Oxidative Stress Defense and Deterioration of Growth-arrested Escherichia coli Cells

Abstract

Analysis of protein carbonylation demonstrates that the stasis-induced catalases and cytoplasmic superoxide dismutases (SOD) have a role in preventing accelerated protein oxidation during growth arrest of Escherichia coli cells. A larger number of proteins are carbonylated in cells lacking cytoplasmic SOD compared with cells lacking catalases, OxyR, or RpoS which, in turn, exhibit a larger number of oxidized proteins than the wild-type parent. Proteins exclusively oxidized during stasis in mutants lacking cytoplasmic SOD include GroEL, EF-G, and the acidic isoform of H-NS indicating that these mutants experience problems in peptide elongation and maintaining protein and DNA architecture. These mutants also survive stasis poorly. Likewise, but to a much lesser extent, mutations in oxyR, an oxidative stress regulator, shorten the life-span of stationary phase cells. The low plating efficiency of cells lacking OxyR is the result of their inability to grow on standard culture plates unless plating is performed anaerobically or with high concentration of catalase. In contrast, cells lacking cytoplasmic SOD appear to die prior to plating. Our data points to the importance of oxidative stress defense in stasis survival, and we also demonstrate that the lifespan of growth-arrested wild-type E. coli cells can be significantly extended by omitting oxygen.Growth arrest of Escherichia coli cells caused by starvation for an essential nutrient triggers the production of catalases and other oxidative stress proteins which render the cell highly resistant to hydrogen peroxide, a phenomenon known as starvation-induced cross-protection (1-5). One model, we could call it the future provision model, suggests that this induction of stress defense proteins including oxidative stress proteins prepares the starved cell for stress conditions that it may encounter in the future. It has been argued that such a response is sensible at the onset of starvation because energy generation will become more and more limited as cells progress into stationary phase making inducible responses less immediate and less effective (6). Another model suggests that the induction of oxidative stress proteins has a role in minimizing damage to target molecules caused by stasis per se (3,7,8). Similarly, it has been proposed that the ubiquitous progressive decline in the functional capacity of aging eukaryotes is a consequence of the accumulation of oxidative damage caused by reactive oxygen species (ROS) 1 produced by normal metabolism (9). This is the postulation of the free radical hypothesis of aging (10). The hypothesis is supported by different experimental data demonstrating that (i) steady-state levels of oxidatively damaged macromolecules increase with age in all species examined thus far (11), (ii) oxidatively modified proteins lose their catalytic activity and structural integrity (12), (iii) there is a close association between oxidative damage of proteins and life expectancy of houseflies (13), and (iv) the life-span of fruitflies can be prolonge...

Lon protease promotes survival of Escherichia coli during anaerobic glucose starvation

“…Wild-type E. coli survived better under anaerobic conditions than aerobic (Fig. 1B and 1C), as reported by Dukan and Nystrom (Dukan and Nystrom, 1999). In contrast, the lon mutant lost viability faster under anaerobic conditions (Figs.…”

“…Dukan and Nystrom (Dukan and Nystrom, 1999) demonstrated that the life-span of wildtype E. coli K-12 MG1655 cells can be extended by omitting oxygen during glucose starvation, consistent with the proposal that aerobically-generated reactive oxygen species decrease the longevity of E. coli cells. We initially attempted to replicate their results, but we used E. coli BL21(DE3) cells rather than the wild-type MG1655 because BL21(DE3) cells are widely used for protein overexpression.…”

“…Oxidatively damaged proteins increase during aerobic carbon starvation (Nystrom, 2004;Dukan and Nystrom, 1999), and cells which fail to control the burden of oxidized proteins lose viability (Desnues et al, 2003). We thus speculate that the aerobically-driven increase in oxidized proteins may be mechanistically important for induction of the adaptive response in Lon deficient cells.…”

“…A sample was taken daily from glucose-starved cultures, serially diluted into M9-G medium, and then plated on Luria-Bertani (LB) agar plates supplemented with 1% glucose (Dukan and Nystrom, 1999). Supplementation with 1% glucose supported more rapid formation of colonies under anaerobic conditions.…”

Mass spectrometry in aging research

“…Nyström and co-workers have used stationary phase bacterial cells as a model for aging somatic cells of higher eukaryotic organisms to elucidate several pathways leading to the accumulation of oxidized proteins (Nyström, 2002). Oxidized proteins were detected based on increased levels of protein-associated carbonyls following derivatization with 2,4-dinitrophenylhydrazine, 2D gel electrophoresis, and Western blotting with anti-2,4-dinitrophenyl (DNP) antibodies (Dukan & Nyström, 1999;Aguilaniu et al, 2001;Ballesteros et al, 2001). The specificity of the anti-DNP antibody has been characterized through specific reaction with DNP conjugated to proteins (Eshhar, Ofarim, & Waks, 1980;Shacter et al, 1994), and a lower limit of detection of protein-associated carbonyls was estimated to 1 pmol (Shacter et al, 1994).…”