Oxidative stress in Escherichia coli: Identification and characterization of new proteins conferring resistance to oxidative stress
Zusammenfassung der Projektergebnisse
Reactive oxygen species (ROS) are generated by the immune response and arise continuously as by-product of aerobic respiration. Increased accumulation of ROS such as hydrogen peroxide (H2O2) and hypochlorite (HOCl) causes oxidative damage that underlies aging and several diseases. HOCl is a powerful oxidant produced by neutrophils to kill invading microorganisms. Despite this important relevance for bacteria, few HOCl-specific protection mechanisms have been identified so far. In this project, I set out to identify novel HOCl-specific protection mechanisms. I applied three strategies: a genomic library to screen for resistant clones, transcriptome analysis to identify HOClup-regulated genes, and evolution of bacteria to maximum stress resistance. The genomic library yielded many genes that confer resistance to HOCl or H2O2, and we focussed on the genes cpdA, yifE, and yjiE (hypT). CpdA confers broad stress resistance via the well-characterized general stress response. YifE confers resistance towards HOCl and H2O2, may interact with ribosomes, and may improved stalled translation under stress. Its exact function, however, could not be unravelled. Lastly, we showed that HypT is the first described HOCl-specific transcription factor. HypT protects cells from HOCl stress by decreasing intracellular iron levels and concomitantly avoiding generation of cytotoxic hydroxyl radicals. HypT is specifically activated by HOCl by oxidation of Met123, Met206, and Met230 to methionine sulfoxide, in contrast to the inactivation of most other proteins by methionine oxidation. HypT reduction and inactivation is facilitated by methionine sulfoxide reductases. Thus, HypT is the first transcription factor that is redox-regulated via methionine oxidation in contrast to the prevailing redox-regulation via cysteine oxidation. Purified HypT forms unusual hexagonal molecules consisting of 12 subunits. Dodecamers dissociate into small oligomers in the presence of DNA and constitute the DNA-binding species. Dimers and tetramers, as generated by artificial dissociation with NaCl, also represent the activation-competent species. Their exposure to stoichiometric amounts of HOCl results in full activation within one minute while HOCl treatment of dodecamers activates HypT 60-times slower. Thus, HypT functions as highly sensitive HOClsensor. Proteins that are aggregation-prone upon HOCl stress are largely overlapping with heat-labile cellular proteins. This observation triggered a new project; E. coli cells were step-wise adapted to non-permissive heat shock temperatures and evolved to grow at a maximum growth temperature of 48.5°C (wild-type E. coli grows until 45.5°C). The central factor for heat-adaptation is the chaperone GroE, whose levels are permanently up-regulated by 16-fold compared to wild-type cells. Supposedly, GroE is required to assist the folding of mutated or heat-labile proteins in evolved cells and thereby acts as mediator of evolution of extremely heat-resistant E. coli cells. Given that the factors described above confer only a low extent of HOCl resistance, maximally HOCl resistant E. coli cells were generated by step-wise adaptation to high HOCl concentrations. The extent of HOCl resistance of evolved cells is several orders of magnitude higher than the HOCl resistance conferred by any factor described so far. Evolved cells do not contain chromosomal mutations, although HOCl is known to damage DNA and cause mutations. Even though the ancestor and the evolved strains show differences in DNA cytosine methylation, they do not correlate with HOCl resistance. HOCl resistance is conferred by a permanently induced OxyR regulon that is normally activated by H2O2; OxyR is partially oxidized and, thus, appears to be a major factor in conferring strong HOCl resistance. Unexpectedly, strong HOCl resistance, partially oxidized OxyR, and constitutive up-regulation of OxyR-regulated genes were maintained in evolved strains even after prolonged cultivation without stress. This suggests that adaptation of bacteria to stressful environments not only occurs by stress-induced mutagenesis but can also manifest in a whole population via other, probably epigenetic, cellular changes.
Projektbezogene Publikationen (Auswahl)
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(2007) The redoxswitch domain of Hsp33 functions as dual stress sensor. Nat. Struct. Mol. Biol. 14, 556-563
Ilbert, M., Horst, J., Ahrens, S., Winter, J., Graf, P.C., Lilie, H. and U. Jakob
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(2008) Bleach Activates A Redox-Regulated Chaperone by Oxidative Protein Unfolding. Cell 135(4), 691-701
Winter, J., Ilbert, M., Graf, P.C., Özcelik, D. and U. Jakob
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(2008) The periplasm of E. coli – oxidative folding of recombinant proteins. In: J. Buchner and L. Moroder (Eds.) Oxidative Folding of Cysteine-rich Peptides and Proteins.
Gebendorfer, K.M. and J. Winter
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(2009) Interplay of Cellular cAMP Levels, σS Activity and Oxidative Stress Resistance in Escherichia coli. Microbiology 155(Pt 5), 1680-1689
Barth, E., Gora, K.V., Gebendorfer, K.M., Settele., F., Jakob, U., and J. Winter
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(2010) Evolution of Escherichia coli for growth at high temperatures. J. Biol. Chem. 285, 19029-19034
Rudolph, B., Gebendorfer, K.M., Buchner, J. and J. Winter
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(2011) Protein disulfide isomerase (PDI) isomerizes non-native disulfide bonds in human proinsulin independent of its peptide binding activity. Protein Sci. 20, 588-596
Winter, J., Gleiter, S., Klappa, P., and H. Lilie
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(2012) Identification of a hypochlorite-specific transcription factor from Escherichia coli. J. Biol. Chem. 287, 6892-6903
Gebendorfer, Katharina M.; Drazic, Adrian; Le, Yan; Gundlach, Jasmin; Bepperling, Alexander; Kastenmüller, Andreas; Ganzinger, Kristina A.; Braun, Nathalie; Franzmann, Titus M. & Winter, Jeannette
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(2013) Methionine oxidation activates a transcription factor in response to oxidative stress. PNAS 110, 9493-9498
Drazic, Adrian; Miura, Haruko; Peschek, Jirka; Le, Yan; Bach, Nina C.; Kriehuber, Thomas & Winter, Jeannette
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(2013) Role of cysteines in the stability and DNA-binding activity of the hypochlorite-specific transcription factor HypT. PLoS One 8, e75683
Drazic, Adrian; Tsoutsoulopoulos, Amelie; Peschek, Jirka; Gundlach, Jasmin; Krause, Maike; Bach, Nina C.; Gebendorfer, Katharina M. & Winter, Jeannette
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(2014) Tetramers are the activation-competent species of the HOCl-specific transcription factor HypT. J. Biol. Chem. 289, 977-986
Drazic, Adrian; Gebendorfer, Katharina M.; Mak, Stefanie; Steiner, Andrea; Krause, Maike; Bepperling, Alexander & Winter, Jeannette
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(2014) The physiological role of reversible methionine oxidation. Review, BBA - Proteins and Proteomics
Drazic, Adrian & Winter, Jeannette
