Vol. 188, No. 3, 1992 November 16, 1992
ESCHERZCHZA
BIOCHEMICAL
COLZ
AND BIOPHYSICAL
GENES INVOLVED
DORMANCY:
ROLE
IN CELL
OF OXIDATIVE
RESEARCH COMMUNICATIONS Pages 1054-1059
SURVIVAL
DURING
STRESS
Abraham Eisenstark*, Cathy Miller, Joyce Jones and Sara L.evCn
Cancer Research Center, Columbia, MO 65201, and Div. Biological Sciences, University of Missouri, Columbia, MO 65211 Received September 2, 1992
When Escherichia coli cells reach stationary phase of growth, specific gene products are “Aged” cells may remain viable in cultures for synthesized that protect cells while dormant. years. For example, agar cultures stored for 38 years still had more thanlOs viable cells/ml. However, when specific mutants were cultured, the population of these mutants dropped sharply after 4-10 days. This defect is termed “Stationary-Phase-Death.” Each mutant strain was hypersensitive to near-ultraviolet radiation and other oxidative agents. Bovine catalase rescued many of the mutants from death in dormancy, suggesting that specific gene products protect “aged” cells against oxidative damage. 0 1992 Academic press, MC.
Escherichia coli and Salmonella typhimurium cells survive long periods of dormancy while in stationary phase. For example, cultures stored during 1954-1970 still contained living cells; in some cases almost a million viable cells/ml. were recovered (Table 1). However, specific mutations render cells less capable of withstanding dormancy. In one case reported to date, the surA mutant (l), the population declines over eight logs within 10 days; usually, not a single colony can be recovered upon plating a sample that earlier contained 108 viable cells. We now report that several additional “stationary-phase-death” (SPD) mutants were identified that underwent catastrophic loss of viability during dormancy (Fig. 1, Table 2). Further, each of the mutant strains were hypersensitive to oxidative stress, including photooxidative nearultraviolet irradiation stress (2).
MATERIALS
AND METHODS
Populations were determined by standard microbiological materials and methods (3). Specific details of experiments are included with Table and Figure legends.
*To whom correspondence should be addressed. 0006-29 1X/92 $4.00 Copyright 0 I992 by Academic Press, Inc. All rights of reproduction in any form reserved.
1054
Vol.
188,
BIOCHEMICAL
No. 3, 1992
AND BIOPHYSICAL
Table 1 - LONG TERM SURVIVAL
RESEARCH COMMUNICATIONS
OF BACTERIAL
CELLS
DESIGNATION/ PHENOTYPE
STOCK DATE
CFU/ml
1980 604 3921 3837 B259 B870 17 2480
thr, ade3, pyr-2,his lys-758 - met, str’ HfrB3 cys-343 cys-759 Hfr261 tryABE 130
l/63 9162 8162 8162 7163 5160 4164
2.1 x 104 1.4 x. 105 8.1 x 104 6.2 x 104 7.1 x 104 9.6 x 10“ 8.6 x 104
4554 4624 9148 4622 5421 8721 9156 5442
pur-5 purE75 ser-172 purB73 cysD22 proB173 ser-180 cysD48
7158 7159 8159 4159 6l54 4156 8159 8154
9.2 x 104 3.1 x 104 4.8 x 104 5.3 x 104 8.2 x 104 7.9 x 104 8.7 x 10“ 6.8 x 105
LAB # E.
coli
Stock cultures were stored (room temp.) between the years 1954-1970 in soft LB agar stabs, and sealedin paraffin. This collection was establishedby Dr. Milaslav Demerec at Cold Spring Harbor and at Brookhaven National Laboratory. After 22-38 yearsof storage,66 vials were unsealed,and the entire agar plug from each was transferred to 2 ml of LB broth. After vigorous vortex mixing, sampleswere serially diluted for colony counts on LB agar plates. To verify that cultures were not contaminants, cultures were checked for susceptibility to appropriate phage, and for appropriate nutritional auxotrophy. Although 66 vials were sampled, representative results are given for only 17.
