Sam Dukan

Institute of Microbiology of the Mediterranean · Laboratoire de Chimie Bactérienne

Almost all living organisms use protein chaperones with a view to preventing proteins from misfolding or aggregation either spontaneously or during cellular stress. This work uses a reaction-diffusion stochastic model to describe the dynamic localization of the Hsp70 chaperone DnaK in E. coli cells during transient proteotoxic collapse characterize...

We recently developed a mathematical model for predicting reactive oxygen species (ROS) concentration and macromolecules oxidation in vivo. We constructed such a model using Escherichia coli as a model organism and a set of ordinary differential equations. In order to evaluate the major defences relative roles against hydrogen peroxide (H2 O2), we...

Superoxide kinetic and evolution. (PDF)

Imlay and Linn show that exposure of logarithmically growing Escherichia coli to hydrogen peroxide (H2O2) leads to two kinetically distinguishable modes of cell killing. Mode one killing is pronounced near 1 mM concentration of H2O2 and is caused by DNA damage, whereas mode-two killing requires higher concentration (>10 mM). The second mode seems t...

Kinetic constants.

Homogeneous model versus compartmentalization.

Currently, identification of pathogenic bacteria present at very low concentration requires a preliminary culture-based enrichment step. Many research efforts focus on the possibility to shorten this pre-enrichment step which is needed to reach the minimal number of cells that allows efficient identification. Rapid microbiological controls are a re...

Legionella pneumophila is a pathogenic bacterium involved in regular outbreaks characterized by a relatively high fatality rate and an important societal impact. Frequent monitoring of the presence of this bacterium in environmental water samples is necessary to prevent these epidemic events, but the traditional culture-based detection and identifi...

Legionella pneumophila is a waterborne pathogen responsible for Legionnaires' disease, an infection which can lead to potentially fatal pneumonia. After disinfection, L. pneumophila has been detected, like many other bacteria, in a "viable but non culturable" state (VBNC). The physiological significance of the VBNC state is unclear and controversia...

Discrimination among viable/active or dead/inactive cells in a microbial community is a vital question to address issues on ecological microbiology or microbiological quality control. It is commonly assumed that metabolically active cells (ChemchromeV6 [CV6] procedure) correspond to viable cells (direct viable count procedure [DVC]), although this...

Clicking bugs: Metabolically active Gram-negative bacteria can specifically incorporate an azide-modified analogue of 3-deoxy-D-manno-octulosonic acid (1; see scheme) into the lipopolysaccharides on their membranes. This process adds a bioorthogonal chemical reporter onto the cell surface and allows the rapid fluorescent labeling of viable cells by...

Adaptation to a solar saltern environment requires mechanisms providing tolerance not only to salinity but also to UV radiation (UVR) and to reactive oxygen species (ROS). We cultivated prokaryote halophiles from two different salinity ponds: the concentrator M1 pond (240 g·L(-1) NaCl) and the crystallizer TS pond (380 g·L(-1) NaCl). We then estima...

The toxicity of carbon dioxide has been established for close to a century. A number of animal experiments have explored both acute and long-term toxicity with respect to the lungs, the cardiovascular system, and the bladder, showing inflammatory and possible carcinogenic effects. Carbon dioxide also induces multiple fetal malformations and probabl...

The fact that Mycobacterium tuberculosis mobilizes lipid bodies (LB) located in the cytosol during infection process has been proposed for decades. However, the mechanisms and dynamics of mobilization of these lipid droplets within mycobacteria are still not completely characterized. Evidence in favour of this characterization was obtained here usi...

The ability of Staphylococcus aureus to adapt to various conditions of stress is the result of a complex regulatory response. Among them, ClpC, belonging to the Hsp100/Clp ATPase family, seems to play an important role. For instance, we previously demonstrated that a functional clpC deletion resulted in enhanced survival in the late stationary phas...

Here, we combined flow cytometry (FCM) and phylogenetic analyses after cell sorting to characterize the dominant groups of the prokaryotic assemblages inhabiting two ponds of increasing salinity: a crystallizer pond (TS) with a salinity of 390 g/L, and the non-crystallizer pond (M1) with a salinity of 200 g/L retrieved from the solar saltern of Sfa...

Reactive oxygen species (ROS) are harmful because they can oxidize biological macromolecules. We show here that atmospheric CO(2) (concentration range studied: 40-1,000 p.p.m.) increases death rates due to H(2)O(2) stress in Escherichia coli in a dose-specific manner. This effect is correlated with an increase in H(2)O(2)-induced mutagenesis and, a...

