Bacterial viability and oxydative stress
1 - Oxidative stress
In previous studies, Sam Dukan & Thomas Nyström have shown that carbonylated proteins (carbonylation is currently used as a marker for irreversible protein oxidative damage), which increases during stationary phase (Dukan and Nystrom, 1998 ; Dukan and Nystrom, 1999), are preferentially detected in VBNC cells (Desnues et al., 2003). Raisons why these carbonylated proteins were preferentially detected in VBNC cells and why only some proteins were specifically carbonylated have been our major focus during these past years.
Protein carbonyl levels in single cells of a growing (A, C) and starving (B, D) population of E. coli. Transmission images of cells (A, B) and fluorescence emission from flour-labeled antibodies (C, D) are shown. The red lines in B point to the cells that exhibit an increased carbonyl signal as shown in D. The inset in C and D shows total carbonyl levels (slot blot analysis) in protein extracts from growing (C) and starving (D) populations. Carbonyl intensity analysis of about 200 growing (green line) and 300 starving (purple line) cells is depicted in E. Cell integrity of growing (F) and starving (G) cells were analyzed using the fluorescent LIVE/DEAD BacLight bacterial viability kit (Molecular Probes Inc.).
We observed that in E. coli, more than 95% of the total carbonyl content consisted of insoluble protein and most were cytosolic proteins. We thereby demonstrate that, in vivo, carbonylated proteins are detectable mainly in an aggregate state. Finally, we show that detectable carbonylated proteins are not degraded in vivo like it was previously proposed by Dukan et al., (2000). Here we propose that some carbonylated proteins escape degradation in vivo by forming carbonylated protein aggregates and thus becoming nondegradable. In light of these findings, we provide evidence that the accumulation of nondegradable carbonylated protein presented in an aggregate state contributes to the increases in carbonyl content observed in VBNC cells (Maisonneuve et al., 2008c).
Diagram shows the separation of the pellets and supernatant
Specific protein carbonylation of SN30, LP, and SP from an exponentially grown E. coli culture. Extracts from a French press CE of an exponentially grown culture of E. coli (OD600= 0.5) obtained after various centrifugation times were processed for resolution on 2D polyacrylamide. Autoradiograms were obtained after carbonyl immunoassay of proteins. Molecular masses (M) in kDa are indicated on the left.
Carbonyl derivatives are mainly formed by direct metal-catalysed oxidation (MCO) attacks on the amino-acid side chains of proline, arginine, lysine and threonine residues. For reasons unknown, only some proteins are prone to carbonylation. We used mass spectrometry analysis to identify carbonylated sites in : BSA that had undergone in vitro MCO, and 23 carbonylated proteins in E. coli. The presence of a carbonylated site rendered the neighbouring carbonylatable site more prone to carbonylation. Most carbonylated sites were present within hot spots of carbonylation. These observations led us to suggest rules for identifying sites more prone to carbonylation. We used these rules to design an in silico model (available at http://www.lcb.cnrs-mrs.fr/CSPD/), allowing an effective and accurate prediction of sites and of proteins more prone to carbonylation in the E. coli proteome. We observed that proteins evolve to either selectively maintain or lose predicted hot spots of carbonylation depending on their biological function. As our predictive model also allows efficient detection of carbonylated proteins in Bacillus subtilis, we believe that our model may be extended to direct MCO attacks in all organisms (Maisonneuve et al., 2009).
The details of the principle of detection are described in Materials and Methods section (Maisonneuve et al., 2009). A predicted HSC (grey box) is defined by two regions. A) An RKPT-enriched region (3 carbonylatable residues within a sequence of 4 amino acids (R, K, P, T ; 3 ; 4) containing at least one proline (P ; 1 ; 0). B) A specific environment around an RKPT-enriched region, enriched in various residues : (i) iron binding sites (D, E, Y, H, C, namely 1 residue within a window of 2 residues (D, E, Y, H, C ; 1 ; 2) and 8 residues within a window of 29 residues (D, E, Y, H, C ; 8 ; 29) ; (ii) hydrophobic amino acids (A, V, G, I, namely 1 residue within a window of 2 (A, V, G, I ; 1 ; 2)) ; (G namely 2 residues within a window of 14 (G ; 2 ; 14)) ; and (iii) (P, T) with 2 residues occurring within a window of 21 residues (P, T ; 2 ; 21). E, environment ; r, right ; l, left and w, window.
