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DNA-damaging agents and DNA-synthesis inhibitors induce luminescence in dark variants of luminous bacteria
Mutation Research, 91 (1981)443-450
443
Elsevier/North-Holland BiomedicalPress
D N A - D A M A G I N G A G E N T S AND DNA-SYNTHESIS INHIBITORS INDUCE L U M I N E S C E N C E IN DARK VARIANTS OF LUMINOUS BACTERIA
IR1TH WEISER, S. ULITZUR and S. YANNAI Department of Food Engineering and Biotechnology Technion, Israel Institute of Technology, Haifa (Israel)
(Received l0 February 1981) (Accepted 14 April 1981)
SUMMARY The DNA-damaging agents mitomycin C and UV irradiation, as well as the DNAsynthesis inhibitors nalidixic acid, novobiocin and coumermycin, induce the de novo synthesis of luciferase and in vivo luminescence in dark variant cells of the luminous bacteria Photobacterium ieiognathi. Mitomycin C and nalidixic acid also cause the induction of luminescence in wild-type cells in the absence of its natural inducer. In spite of the high level of in vivo luminescence of the treated dark-variant cells, none of these agents result in the appearance of genetically luminous revertants. The possibility is discussed that these agents phenotypically induce luminescence through their ability to trigger 'SOS functions', which in turn leads to the transitory inactivation of certain repressors.
Naturally-occurring dark variants of luminous bacteria are characterized by their very low levels of in vivo luminescence and cellular luciferase content (Nealson and Hastings, 1979). The effect of different chemical agents on the reversion frequency o f these dark variants to the luminescent state has been recently studied by Ulitzur et al. (1980), and Ulitzur and Weiser (1981). 2 distinct groups of active chemicals were characterized. The first group that included mutagens showing either basesubstitution or frameshift capability increased the reversion frequency o f the dark variants to luminescent cells. The second group of chemicals share the common feature o f being DNA-intercalating agents (e.g. acridine dyes, ethidium bromide, caffeine and theophylline) caused almost complete restortion of the in vivo luminescence o f the dark-variant cells, but failed to increase the reversion rate at the genetic level. This second group o f chemicals was also shown to be able to induce luminescence in wild-type cells in the absence of an added inducer. 0165-7992/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
444
In the present paper we show that DNA-damaging agents and DNA-synthesis inhibitors are also capable of inducing phenotypic reversion of dark-variant cells, as well as in the wild-type cells prior to induction. The possibility that these agents cause inactivation of the repressor of the luciferase operon is discussed. MATERIALS AND METHODS
Bacterial strains and conditions o f growth. Photobacterium leiognathi, strain BE8, and its spontaneous dark variant (8SD18) were described by Ulitzur et al. (1980). The cells were grown with shaking at 30°C, in ASWRP liquid medium as previously described (Ulitzur et al., 1980). The solid medium consisted of ASWRP medium with 0.3°70 yeast extract (Difco) and 1.7070 agar (Difco). Luminescence determination. In vivo and in vitro luminescence were determined as described by Ulitzur and Weiser (1981).
E
= O"
d,z z
Control
"~ I0 0
5
I0
~ 15
-
cm~ MC. 20
TIME (hrs.)
Fig. 1. Effect o f mitomycin C on the luminescence o f P. leiognathi 8SD 18 cells. P. leiognathi 8SD 18 cells were grown in A S W R P liquid m e d i u m to a final cell density of about 5 x l07 cells- m l - i. 0.1-ml aliquots o f this culture were added to scintillation vials, containing 1 ml o f A S W R P , without (control) or with m itomycin C (MC 5 #g. m l - 1) or mitomycin (5 #g. m l - l) with chloramphenicol (10 #g- m l - l). The vials were incubated at 28°C in a scintillation counter and their luminescence was determined with time.
