Adaptation proceeds through the selection of mutations. The distribution of mutant fitness effect and the forces shaping this distribution are therefore keys to predict the evolutionary fate of organisms and their constituents such as enzymes. Here, by producing and sequencing a comprehensive collection of 10,000 mutants, we explore the mutational landscape of one enzyme involved in the spread of antibiotic resistance, the beta-lactamase TEM-1. We measured mutation impact on the enzyme activity through the estimation of amoxicillin Minimum Inhibitory Concentration (MIC) on a subset of 990 mutants carrying a unique missense mutation, representing 64% of possible amino acid changes in that protein reachable by point mutation. We established that mutation type, solvent accessibility of residues and the predicted effect of mutations on protein stability primarily determined alone or in combination changes in MIC of mutants. Moreover, we were able to capture the drastic modification of the mutational landscape induced by a single…

Dozens of genes are known to control the pathways of crossover formation between homologs during meiosis. In most species, crossovers interfere, a phenomenon first discovered in the Morgan lab nearly a century ago. Nevertheless the mechanisms producing crossover interference remain unknown. I will show how quantitative modeling has been used, what has been learned, and the road still ahead.

Microbes engage in a remarkable array of cooperative behaviors, secreting shared proteins that are essential for foraging, shelter, microbial warfare and virulence. These proteins are costly, rendering populations of cooperators vulnerable to exploitation by non-producing cheaters arising by gene loss or migration. In such conditions, how can cooperation persist? Our model predicts that more mobile loci generate higher relatedness, and therefore allow the maintenance of more cooperative traits via kin selection. By analyzing bacterial genomes we observe a massive amount of horizontal transfer and show that this is the predominant mechanism of innovation in bacterial genomes. Additionally, this shows that we are far from knowing the diversity of functions encoded in the species. We confirm that genes coding for secreted proteins - the secretome – are very frequently lost and gained and are associated with mobile elements, supporting our major prediction that gene mobility drives bacterial cooperation. We demonstrate that mobile elements…

To sample their environment and coordinate appropriate adaptation responses bacteria require a flurry of signal transduction systems. Among the most commonly utilized are the so-called two-component systems. These systems comprise (1) a sensor protein, the sensor histidine kinase which modulates an intrinsic autophosporylation activity in response to changing cellular and environmental clues and (2) a response regulator protein that interacts with DNA, RNA or protein targets to coordinate an appropriate response. This talk will discuss our recent findings studying selected systems in the Gram-positive model organism Bacillus subtilis by integrating information derived from a diverse set of experimental approaches such as genetics, enzymology and structural biology and in silico approaches such as Molecular Dynamics Simulations and Bioinformatic techniques. These studies identified some general rules by which these proteins coordinate a response on the molecular level, of relevance to all two-component systems and beyond. One system in particular, the YycFG system is conserved…

Aptamers are short oligonucleotides (< 100 bases) selected from large combinatorial pools of sequences (from 1012 to 1015) for their capacity to bind a target (amino acids, antibiotic, proteins…). Such ligands are isolated by a method of directed molecular evolution usually named SELEX (Systematic Evolution of Ligands by Exponential enrichment). Since 2005, our group and others have adapted the SELEX against whole living cells. Using this strategy, we selected several nuclease resistant 2'-Fluoro-pyrimidines (2'-F-Py) RNA aptamers against cell surface biomarkers that can represent surrogate markers of cancer cells (1-3). This technique, usually named cell-SELEX, allows the parallel selection of several aptamers against membrane proteins in a native conformation. However, although these aptamers are very important tools for in vitro experiments, their translation for in vivo uses is still difficult to predict. To solve this problem, we have developed fluorescence imaging techniques to investigate their biodistribution and tumour targeting capacities in small animal…

It has been recently proposed that competitive endogenous RNAs (ceRNAs) sequester microRNAs to regulate mRNA transcripts containing common microRNA recognition elements (MREs). The ceRNA hypothesis stems from the observation that RNA transcripts can communicate with each other through a recently discovered mechanism. MicroRNAs are tiny snippets of RNA (~22nt long) which negatively regulate target gene expression via translational inhibition or transcript cleavage. A microRNA may target many different transcripts, and conversely, individual transcripts may be bound by multiple different microRNAs. In addition to this conventional microRNA → RNA function, it has recently been established that a reversed RNA → microRNA dimension exists, whereby ceRNAs regulate transcript expression via competition for common microRNAs, with microRNA response elements (abbreviated MREs) as the building blocks of this ‘RNA language’. Phenomenological, theoretical, and experimental aspects will be addressed.

ProteinProtein-protein interactions (PPIs) and the formation of protein-protein complexes are central to many biological processes. Protein docking is the task of calculating the three-dimensional (3D) structure of a protein complex starting from the two individual structures of the component proteins. In this talk, I will give an overview of my "Hex" polar Fourier correlation docking algorithm, and I will briefly describe how the calculations may be accelerated using modern graphics processor units (GPUs). Then I will describe some recent work on using a knowledge-based approach to the docking problem called “KBDOCK”. Briefly, KBDOCK collects all known hetero domain interactions from the Protein DataBank and combines and clusters them by Pfam family. This allows known protein binding sites to be re-used as 3D docking “templates” in what is effectively a case-based reasoning approach to the docking problem. We find that protein domains belonging to the same Pfam family often interact in similar ways.…