MarA, SoxS and Rob of Escherichia coli – Global Regulators of Multidrug Resistance, Virulence and Stress Response

Bacteria have a great capacity for adjusting their metabolism in response to environmental changes by linking extracellular stimuli to the regulation of genes by transcription factors. By working in a co-operative manner, transcription factors provide a rapid response to external threats, allowing the bacteria to survive. This review will focus on transcription factors MarA, SoxS and Rob in Escherichia coli, three members of the AraC family of proteins. These homologous proteins exemplify the ability to respond to multiple threats such as oxidative stress, drugs and toxic compounds, acidic pH, and host antimicrobial peptides. MarA, SoxS and Rob recognize similar DNA sequences in the promoter region of more than 40 regulatory target genes. As their regulons overlap, a finely tuned adaptive response allows E. coli to survive in the presence of different assaults in a co-ordinated manner. These regulators are well conserved amongst Enterobacteriaceae and due to their broad involvement in bacterial adaptation in the host, have recently been explored as targets to develop new anti-virulence agents. The regulators are also being examined for their roles in novel technologies such as biofuel production

Advantages and drawbacks of nanospray for studying noncovalent protein-DNA complexes by mass spectrometry

The noncovalent complexes between the BlaI protein dimer (wild-type and GM2 mutant) and its double-stranded DNA operator were studied by nanospray mass spectrometry and tandem mass spectrometry (MS/MS). Reproducibility problems in the nanospray single-stage mass spectra are emphasized. The relative intensities depend greatly on the shape of the capillary tip and on the capillary-cone distance. This results in difficulties in assessing the relative stabilities of the complexes simply from MS' spectra of protein-DNA mixtures. Competition experiments using MS/MS are a better approach to determine relative binding affinities. A competition between histidine-tagged BlaIWT (BlaIWTHis) and the GM2 mutant revealed that the two proteins have similar affinities for the DNA operator, and that they co-dimerize to form heterocomplexes. The low sample consumption of nanospray allows MS/MS spectra to be recorded at different collision energies for different charge states with 1 muL of sample. The MS/MS experiments on the dimers reveal that the GM2 dimer is more kinetically stable in the gas phase than the wild-type dimer. The MS/MS experiments on the complexes shows that the two proteins require the same collision energy to dissociate from the complex. This indicates that the rate-limiting step in the monomer loss from the protein-DNA complex arises from the breaking of the protein-DNA interface rather than the protein-protein interface. The dissociation of the protein-DNA complex proceeds by the loss of a highly charged monomer (carrying about two-thirds of the total charge and one-third of the total mass). MS/MS experiments on a heterocomplex also show that the two proteins BlaIWTHis and BlaIGM2 have slightly different charge distributions in the fragments. This emphasizes the need for better understanding the dissociation mechanisms of biomolecular complexes

Large ring 1,3-bridged 2-azetidinones: experimental and theoretical studies

The relationship between angular strain and (re)activity of bicyclic 2-azetidinones is still an open question of major concern in the field of penicillin antibiotics. Our study deals with original 13-membered-ring 1,3-bridged 2-azetidinones related to the carbapenem family, and featuring a "planar amide" instead of the "twisted amide" typical of penam derivatives. The bicycles 11 and 12 were obtained from acetoxy-azetidinone 7, via the key-intermediate 10, by using the RCM (ring closing metathesis) strategy. Theoretical predictions and experimental results of hydrolysis showed that the large bicycle 12, endowed with high conformational flexibility, is more reactive than the bicycle 11, including a C=C bond of E configuration, and the monocyclic 2-azetidinone precursor 10. The processing of 2-azetidinones 10-12 in the active site of serine enzymes has been computed by ab initio methods, considering three models. Due to geometrical parameters of the enzymic cavity (nucleophilic attack from the alpha-face), precursor 10 was predicted more active than 11 and 12 in the acylation step by Ser-OH. Indeed, bicycles 11 and 12 are modest inhibitors of PBP2a, while 10 is a good to excellent inhibitor of PBP2a and R39 bacterial enzymes. (C) 2008 Elsevier Masson SAS. All rights reserved

