average molecular weight of the untreated single-stranded DNA after heat denaturation was 2 x 106. Thus one cross-link should cause 4 x 106 daltons of DNA or 0.25% of the genome to become resistant to Si nuclease, assuming a genome size of 1.6 x 109 for H. influenzae (9). When cross-links were measured after 10 min of treatment of strains more resistant than uvr-2, the result was the same as in Fig. 3. However, after longer times there were fewer cross-links than in uvr-2. MC-induced cross-links disappear when wildtype cells are incubated in growth medium after treatment with MC; 90% are removed after 60. min (Fig. 4). The excision-defective strains uvr-1 and uvr-2 remove few or none of the cross-links during the same time. KW31, an Add- mutant, removes the cross-links slightly more slowly than the wild type, whereas the
J. BACTERIOL.
rec-1 mutant removes the cross-links at the same rate as the wild type. Degradation of DNA after MC treatment. Figure 5 shows the degradation of DNA in various strains after 10 min of treatment with 5 jig of MC per ml. It is seen that the uvr-2 gene is required for breakdown, but the uvr-1 gene is not, although both are defective in cross-link removal as well as in excision of pyrimidine dimers induced by UV irradiation. The Addmutant KW31 does not exhibit degradation after MC treatment. Degradation is more extensive in the rec-1 mutant than in the wild type, but is absent in the rec-1 uvr-2 double mutant. Single-strand breaks in DNA after MC treatment. Figure 6 illustrates that singlestrand breaks are produced in DNA upon incubation of uvr-2 cells after MC treatment, which does not remove cross-links or exhibit MCinduced DNA degradation (Fig. 5). No breaks are observed if the cells are kept on ice for 80 min after MC treatment. Similar results were obtained with strain uvr-1 and the wild type. After long incubation times, the DNA sedimentation pattern returned towards the control pattern. Note that at this MC dose (0.5 jig/ml for 5 min) there is little or no evidence for cross-links, which would be seen as a displacement of the sedimentation pattern towards the left. Influence of prophage on MC sensitivity. An HPlcl prophage in the add-i strain has only a small effect on the MC sensitivity (Fig. 7). Similarly, a HPlcl lysogen of the wild type showed approximately the same MC sensitivity
incubation after MC (min) FIG. 2. DNA synthesis measured as uptake of [3H]thymidine after treatment of exponentially growing cells by 0.5 jig of MC per ml for the times shown in minutes.
VOL. 125, 1976
EFFECT OF MITOMYCIN C ON H. INFLUENZAE
647
w80 C
O
60
0
E 40 4
\~~ ~
~
~
~~~~~--
0
20
Rd or rec\
z
rn 0
0 -0
10 20 30 40 50 60 INCUBATION AFTER MC TREATMENT (min)
FIG. 4. Disappearance of cross-links induced by 5 of MC per ml for 10 min as a function of post-treatment incubation of cells treated in exponential phase. Of the DNA, 20 to 25% was resistant to Sl nuclease digestion after MC treatment.
4r)
ug
4 z 0
0
5
10 15 reaction with MC (min)
20
FIG. 3. Cross-links induced by 5,ug of MC per ml in the DNA of exponentially growing uvr-2.
the corresponding nonlysogenized strain (data not shown). Since all these strains carry a defective prophage (3), it seemed possible that an additional prophage might not affect the MC sensitivity, because with the exception of the rec-1 mutant (23), these strains are already inducible for defective phage. Therefore a comparison was made of the wild type with strain BC200 (2), which is not inducible for the defective phage (22). Table 2 shows that the sensitivity of the wild-type strain is only slightly greater than that of BC200. If an inducible prophage is present in BC200, the sensitivity becomes the same as in the wild type. Thus neither induction of HPlcl nor the defective phage has much influence on the lethal effect of MC. as
Influence
of growth
stage
on
MC
sensitivity. Figure 8 shows that not only the wild type, but also a more sensitive excisiondefective mutant, are more sensitive in stationary phase than in a stage of exponential growth. The ratio of the sensitivities in the two stages (calculated from the time required to inactivate the cells down to 10-3) is 2.4 for uvr-2 and 2.5 for the wild type. Similar differences were obtained with strains rec-1 uvr-2, rec-1, add-1, and add-8. These data suggest that whatever the explanation for the growth stage difference, it does not involve differences in repair. To determine whether the amount of MC taken up by the cell varies with growth stage, a crude assay for MC uptake was devised, based on the idea that a large number of cells should remove enough MC from the medium so that the lethal effect of the MC solution is decreased. A calibration curve was constructed (Fig. 9), in which the survival of cells treated for 10 min was plotted against MC concentration. Figure 10 shows the inactivation of wild-type cells by an MC solution in which exponentially growing or stationary-phase cells, concentrated to around 3 x 1010/ml, had been previously incubated for 20 min and then centrifuged down. The control MC solution was incubated in growth medium for the same time. It is clear that there is much less inactivation by the media in which cells had been incubated, and the stationary-phase cells appear to remove more MC than the exponential-phase cells. Because it was necessary to use a large number
648
J. BACTERIOL.
SMALL ET AL.
