Mutations, i.e. changes in the genetic sequence of organisms, are the driving force of evolution. They also have important consequences for human health. Our fight against infectious diseases is underpinned by an evolutionary arms race with pathogens, in which mutations fuel the evolution of virulence, antibiotic resistance and bacteriophage escape. Mutations have been intensively studied for more than a century, shedding light on the mechanisms underlying their production and their effects on individuals and populations. Our group contributes to this body of knowledge by addressing some of the unexplored questions about mutagenesis in phages and bacteria. Some of these require the development of new experimental approaches. Notably, in 2018, we developed a novel, single-cell level approach that combines microscopy, microfluidics, and a fluorescent reporter of replication errors allowing us to follow mutations and their effects directly in single bacteria.
Many spontaneous mutations are due to DNA replication errors. Such errors can be repaired by a dedicated system called Mismatch Repair (MMR). Therefore, mutations occur in two steps: the production of an erroneous DNA sequence by the polymerase, and the failure of its repair by MMR.
Recently, we have used our mutation visualization approach to investigate the dynamics of error production, i.e. the first step of mutation occurrence, revealing moderate cell-to-cell variations in error rate during normal growth. Now, we extend our method to visualize simultaneously replication error production and repair by MMR. The second step (i.e. fluctuations of error repair efficiency and the dynamics of mutations caused by repair failures) allows us to fully characterize the mutation dynamics in growing cells.
Research leads: Lydia ROBERT & Marina ELEZ
In this project, we address questions related to the occurrence of mutations during phage infections. Why do DNA phages, even those that use the replication machinery of their hosts, have a mutation rate about two orders of magnitude higher than that of their hosts? Can we modulate the mutation rate of phages? Can we slow down the diversification and evolution of phages? What is the impact of infection on the host mutation rate? To investigate these questions, we use E. coli and its bacteriophages lambda, T4 and M13 as model systems. We address these questions using, in addition to video microscopy and the microfluidics-based mutation visualization experiment originally developed in the group, molecular biology, microbiology, genetics, and genome-wide approaches such as Duplex Sequencing and Chip-Seq.
Research leads: Marianne DE PAEPE & Marina ELEZ
Data accumulated over the last decades suggest that under stressful conditions, some cells could trigger specific molecular mechanisms that increase their mutation rate. In particular, bacteria could increase their mutation rate in presence of sublethal concentrations of antibiotics. Traces of antibiotics are often found in natural environments and could increase the rate of adaptation of bacteria and thus their capacity to acquire mutations conferring antibiotic resistance.
Previous studies investigating the effect of stress on mutagenesis were hampered by the limitations of classical experimental approaches. Therefore, we use our new approach to characterize mutagenesis in E.coli in stressful environments. We will visualize replication errors, assessing replication fidelity and repair capacity at the single cell level, and we will also develop a new method to estimate the rate, spectrum and localization of all types of mutations.
Research leads: Marina ELEZ & Lydia ROBERT
All the team publications are available in the MUSE-MICALIS HAL collection
Application n° EP21305834
