Jun 14, 1991 - When exposed to high temperatures, Escherichia coli induces a set of proteins (heat shock proteins), including. GroES, GroEL, DnaK, DnaJ, ...
Vol. 174, No. 2
JOURNAL OF BACTERIOLOGY, Jan. 1992, p. 630-632 0021-9193/92/020630-03$02.00/0 Copyright C 1992, American Society for Microbiology
Sensitization of Escherichia coli Cells to Oxidative Stress by Deletion of the rpoH Gene, Which Encodes the Heat Shock Sigma Factor TOKIO KOGOMAl 2* AND TAKASHI YURA' Institute for Virus Research, Kyoto University, Kyoto 606, Japan,1 and Departments of Cell Biology and Microbiology, University of New Mexico Medical Center, Albuquerque, New Mexico 871312* Received 14 June 1991/Accepted 9 November 1991
A deletion in the rpoH gene greatly increased the sensitivity of Escherichia coli sodA sodB mutants to oxidative stress. The effect of the rpoH deletion on sodA+ sodB+ cells was only marginal. Mutations in heat shock genes singly sensitized sodA sodB double mutant cells to plumbagin. sodA sodB double mutants were neither more sensitive nor more resistant to thermal stress than the wild type.
mutation was introduced by P1-mediated transduction. P1 lysate for ArpoH30::kan transduction was prepared from a derivative of KY1612 (ArpoH30::kan zhp5O::TnJO [21]) harboring pKV3, which is a derivative of pBR322 carrying an insert containing an rpoH+ gene (17). These mutants were tested for their sensitivity to oxidative stress by growth inhibition with paraquat, plumbagin, and hydrogen peroxide (Table 1). Oxidative stress sensitivity was assayed as follows. A 0.8-ml sample of overnight culture that had been diluted twofold with fresh broth was applied to an L plate. After the cells were spread evenly on the plate, excess cell suspension was removed. A paper filter disk (7 mm in diameter) was then placed in the center of the plate, and 5 ,ul of 1 M paraquat or 50 mM plumbagin in dimethyl sulfoxide or 30% H202 was applied onto the disk. Plates were incubated at 34°C for 48 h or at 20°C for 72 h before the diameter of the zone of inhibition was determined. The results of seven separate experiments are summarized in Table 1. The salient results of the study with paraquat are as follows. (i) The ArpoH mutation drastically enhanced the sensitivity of SOD- cells, whereas the effect on SOD' cells was only marginal. (ii) The heat shock mutations singly had little effect on sensitivity to paraquat regardless of the presence or absence of SOD, with the possible exception of the effect of the groEL44 mutation on SOD- cells. (iii) SODcells were sensitive to paraquat, as previously shown (3). (iv) Incubation at low temperatures sensitized wild-type cells to paraquat. Inhibition rings with plumbagin on SOD' strains were not consistently seen for a reason which is not understood. Unlike paraquat, however, plumbagin had a significant inhibitory effect on the growth of heat shock mutants in the SOD- background, including the ArpoH derivative of SOD-. The sensitivity to hydrogen peroxide was not increased by the heat shock mutations. However, the ArpoH mutation significantly enhanced the sensitivity of SODcells to hydrogen peroxide (Table 1). The results described above indicate that the rpoH deletion, which prevents induction of heat shock genes (21), sensitizes cells to both peroxide- and superoxide-mediated oxidative stress, particularly in the absence of SOD activity. This suggests that heat shock proteins play some role in protection against oxidative stress when the stress becomes very severe. Since heat shock proteins are induced under several different stress conditions (6), these proteins may