Rd M
.--
0
o
I-
z
TlYl
4020 20
recId _
0
0 o
+MC
±+MC
0 80
-r60ec uvr2 4 40 e D- 100 20-
uvrKW3
-6M
-
100
±MC ±oM CD
Vol. 125, No. 2 Printed in U.S.A.
Lethal Effect of Mitomycin C on Haemophilus influenzae GARY D. SMALL,I JANE K. SETLOW,* JAN KOOISTRA, AND ROSLYN SHAPANKA Biology Department, Brookhaven National Laboratory, Upton, New York 11973
Received for publication 10 November 1975
The sensitivity of ultraviolet-sensitive strains to inactivation by mitomycin C (MC) is at the most only a factor of two greater than that of the wild type. The presence of inducible prophage has very little effect on the sensitivity. Genes which control excision of ultraviolet-induced pyrimidine dimers also control repair of MC-induced cross-links, as measured by resistance of denatured deoxyribonucleic acid (DNA) from treated cells to S1 nuclease digestion. However, endonucleolytic breaks in MC-damaged DNA, as judged by decreased single-strand molecular weight upon incubation of treated cells, are independent of these genes and probably are caused by monoadducts. After long periods of incubation there is a return to the molecular weight of untreated DNA. DNA degradation after MC treatment of various strains is not correlated with sensitivity to inactivation. Stationary-phase cells of all strains are more than twice as sensitive to MC as exponentially growing cells, and the sensitivity difference agrees with the measured difference in the number of cross-links after MC treatment of cells in the two growth stages. Evidence has been obtained that these phenomena result from differences in uptake of MC, which can be influenced by cyclic adenosine monophosphate. Small deviations in MC sensitivity from that of the wild type observed in mutants lacking the adenosine 5'-triphosphate-dependent nuclease are postulated to result from differences in MC uptake. These mutants, although no more ultraviolet sensitive than the wild type, are more sensitive to streptomycin, which also must be taken up by the cell to be effective.
Mitomycin C (MC) has lethal and mutagenic effects on bacteria (10) and induces phage in lysogenic strains (21). After undergoing enzymatic reduction inside the cell, or chemical reduction in vitro, MC can induce cross-links in deoxyribonucleic acid (DNA) (11, 12). Strains of Escherichia coli or Bacillus subtilis which are able to excise pyrimidine dimers from their DNA are also able to remove MC-induced cross-links (18, 26). Excision-defective strains are much more sensitive to killing by MC, suggesting that cross-links are important as lethal lesions. However, there are differences in sensitivity in strains of E. coli that cannot be explained by differences in the ability to excise cross-links (26). Sensitivity to MC has been used for screening in the isolation of mutants of B. subtilis (20) and Haemophilus influenzae (13-15), some of which are not excision defective. In an effort to clarify the molecular basis of MC sensitivity, we determined the MC sensitivity of the wild type and mutant strains of H. influenzae and determined cross-links as a func' Present
address: Department of Biochemistry, University of South Dakota, Vermillion, S. D. 57069.
tion of post-treatment incubation. We found that the disappearance of cross-links requires a functional excision mechanism. However, the absence of such a repair system makes relatively little difference to the survival, indicating that cross-links are not the only biologically important MC damage. Endonucleolytic nicks are made and sealed after MC treatment even in strains defective in dimer excision, suggesting that MC damage other than cross-links may be repaired by an enzyme system that does not work on ultraviolet (UV)-induced pyrimidine dimers. We have also obtained evidence that differences in uptake of MC can account for some of the differences in sensitivity, and that cyclic adenosine monophosphate (cAMP) may play a role in enhanced uptake and sensitivity. We postulate that mutants defective in adenosine 5'-triphosphate (ATP)-dependent nuclease are MC sensitive because they are more permeable to the compound. MATERIALS AND METHODS Microorganisms. Table 1 gives a list of strains used, with their relevant phenotypes. Two of the