An extremely halophilic archaeon, strain ETD6, was isolated from a marine solar saltern in Sfax, Tunisia. Analysis of the 16S rRNA gene sequence showed that the isolate was phylogenetically related to species of the genus Halorubrum among the family Halobacteriaceae, with a close relationship to Hrr. xinjiangense (99.77% of identity). However, valu...

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...

MS spectra from MALDI TOF analysis of BSA at different MCO levels. Arrows point out the appearance of BSA fragmentation and the BSA dimer disappearance as the MCO level increases. (0.11 MB DOC)

Carbonyl content 2D electrophoresis analysis of unsoluble cell fractions from exponentially grown E. coli. (A) Specific pattern of carbonylation in unsoluble cell fractions from exponentially grown E. coli, as determined by two-dimensional western blot immunoassays, carried out as previously described [1]. (B) PVDF membrane after 2D western blot st...

Parameters for the identification of RKPT-enriched regions The primary sequence of BSA contains an average of one carbonylatable site within a sequence of 4 amino acids (23.5% of carbonylatable sites); thus, we tested how to define an RKPT-enriched region by analysing the specificity, the sensitivity and the positive predictive value of four enrich...

Site-specific carbonylation in E. coli proteins. Amino acid sequences of E. coli CP were obtained from the NCBI database. CS, based on mass spectrometry analysis, are highlighted in yellow. The regions of the sequence identified by mass spectrometry are shown in red. RPKT-enriched regions (3 carbonylatable sites within a sequence of 4 amino acids)...

Analysis of the amino acid environment around predicted HSC. Occurence of amino acid subgroups in regions flanking a predicted HSC. Amino acid subgroups are (D, E, H, Y, C) (violet), (A, V, G, I) (cyan) (P, T) (pink), and (G) (yellow). The dotted line shows the average occurence of the corresponding amino acid subgroups in the E. coli proteome. (0....

MS/MS spectra from peptide R459 to L483. MS/MS spectrum of the five peptides, containing 0 to 7 oxidations, with overlapping RKPT-enriched regions (T462 to T472). (A) MS/MS spectrum for peptide P464 to R482 and for peptide P464 until R482 (B) confirming that P464 and P470 were oxidised and DNP-labelled with a mass difference of +196 Da. (C) MS/MS s...

MS data for MALDI-TOF global mass analysis of BSA (*) M/Z of BSA monomer were obtained with standard deviations of 1000 ppm (▒) As the ionisation energy was the same for each sample, the half height width of the BSA monomer accounted for the BSA molecule heterogeneity. However, due to the quantitative decrease of BSA monomer (for instance at MCO le...

Set of E. coli proteins specifically carbonylated or uncarbonylated. The table shows three sets of proteins used to test the efficiency of the CSPD model. CP from exponentially grown cells were provided from this study. CP from the stationary phase were obtained from several studies already carried out on E. coli [1], [2]. Non-CP were provided from...

Data analysis of CS found in the proteins analysed by nano-LC ESI MS/MS. List of all carbonylated peptides containing at least one CS as judged by mass spectrometry analysis. CS and oxidised methionines are shaded in gray. Whenever possible, assessment of the structural environment and of the solvent exposure of isolated CS was done by visual inspe...

Most time lapse microscopy experiments studying bacterial processes ie growth, progression through the cell cycle and motility have been performed on thin nutrient agar pads. An important limitation of this approach is that dynamic perturbations of the experimental conditions cannot be easily performed. In eukaryotic cell biology, fluidic approache...

Transient SodA:mcherry expression among cell division. Left: Sequence of images showing an overlay of phase contrast and YFP fluorescence of a colony from a cell that carry a foci of the protein aggregation marker ibpA-YFP; right: Sequence of images showing an overlay of mcherry fluorescence and cell contours as retrieved by the annotation software...

Overall principle of the setup. A) The hybrid flow chamber. B) Diagram of the automated flow networks. The 12 separate valves are connected to a bubble detector (BS) that isolates the flow chamber from potential bubbles in the flow network by triggering two additional valves. Aspiration of the different solutions is accomplished by a syringe pump (...

Tracking of reversion events of a moving cell. Up: Sequence of images showing phase contrast of a single moving cell that carry two reversion during 21 min; down: cell contours with cell number (green), cell's track (red) and reversion (blue square) as retrieved by the annotation software. Each frame was taking every 30 sec. (1.89 MB MOV)

The existence of Salmonella enterica serovar Typhimurium viable-but-nonculturable (VBNC) cells is a public health concern since they could constitute unrecognized sources of infection if they retain their pathogenicity. To date, many studies have addressed the ability of S. Typhimurium VBNC cells to remain infectious, but their conclusions are conf...