445
Chemicals. Mitomycin C, novobiocin, nalidixic acid and chloramphenicol were purchased from Sigma Chemicals Co., St. Louis, MO. Coumermycin and antipain were obtained as a gift from Prof. R. Ben-Ishai, Department of Biology, Technion (Israel). RESULTS
The effect of mitomycin C on the in vivo luminescence in dark cells of P. leiognathi strain 8SD18, is shown in Fig. 1. As can be seen, about 120 min after addition of mitomycin C (5 #g- ml- l), in vivo luminescence began to increase over the control
I0 0
107
io6
tirol
,o
d
'°o
!
i
3 TIME (hrs.)
Fig. 2. Effect o f UV-irradiation on the luminescence of P. leiognathi 8SD18 cells. P. leiognathi 8SD18 cells were suspended from 20-h old plates into A S W R P liquid m e d i u m (without peptone) to give a final cell density of 5 x 107 cells, m l - i. UV-irradiation was applied in a petri dish (9-cm diameter) containing l0 ml of the culture. A quartz lamp ( H a n a u ) was used at a distance of 60 cm. The cell suspension was irradiated with 400 e r g / m m 2 followed by dilution o f 0.2 ml of the treated culture into 1 ml of A S W R P liquid medium. UV-treated cells as well as the control culture, were incubated at 28°C and their luminescence was determined as a function o f time.
446
level, and finally attained (after 15 h) a level of up to 1000 times greater than that of the untreated cells. The final in vitro luciferase activity is also elevated, and is about 50 times higher than that of the control cells. The increase of both in vivo and in vitro luciferase activity by mitomycin C, is completely inhibited by chloramphenicol (10 #g.ml-l). In spite of the high level of the in vivo luminescence, no luminous revertants were observed among 2- 104 colonies of mitomycin C-treated cells. A similar picture was observed when 8SD18 cells were irradiated with UV light (Fig. 2). As with mitomycin C, the onset of luminescence appears about 100 min after the treatment and it reaches a level 1000 times higher than that of the control culture. The number of stable luminous revertants in the UV-treated culture was also less than 1 per 104. As both mitomycin C and UV irradiation cause cessation of DNA replication, we tested the activity of some known DNA-synthesis inhibitors on the induction of luminescence in the dark-variant cells. Fig. 3 shows that of all DNA-synthesis inhibitors tested, nalidixic acid, novobiocin and coumermycin, are very active in restoring the in vivo luminescence of the dark-variant cells. The onset of luminescence with these inhibitors appears about 3 h after the administration of the
tO to
10 9
10 8
-r
=
,oT
~r i.i ¢O Z W (/) i=1 Z
10 6
o
i .J
105
I0d
I
I
I
I
0 TIME
(hrl.)
Fig. 3. Effect o f novobiocin, coumermycin and nalidixic acid on the luminescence o f P. leiognathi 8SD18 cells. Novobiocin (0.2 #g), coumermycin (7.5/~g) and nalidixic acid (0.7/zg) were added to a set o f scintillation vials containing 1 mi of A S W R P liquid medium. To each vial 0.1 ml o f a logarithmic culture o f 8SD18 cells (107 cell. m l - n) was added and the luminescence was determined at different times at 28°C.
447
active agent. As with mitomycin C and UV light, no increase in genetic reversion to luminous colonies (less than 1 per 104) was observed in the cultures treated with the DNA-synthesis inhibitors. The luminescence system is synthesized de novo only towards the end of the logarithmic phase of growth, upon an accumulation of 'autoinducer' in the growing culture (Nealson and Hastings 1979). Ulitzur and Weiser (1981) have recently shown that young cultures of the wild-type cells of P. leiognathi prior to inducton, behave similarly to dark-variant cells; the luciferase synthesis in both kinds of cells is under repression, which can be overcome by different DNA-intercalating agents, such as acridine dyes. We have found (Fig. 4) that the DNA-damaging agent, mitomycin C and the DNA-synthesis inhibitor nalidixic acid, induce luminescence in wild-type cells prior to induction.
108
NAL. E m O"
Conh'ol
I
0
O
I
V 2 TIME
3
~
4
5
6
(hrs.)
Fig. 4. Effect o f mitomycin C a n d nalidixic acid on the induction of the luminescence o f P. leiognathi BE8 cells. P. leiognathi BE8 wild-type cells were incubated into A S W R P liquid m e d i u m to a final cell density of about l0 cells, m l - i. W h e n the culture reached a cell density of l0 s cells- m l - l, it was diluted 100-fold in 1 ml of A S W R P m e d i u m containing either mitomycin C (5 #g. m l - L), nalidixic acid (2.5 jzg. ml-m) or none (control). The vials were incubated at 28°C and the luminescence was determined with time.