RESULTS
AND
DISCUSSION
After cultures of mutants (Table 2) reached ca 2-8 X 109 cells/ml, they maintained a high population for 3-6 days with a loss of less than two logs. This was followed by a precipitous drop in viable cells within a few days (Table 2 and Fig. 1).
10i 2 ai! . b .
-
6-
: 3
4-
o.,.I.,.,.,.,.,.,.,.,, 1 2
3
4 5 6 Time (days)
7
a
9
10
Fig. 1. Survival of cells after reaching stationary phase of growth. After one day of growth in LB, colony counts were made daily. Upward arrow indicates that in some cultures, even after population drops 8 logs, cultures may eventually grow back to reach 10s colony farmers/ml. The genetic background of these suppressors(?) are currently under study. Data are given only for wildtype (WT), katF and the double mutant sodAsodB. 1055
Vol.
188,
No.
Table Lib
3,
BIOCHEMICAL
2 - STRAINS
Main GenotvDe(sJ
apaH::minikan
10.54
ksgA
619 568 1139 1136 887 858 913 1037
nur (katF) katF13::TnlO rpoS (katF) katF::minikan oxys-1 I sodAB
f@ fnr
1042
ti
892 895 969 612
pyrG51 cysH56 nuvA1 PM
AND
EXHIBITED
catalase HP1 & HPII; Mn superoxide dismutase survival (unknown product) diadenosine tetraphosphatase S-adenosylmethioninedimethyluansferase Regulator (see text) Regulator (see text) Regulator (see text) Regulator (see text) oxygen sensitive Superoxide dismutase Enterobactin receptor Regulator, nitrate reductase Phosphatase isozyme conversion CTPsynthetase Adenylsulfate reductase Uridine thiolation factor peptidase
surAl::TnlO 963
THAT
Phenotvne
katEGsodA
802
1992
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
STATIONARY-PHASE-DEATH
(SPD)
Additional Strain Desiqmtion
=
NUV
CSH57
>10*
sens.
-I
sens.
(18)
ZK126; W3110 (kant)
>10*
sens.
sew
ix
(1)
AB1157
>I06
sens.
sens.
+
(19)
AB 1157; SF224
>106
sens.
sens.
2
(19)
KL 16-99 MP180 RHlOO MC4100 (let’) ZKlOOO (kan’) AB 1157
>1os
sens. sens. sens. sens. sens.
* + sens. + sens. sens. + _+
(20)
(18)
Sensitivitv HOOH
Referent PO
AB1515; kanrcassette JRG861a; CGSC 5519
>106 >107 >104 2.107 >106 >106 >107
sens. sens.
sens. sens. sens. sens. sens. sens. f sens.
ANSSI (KL-267-srl)
>107
sens.
sens.
C
(18)
JF619 CGSC 5566 JM96; CGSC5746
>106 >106
>10*
sens. sens. sens. sens.
* f + +
(18) (18)
S. typhimurium S. typhimurium
sens. sens. sens. sens.
AQ 634
>106
sens.
(4)
(2.1) 6)
I:;; (23)
(15) (24)
Examples of mutants that undergo stationary-phase-death (SPD), and sensitivity to near-UV (NUV), hydrogen peroxide (HOOH) and paraquat (PQ). The designation, kutF, is the most commonly used allelic term. However, other allelic designations have been used, including nur (20), the original isolate of a near-UV sensitive mutant; rpoS, sigma factor (6), and appR (25). Results of sensitivity to other oxidative agents (e.g., cumeine hydroperoxide and menadione) are not shown. The numbers under SPD represent the drop in population (colony formers, in logs) when approximately 5 x 108 cells were plated 10 days beyond reaching stationary phase of growth. In many cases, the sharp drop occurred earlier (data not shown). Data in this table are not identical to data in Tables 3 and 4. Experiments were done at different times, and samples were also taken at different times after cells reached stationary phase.