643
644
J. BACTERIOL.
SMALL ET AL. TABLE 1. Strains of H. influenzae Mutation
Strain
transformed into strain Rd
Phenotype
KW31 (add-i) KW17 (add-6)
Wild type Defective in excision of pyrimidine dimers Defective in excision of' primidine dimers Recombination defective Recombination defective Recombination and excision def'ective Lacks ATP-dependent nuclease Lacks ATP-dependent nuclease
JK30 (add-6) JK57 (add-7) JK43 (add-8) BC200 BC200 (HP1C1) KW31 (HPlc1) Rd (HP1c1)
Lacks ATP-dependent nuclease Lacks ATP-dependent nuclease Lacks ATP-dependent nuclease Lacks inducible prophage BC200 lysogen of phage HP1cl KW31 lysogen of phage HP1c1 Rd lysogen of'phage HPlc1
Rd uvr-I uvr-2
rec-1 rec-2
rec-1 uvr-2
mutants defective in ATP-dependent nuclease (add) the gift of Kent W. Wilcox and had been originally selected on the basis of enzyme deficiency (29). Since one of these (KW31) was already a wild-type transformant, it was used as is. A lysate of' the other (KW17) was made, and the enzyme defect was transformed into strain Rd with the use of MC (3 X 10-2 ug /ml in agar plates) as a screening agent. Two other Add- strains were originally selected as MCwere
sensitive strains and subsequently were found lacking in the enzyme. Both of these mutations were transformed into strain Rd, selected for MC sensitivity and assayed for enzyme (JK57 and JK43). The lysogen of KW31 was made and tested as described before (22). Growth of cells. Media have been described (24). Growth stage was monitored by turbidity read at 675 nm in a Bausch and Lomb spectrophotometer. Exponentially growing cells were taken at a turbidity of 0.4, and stationary-phase cells were taken at a turbidity of 1.0. Cells were diluted in 3% Eugonbroth (Difco). ATP-dependent nuclease assay. Extracts were prepared by sonic treatment followed by centrifugation to remove cell debris. The enzyme was assayed essentially by the method of Friedman and Smith (8), except that the pH of the tris(hydroxymethyl)aminomethane buffer was 8.3 and the mercaptoethanol was omitted. Mitomycin C treatment and UV irradiation. Cells were treated at 37 C with MC for various times in the brain heart infusion growth medium in which they were grown, unless otherwise stated, and immediately diluted in the cold by a factor of at least 100 before they were plated. UV irradiation was performed as described previously (24). Because we were attempting to measure small differences in sensitivity, it was convenient to eliminate many of the experimental errors in the comparison of different
Reference
+
17
+
17
+
23 23 17
+ +
+ 4
+
+
29 K. W. Wilcox, unpublished data This paper This paper This paper 2, 22 22 This paper 4
strains by treating three or four strains with MC or UV irradiation in the same container. This method was made possible when each of' the strains was made resistant to a different antibiotic by transformation. One set of dilutions was made for each time point. The cells were plated in agar without antibiotic and then layered with the appropriate antibiotic-containing agar. By individual treatment of strains with and without markers conferring resistant to streptomycin (250 ,g/ml), novobiocin (2.5 gg/ml), erythromycin (10 gg/ml), and nalidixic acid (2 4g/ml), it was determined that the sensitivity was not altered by the antibiotic markers or the plating with antibiotics. A big advantage of the simultaneous treatment method is that it is labor saving. Measurement of DNA cross-links. Reaction of MC with DNA yields interstrand cross-links that prevent the DNA f'rom becoming irreversibly denatured (11). The amount of cross-linked DNA was determined by measuring the amount of DNA resistant to S1 nuclease (1), a single-strand-specific nuclease, after heat denaturation of the DNA. The DNA was labeled by growing cells in medium containing 5 MCi of [3H]thymidine per ml (20 Ci/mmol) for two or three generations to a final turbidity of around 0.4 or 1.0. Cells were centrifuged and suspended in fresh medium at the same concentration unless otherwise stated. MC was added, and the mixture was incubated at 37 C for varying times. The MC was removed by centrifuging and washing the cells in fresh medium. After the appropriate post-treatment incubation, portions were centrifuged and washed with 0.01 M tris(hydroxymethyl)aminomethane-0.01 M ethylenediaminetetraacetate, pH 7.4. The cells were suspended at a concentration of 109 to 5 x 109/ml in 0.2 ml of' the same buffer plus 0.09 ml of lysozyme (10 mg/ml). After 10 min on ice, 0.06 ml of' 5Nc
VOL. 125, 1976
EFFECT OF MITOMYCIN C ON H. INFLUENZAE
645