When exposed to high temperatures, Escherichia coli induces a set of proteins (heat shock proteins), including GroES, GroEL, DnaK, DnaJ, GrpE, HtpG, and Lon (for reviews, see references 8 and 15). These proteins contribute to cell survival under thermal stress. Thus, mutations in the heat shock genes render cells temperature sensitive. The induction of heat shock proteins involves recognition of heat shock promoters by RNA polymerase (Er32) equipped with a32, which is encoded by the rpoH gene (5, 9). rpoH deletion mutants completely lacking C32 protein are incapable of inducing heat shock proteins. Consequently, these mutants are very sensitive to thermal stress and are unable to grow at temperatures above 20°C (21). E. coli possesses two superoxide dismutases (SODs): an inducible MnSOD encoded by sodA and a constitutively produced FeSOD encoded by sodB (reviewed in reference 6). SOD catalyzes dismutation of 02 to hydrogen peroxide and molecular oxygen. Therefore, sodA sodB double mutants lack SOD activity altogether and are very sensitive to superoxide generators such as paraquat and plumbagin (6). H202 and 2- each induce a set of some 30 proteins; there is a slight overlap between the two sets of proteins. The induction of a subset of the proteins by H202 and 02stresses is regulated by the control loci oxyR and soxRS, respectively (6). Effects of heat shock mutations on oxidative stress sensitivity. Among those proteins induced under 02- stress and H202 stress are GroEL, GroES, and DnaK (reviewed in reference 6). We examined the possibility that heat shock proteins play roles in defense against oxidative stress. To examine the effects of heat shock mutations on susceptibility to oxidative stress, two otherwise isogenic sets of heat shock mutants, one with mutations in the sodA+ sodB+ (SOD') genetic background and the other with mutations in the sodA sodB (SOD-) genetic background, were constructed. The groEL44 (12), dnaK756 (7), grpE280 (1), and ArpoH30::kan (21) mutants have been previously described. The groES72 mutant was isolated by localized mutagenesis (lOa). The groEL, groES, dnaK, and grpE mutations were introduced into QC779 (sodA sodB double mutant) and the parental strain GC4468 (3) by T4GT7-mediated transduction (18) by virtue of their linkage to a TnJO insertion. The ArpoH30::kan
*
Corresponding author. 630
NOTES
VOL. 174, 1992
631
TABLE 1. Sensitivity of heat shock mutants to oxidative stress Expt. no.
Strain
5 6 7
Relevant genotype
Inhibition ring diam (mm)a
Temp (OC)
PQ (Avg + SEM)
+ +
34 34 34
17.5 (20.8 ± 1.4) 22.0 23.0
9.0 8.0
21.0 23.0 21.0
+ +
+ +
34 34
22.0 (22.0 ± 0.0) 22.0
0 0
22.0 22.0
+ +
+ +
+ +
34 34
13.3 (15.4 ± 0.4) 17.5
0 8.0
ND 14.0
+ +
+ +
-
+ +
34 34
18.5 (18.3 ± 0.5) 18.0
10.5 0
ND 18.0
+ +
+ +
-
+ +
+ +
34 34
15.5 (16.3 ± 0.4) 17.0
0 10.0
ND 19.0
-
+ + +
+ + +
+ + +
+ + +
+ + +
34 34 34
21.5 (25.5 ± 1.7) 27.0 28.5
25.5 (21.5 21.0 18.0
+
1.8)
23.0 25.0 24.0
-
-
_ -
+ +
+ +
+ +
+ +
34 34
35.0(34.0 ± 0.7) 33.0
30.0 (28.0 26.0
+
1.4)
26.0 26.0
-
-
+ +
-
+ +
+ +
+ +
34 34
20.5 (20.0 19.5
0.5)
-
29.5 (34.0 ± 0.9) 38.5
ND 20.0
-
-
+ +
+ +
+ +
-
+ +
34 34
20.0 (20.3 ± 0.6) 20.5
32.0 (31.8 ± 0.9) 31.5
ND 20.5
TKY38 TKY38
-
-
+ +
+ +
-
+ +
+ +
34 34
21.0 (20.8 ± 0.7) 20.5
30.0 (28.8 27.5
ND 21.0
4 5
TKY39 TKY39
+ +
+ +
+ +
+ +
+ +
+ +
+ +
20 20
27.5 (27.0 ± 0.7) 26.5
0 0
ND 21.5
3 4 5
TKY45 TKY45 TKY45
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
A A A
20 20 20
31.5(30.7±0.8) 32.5 28.0
0 0 0
ND ND 20.0
4 5
TKY40 TKY40
-
-
+ +
+ +
+ +
+ +
+ +
20 20
28.5 (28.5 ± 0.8) 28.5
20.5 (21.5 ± 0.6) 22.5
ND 21.5
3 4 5
TKY46 TKY46 TKY46
_ -
_ -
+
+
+
+
+
+
+
+
+
+
+
A A A
20 20 20
47.5 (45.8 ± 1.2) 47.0 43.0
34.5 (33.0 ± 0.9) 32.5 33.0
ND ND 32.5
a
Values in parentheses
sodA
sodB
groEL
groES
dnaK
grpE
rpoH
TKY39 TKY39 TKY39
+
+ +
+ +
+ +
+ +
+
+
+
+
+
+ + +
+
+
6 7
TKY69 TKY69
+
+ +
-
+
-
+ +
+ +
1 5
TKY29 TKY29
+ +
+ +
+ +
-
2 5
TKY35 TKY35
+ +
+ +
+ +
2 5
TKY36 TKY36
+ +
+ +
5 6 7
TKY40
.TKY40
-
6 7
TKY70 TKY70
1 5
TKY30 TKY30
2 5
TKY37 TKY37
2 5
TKY40
are
+
for each set of two
or
-
PM (Avg ± SEM)
8.0
+
0.8)
H202
three values. PQ, paraquat; PM, plumbagin; ND, not determined.