As may be noted (Table 2), karF::TNlO phenotype. grown
Mulvey,
in minimal
that kutF 13::TnlO
et al (4) reported plus glucose
and alleles are among mutants with SPD
medium
cells lose about 95% of population
when
for 4-5 days. When katF strains were grown in highly
enriched media, the loss in population was precipitous (Table 2, Fig. 1). We found differences in various kufF::TnlO cultures by SPD; e.g., one katF::TnlO culture started from a single colony showed 2-log drop in population, but another kafF::TnlO
dropped
consistently from 109 to less than 100 CFU/ml. Possibly, either upon subculturing, or during dormancy, a second mutation may have occurred in one but not in another culture that would account for the difference. (unpublished),
which
could
We have other evidence that katF is a highly mutable gene explain
why some katF isolates
and alleles
did not show the sharp
SPD that are noted in Table 2 and Fig. 1. This anomaly may be clarified as we identify additional sequence changes in katF. We checked whether some strains in Table 2 could have a previously unknown katF mutation, thus accounting for the SPD phenotype. Indeed, as may be noted (Fig. 3, Table 3), putting the katF+ plasmid into strains rescued pyrG and sodAkatEG, as well as all katF strains. Further tests revealed that pyrG and soaM.ztEG were katF mutants as well. 1056
Vol.
188, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Table 3 - RESCUE BY katF PLASMID and/or CATALASE OF STRAINS DESTINED FOR STATIONARY-PHASE-DEATH Lab& 892 1139 1106 802 887 1054
915 913 858 612 1011 568 1136 1015 619 986
Genotvw PyrG ArpoS katF katEGsodA oxys- 11 ksg
yes yes (partial)
yes yes
yes (partial)
yes
yes
yes (slight) lethal
ppz e
no no
sodAB PepT
lethal yes
WT(1157)
control
katF::TnlO katF::kn WT
yes @tial) no control yes control
KB,r
yes
killed by catalase Hled by catalase no no yes yes killed by catalase yes
killed b;(datalase yes control
Examples of rescue of cells from Stationary-Phase-Death by bovine catalase and by a katF+carrying plasmid. Seelegend of Table 2 concerning survivors after SPD. Other strain listed in Table 2 gave similar results.
About 32 proteins may be regulated by karF (5,6). Many, if not all, are maximally synthesized when cells enter stationary phase of growth (4,7,8). Thus, the role of kurF protein may be to initiate a signal that alerts the cell that adversity (including oxidative stress) awaits, and certain protective proteins will he needed during dormancy. This is reminiscent of “checkpoint” genes of Saccharomyces cerevisiae (9) and of pre-sporulation genes of Bacillus subtilis (10) whose products signal cells that survival may depend on arresting chromosome synthesis at precise
times. Although the mechanism of death is unknown, alteration of certain environmental conditions did rescue cells from SPD. Among these were anaerobic growth, growth at low temperature, and growth in minimal medium versus growth in rich medium. In all of these environmental changes, growth was much slower than under conditions in which we observe SPD. It has long been known that, when cells are injured, recovery is increased under sub-optimal growth conditions (1 I). Perhaps slower growing cells deal with oxidative molecules better as a result of increase in antioxidants and repair enzymes. SPD mutants, including SW-AI, were sensitive to near-W (Fig. 2) and to at least one other oxidative agent (Table 2). SPD might be anticipated in those mutants that had known defects in antioxidant enzymes, such as sodAB double mutants that lack both of the superoxide dismutases (12). In other cases, the failure to recover from oxidative stress is less clear. In support of a role for oxidative damage as a cause of SPD, note that the peroxide-hypersensitive dps mutation results in large alteration of a DNA binding protein from starved cells (8). To test our hypothesis that oxidative stress during dormancy might be a factor in SPD, we added bovine catalase to parallel cultures to see if the enzyme would rescue cells, Indeed, this was the case for several SPD strains (Fig. 4, Table 3). Surprisingly, catalase was toxic for four strains, two of which are wildtype. 1057
Vol.
188, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
a-
tE
(p/katF)
-
0’ -6
T;r;oS (pikatF) 4
rp4-S ,.~,~.,.*,*.,..I..,
02
20
Near-UV
40
60
fluence
80
100120
(kJ/m’)
0
6-
no catalase
2
0
k
+ catalase
ah
katEGsodA
i 0
2
4
6
8
Time (days)
10
12
4 0 varying doses of
Fig. j?. Survival of cells after exposure to radiation (320-400 nm). The procedure has been described (26). Fi . 3. Restoration of survival ability upon transformation sgee note in legend of Table 2 concerning survivorsafter SPD.
4
6
8
10
Time (days)
near-ultraviolet
with k&F+
plasmid.
Fig. 4. Survival of katF cells with and without bovine catalase in LB. in legend of Table 1 concerning survivors after SPD.
See note
There are resemblances between death by near-UV irradiation (2) and death of mutants in stationary phase. When wildtype E. coli cells are irradiated by near-UV at sub-lethal fluences, growth ceases abruptly, known as “growth delay (13,14,15).” Cells remain alive for a few days under non-lethal fluences, but then cells abruptly die as if they suddenly age. In nature, E. coli cells plunge back and forth between anaerobic and aerobic conditions, nutritional feast and famine, warmth and cold, darkness and high solar radiation. However, most of the cell’s natural life is in dormancy. Dormant cells may be protected from oxidative stress by katF-regulated gene products, such as an anti-oxidant (catalase HPII; 4), and a DNA repair enzyme (exonuclease III; 7). We have tested several mutants known to be regulated by kafF (karE, &A, and apA). While these mutants are less viable than wildtype during dormancy, the population drop is mild compared to that of karF mutants. No karF-regulated product is known to be a full protector of cells. After drastic population loss, cultures occasionally return to full population. We are studying these putative suppressors to determine if this reversion of SPD phenotype has a genetic basis (16,17). In summary, we describe (a) cell survival after decades of dormancy, (b) mutations that render cells incapable (or sharply less capable) of survival during dormancy, (c) a role for certain gene products to protect dormant cells against oxidative stressincluding near-UV radiation.
ACKNOWLEDGMENTS These investigations
were supported
by a grant from the National
Institute
of
Environmental Health (Grant No. ES04889), and by Cancer Research Center, Columbia, Missouri. We thank 3. Bachmann, P. Loewen, D. Touati, R. Tuveson, B. Weiss, A. Matin, R. Kolter, H. Adler, M. McIntosh, A. Nakata, P. Boquet, R. Hengge-Aronis, B. Ames, C.A. Miller, and S. Farr for strains. 1058
Vol.
188, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
REFERENCES :: ::
ilb. :: 13: 14. 15. 16. 17. 18. 19. 20. 21. 2 24. 25. 26.
Tormo, A., Almiron, M., and Kolter, R. (1990) J. Bacreriof. 172, 4339-4347. Eisenstark, A. (1989) Advances in Genetics 26,99-147. Miller, J.H. (1972) Cold Spring Harbor Lab., Cold Spring Harbor Press, NY. 172, Mulvey, M.R., Switala, J., Borys, A., and Loewen, P.C. (1990) J. Bacreriol. 6713-6720. McCann, M.P., Kidwell, J.P. and Matin A. (1990) J. Bacreriol. 173, 4188-4194. Lange, R., and Hennge-Aronis, R. (1991) Molec. Microbial. 5, 49-59. Sak, B.D., Eisenstark, A. and Touati, D. (1989) Proc. Natf. Acad. Sci., USA, 86, 327 l3275. Siegele, D. Aand Kolter, R. (1989) J. Bacreriol. 174, 345-348. Hartwell, L.H., and Weinert, T.E. (1990) Science 246, 629-634. Setlow, P. (1992) J. Bacterial. 