Toward an Enhanced Mutual Awareness in Asymmetric CVE

International audience—Collaborative Virtual Environments (CVEs) aim at providing several users with a consistent shared virtual world. In this work, we focus on the lack of mutual awareness that may appear in many situations and we evaluate different ways to present the distant user and his actions in the Virtual Environment (VE) in order to understand his perception and cognitive process. Indeed, an efficient collaboration involves not only the good perception of some objects but their meaning too. This second criterion introduces the concept of distant analysis that could be a great help in improving the understanding of distant activities. For this work, we focus on a common case consisting in estimating accurately the time at which a distant user analyzed the meaning of a remotely pointed object. Thus, we conduct some experiments to evaluate the concept and compare different techniques for implementing this new awareness feature in a CVE. Amongst others, results show that expertise of the users influences on how they estimate the distant activity and the type of applied strategies

Conformational and thermodynamic changes of the repressor/DNA operator complex upon monomerization shed new light on regulation mechanisms of bacterial resistance against β-lactam antibiotics

In absence of β-lactam antibiotics, BlaI and MecI homodimeric repressors negatively control the expression of genes involved in β-lactam resistance in Bacillus licheniformis and in Staphylococcus aureus. Subsequently to β-lactam presence, BlaI/MecI is inactivated by a single-point proteolysis that separates its N-terminal DNA-binding domain to its C-terminal domain responsible for its dimerization. Concomitantly to this proteolysis, the truncated repressor acquires a low affinity for its DNA target that explains the expression of the structural gene for resistance. To understand the loss of the high DNA affinity of the truncated repressor, we have determined the different dissociation constants of the system and solved the solution structure of the B. licheniformis monomeric repressor complexed to the semi-operating sequence OP1 of blaP (1/2OP1blaP) by using a de novo docking approach based on inter-molecular nuclear Overhauser effects and chemical-shift differences measured on each macromolecular partner. Although the N-terminal domain of the repressor is not subject to internal structural rearrangements upon DNA binding, the molecules adopt a tertiary conformation different from the crystallographic operator–repressor dimer complex, leading to a 30° rotation of the monomer with respect to a central axis extended across the DNA

From 3D Bimanual Toward Distant Collaborative Interaction Techniques: An Awareness Issue

International audienceThis paper aims to raise the question : "How much 3D bimanual interaction techniques can be useful to the design of collaborative interaction techniques in the field of Collaborative Virtual Environment (CVE)?". Indeed, CVE involve the use of complex interaction techniques based on specific collaborative metaphors. The design of these metaphors may be a difficult task because it has to deal with collaborative issues that came from sparse research areas (Human-Computer Interfaces, Human-Human Interactions, Networking, Physiology and Social Psychology). Metaphors for bimanual interactions have been developed for a while essentially because it is a widely spread area of interest for common tasks. Bimanual interactions involve the simultaneous use of both hands of the user in order to achieve a goal with better performances compared to uni-manual interactions thanks to a natural skill that is proprioception. This collaborative aspect could certainly be a helpful entry point in the design of efficient collaborative interaction techniques extended from improved bimanual metaphors. However, the proprioceptive sense cannot be considered in the same way, and additional features must be proposed to be able to collaborate efficiently. Thus, awareness is a key to let CVE be usable and the availability of collaborative feedbacks is essential to extend bimanual interactions toward collaborative ones. In this paper, we based our study on existing work on bimanual and collaborative interaction techniques trying to draw similarities between them. We emphasize common points between both fields that could be useful to better design both metaphors and awareness in CVE

The Stretchable Arms for Collaborative Remote Guiding

International audienceFigure 1: The stretchable guiding arms, controlled by the remote expert's location, from the agent's viewpoint. On the left, the arms are extended and red because the guide is ahead of the agent. On the right, the agent has reached the guide's location and the guiding arms retrieve their initial length and green color. Abstract The help of a remote expert to guide an agent in performing a physical task can be advantageous in many ways: saving time and money by avoiding travel, and thus increasing the rate of intervention. In many situations, the remote expert wishes to guide the agent by first placing him in the correct location to achieve the task. However, as the agent is not a robot, the expert can not use a location controller to place the agent. Instead, interaction techniques must enable the expert to achieve this task before physical manipulation guidance. In this paper, we propose a novel interaction technique for remote guiding based on arm gestures. First, the remote expert (using a VR setup) virtually collocates himself with the agent (using an AR setup), then controls virtual arms collocated with both users' shoulders. Second, if the expert starts to move forward to grasp a virtual object, the virtual arms start to stretch in order to keep the shoulders' collocation on the agent's side. This metaphor allows the agent to understand the direction of the expert's motion easily while preserving the naturalness of the interaction and avoiding the use of a frustum to represent the expert's head location. Categories and Subject Descriptors (according to ACM CCS): Information Interfaces and Presentation (e.g. HCI) [H.5.2]: User Interfaces—Computer Graphics [I.3.6]: Methodology and Techniques—

