JOURNAL OF BACTERIOLOGY, Aug. 1977, p. iO7-709 Copyright C 1977 American Society for Microbiology
Vol. 131, No. 2 Printed in U.S.A.
Role of Cyclic Adenosine 3',5'-Monophosphate on Cessation of Respiration in Ultraviolet-Irradiated Escherichia coli P. A. SWENSON,* R. L. SCHENLEY AND J. G. JOSHI Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830,* and Biochemistry Department, University of Tennessee, Knoxville, Tennessee 37916
Received for publication 31 March 1977
The addition of cyclic adenosine 3',5'-monophosphate (cAMP) to ultravioletirradiated Escherichia coli B/r cultures causes additional cells to cease respiring and to die. These effects of cAMP are greater on glucose-grown cells, where the effects of ultraviolet radiations alone are smaller and where the intracellular concentrations of cAMP are known to be lower.
Ultraviolet radiations (UV) cause the respiration ofsome Escherichia coli cells in a culture to cease irreversibly beginning 30 min after irradiation (9). The shutoff process requires protein synthesis (6) and the recA + and exrA+(lex+) gene products (10). A number of rec/lex responses to UV, including cessation of respiration (8), are thought to involve derepression of an operon (9, 11). We have shown that more irradiated cells stop respiring (6) and are killed (7) when the cells are grown on glycerol than when they are grown on glucose. Since glycerol-grown cells have higher intracellular cyclic adenosine 3',5'-monophosphate (cAMP) concentrations than cells grown on glucose (1), we investigated the effect of exogenous cAMP on respiration and viability of UV-irradiated cells grown on the two carbon sources. Figure 1 shows the effects of various concentrations of cAMP on the respiration of irradiated (52 J/m2) E. coli B/r cultures grown on glucose. An exogenous concentration of 5 mM cAMP added immediately after UV causes respiration of the culture to cease almost completely for several hours beginning 60 min after irradiation. The effects are less pronounced with lower concentrations of cAMP. The respiratory response of the cultures to which cAMP was added at a concentration of 5 mM is the same as that of glycerol-grown cultures without cAMP at the same UV fluence (6). No modifying effect of respiration of unirradiated or irradiated cultures was brought about by 5 mM concentrations of adenosine 5'-monophosphate, adenosine, adenine, or guanosine 3',5'-monophosphate. Figure 2 shows viability timecourse curves for UV-irradiated (52 J/m2), glucose-grown cells with and without cAMP in the liquid growth medium. When cAMP is present (Fig. 2), the viability decreases rapidly 30 min
after UV, but the exponential increase in viability begins at the same time (90 min) as for untreated cells. The effects of cAMP on the respiration of UVirradiated, glycerol-grown cultures are shown in Fig. 3A. For two fluences, 16.5 and 27 J/m2, cAMP causes a more complete inhibition of respiration, but in each case the effect is less than that seen for the glucose-grown cultures; the effect on viability is also smaller (data not shown). As shown in Fig. 1 and 3A, appropriate fluences of UV cause transitory cessation of respiration in both glucose- and glycerol-grown cultures. We have established by the post-UV use of the detergent Triton X-100 that only the nonviable cells in UV-irradiated cultures cease respiring; the cell membranes of these cells are dissolved by the detergent, thus converting them to small entities and causing a decrease in turbidity of the culture (9); all of the large unconverted cells remain viable (4). Figure 3B shows the effect of Triton X-100 on the turbidity of UV-irradiated (16.5 J/m2), glycerol-grown cultures in the presence and absence of cAMP. The turbidity of the non-detergent-treated cultures parallels the respiration curves for the corresponding radiation fluence shown in Fig. 3A and, after maximum turbidity losses, the curves of the detergent-treated cultures parallel the corresponding curves of those not receiving the treatment. The increases in turbidity after maximum losses are presumably caused by growth of surviving cells. Since Triton X-100 does not affect respiration or viability (4, 9), it appears that cAMP causes additional cells to cease respiring. The functioning of a number of genes in E. coli is under transcriptional control by cAMP (3). In addition, this cyclic nucleotide appears to regulate a number of membrane functions, 707