448 DISCUSSION The action of the DNA-damaging agents and the DNA-synthesis inhibitors on the induction of luminescence is characterized by 3 features: (1) All agents increase the de novo synthesis of luciferase. (2) No genetic luminous revertants (less than one per l04 colonies) are observed among the offspring of the treated cultures. (3) Mitomycin C, a DNA-damaging agent, and nalidixic acid, a DNA-synthesis inhibitor, also induce the luminescence in young culture of wild-type cells prior to induction. These features also characterize the action of the DNA-intercalating agents that restore luminescence in dark and wild-type cells of luminous bacteria (Ulitzur and Weiser, 1981). We have previously suggested that these agents act through their ability to intercalate into DNA, which causes configurational changes and results in the derepressed transcription of the luminescence operon (Ulitzur and Weiser, 1981). The great similarity between the effect produced by intercalating agents and those produced by DNA-damaging agents and DNA-synthesis inhibitors leads us to believe that these latter compounds also act through their ability to derepress the transcription of the luminescence operon. It is well documented that when bacterial DNA replication is inhibited either by specific inhibitors or by damage to the DNA, a sequence of events known as 'SOS functions' are initiated in procaryotic and eucaryotic cells (Witkin, 1974, 1976). A typical example of 'SOS function' is the induction of the ~,-prophage. The crucial step in h-prophage induction appears to be the proteolytic cleavage of the ~,repressor by the recA protein in Escherichia coli cells (Roberts et al., 1978). Recently, Kenyon and Walker (1980) have shown that DNA-damaging agents such as mitomycin C and UV irradiation stimulate gene expression not only of )~prophage but of at least 5 other different bacterial loci in E. coli. Similarly, Cowlishawa and Ginoza (1970) have shown that nalidixic acid and novobiocin induce lysogenic cultures of h-prophage. McCoy et al. (1980) postulated that this process is initiated by the interaction of these antibiotics with DNA-gyrase, and that this interaction is capable of triggering the SOS process. On the basis of these observations we speculated that DNA-synthesis inhibitors and DNA-damaging agents, through their induction of the 'SOS functions' might induce a protease that inactivates the repressor of the luminescence system. A direct approach to the problem is to inhibit protease activity by the use of inhihitors. Our results with one such inhibitor, antipain, were equivocal; although antipain (2 mg. ml- 1) strongly inhibited the induction of luminescence by mitomycin C, it was also highly toxic to mitomycin C-treated cells but not to the control cells (data not shown). This synergistic effect between mitomycin C and antipain could indicate that protease plays a positive role in Photobacterium cells that are treated with DNA-damaging agents. Since we lack the relevant mutant such as recA- or lexA- of E.coli, it was quite
449
P( ~
LUCIFERASECISTRONI
~ / R 7
~1
/sos
FUNCTIONSX
/
Fig. 5. See text for details.
difficult to obtain direct results to support our hypothesis. A preliminary approach was to show induction o f the luminescence by oligodeoxynucleotides (Oishi and Smith, 1978). Digested DNA increased the in vivo luminescence of dark-variant ceils of Beneckea harveyi by more than 1000-fold (unpublished observation). The influence of oligodeoxynucleotides on the luminescence of P. leiognathi cells is presently being studied. We can now postulate 3 groups of agents that induce luminescence in dark variants of luminous bacteria (Fig. 5): (a) Direct mutagens: these agents are either base-substitution or frameshift agents and directly revert the dark variants to genetically stable luminous cells. This may be due to changes in the repressor or the operator (Ulitzur et al., 1980). (b) DNA-intercalating agents: these agents cause phenotypic and almost complete reversion o f luminescence in dark variants through altering the ability of the repressor to bind DNA (Ulitzur and Weiser, 1981). (c) DNA-damaging agents and DNA-synthesis inhibitors that phenotypically revert the luminescence by triggering the 'SOS functions', which in turn inactivate the repressor o f the luminescence system. Thus, the dark variants of luminous bacteria offer a potentially valuable tool for studying the activity o f different physicochemical agents that interact with DNA. This system can also be used as a simple and sensitive test for detecting chemical agents that interact with DNA or its synthesis and thus serve as a prescreening test for suspected carcinogens. REFERENCES Cowlishawa, J., and W. Ginoza (1970) Induction ofh prophage by nalidixicacid, Virology,41,244-255.
