Functional characterization of the genome of the Bacillus subtilis phage SPP1 using Synthetic Biology approaches
Résumé
A strategy to foster innovation in biotechnology relies on constructing cellular chassis strains with genomes appropriately streamlined for the desired application. Streamlining genomes requires the development of efficient and robust genetic tools for genome engineering. While Gram-negative model bacteria have multiple genetic tools of phage origin, this is not the case for Gram-positive bacteria. This work aims to pioneers methods and techniques for investigating and manipulating bacteriophage genomes, with a particular focus on the SPP1 phage from Bacillus subtilis. SPP1 is one of the best-characterized lytic bacteriophages in the siphovirus family. However, numerous questions persist regarding the function and essentiality of its genes, as well as the processes of SPP1 transcription, replication, and encapsidation. Two complementary libraries of mutants have been constructed. The first one is a library of B. subtilis mutant strains, each carrying one or more phage genes integrated into the bacterial chromosome, with inducible expression. The toxicity of viral proteins to B. subtilis was tested for 82 mutants. Approximately 23% of the mutants displayed altered phenotypes due to the expression of phage genes. For instance, regarding genes of unknown function, the expression of gp29.1 led to a dose-dependent reduction in growth rate, while gp37.1-37.2 expression induced cell filamentation. The second library is composed of semi-synthetic SPP1 phages, each deleted for one or more essential and non-essential genes. A deletion method by in vitro assembly of SPP1 genome fragments followed by host cell transformation was developed. Each phage mutant was built and propagated in the corresponding B. subtilis mutant strain from the first collection to allow for trans-complementation of the phage mutation. The fitness of 36 mutants was characterized during B. subtilis infection, revealing that around 25% of phage genes were found to be essential or nearly essential for phage propagation. For instance, the mutant SPP1 ∆gp22, involved in the tail assembly but of unknown function, exhibited significantly reduced capacity for multiplication. Lastly, an in vivo engineering method of genomes of phages from Gram-positive bacteria using CRISPR-Cas9 was dnd validated. These results have helped decipher some interactions between SPP1 and B. subtilis and will ultimately contribute to the design of new genetic engineering tools.