174, 2737-2741. Alper, T. and Gillies, N. E. (1958) J. Gen. Microbial. 18,461-468. Carlioz, A. and Touati, D. (1986) EMBO J., 5,623-630. Favre, A., Hajnsdorf, E., Thiam, K. and Caldeira de Araujo, A. (1985) Biochimie 67, 335-342. Hoerter, J. and Eisenstark, A. (1990) J. Phorochem. Photobiof. 6,283-289. Kramer, G.F., Baker, J.C. and Ames, B.N. (1988) J. Bacreriof. 170, 2344-2351. Cairns, J., Overbaugh, J., and Miller, S. (1988) Nature 335, 142-145. Hall, B.G. (1991) The New Biol., 3, 729-733. Bachmann, B.J. (1990) Microbial. Reviews 54, 130-197. Fat-r, S.B., Arnosti, D.N., Chamberlin, M.J. and Ames, B.N. (1989) Proc. rd., Acad. Sci. USA, 86, 5010-5014. Tuveson, R. W. (1980) Photochem. Photobiol. 32,703-705. Lange, R. and Hengge-Aronis, R. (1991) J. Bacreriof. 173, 4474-4481. Jamison, C.S. and Adler, H. (1987) J. Bacreriol. 169, 5087-5094. Ozenberger, B.A., Nahlik, M.S. and McIntosh M.A. (1987) J. Bacterial. 169, 36383646). Strauch, K.L., Lenk, J.B., Gamble, B.L. and Miller, C.G. (1985) J. Bacreriol. 16 1, 673-680. Touati, E., Dassa, E., and Boquet, P. L. (1986) Mol. Gen. Genet. 202, 257-264. Hartman, P.S. and Eisenstark, A. (1978) J. Bacteriof. 131, 769-779.
1059
ESCHERZCHZA
BIOCHEMICAL
COLZ
AND BIOPHYSICAL
GENES INVOLVED
DORMANCY:
ROLE
IN CELL
OF OXIDATIVE
RESEARCH COMMUNICATIONS Pages 1054-1059
SURVIVAL
DURING
STRESS
Abraham Eisenstark*, Cathy Miller, Joyce Jones and Sara L.evCn
Cancer Research Center, Columbia, MO 65201, and Div. Biological Sciences, University of Missouri, Columbia, MO 65211 Received September 2, 1992
When Escherichia coli cells reach stationary phase of growth, specific gene products are “Aged” cells may remain viable in cultures for synthesized that protect cells while dormant. years. For example, agar cultures stored for 38 years still had more thanlOs viable cells/ml. However, when specific mutants were cultured, the population of these mutants dropped sharply after 4-10 days. This defect is termed “Stationary-Phase-Death.” Each mutant strain was hypersensitive to near-ultraviolet radiation and other oxidative agents. Bovine catalase rescued many of the mutants from death in dormancy, suggesting that specific gene products protect “aged” cells against oxidative damage. 0 1992 Academic press, MC.
Escherichia coli and Salmonella typhimurium cells survive long periods of dormancy while in stationary phase. For example, cultures stored during 1954-1970 still contained living cells; in some cases almost a million viable cells/ml. were recovered (Table 1). However, specific mutations render cells less capable of withstanding dormancy. In one case reported to date, the surA mutant (l), the population declines over eight logs within 10 days; usually, not a single colony can be recovered upon plating a sample that earlier contained 108 viable cells. We now report that several additional “stationary-phase-death” (SPD) mutants were identified that underwent catastrophic loss of viability during dormancy (Fig. 1, Table 2). Further, each of the mutant strains were hypersensitive to oxidative stress, including photooxidative nearultraviolet irradiation stress (2).
MATERIALS
AND METHODS
Populations were determined by standard microbiological materials and methods (3). Specific details of experiments are included with Table and Figure legends.
*To whom correspondence should be addressed. 0006-29 1X/92 $4.00 Copyright 0 I992 by Academic Press, Inc. All rights of reproduction in any form reserved.
1054
Vol.