450 Kenyon, C.J., and G.C. Walker (1980) DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli, Proc. Natl. Acad. Sci. (U.S.A.), 77, 2819-2823. McCoy, E.C., L.A. Petrullo and H.S. Rosenkranz (1980) Non-mutagenic genotoxicants: novobiocin and nalidixic acid, 2 inhibitors of DNA gyrase, Mutation Res., 79, 33-43. Nealson, K.H., and J.W. Hastings (1979) Bacterial bioluminescence: Its control and ecological significance, Microbiol. Rev., 43,496-518. Oishi, M., and C.L. Smith (1978) Inactivation of phage repressor in a permeable cell system: Role of recBC DNAase in induction, Proc. Natl. Acad. Sci. (U.S.A.), 75, 3569-3573. Roberts, J., C. Roberts and N. Craig (1978) E. coli rec A gene product inactivates phage lambda repressor, Proc. Natl. Acad. Sci, (U.S.A.), 75, 4714-4718. Ultizur, S., and I. Weiser (1981) Acridine dyes and other DNA-intercalating agents induce the luminescence system of luminous bacteria and their dark variants, Proc. Natl. Acad. Sci. (U.S.A.), in press. Ulitzur, S., I. Weiser and S. Yannai (1980) A new, sensitive and simple bioluminescence test for mutagenic compounds, Mutation Res., 74, 113-124. Witkin, E. (1974) Thermal enhancement of ultraviolet mutability in a tif-I uvrA derivative o f Escherichia coli: B/r: Evidence that ultraviolet mutagenesis depends upon an inducible function, Proc. Natl. Acad. Sci. (U.S.A.), 71, 1930-1934. Witkin, E. (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli, Bacteriol. Rev., 40, 869-907.
443
Elsevier/North-Holland BiomedicalPress
D N A - D A M A G I N G A G E N T S AND DNA-SYNTHESIS INHIBITORS INDUCE L U M I N E S C E N C E IN DARK VARIANTS OF LUMINOUS BACTERIA
IR1TH WEISER, S. ULITZUR and S. YANNAI Department of Food Engineering and Biotechnology Technion, Israel Institute of Technology, Haifa (Israel)
(Received l0 February 1981) (Accepted 14 April 1981)
SUMMARY The DNA-damaging agents mitomycin C and UV irradiation, as well as the DNAsynthesis inhibitors nalidixic acid, novobiocin and coumermycin, induce the de novo synthesis of luciferase and in vivo luminescence in dark variant cells of the luminous bacteria Photobacterium ieiognathi. Mitomycin C and nalidixic acid also cause the induction of luminescence in wild-type cells in the absence of its natural inducer. In spite of the high level of in vivo luminescence of the treated dark-variant cells, none of these agents result in the appearance of genetically luminous revertants. The possibility is discussed that these agents phenotypically induce luminescence through their ability to trigger 'SOS functions', which in turn leads to the transitory inactivation of certain repressors.