Knowledge of the impacts of clay minerals on microorganisms is essential to a complete understanding of microbially-mediated processes. Information available in this regard remains scarce. Using Escherichia coli (E. coli) as a model bacterium, we investigated the effect of kaolinite on various growth parameters. This clay mineral significantly affe...

Carbonylation is currently used as a marker for irreversible protein oxidative damage. Several studies indicate that carbonylated proteins are more prone to degradation than their nonoxidized counterparts. In this study, we observed that in Escherichia coli, more than 95% of the total carbonyl content consisted of insoluble protein and most were cy...

In a previous study, we demonstrated the presence of protein aggregates in an exponentially grown Escherichia coli culture. In light of these observations, protein aggregates could be considered damage to cells that is able to pass from one generation to the next. Based on the assumption that the amount of aggregate protein could represent an aging...

Protein aggregation is a phenomenon observed in all organisms and has often been linked with cell disorders. In addition, several groups have reported a virtual absence of protein aggregates in healthy cells. In contrast to previous studies and the expected outcome, we observed aggregated proteins in aerobic exponentially growing and “healthy” Esch...

Here, we show that resistance of Escherichia coli to TiO2 photocatalysis involves defenses against reactive oxygen species. Results support the idea that TiO2 photocatalysis generates damage which later becomes deleterious during recovery. We found this to be partly due to DNA attack via hydroxyl radicals generated by the Fenton reaction during rec...

We have investigated the first events that occur when exponentially grown cells are transferred from a liquid medium (Luria-Bertani [LB]) to a solid medium (LB agar [LBA]). We observed an initial lag phase of 180 min for the wild type MG1655 without any apparent growth. This lack of growth was independent of the bacterial physiological state (eithe...

The bactericidal effect of photocatalysis with TiO2 is well recognized, although its mode of action is still poorly characterized. It may involve oxidation, as illuminated TiO2 generates reactive oxygen species. Here we analyze the bactericidal effect of illuminated TiO2 in NaCl-KCl or sodium phosphate solutions. We found that adsorption of bacteri...

The response of Staphylococcus aureus to hypochlorous acid (HOCl) exposure was investigated. HOCl challenges were performed on cultures interrupted in the exponential phase. Pretreatment with HOCl conferred resistance to hydrogen peroxide in a PerR-dependent manner. Derepression of the PerR regulon was observed at low HOCl concentration (survival >...

The Escherichia coli rpsD12 allele, which reduces translational fidelity and elevates expression of heat shock protein (Hsp) genes, only enhanced Hsp gene expression in the presence of oxygen. Similarly, the rpsL141 allele, which reduces mistranslation and Hsp gene expression, failed to affect the Hsp regulon in cells grown anaerobically. Increased...

Protein carbonylation is an irreversible oxidative modification that increases during organism aging and bacterial growth arrest. We analyzed whether the heat shock regulon has a role in defending Escherichia coli cells against this deleterious modification upon entry into stationary phase. Providing the cell with ectopically elevated levels of the...

In previous experiments we were able to separate, using a nondestructive separation technique, culturable and nonculturable bacteria, from a Luria-Bertani (LB) medium culture of Escherichia coli incubated for 48 h. We observed in the nonculturable bacterial population an increase in oxidative damage and up-induction of most defenses against reactiv...

To investigate the fate of Aeromonas hydrophila pathogenicity when cells switch, in nutrient-poor filtered sterilized seawater, between the culturable and nonculturable state. Aeromonas hydrophila ATCC 7966, rendered non culturable within 50-55 days of exposure to marine stress conditions, was tested for its ability to maintain haemolysin and to ad...

The behaviour of Aeromonas hydrophila in nutrient-poor filter-sterilized seawater was investigated at 23 and 5 degrees C with respect to its growth phase. At both temperatures, the culturable A. hydrophila population declined below the detection level (0.1 c.f.u. ml(-1)) after 3-5 weeks, depending on the initial physiological state of the cells. Du...

Seasonal dynamics of motile Aeromonas in a treated urban effluent and in natural seawater along the Sfax coast (Mediterranean sea, Tunisia) were measured over a year concurrently with seven environmental factors, and compared with those of faecal coliforms. Counts for Aeromonas from a standard plate count method, ranged from 1.48×10⁵ CFU.100 ml⁻¹ t...