188,
BIOCHEMICAL
No. 3, 1992
AND BIOPHYSICAL
Table 1 - LONG TERM SURVIVAL
RESEARCH COMMUNICATIONS
OF BACTERIAL
CELLS
DESIGNATION/ PHENOTYPE
STOCK DATE
CFU/ml
1980 604 3921 3837 B259 B870 17 2480
thr, ade3, pyr-2,his lys-758 - met, str’ HfrB3 cys-343 cys-759 Hfr261 tryABE 130
l/63 9162 8162 8162 7163 5160 4164
2.1 x 104 1.4 x. 105 8.1 x 104 6.2 x 104 7.1 x 104 9.6 x 10“ 8.6 x 104
4554 4624 9148 4622 5421 8721 9156 5442
pur-5 purE75 ser-172 purB73 cysD22 proB173 ser-180 cysD48
7158 7159 8159 4159 6l54 4156 8159 8154
9.2 x 104 3.1 x 104 4.8 x 104 5.3 x 104 8.2 x 104 7.9 x 104 8.7 x 10“ 6.8 x 105
LAB # E.
coli
Stock cultures were stored (room temp.) between the years 1954-1970 in soft LB agar stabs, and sealedin paraffin. This collection was establishedby Dr. Milaslav Demerec at Cold Spring Harbor and at Brookhaven National Laboratory. After 22-38 yearsof storage,66 vials were unsealed,and the entire agar plug from each was transferred to 2 ml of LB broth. After vigorous vortex mixing, sampleswere serially diluted for colony counts on LB agar plates. To verify that cultures were not contaminants, cultures were checked for susceptibility to appropriate phage, and for appropriate nutritional auxotrophy. Although 66 vials were sampled, representative results are given for only 17.
RESULTS
AND
DISCUSSION
After cultures of mutants (Table 2) reached ca 2-8 X 109 cells/ml, they maintained a high population for 3-6 days with a loss of less than two logs. This was followed by a precipitous drop in viable cells within a few days (Table 2 and Fig. 1).
10i 2 ai! . b .
-
6-
: 3
4-
o.,.I.,.,.,.,.,.,.,.,, 1 2
3
4 5 6 Time (days)
7
a
9
10
Fig. 1. Survival of cells after reaching stationary phase of growth. After one day of growth in LB, colony counts were made daily. Upward arrow indicates that in some cultures, even after population drops 8 logs, cultures may eventually grow back to reach 10s colony farmers/ml. The genetic background of these suppressors(?) are currently under study. Data are given only for wildtype (WT), katF and the double mutant sodAsodB. 1055
Vol.
188,
No.
Table Lib
3,
BIOCHEMICAL
2 - STRAINS
Main GenotvDe(sJ
apaH::minikan
10.54
ksgA
619 568 1139 1136 887 858 913 1037
nur (katF) katF13::TnlO rpoS (katF) katF::minikan oxys-1 I sodAB
f@ fnr
1042
ti
892 895 969 612
pyrG51 cysH56 nuvA1 PM
AND
EXHIBITED
catalase HP1 & HPII; Mn superoxide dismutase survival (unknown product) diadenosine tetraphosphatase S-adenosylmethioninedimethyluansferase Regulator (see text) Regulator (see text) Regulator (see text) Regulator (see text) oxygen sensitive Superoxide dismutase Enterobactin receptor Regulator, nitrate reductase Phosphatase isozyme conversion CTPsynthetase Adenylsulfate reductase Uridine thiolation factor peptidase
surAl::TnlO 963
THAT
Phenotvne
katEGsodA
802
1992
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
STATIONARY-PHASE-DEATH
(SPD)
Additional Strain Desiqmtion
=
NUV
CSH57
>10*
sens.
-I
sens.
(18)
ZK126; W3110 (kant)
>10*
sens.
sew
ix
(1)
AB1157
>I06
sens.
sens.
+
(19)
AB 1157; SF224
>106
sens.
sens.
2
(19)
KL 16-99 MP180 RHlOO MC4100 (let’) ZKlOOO (kan’) AB 1157
>1os
sens. sens. sens. sens. sens.
* + sens. + sens. sens. + _+
(20)
(18)
Sensitivitv HOOH
Referent PO
AB1515; kanrcassette JRG861a; CGSC 5519
>106 >107 >104 2.107 >106 >106 >107
sens. sens.
sens. sens. sens. sens. sens. sens. f sens.