Naturally-occurring dark variants of luminous bacteria are characterized by their very low levels of in vivo luminescence and cellular luciferase content (Nealson and Hastings, 1979). The effect of different chemical agents on the reversion frequency o f these dark variants to the luminescent state has been recently studied by Ulitzur et al. (1980), and Ulitzur and Weiser (1981). 2 distinct groups of active chemicals were characterized. The first group that included mutagens showing either basesubstitution or frameshift capability increased the reversion frequency o f the dark variants to luminescent cells. The second group of chemicals share the common feature o f being DNA-intercalating agents (e.g. acridine dyes, ethidium bromide, caffeine and theophylline) caused almost complete restortion of the in vivo luminescence o f the dark-variant cells, but failed to increase the reversion rate at the genetic level. This second group o f chemicals was also shown to be able to induce luminescence in wild-type cells in the absence of an added inducer. 0165-7992/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
444
In the present paper we show that DNA-damaging agents and DNA-synthesis inhibitors are also capable of inducing phenotypic reversion of dark-variant cells, as well as in the wild-type cells prior to induction. The possibility that these agents cause inactivation of the repressor of the luciferase operon is discussed. MATERIALS AND METHODS
Bacterial strains and conditions o f growth. Photobacterium leiognathi, strain BE8, and its spontaneous dark variant (8SD18) were described by Ulitzur et al. (1980). The cells were grown with shaking at 30°C, in ASWRP liquid medium as previously described (Ulitzur et al., 1980). The solid medium consisted of ASWRP medium with 0.3°70 yeast extract (Difco) and 1.7070 agar (Difco). Luminescence determination. In vivo and in vitro luminescence were determined as described by Ulitzur and Weiser (1981).
E
= O"
d,z z
Control
"~ I0 0
5
I0
~ 15
-
cm~ MC. 20
TIME (hrs.)
Fig. 1. Effect o f mitomycin C on the luminescence o f P. leiognathi 8SD 18 cells. P. leiognathi 8SD 18 cells were grown in A S W R P liquid m e d i u m to a final cell density of about 5 x l07 cells- m l - i. 0.1-ml aliquots o f this culture were added to scintillation vials, containing 1 ml o f A S W R P , without (control) or with m itomycin C (MC 5 #g. m l - 1) or mitomycin (5 #g. m l - l) with chloramphenicol (10 #g- m l - l). The vials were incubated at 28°C in a scintillation counter and their luminescence was determined with time.
445
Chemicals. Mitomycin C, novobiocin, nalidixic acid and chloramphenicol were purchased from Sigma Chemicals Co., St. Louis, MO. Coumermycin and antipain were obtained as a gift from Prof. R. Ben-Ishai, Department of Biology, Technion (Israel). RESULTS
The effect of mitomycin C on the in vivo luminescence in dark cells of P. leiognathi strain 8SD18, is shown in Fig. 1. As can be seen, about 120 min after addition of mitomycin C (5 #g- ml- l), in vivo luminescence began to increase over the control
I0 0
107
io6
tirol
,o
d
'°o
!
i
3 TIME (hrs.)
Fig. 2. Effect o f UV-irradiation on the luminescence of P. leiognathi 8SD18 cells. P. leiognathi 8SD18 cells were suspended from 20-h old plates into A S W R P liquid m e d i u m (without peptone) to give a final cell density of 5 x 107 cells, m l - i. UV-irradiation was applied in a petri dish (9-cm diameter) containing l0 ml of the culture. A quartz lamp ( H a n a u ) was used at a distance of 60 cm. The cell suspension was irradiated with 400 e r g / m m 2 followed by dilution o f 0.2 ml of the treated culture into 1 ml of A S W R P liquid medium. UV-treated cells as well as the control culture, were incubated at 28°C and their luminescence was determined as a function o f time.
446
level, and finally attained (after 15 h) a level of up to 1000 times greater than that of the untreated cells. The final in vitro luciferase activity is also elevated, and is about 50 times higher than that of the control cells. The increase of both in vivo and in vitro luciferase activity by mitomycin C, is completely inhibited by chloramphenicol (10 #g.ml-l). In spite of the high level of the in vivo luminescence, no luminous revertants were observed among 2- 104 colonies of mitomycin C-treated cells. A similar picture was observed when 8SD18 cells were irradiated with UV light (Fig. 2). As with mitomycin C, the onset of luminescence appears about 100 min after the treatment and it reaches a level 1000 times higher than that of the control culture. The number of stable luminous revertants in the UV-treated culture was also less than 1 per 104. As both mitomycin C and UV irradiation cause cessation of DNA replication, we tested the activity of some known DNA-synthesis inhibitors on the induction of luminescence in the dark-variant cells. Fig. 3 shows that of all DNA-synthesis inhibitors tested, nalidixic acid, novobiocin and coumermycin, are very active in restoring the in vivo luminescence of the dark-variant cells. The onset of luminescence with these inhibitors appears about 3 h after the administration of the
tO to
10 9
10 8
-r
=
,oT
~r i.i ¢O Z W (/) i=1 Z
10 6
o
i .J
105
I0d
I
I
I
I
0 TIME
(hrl.)