Protein quality control and DnaK localization
CO2 modulate oxidative stress
During these last four years we have developed a new axis of research dealing with CO2 impact on oxidative stress. This new axis was based on a recent paper from I. Fridovich lab’s indicating a link between Reactive oxygen species (ROS) and CO2 in vitro (Liochev and Fridovich, 2004). For this purpose we have designed and developed (in relation with Jacomex Company) a chamber allowing us to control the atmospheric CO2 level within it. Benjamin Ezraty (CR2) is in charge of this project since 2007 (in association with Maïalène Chabalier (tech.)) and he is responsible for major advances of this topic. ROS, which encompass the superoxide anion (O2•-), hydrogen peroxide (H2O2) and the hydroxyl radical (HO•), are harmful as they can oxidize all biological macromolecules (Finkel and Holbrook, 2000). in vitro metal-catalyzed reactions between CO2 and ROS can generate the harmful carbonate radical (CO3•-) (Liochev and Fridovich, 2004). In vivo, CO2 is both a major by-product of metabolism and the major pH buffer system in higher eukaryotes. CO2 is also required for the growth of many microorganisms (Walker, 1932). We thus tested whether atmospheric CO2 (current value 389 ppm) could exacerbate oxidative stress. We used E. coli as model organism to evaluate whether atmospheric CO2 influenced oxidative stress. We established that the minimal atmospheric CO2 concentration required for optimal growth was 40 ppm. We show that atmospheric CO2 (range studied : 40 to 1,000 ppm) increases the death of Escherichia coli caused by peroxide stress in a dose-specific fashion. This effect correlates with increases in H2O2-induced mutagenesis rates and DNA bases oxidation as measured by the amount of 8-oxo-guanine in the cell. Moreover, survival of mutants sensitive to aerobic growth (Hpx- dps and recA fur) (Park and Imlay, 2005 ; Touati et al., 1995), presumably due to their inability to tolerate oxygen-derived ROS, appear to be CO2 level-dependent (range studied : 40 to 1,000 ppm). The aerobic viability defect of these strains has been attributed to DNA damage caused by a Fenton reaction-based hydroxyl radical (HO•) production (Finkel and Holbrook, 2000 ; Park and Imlay, 2005 ; Touati et al., 1995). The higher oxygen toxicity at higher CO2 concentrations thus indicates that CO2 exacerbates HO•–induced DNA damage. Altogether these results indicate that atmospheric CO2 exacerbates ROS toxicity by increasing oxidative cellular lesions. This study provides the first evidence that oxidative stress is exacerbated by atmospheric CO2. CO2 have been a major point of focus, due to their contribution to the greenhouse effect (Cox et al., 2000), and increases in these levels are thought to be associated with global warming. In the light of the Special Report on Emissions Scenario, predicting that the atmosphere in 2100 will contain 1,000 ppm CO2 (Schneider, 2009) our results suggest that increases in atmospheric CO2 concentration have toxic effects over and above those of global warming (Ezraty et al., submitted).
Archae adaptative response
Since several years our team is in collaboration with Sami Maalej group in Sfax University (Tunisia). In this sense, in 2007, a new thesis sudent is partially in our Lab and work specifically on trying to identify new defences against oxidative stress from Tunisian solar saltern ecosystem. The first step was to better characterize this extreme environment. The solar saltern of Sfax, central-eastern coast of Tunisia, is an artificial system consisting of interconnecting shallow ponds (20 – 70 cm depth) which extend over 1500 hectares. Adaptation to this extreme environment requires coping mechanisms providing tolerance to variable salinities from near zero to saturated and unshaded exposure to the solar UV radiation. The purpose of this study was to determine the culturable bacteria diversity in two different salt concentration ponds : the crystallizer pond (TS18 with 380 g/l NaCl) and in a concentrator pond M1 (with 240 g/l NaCl). Characterization of isolated bacteria was performed via both phenotypic and phylogenetic approaches. We observed a higher biodiversity in term of culturable bacteria in M1 compared to TS18. Moreover, whereas all TS18 isolated strains appear to be extreme halophilic archaea (genus Halorubrum), culturable strains isolated from M1 could be ranged from extreme halophilic archaea (genus Halloterigena or Halorubrum) to moderate halophilic bacteria (Pseudomendales group). In addition, UVB and H2O2 resistances of two characterized haloarchaeal and two eubacterial strains were evaluated at varied salt concentrations. We observed that archaea strains were much more resistant than eubacterial strains. Moreover, all tested strains appear to reach their optimal resistance (UVB or H2O2) for the NaCl concentration leading to the optimum growth. Altogether, our results indicated that all culturable bacteria isolated from TS18 were well adapted to this extreme environment, whereas it s was partially the case for culturable bacteria from M1.