CE12 - Génétique, génomique et ARN

Phage-Bacteria Reciprocal Modulation of Mutation Rate and Its Consequences – MUMI

We address these questions using a variety of methodologies: in addition to video microscopy and the microfluidic-based mutation visualization experiment originally developed by the team, we use molecular biology, microbiology, genetics, and approaches based on whole genome sequencings such as Dup Seq and Chip Seq.

We performed Dup Seq experiments to obtain the first estimate of lambda's spontaneous mutation rate from whole genome data. Our results show that lambda mutates more than three orders of magnitude more than its host E. coli, with a mutation rate 2-20 times higher than previously estimated using a single locus test. We found that the difference in mutation rate between lambda infecting E. coli competent in replication error repair, i.e., mismatch repair (MMR), and MMR-deficient E. coli is small, 2-fold. This result is in contradiction with the 150-fold difference in mutation rate between E. coli capable of replication error repair and MMR-deficient E. coli. This suggests that MMR may be inefficient during lambda replication. Our experiments on visualization of replication errors (using the fluorescently tagged MMR protein MutL and microfluidics) suggest that this is not due to inefficient recognition of replication errors by the MMR. This suggests that the MMR inefficiency could result from a problem at a downstream step (e.g., at strand incision, due to the under-methylation of GATC sites on lambda genomes). Alternatively, the MMR defect could result from excessive error production by the Pol III replicative DNA polymerase. To investigate this hypothesis, we analyzed the spectrum of lambda mutations obtained by Dup Seq. Comparison of the spectrum of lambda mutations to that of E. coli i) expressing a defective DNA polymerase proofreading subunit ii) inactivated for MMR or iii) inactivated for MMR and with a DNA polymerase proofreading defect, suggests a strong proofreading defect and an incomplete MMR defect during lambda proliferation.

Future work will aim to study how altering the level of heat shock chaperones, the level of Pol III proofreading, and the mode and tempo of lambda replication affect the lambda mutation rate. We will also characterize mutation occurrences (rate and origin of mutations) during infection of E. coli with T4 and M13 phages.

Submission summary

Mutations were for a long time difficult to witness directly in single cells. This prevented the direct characterization of their dynamics and fitness effects, which is critical for most evolutionary studies. We recently developed a method that allows visualizing the emergence of spontaneous mutations in real-time in single bacterial cells. Here, we propose to use this approach to characterize spontaneous mutation occurrences in the genomes of bacteria and phages during infection and determine whether infection with filamentous phages modulates bacterial mutation rate. Then, we will track the fitness of infected bacteria at the single-cell level during hundreds of generations. This will allow us to determine how infection may affect the adaptation ability of the host cells, for instance through a transient modulation of their mutation rate. These results are relevant for the evolution and adaptation of phages and bacteria and thus, with respect to human health, for antibioresistance and phage therapy.

Project coordination

Marina Elez (Microbiologie de l'alimentation au service de la santé)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


LJP Laboratoire Jean PERRIN
MICALIS Microbiologie de l'alimentation au service de la santé
MICALIS MICrobiologie de l'ALImentation au service de la Santé

Help of the ANR 379,619 euros
Beginning and duration of the scientific project: October 2020 - 48 Months

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