Sarkosyl was added, and the DNA was denatured by ied. Table 2 gives a summary of some of the heating at 100 C for 6 min followed by cooling on survival data. MC sensitivity is arbitrarily deice. To the cooled samples was added 0.6 ml of a solution containing 50 mM sodium acetate, pH 4.6, fined as the reciprocal of the number of minutes 8.3 mM ZnSO4, 0.23 M NaCl and 33 Mg of sonically of treatment required to inactivate the culture treated denatured calf thymus DNA per ml. Dupli- down to 10-3. It is seen that mutations in the cate 0.1-ml aliquots were removed to determine the uvr-1, uvr-2, and rec-1 loci increase the MC total amount of DNA present. To the remainder was added 5 ul of Si nuclease prepared as previously described (25), and the mixture was incubated at 50 C. Samples (0.1 ml) were removed after 10, 15, 20 and 25 min and absorbed on 2.3-cm Whatman 3MM filter disks. The disks were washed twice in 5% trichloroacetic acid and twice in 95% ethanol, dried, and counted with a toluene-based scintillation fluid. DNA synthesis and degradation. Synthesis was measured as uptake of [3H ]thymidine (19). For degradation the DNA was labeled by growing the cells in medium containing 5 MCi of [3H ]thymidine per ml for 90 min to a turbidity of around 0.4. The cells were washed with growth medium and divided into two portions. One portion was treated with 5Sug of MC per ml for 10 min at 37 C. The treated cells were washed to remove the excess MC and then suspended in fresh growth medium. Both the MC-treated and untreated control samples were incubated at 37 C, and 0.05-ml portions were removed at various times, placed on 2.3-cm Whatman 3MM filter disks, and submersed in cold 5% trichloroacetic acid. The disks were processed as above. Sedimentation of DNA. The single-strand molecular weight of cellular DNA after reaction with MC was determined by sedimentation in alkaline sucrose gradients. The DNA was labeled by growing the cells in medium containing 10 iCi of [3H ]thymidine per ml for 90 min to a turbidity of approximately 0.4. The cells were then washed, suspended in fresh growth medium, treated with 0.5 Mg of MC per ml for 5 min at 37 C, washed, and suspended in fresh medium at the same density. Portions (0.3-ml) were removed at various times of further incubation at 37 C, centrifuged, and suspended in 0.15 ml of 0.01 M tris(hydroxymethyl)aminomethane-0.01 M ethylenediamine6 8 10 tetraacetate, pH 7.4. A 0.1-ml amount of the cell M%itomycin C (min) suspension was lysed in 0.1 ml of 1 N NaOH on top of 5 to 20% sucrose gradients containing 0.5 M NaCl, FIG. 1. Inactivation of various strains of H. in0.2 N NaOH and 1 mM ethylenediaminetetra- fluenzae in stationary phase by MC at 0.5 Mg/ml. acetate. Centrifugation was for 90 min at 30,000 rpm in an SW50.1 rotor. Approximately 30 fractions were collected and processed as described previously TABLE 2. Relative MC sensitivities of various strains (5). Molecular weights were estimated with the use of of H. influenzae in stationary phase T2 as a reference. The single-strand size of DNA synthesized after Relative Strain MC treatment was determined as previously desensitivity scribed (16). 1.0 Rd 1.0 rec-2 RESULTS rec-1 1.5 1.5 uvr-2 Influence of excision and Rec genes on MC 1.6 uvr-1 sensitivity. Figure 1 shows typical MC dose2.1 rec-1 uvr-2 effect curves for various mutants and the wild 0.8 BC200 type (strain Rd), in which the time of treatment 1.0 BC200 (HP1C1) with a single MC concentration had been var-
646
SMALL ET AL.
sensitivity, whereas the rec-2 mutant has the same sensitivity as the wild type. The double mutant rec-1 uvr-2 is somewhat more sensitive than those of the corresponding single mutants. The rec-1 and uvr-2 mutations affect sensitivity to the same extent. Synthesis of DNA after MC treatment. Figure 2 shows DNA synthesis in three strains after MC treatment (0.5 ,g/ml) for various times (0, 3, and 6 min). The responses of the three strains are not very different. There is no evidence of a resumption of synthesis after delay. DNA cross-links induced by MC. The amount of cross-linking increases linearly with time of reaction with MC in uvr-2 (Fig. 3). The number average molecular weight of the untreated single-stranded DNA after heat denaturation was 2 x 106. Thus one cross-link should cause 4 x 106 daltons of DNA or 0.25% of the genome to become resistant to Si nuclease, assuming a genome size of 1.6 x 109 for H. influenzae (9). When cross-links were measured after 10 min of treatment of strains more resistant than uvr-2, the result was the same as in Fig. 3. However, after longer times there were fewer cross-links than in uvr-2. MC-induced cross-links disappear when wildtype cells are incubated in growth medium after treatment with MC; 90% are removed after 60. min (Fig. 4). The excision-defective strains uvr-1 and uvr-2 remove few or none of the cross-links during the same time. KW31, an Add- mutant, removes the cross-links slightly more slowly than the wild type, whereas the