serve as general antistress proteins. It would be of great interest to determine whether overproduction of GroE proteins suppresses the hypersensitivity of SOD- cells to oxidative stress, as it effectively alleviates the temperature sensitivity defect of the ArpoH mutants (11). On the other hand, the possibility that 32 protein is involved in expression of certain genes essential specifically for survival against oxidative stress cannot at present be ruled out. Heat tolerance of SOD- cells. The heat tolerance of sodA sodB double mutants was examined by comparing the surviving fractions of SOD' (TKY6) and SOD- (TKY11) cells that were subjected to heat shock at 53°C for up to 6 min. There was no difference in the overall killing rates at this temperature (Fig. 1). The difference seen at the 1-min point in this experiment was not reproducible when the experiment was repeated at 50 or 55°C (data not shown).
The growth rate, a parameter of heat shock sensitivity (19), was also compared. Cultures growing at 30°C were shifted to high temperatures, and the growth was monitored by the increase in optical density at 600 nm. The growth of SOD' and SOD- cultures was exponential for at least 80 min after the temperature shift (data not shown). The growth rates of SOD' cells at 30, 43, and 46°C were 2.12, 3.80, and 2.83 doublings per h, respectively. On the other hand, those of SOD- cells were 1.40, 1.52, and 1.43 doublings per h, respectively, at these temperatures. Thus, SOD- cells appeared not to be more sensitive to thermal stress than SOD' cells. Pretreatment of Salmonella typhimurium with adaptive doses of H202, which induces five heat shock proteins, including DnaK (14), leads to markedly increased resistance to thermal stress (4). In SOD- cells which were chronically
632
J. BACTERIOL.
NOTES synthesized
as a
consequence of oxidative stress. Cell 37:225-
232
0
(53
10
0) c
U)
10
162
0
2 Minutes
4
6
FIG. 1. TKY6 (0) and TKY11 (0) were grown to mid-log phase at 30°C with vigorous aeration. TKY6 and TKY11 were ArecA derivatives of GC4468 and QC779, respectively. The cultures were subjected to heat shock at 53°C for the periods indicated. The samples were immediately diluted, plated in duplicate, and incubated for 44 h at 30°C before the colonies were counted.
stressed by high levels of 02- due to the sodA sodB mutations (10), the promoter activity of several heat shock genes, including groE and htpG, was found to be constitutively elevated (unpublished results). However, the present study indicates that sodA sodB double mutants are no more resistant to heat shock than the wild-type cells. This would be accounted for, at least in part, by the observation that the double mutants are defective in the induction of heat shock genes upon temperature shift (unpublished data). Our result indicates that SOD does not play a role in protection against thermal stress because the sodA and sodB mutations did not sensitize cells to heat shock. However, MnSOD has been reported to be induced by heat shock only in aerobically growing cells (16). This observation suggests that heat shock generates 02 (20) or another signal that is shared by heat shock and oxidative stress responses. The unusual nucleotides AppppN and ApppN (N = A, C, G, or U) accumulate in cells stressed by heat shock or oxidative stress (13). It has been proposed that these nucleotides ("alarmones") are the alarm signaling the onset of the stress (2). However, the results of recent studies appear to rule out this possibility (reviewed in reference 6). A major part of this work was performed when T.K. visited the Institute for Virus Research in Kyoto. T.K. greatly appreciates the hospitality of the members of the laboratory of T. Yura. This work was supported by grants from the Ministry of Education, Science, and Culture of Japan to T.Y., a JSPS Fellowship from the Japan Society for the Promotion of Science to T.K., and a grant from the National Science Foundation (DMB-8613990) to T.K. REFERENCES 1. Ang, D., G. N. Chandrasekhar, M. Zylicz, and C. Georgopoulos. 1986. Escherichia coli grpE gene codes for the heat shock protein B25.3, essential for both A DNA replication at all temperatures and host growth at high temperature. J. Bacteriol. 167:25-29. 2. Bochner, B. R., P. C. Lee, S. W. Wilson, C. W. Cutler, and B. N. Ames. 1984. AppppA and related adenylylated nucleotides are
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