Fig. 3. Effect o f novobiocin, coumermycin and nalidixic acid on the luminescence o f P. leiognathi 8SD18 cells. Novobiocin (0.2 #g), coumermycin (7.5/~g) and nalidixic acid (0.7/zg) were added to a set o f scintillation vials containing 1 mi of A S W R P liquid medium. To each vial 0.1 ml o f a logarithmic culture o f 8SD18 cells (107 cell. m l - n) was added and the luminescence was determined at different times at 28°C.
447
active agent. As with mitomycin C and UV light, no increase in genetic reversion to luminous colonies (less than 1 per 104) was observed in the cultures treated with the DNA-synthesis inhibitors. The luminescence system is synthesized de novo only towards the end of the logarithmic phase of growth, upon an accumulation of 'autoinducer' in the growing culture (Nealson and Hastings 1979). Ulitzur and Weiser (1981) have recently shown that young cultures of the wild-type cells of P. leiognathi prior to inducton, behave similarly to dark-variant cells; the luciferase synthesis in both kinds of cells is under repression, which can be overcome by different DNA-intercalating agents, such as acridine dyes. We have found (Fig. 4) that the DNA-damaging agent, mitomycin C and the DNA-synthesis inhibitor nalidixic acid, induce luminescence in wild-type cells prior to induction.
108
NAL. E m O"
Conh'ol
I
0
O
I
V 2 TIME
3
~
4
5
6
(hrs.)
Fig. 4. Effect o f mitomycin C a n d nalidixic acid on the induction of the luminescence o f P. leiognathi BE8 cells. P. leiognathi BE8 wild-type cells were incubated into A S W R P liquid m e d i u m to a final cell density of about l0 cells, m l - i. W h e n the culture reached a cell density of l0 s cells- m l - l, it was diluted 100-fold in 1 ml of A S W R P m e d i u m containing either mitomycin C (5 #g. m l - L), nalidixic acid (2.5 jzg. ml-m) or none (control). The vials were incubated at 28°C and the luminescence was determined with time.
448 DISCUSSION The action of the DNA-damaging agents and the DNA-synthesis inhibitors on the induction of luminescence is characterized by 3 features: (1) All agents increase the de novo synthesis of luciferase. (2) No genetic luminous revertants (less than one per l04 colonies) are observed among the offspring of the treated cultures. (3) Mitomycin C, a DNA-damaging agent, and nalidixic acid, a DNA-synthesis inhibitor, also induce the luminescence in young culture of wild-type cells prior to induction. These features also characterize the action of the DNA-intercalating agents that restore luminescence in dark and wild-type cells of luminous bacteria (Ulitzur and Weiser, 1981). We have previously suggested that these agents act through their ability to intercalate into DNA, which causes configurational changes and results in the derepressed transcription of the luminescence operon (Ulitzur and Weiser, 1981). The great similarity between the effect produced by intercalating agents and those produced by DNA-damaging agents and DNA-synthesis inhibitors leads us to believe that these latter compounds also act through their ability to derepress the transcription of the luminescence operon. It is well documented that when bacterial DNA replication is inhibited either by specific inhibitors or by damage to the DNA, a sequence of events known as 'SOS functions' are initiated in procaryotic and eucaryotic cells (Witkin, 1974, 1976). A typical example of 'SOS function' is the induction of the ~,-prophage. The crucial step in h-prophage induction appears to be the proteolytic cleavage of the ~,repressor by the recA protein in Escherichia coli cells (Roberts et al., 1978). Recently, Kenyon and Walker (1980) have shown that DNA-damaging agents such as mitomycin C and UV irradiation stimulate gene expression not only of )~prophage but of at least 5 other different bacterial loci in E. coli. Similarly, Cowlishawa and Ginoza (1970) have shown that nalidixic acid and novobiocin induce lysogenic cultures of h-prophage. McCoy et al. (1980) postulated that this process is initiated by the interaction of these antibiotics with DNA-gyrase, and that this interaction is capable of triggering the SOS process. On the basis of these observations we speculated that DNA-synthesis inhibitors and DNA-damaging agents, through their induction of the 'SOS functions' might induce a protease that inactivates the repressor of the luminescence system. A direct approach to the problem is to inhibit protease activity by the use of inhihitors. Our results with one such inhibitor, antipain, were equivocal; although antipain (2 mg. ml- 1) strongly inhibited the induction of luminescence by mitomycin C, it was also highly toxic to mitomycin C-treated cells but not to the control cells (data not shown). This synergistic effect between mitomycin C and antipain could indicate that protease plays a positive role in Photobacterium cells that are treated with DNA-damaging agents. Since we lack the relevant mutant such as recA- or lexA- of E.coli, it was quite
449
P( ~
LUCIFERASECISTRONI
~ / R 7
~1
/sos
FUNCTIONSX
/
Fig. 5. See text for details.