Potentially pathogenic bacteria, such as Escherichia coli and Vibrio cholerae, become non-culturable during stasis. The analysis of such cells has been hampered by difficulties in studying bacterial population heterogeneity. Using in situ detection of protein oxidation in single E. coli cells, and using a density-gradient centrifugation technique t...

Mesophilic, hydrogenotrophic, sulfate-reducing bacteria were isolated from a deep-sea hydrothermal chimney sample collected at 13 degrees N on the East-Pacific Rise at a depth of 2,600 m. Two strains (BL5 and H9) were found to be phylogenetically similar to Desulfovibrio profundus (similarity >99%), whereas two other strains (H1 and AM13T) were fou...

In this study, we show a correlation between synthesis of aberrant proteins and their oxidative modification. The level of aberrant proteins was elevated in Escherichia coli cultures by decreasing transcriptional or translational fidelity using specific mutations or drugs. Protein carbonylation, an oxidative modification, increased in parallel to t...

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...

Hypochlorous acid (HOCl) is probably the most widely used disinfectant worldwide and has an important role in inflammatory reaction and in human resistance to infection. However, the nature and mechanisms of its bactericidal activity are still poorly understood. Bacteria challenged aerobically with HOCl concentrations ranging from 9.5 to 76 microM...

Aging, or senescence, is the progressive deterioration of every bodily function over time. A fundamental question that applies to all life forms, including growth-arrested bacteria, is why growing older by necessity causes organisms to grow more fragile. In this work, we demonstrate that the levels of oxidized proteins is correlated to the age of a...

Because on-site experimentation raises numerous problems, the study and the modelling of bacterial regrowth phenomena in drinking water distribution systems has been performed using a pipe loop pilot under various operating conditions. As a result, experiments have shown that inlet bacterial counts have little influence on the biofilm behavior whic...

A protocol including 2D SDS-PAGE, electroblotting proteins onto nitrocellulose membranes, and CNBr cleavage, followed by MALDI-MS analysis of intact proteins and peptide fragments and a database search, has been optimized and applied to the rapid identification of the Escherichia coli response to hypochlorous acid. The methodology has proved to be...

To control bacterial regrowth in distribution systems, chlorine is often added at the outlet of the treatment plant. The solution to meet this objective depends on the quality of the water leaving the treatment plant and also network characteristics. Thus the management of bacteriological water quality, which means to define the better conditions t...

Of the many causes of drinking water quality deterioration in distribution systems, biological phenomena are undoubtedly the subject of the most study. They are also the most closely monitored because of short-term public health risks. A determinist model was developed to predict bacterial growth in the network and to locate the zones where the ris...

An Escherichia coli population harvested in exponential phase at about 10(8) cells/ml was treated in phosphate buffer with HOCl at concentrations ranging from 0.4 to 1 mg/liter (7.7 to 19 microM). The HOCl stress resulted in the appearance of three cell subpopulations: a majority of dead (nonrespiring) cells, a few culturable cells (10(2) to 10(4))...

In view of the complexity of the different phenomena involved, mathematical modelling is necessary in order to take all parameters into account. A determinist type modelling was developed to predict bacterial variations during distribution. The Piccobio model takes into account: the fate of available nutrients consumed for the growth of suspended a...

A series of plasmids, containing fusions of different stress promoters to lux reporter genes, was used in an attempt to monitor the defense circuits activated upon exposure of Escherichia coli to sublethal doses of free chlorine. A significant level of activation was exhibited by promoters of three heat shock genes (grpE, dnaK, and lon), in an rpoH...

We have investigated the mechanisms of killing of Escherichia coli by HOCl by identifying protective functions. HOCl challenges were performed on cultures arrested in stationary phase and in exponential phase. Resistance to HOCl in both cases was largely mediated by genes involved in resistance to hydrogen peroxide (H2O2). In stationary phase, a mu...

Numerous biological and physicochemical reactions take place in drinking water distribution systems, and give rise to phenomena whereby the organoleptic or bacteriological characteristics of the distributed water are modified. Drinking water may contain residual biodegradable dissolved organic compounds which provide a primary source for the format...

La prolifération bactérienne en réseaux de distribution d'eau potable est un souci majeur des distributeurs d'eau. La complexité des phénomènes impliqués dans la croissance bactérienne en réseaux nécessite une modélisation mathématique pour définir l'impact des différents paramètres de la qualité de l'eau et généraliser ces résultats à l'échelle du...