708
400
cAMP 1
4
~~~5mM
LLJ
O~~~~~TM A
28035
7,A
*-*~~~~~~~~0
S ~~~~~~~
UV(mn AFE
25 E esprto 1 Effects ~.of cAPo0h /2 nalcassteel UVfunews5 Th FIG. eprto ofE 1. Effect ofcM5nte FIG.
ben decrbe (4). coli Bir cells grown on glucose minimal medium. The UV fluence was 52 JIM2. In all cases the cells were grown in the absence of cAMP and were suspended at 37'C in liquid growth medium in the presence of the nucleotide immediately after irradiation. The composition of minimal medium and the methodologies for irradiation and respirometry have been described (4).
A NO ADD
060
o/ NO TRITON
E 0 0
to
/
+cAM8"
_+ cAMP roA
-A
A/
cAMP
UV
NO UV
08- B
0.2-
E
BACTERIOL.
J.
NOTES
120
1_
1_
180
240
300
360
TIME AFTER UV (mnin)
FIG. 2. Viability time-course curves for UV-irradiated E. coli Bir cells in the presence and absence of cAMP. The carbon source was glucose; the UV fluence was 52 J/m2; the concentration of cAMP was 5 mM. Cell dilutions were placed on glucose minimal media solidified with 1.2% agar and incubated at 37°C for 48 h.
including transport (2) and energetic systems (S. S. Dills and W. A. Dobrogosz, Abstr. Annu. Meet. Am. Soc. Microbiol. 1977, K13, p. 188). The results in this paper show that cessation of respiration and cell killing after UV are also regulated by cAMP. We suggest, in line with
This research was supported by the Energy Research and Development Administration under contract with the Union Carbide Corp. LITERATURE CITED 1. Epstein, W., L. B. Rothman-Denes, and J. Hesse. 1975. Adenosine 3':5' cyclic monophosphate as mediator of catabolite repression in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 72:2300-2304. 2. Ezzell, J. W., and W. J. Dobrogosz. 1975. Altered hexose transport and salt sensitivity in cyclic adenosine 3',5'-monophosphate-deficient Escherichia coli. J. Bacteriol. 124:815-824. 3. Pastan, I., and S. Adhya. 1976. Cyclic adenosine 5'monophosphate in Escherichia coli. Bacteriol. Rev.
40:527-551.
4. Schenley, R. L., W. D. Fisher, P. A. Swenson, and G. G. Khachatourians. 1976. Centrifugal separation of
irradiated cultures ofEscherichia coli cells into viable
VOL. 131, 1977
and nonviable populations. J. Bacteriol. 126:977-984. 5. Swenson, P. A. 1976. Physiological responses of Escherichia coli to far ultraviolet radiation, p. 269-387. In K. C. Smith (ed.), Photochemical and photobiological reviews, vol. 1. Plenum Press, New York. 6. Swenson, P. A., and R. L. Schenley. 1970. Evidence for the control of respiration by DNA in ultraviolet-irradiated Escherichia coli B/r cells. Mutat. Res. 9:443453. 7. Swenson, P. A., and R. L. Schenley. 1970. Role of pyridine nucleotides in the control of respiration in ultraviolet-irradiated Escherichia coli B/r cells. J. Bacteriol. 104:1230-1235. 8. Swenson, P. A., and R. L. Schenley. 1972. Death
NOTES
709
through respiratory failure of a fraction of ultraviolet-irradiated Escherichia coli B/r cells. J. Bacteriol. 111:658-663. 9. Swenson, P. A., and R. L. Schenley. 1974. Evidence relating cessation of respiration, cell envelope changes, and death in ultraviolet-irradiated Escherichia coli B/r cells. J. Bacteriol. 117:551-559. 10. Swenson, P. A., and R. L. Schenley. 1974. Respiration, growth and viability of repair-deficient mutants of Escherichia coli after ultraviolet irradiation. Int. J. Radiat. Biol. 74:51-60. 11. Witkin, E. M. 1976. Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol. Rev. 40:869-907.