Specificity of the metabolic signatures of fish from cyanobacteria rich lakes

International audienceThe liver metabolomes of fish from cyanobacterial-dominated ponds were investigated. Cyanobacterial metabolites were only be detected in cyanobacterial dominated ponds. The metabolomes of the 2 fish species exhibit similar correlation with cyanobacteria occurrence. Correlations between the levels of some metabolites and phycocyanin or pH were observed. a b s t r a c t With the increasing impact of the global warming, occurrences of cyanobacterial blooms in aquatic ecosystems are becoming a main worldwide ecological concern. Due to their capacity to produce potential toxic metabolites, interactions between the cyanobacteria, their cyanotoxins and the surrounding freshwater organisms have been investigated during the last past years. Non-targeted metabolomic analyses have the powerful capacity to study simultaneously a high number of metabolites and thus to investigate in depth the molecular signatures between various organisms encountering different environmental scenario, and potentially facing cyanobacterial blooms. In this way, the liver metabolomes of two fish species (Perca fluviatilis and Lepomis gibbosus) colonizing various peri-urban lakes of theÎle-de-France region displaying high biomass of cyanobacteria, or not, were investigated. The fish metabolome hydrophilic fraction was analyzed by 1 H NMR analysis coupled with Batman peak treatment for the quantification and the annotation attempt of the metabolites. The results suggest that similar metabolome profiles occur in both fish species, for individuals collected from cyanobacterial blooming lakes compared to organism from non-cyanobacterial dominant environments. Overall, such environmental metabolomic pilot study provides new research perspectives in ecology and ecotoxicology fields, and may notably provide new information concerning the cyanobacteria/fish eco-toxicological interactions

Backbone 1H, 13C, and 15N resonance assignments for lysozyme from bacteriophage lambda

Lysozyme from lambda bacteriophage (λ lysozyme) is an 18 kDa globular protein displaying some of the structural features common to all lysozymes; in particular, λ lysozyme consists of two structural domains connected by a helix, and has its catalytic residues located at the interface between these two domains. An interesting feature of λ lysozyme, when compared to the well-characterised hen egg-white lysozyme, is its lack of disulfide bridges; this makes λ lysozyme an interesting system for studies of protein folding. A comparison of the folding properties of λ lysozyme and hen lysozyme will provide important insights into the role that disulfide bonds play in the refolding pathway of the latter protein. Here we report the 1H, 13C and 15N backbone resonance assignments for λ lysozyme by heteronuclear multidimensional NMR spectroscopy. These assignments provide the starting point for detailed investigation of the refolding pathway using pulse-labelling hydrogen/deuterium exchange experiments monitored by NMR

A Tale of Two Oxidation States: Bacterial Colonization of Arsenic-Rich Environments

Microbial biotransformations have a major impact on contamination by toxic elements, which threatens public health in developing and industrial countries. Finding a means of preserving natural environments—including ground and surface waters—from arsenic constitutes a major challenge facing modern society. Although this metalloid is ubiquitous on Earth, thus far no bacterium thriving in arsenic-contaminated environments has been fully characterized. In-depth exploration of the genome of the β-proteobacterium Herminiimonas arsenicoxydans with regard to physiology, genetics, and proteomics, revealed that it possesses heretofore unsuspected mechanisms for coping with arsenic. Aside from multiple biochemical processes such as arsenic oxidation, reduction, and efflux, H. arsenicoxydans also exhibits positive chemotaxis and motility towards arsenic and metalloid scavenging by exopolysaccharides. These observations demonstrate the existence of a novel strategy to efficiently colonize arsenic-rich environments, which extends beyond oxidoreduction reactions. Such a microbial mechanism of detoxification, which is possibly exploitable for bioremediation applications of contaminated sites, may have played a crucial role in the occupation of ancient ecological niches on earth