J. BACTERIOL.
rec-1 mutant removes the cross-links at the same rate as the wild type. Degradation of DNA after MC treatment. Figure 5 shows the degradation of DNA in various strains after 10 min of treatment with 5 jig of MC per ml. It is seen that the uvr-2 gene is required for breakdown, but the uvr-1 gene is not, although both are defective in cross-link removal as well as in excision of pyrimidine dimers induced by UV irradiation. The Addmutant KW31 does not exhibit degradation after MC treatment. Degradation is more extensive in the rec-1 mutant than in the wild type, but is absent in the rec-1 uvr-2 double mutant. Single-strand breaks in DNA after MC treatment. Figure 6 illustrates that singlestrand breaks are produced in DNA upon incubation of uvr-2 cells after MC treatment, which does not remove cross-links or exhibit MCinduced DNA degradation (Fig. 5). No breaks are observed if the cells are kept on ice for 80 min after MC treatment. Similar results were obtained with strain uvr-1 and the wild type. After long incubation times, the DNA sedimentation pattern returned towards the control pattern. Note that at this MC dose (0.5 jig/ml for 5 min) there is little or no evidence for cross-links, which would be seen as a displacement of the sedimentation pattern towards the left. Influence of prophage on MC sensitivity. An HPlcl prophage in the add-i strain has only a small effect on the MC sensitivity (Fig. 7). Similarly, a HPlcl lysogen of the wild type showed approximately the same MC sensitivity
incubation after MC (min) FIG. 2. DNA synthesis measured as uptake of [3H]thymidine after treatment of exponentially growing cells by 0.5 jig of MC per ml for the times shown in minutes.
VOL. 125, 1976
EFFECT OF MITOMYCIN C ON H. INFLUENZAE
647
w80 C
O
60
0
E 40 4
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~
~
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0
20
Rd or rec\
z
rn 0
0 -0
10 20 30 40 50 60 INCUBATION AFTER MC TREATMENT (min)
FIG. 4. Disappearance of cross-links induced by 5 of MC per ml for 10 min as a function of post-treatment incubation of cells treated in exponential phase. Of the DNA, 20 to 25% was resistant to Sl nuclease digestion after MC treatment.
4r)
ug
4 z 0
0
5
10 15 reaction with MC (min)
20
FIG. 3. Cross-links induced by 5,ug of MC per ml in the DNA of exponentially growing uvr-2.
the corresponding nonlysogenized strain (data not shown). Since all these strains carry a defective prophage (3), it seemed possible that an additional prophage might not affect the MC sensitivity, because with the exception of the rec-1 mutant (23), these strains are already inducible for defective phage. Therefore a comparison was made of the wild type with strain BC200 (2), which is not inducible for the defective phage (22). Table 2 shows that the sensitivity of the wild-type strain is only slightly greater than that of BC200. If an inducible prophage is present in BC200, the sensitivity becomes the same as in the wild type. Thus neither induction of HPlcl nor the defective phage has much influence on the lethal effect of MC. as
Influence
of growth
stage
on
MC
sensitivity. Figure 8 shows that not only the wild type, but also a more sensitive excisiondefective mutant, are more sensitive in stationary phase than in a stage of exponential growth. The ratio of the sensitivities in the two stages (calculated from the time required to inactivate the cells down to 10-3) is 2.4 for uvr-2 and 2.5 for the wild type. Similar differences were obtained with strains rec-1 uvr-2, rec-1, add-1, and add-8. These data suggest that whatever the explanation for the growth stage difference, it does not involve differences in repair. To determine whether the amount of MC taken up by the cell varies with growth stage, a crude assay for MC uptake was devised, based on the idea that a large number of cells should remove enough MC from the medium so that the lethal effect of the MC solution is decreased. A calibration curve was constructed (Fig. 9), in which the survival of cells treated for 10 min was plotted against MC concentration. Figure 10 shows the inactivation of wild-type cells by an MC solution in which exponentially growing or stationary-phase cells, concentrated to around 3 x 1010/ml, had been previously incubated for 20 min and then centrifuged down. The control MC solution was incubated in growth medium for the same time. It is clear that there is much less inactivation by the media in which cells had been incubated, and the stationary-phase cells appear to remove more MC than the exponential-phase cells. Because it was necessary to use a large number
648
J. BACTERIOL.
SMALL ET AL.
Rd M
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0
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