Vol. 131, No. 2 Printed in U.S.A.
Role of Cyclic Adenosine 3',5'-Monophosphate on Cessation of Respiration in Ultraviolet-Irradiated Escherichia coli P. A. SWENSON,* R. L. SCHENLEY AND J. G. JOSHI Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830,* and Biochemistry Department, University of Tennessee, Knoxville, Tennessee 37916
Received for publication 31 March 1977
The addition of cyclic adenosine 3',5'-monophosphate (cAMP) to ultravioletirradiated Escherichia coli B/r cultures causes additional cells to cease respiring and to die. These effects of cAMP are greater on glucose-grown cells, where the effects of ultraviolet radiations alone are smaller and where the intracellular concentrations of cAMP are known to be lower.
Ultraviolet radiations (UV) cause the respiration ofsome Escherichia coli cells in a culture to cease irreversibly beginning 30 min after irradiation (9). The shutoff process requires protein synthesis (6) and the recA + and exrA+(lex+) gene products (10). A number of rec/lex responses to UV, including cessation of respiration (8), are thought to involve derepression of an operon (9, 11). We have shown that more irradiated cells stop respiring (6) and are killed (7) when the cells are grown on glycerol than when they are grown on glucose. Since glycerol-grown cells have higher intracellular cyclic adenosine 3',5'-monophosphate (cAMP) concentrations than cells grown on glucose (1), we investigated the effect of exogenous cAMP on respiration and viability of UV-irradiated cells grown on the two carbon sources. Figure 1 shows the effects of various concentrations of cAMP on the respiration of irradiated (52 J/m2) E. coli B/r cultures grown on glucose. An exogenous concentration of 5 mM cAMP added immediately after UV causes respiration of the culture to cease almost completely for several hours beginning 60 min after irradiation. The effects are less pronounced with lower concentrations of cAMP. The respiratory response of the cultures to which cAMP was added at a concentration of 5 mM is the same as that of glycerol-grown cultures without cAMP at the same UV fluence (6). No modifying effect of respiration of unirradiated or irradiated cultures was brought about by 5 mM concentrations of adenosine 5'-monophosphate, adenosine, adenine, or guanosine 3',5'-monophosphate. Figure 2 shows viability timecourse curves for UV-irradiated (52 J/m2), glucose-grown cells with and without cAMP in the liquid growth medium. When cAMP is present (Fig. 2), the viability decreases rapidly 30 min
after UV, but the exponential increase in viability begins at the same time (90 min) as for untreated cells. The effects of cAMP on the respiration of UVirradiated, glycerol-grown cultures are shown in Fig. 3A. For two fluences, 16.5 and 27 J/m2, cAMP causes a more complete inhibition of respiration, but in each case the effect is less than that seen for the glucose-grown cultures; the effect on viability is also smaller (data not shown). As shown in Fig. 1 and 3A, appropriate fluences of UV cause transitory cessation of respiration in both glucose- and glycerol-grown cultures. We have established by the post-UV use of the detergent Triton X-100 that only the nonviable cells in UV-irradiated cultures cease respiring; the cell membranes of these cells are dissolved by the detergent, thus converting them to small entities and causing a decrease in turbidity of the culture (9); all of the large unconverted cells remain viable (4). Figure 3B shows the effect of Triton X-100 on the turbidity of UV-irradiated (16.5 J/m2), glycerol-grown cultures in the presence and absence of cAMP. The turbidity of the non-detergent-treated cultures parallels the respiration curves for the corresponding radiation fluence shown in Fig. 3A and, after maximum turbidity losses, the curves of the detergent-treated cultures parallel the corresponding curves of those not receiving the treatment. The increases in turbidity after maximum losses are presumably caused by growth of surviving cells. Since Triton X-100 does not affect respiration or viability (4, 9), it appears that cAMP causes additional cells to cease respiring. The functioning of a number of genes in E. coli is under transcriptional control by cAMP (3). In addition, this cyclic nucleotide appears to regulate a number of membrane functions, 707