ANSSI (KL-267-srl)
>107
sens.
sens.
C
(18)
JF619 CGSC 5566 JM96; CGSC5746
>106 >106
>10*
sens. sens. sens. sens.
* f + +
(18) (18)
S. typhimurium S. typhimurium
sens. sens. sens. sens.
AQ 634
>106
sens.
(4)
(2.1) 6)
I:;; (23)
(15) (24)
Examples of mutants that undergo stationary-phase-death (SPD), and sensitivity to near-UV (NUV), hydrogen peroxide (HOOH) and paraquat (PQ). The designation, kutF, is the most commonly used allelic term. However, other allelic designations have been used, including nur (20), the original isolate of a near-UV sensitive mutant; rpoS, sigma factor (6), and appR (25). Results of sensitivity to other oxidative agents (e.g., cumeine hydroperoxide and menadione) are not shown. The numbers under SPD represent the drop in population (colony formers, in logs) when approximately 5 x 108 cells were plated 10 days beyond reaching stationary phase of growth. In many cases, the sharp drop occurred earlier (data not shown). Data in this table are not identical to data in Tables 3 and 4. Experiments were done at different times, and samples were also taken at different times after cells reached stationary phase.
As may be noted (Table 2), karF::TNlO phenotype. grown
Mulvey,
in minimal
that kutF 13::TnlO
et al (4) reported plus glucose
and alleles are among mutants with SPD
medium
cells lose about 95% of population
when
for 4-5 days. When katF strains were grown in highly
enriched media, the loss in population was precipitous (Table 2, Fig. 1). We found differences in various kufF::TnlO cultures by SPD; e.g., one katF::TnlO culture started from a single colony showed 2-log drop in population, but another kafF::TnlO
dropped
consistently from 109 to less than 100 CFU/ml. Possibly, either upon subculturing, or during dormancy, a second mutation may have occurred in one but not in another culture that would account for the difference. (unpublished),
which
could
We have other evidence that katF is a highly mutable gene explain
why some katF isolates
and alleles
did not show the sharp
SPD that are noted in Table 2 and Fig. 1. This anomaly may be clarified as we identify additional sequence changes in katF. We checked whether some strains in Table 2 could have a previously unknown katF mutation, thus accounting for the SPD phenotype. Indeed, as may be noted (Fig. 3, Table 3), putting the katF+ plasmid into strains rescued pyrG and sodAkatEG, as well as all katF strains. Further tests revealed that pyrG and soaM.ztEG were katF mutants as well. 1056
Vol.
188, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Table 3 - RESCUE BY katF PLASMID and/or CATALASE OF STRAINS DESTINED FOR STATIONARY-PHASE-DEATH Lab& 892 1139 1106 802 887 1054
915 913 858 612 1011 568 1136 1015 619 986
Genotvw PyrG ArpoS katF katEGsodA oxys- 11 ksg
yes yes (partial)
yes yes
yes (partial)
yes
yes
yes (slight) lethal
ppz e
no no
sodAB PepT
lethal yes
WT(1157)
control
katF::TnlO katF::kn WT
yes @tial) no control yes control
KB,r
yes
killed by catalase Hled by catalase no no yes yes killed by catalase yes
killed b;(datalase yes control
Examples of rescue of cells from Stationary-Phase-Death by bovine catalase and by a katF+carrying plasmid. Seelegend of Table 2 concerning survivors after SPD. Other strain listed in Table 2 gave similar results.