difficult to obtain direct results to support our hypothesis. A preliminary approach was to show induction o f the luminescence by oligodeoxynucleotides (Oishi and Smith, 1978). Digested DNA increased the in vivo luminescence of dark-variant ceils of Beneckea harveyi by more than 1000-fold (unpublished observation). The influence of oligodeoxynucleotides on the luminescence of P. leiognathi cells is presently being studied. We can now postulate 3 groups of agents that induce luminescence in dark variants of luminous bacteria (Fig. 5): (a) Direct mutagens: these agents are either base-substitution or frameshift agents and directly revert the dark variants to genetically stable luminous cells. This may be due to changes in the repressor or the operator (Ulitzur et al., 1980). (b) DNA-intercalating agents: these agents cause phenotypic and almost complete reversion o f luminescence in dark variants through altering the ability of the repressor to bind DNA (Ulitzur and Weiser, 1981). (c) DNA-damaging agents and DNA-synthesis inhibitors that phenotypically revert the luminescence by triggering the 'SOS functions', which in turn inactivate the repressor o f the luminescence system. Thus, the dark variants of luminous bacteria offer a potentially valuable tool for studying the activity o f different physicochemical agents that interact with DNA. This system can also be used as a simple and sensitive test for detecting chemical agents that interact with DNA or its synthesis and thus serve as a prescreening test for suspected carcinogens. REFERENCES Cowlishawa, J., and W. Ginoza (1970) Induction ofh prophage by nalidixicacid, Virology,41,244-255.
450 Kenyon, C.J., and G.C. Walker (1980) DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli, Proc. Natl. Acad. Sci. (U.S.A.), 77, 2819-2823. McCoy, E.C., L.A. Petrullo and H.S. Rosenkranz (1980) Non-mutagenic genotoxicants: novobiocin and nalidixic acid, 2 inhibitors of DNA gyrase, Mutation Res., 79, 33-43. Nealson, K.H., and J.W. Hastings (1979) Bacterial bioluminescence: Its control and ecological significance, Microbiol. Rev., 43,496-518. Oishi, M., and C.L. Smith (1978) Inactivation of phage repressor in a permeable cell system: Role of recBC DNAase in induction, Proc. Natl. Acad. Sci. (U.S.A.), 75, 3569-3573. Roberts, J., C. Roberts and N. Craig (1978) E. coli rec A gene product inactivates phage lambda repressor, Proc. Natl. Acad. Sci, (U.S.A.), 75, 4714-4718. Ultizur, S., and I. Weiser (1981) Acridine dyes and other DNA-intercalating agents induce the luminescence system of luminous bacteria and their dark variants, Proc. Natl. Acad. Sci. (U.S.A.), in press. Ulitzur, S., I. Weiser and S. Yannai (1980) A new, sensitive and simple bioluminescence test for mutagenic compounds, Mutation Res., 74, 113-124. Witkin, E. (1974) Thermal enhancement of ultraviolet mutability in a tif-I uvrA derivative o f Escherichia coli: B/r: Evidence that ultraviolet mutagenesis depends upon an inducible function, Proc. Natl. Acad. Sci. (U.S.A.), 71, 1930-1934. Witkin, E. (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli, Bacteriol. Rev., 40, 869-907.