708
400
cAMP 1
4
~~~5mM
LLJ
O~~~~~TM A
28035
7,A
*-*~~~~~~~~0
S ~~~~~~~
UV(mn AFE
25 E esprto 1 Effects ~.of cAPo0h /2 nalcassteel UVfunews5 Th FIG. eprto ofE 1. Effect ofcM5nte FIG.
ben decrbe (4). coli Bir cells grown on glucose minimal medium. The UV fluence was 52 JIM2. In all cases the cells were grown in the absence of cAMP and were suspended at 37'C in liquid growth medium in the presence of the nucleotide immediately after irradiation. The composition of minimal medium and the methodologies for irradiation and respirometry have been described (4).
A NO ADD
060
o/ NO TRITON
E 0 0
to
/
+cAM8"
_+ cAMP roA
-A
A/
cAMP
UV
NO UV
08- B
0.2-
E
BACTERIOL.
J.
NOTES
120
1_
1_
180
240
300
360
TIME AFTER UV (mnin)
FIG. 2. Viability time-course curves for UV-irradiated E. coli Bir cells in the presence and absence of cAMP. The carbon source was glucose; the UV fluence was 52 J/m2; the concentration of cAMP was 5 mM. Cell dilutions were placed on glucose minimal media solidified with 1.2% agar and incubated at 37°C for 48 h.
including transport (2) and energetic systems (S. S. Dills and W. A. Dobrogosz, Abstr. Annu. Meet. Am. Soc. Microbiol. 1977, K13, p. 188). The results in this paper show that cessation of respiration and cell killing after UV are also regulated by cAMP. We suggest, in line with
This research was supported by the Energy Research and Development Administration under contract with the Union Carbide Corp. LITERATURE CITED 1. Epstein, W., L. B. Rothman-Denes, and J. Hesse. 1975. Adenosine 3':5' cyclic monophosphate as mediator of catabolite repression in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 72:2300-2304. 2. Ezzell, J. W., and W. J. Dobrogosz. 1975. Altered hexose transport and salt sensitivity in cyclic adenosine 3',5'-monophosphate-deficient Escherichia coli. J. Bacteriol. 124:815-824. 3. Pastan, I., and S. Adhya. 1976. Cyclic adenosine 5'monophosphate in Escherichia coli. Bacteriol. Rev.
40:527-551.
4. Schenley, R. L., W. D. Fisher, P. A. Swenson, and G. G. Khachatourians. 1976. Centrifugal separation of
irradiated cultures ofEscherichia coli cells into viable
VOL. 131, 1977
and nonviable populations. J. Bacteriol. 126:977-984. 5. Swenson, P. A. 1976. Physiological responses of Escherichia coli to far ultraviolet radiation, p. 269-387. In K. C. Smith (ed.), Photochemical and photobiological reviews, vol. 1. Plenum Press, New York. 6. Swenson, P. A., and R. L. Schenley. 1970. Evidence for the control of respiration by DNA in ultraviolet-irradiated Escherichia coli B/r cells. Mutat. Res. 9:443453. 7. Swenson, P. A., and R. L. Schenley. 1970. Role of pyridine nucleotides in the control of respiration in ultraviolet-irradiated Escherichia coli B/r cells. J. Bacteriol. 104:1230-1235. 8. Swenson, P. A., and R. L. Schenley. 1972. Death
NOTES
709
through respiratory failure of a fraction of ultraviolet-irradiated Escherichia coli B/r cells. J. Bacteriol. 111:658-663. 9. Swenson, P. A., and R. L. Schenley. 1974. Evidence relating cessation of respiration, cell envelope changes, and death in ultraviolet-irradiated Escherichia coli B/r cells. J. Bacteriol. 117:551-559. 10. Swenson, P. A., and R. L. Schenley. 1974. Respiration, growth and viability of repair-deficient mutants of Escherichia coli after ultraviolet irradiation. Int. J. Radiat. Biol. 74:51-60. 11. Witkin, E. M. 1976. Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol. Rev. 40:869-907.