About 32 proteins may be regulated by karF (5,6). Many, if not all, are maximally synthesized when cells enter stationary phase of growth (4,7,8). Thus, the role of kurF protein may be to initiate a signal that alerts the cell that adversity (including oxidative stress) awaits, and certain protective proteins will he needed during dormancy. This is reminiscent of “checkpoint” genes of Saccharomyces cerevisiae (9) and of pre-sporulation genes of Bacillus subtilis (10) whose products signal cells that survival may depend on arresting chromosome synthesis at precise
times. Although the mechanism of death is unknown, alteration of certain environmental conditions did rescue cells from SPD. Among these were anaerobic growth, growth at low temperature, and growth in minimal medium versus growth in rich medium. In all of these environmental changes, growth was much slower than under conditions in which we observe SPD. It has long been known that, when cells are injured, recovery is increased under sub-optimal growth conditions (1 I). Perhaps slower growing cells deal with oxidative molecules better as a result of increase in antioxidants and repair enzymes. SPD mutants, including SW-AI, were sensitive to near-W (Fig. 2) and to at least one other oxidative agent (Table 2). SPD might be anticipated in those mutants that had known defects in antioxidant enzymes, such as sodAB double mutants that lack both of the superoxide dismutases (12). In other cases, the failure to recover from oxidative stress is less clear. In support of a role for oxidative damage as a cause of SPD, note that the peroxide-hypersensitive dps mutation results in large alteration of a DNA binding protein from starved cells (8). To test our hypothesis that oxidative stress during dormancy might be a factor in SPD, we added bovine catalase to parallel cultures to see if the enzyme would rescue cells, Indeed, this was the case for several SPD strains (Fig. 4, Table 3). Surprisingly, catalase was toxic for four strains, two of which are wildtype. 1057
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a-
tE
(p/katF)
-
0’ -6
T;r;oS (pikatF) 4
rp4-S ,.~,~.,.*,*.,..I..,
02
20
Near-UV
40
60
fluence
80
100120
(kJ/m’)
0
6-
no catalase
2
0
k
+ catalase
ah
katEGsodA
i 0
2
4
6
8
Time (days)
10
12
4 0 varying doses of
Fig. j?. Survival of cells after exposure to radiation (320-400 nm). The procedure has been described (26). Fi . 3. Restoration of survival ability upon transformation sgee note in legend of Table 2 concerning survivorsafter SPD.
4
6
8
10
Time (days)
near-ultraviolet
with k&F+
plasmid.
Fig. 4. Survival of katF cells with and without bovine catalase in LB. in legend of Table 1 concerning survivors after SPD.
See note
There are resemblances between death by near-UV irradiation (2) and death of mutants in stationary phase. When wildtype E. coli cells are irradiated by near-UV at sub-lethal fluences, growth ceases abruptly, known as “growth delay (13,14,15).” Cells remain alive for a few days under non-lethal fluences, but then cells abruptly die as if they suddenly age. In nature, E. coli cells plunge back and forth between anaerobic and aerobic conditions, nutritional feast and famine, warmth and cold, darkness and high solar radiation. However, most of the cell’s natural life is in dormancy. Dormant cells may be protected from oxidative stress by katF-regulated gene products, such as an anti-oxidant (catalase HPII; 4), and a DNA repair enzyme (exonuclease III; 7). We have tested several mutants known to be regulated by kafF (karE, &A, and apA). While these mutants are less viable than wildtype during dormancy, the population drop is mild compared to that of karF mutants. No karF-regulated product is known to be a full protector of cells. After drastic population loss, cultures occasionally return to full population. We are studying these putative suppressors to determine if this reversion of SPD phenotype has a genetic basis (16,17). In summary, we describe (a) cell survival after decades of dormancy, (b) mutations that render cells incapable (or sharply less capable) of survival during dormancy, (c) a role for certain gene products to protect dormant cells against oxidative stressincluding near-UV radiation.
ACKNOWLEDGMENTS These investigations
were supported
by a grant from the National
Institute
of
Environmental Health (Grant No. ES04889), and by Cancer Research Center, Columbia, Missouri. We thank 3. Bachmann, P. Loewen, D. Touati, R. Tuveson, B. Weiss, A. Matin, R. Kolter, H. Adler, M. McIntosh, A. Nakata, P. Boquet, R. Hengge-Aronis, B. Ames, C.A. Miller, and S. Farr for strains. 1058
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REFERENCES :: ::
ilb. :: 13: 14. 15. 16. 17. 18. 19. 20. 21. 2 24. 25. 26.
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