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. 2008 Jan;67(1):2-14.
doi: 10.1111/j.1365-2958.2007.05988.x. Epub 2007 Nov 25.

Mutations in two global regulators lower individual mortality in Escherichia coli

Fanette Fontaine  1 , Eric J Stewart, Ariel B Lindner, François Taddei
Affiliations

Mutations in two global regulators lower individual mortality in Escherichia coli

Fanette Fontaine et al. Mol Microbiol. 2008 Jan.

Abstract

There has been considerable investigation into the survival of bacterial cells under stress conditions, but little is known about the causes of mortality in the absence of exogenous stress. That there is a basal frequency of cell death in such populations may reflect that it is either impossible to avoid all lethal events, or alternatively, that it is too costly. Here, through a genetic screen in the model organism Escherichia coli, we identify two mutants with lower frequencies of mortality: rssB and fliA. Intriguingly, these two genes both affect the levels of different sigma factors within the cell. The rssB mutant displays enhanced resistance to multiple external stresses, possibly indicating that the cell gains its increased vitality through elevated resistance to spontaneous, endogenous stresses. The loss of fliA does not result in elevated stress resistance; rather, its survival is apparently due to a decreased physical stress linked to the insertion of the flagellum through the membrane and energy saved through the loss of the motor proteins. The identification of these two mutants implies that reducing mortality is not impossible; rather, due to its cost, it is subject to trade-offs with other traits that contribute to the competitive success of the organism.

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Figures

Fig. 1
Fig. 1. Qualitative screen for mutants with fewer dead cells. Cultures of the mutants, WT and controls were spotted onto agar plates containing PI, and screened visually in the microscope. Images are 40× magnification grey-scale pictures taken with simultaneous phase contrast and fluorescent light. Bright cells represent dead cells stained with PI; mutants with altered numbers of dead cells are identifiable by this method.
A. WT control strain. B. Control rpoS mutant showing increased cell death. C. Decreased death mutant fliA. D. Decreased death mutant rssB.
Fig. 2
Fig. 2
Quantification of dead cells in liquid cultures by FC analysis. Measurements were performed on WT (white bars), fliA (striped bars), rssB (black bars) and fliArssB (grey bars) cells grown at 30°C, on LB (A) or in Minimal medium (B) stained with SYTOX Green. Values correspond to 5–20 independent repeats. Error bars represent the standard error. The asterisk corresponds to P-values between < 0.05 and < 0.001, relative to WT by unpaired t-test. The double asterisks correspond to P-values < 0.001, relative to WT by unpaired t-test.
Fig. 3
Fig. 3. Gene regulation pathways and quantification of dead cells.
A. Simplified schematic of the motility regulon. B. The general stress-response regulon. For both schematics, arrows indicate a positive effect, while blocked lines indicate a negative effect. C. Quantification of dead cells by FC analysis. Measurements were performed on early stationary-phase cultures grown at 30°C, stained with SYTOX Green. White bars represent cultures grown in M9; striped bars represent cultures grown in LB. The values reported are the ratio of the fraction of dead cells in the mutant strain compared with the fraction of dead cells in WT. Each bar represents mean of 5–15 repeats, each in turn consisting of three parallel cultures. The total number of cells assayed per condition is at least 3 × 106. The only strains not significantly different from WT are fliC and flgJ (fliC in LB P = 0.416; in M9 P = 0.342; flgJ in M9, P = 0.926).
Fig. 4
Fig. 4
Resistance to external stress. Strains were tested for their resistance to (A) osmotic stress (2 M NaCl, 24 h), (B) oxidative stress (17 mM H2O2, 30 min), and (C) heat shock (55°C, 30 min). Error bars represent the standard error of the mean of six experiments.
Fig. 5
Fig. 5. Competitions between mutants and WT. All competitions were performed in LB (solid symbols) and M9 (hollow symbols).
A. The fliA mutant in liquid media with shaking. B. The rssB mutant in liquid media with shaking. C. The fliA mutant in structured media; dashed lines 3 g l−1 agar, solid lines 7.5 g l−1 agar. D. The rssB mutant in structured media; dashed lines 3 g l−1 agar, solid lines 7.5 g l−1 agar. All data have been log (base 10) transformed. The error bars represent the standard error of the mean of four experiments. All competition experiments were repeated with the markers swapped between strains; no significant differences were observed (data not shown).
Fig. 6
Fig. 6. Relative gene expression levels controlled by three σ factors.
A. Images of one field of strains carrying fluorescent reporter proteins under the control of promoters responsive to σ70 (blue, left), σS (green, middle) and σ32 (red, right). Each graph corresponds to the mean fluorescence intensity for each reporter. B. Normalized expression from the σ32 reporter in the mutant strains and WT. This reporter is significantly different from WT in the rssB strain (P = 0.007), but not in the fliA strain (P = 0.344), by unpaired data t-test. C. Normalized expression from the σS reporter in the mutant strains and WT. Both strains are significantly different from WT for this reporter (rssB, P < 0.001; fliA, P = 0.007) by unpaired data t-test. Each point represents one experiment; the black bar represents the mean of each strain.
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References

    1. Blattner FR, Plunkett G, Bloch CA, Perna NT, Burland V, et al. The complete genome sequence of Escherichia coli K-12. Science. 1997;277:1453–1474. - PubMed
    1. Barer MR, Harwood CR. Bacterial viability and culturability. Adv Microb Physiol. 1999;41:93–137. - PubMed
    1. Caetano-Anolles G. Amplifying DNA with arbitrary oligonucleotide primers. PCR Methods Appl. 1993;3:85–94. - PubMed
    1. Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, Tsien RY. A monomeric red fluorescent protein. Proc Natl Acad Sci USA. 2002;99:7877–7882. - PMC - PubMed
    1. Chilcott GS, Hughes KT. Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar typhimurium and Escherichia coli. Microbiol Mol Biol Rev. 2000;64:694–708. - PMC - PubMed

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