Spontaneous phage-resistant mutants in Bacillus thuringiensis: the tectivirus-bacteria interplay
(2018) BSPIT 2018 colloque «Bactéries sporulantes pathogènes ou d’intérêt technologique» — Location: Paris, France
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- Phages and bacteria are constantly involved in a dynamic evolutionary process in which bacteria develop resistance to phages, and phages counter-evolve to fight back the resistant bacteria. Despite the importance of this dynamic process, phage-resistance in members of the Bacillus cereus group and the mechanism(s) involved are still poorly understood. Therefore, this work aimed to depict bacterial resistance triggered by the presence of tectiviruses, a relative rare group of non-enveloped tail-less phages that are able to replicate as linear plasmids in their B. cereus sensu lato host. To this end, lytic variants of tectiviruses GIL01 and GIL16 where obtained by selecting clear plaques that emerged among the turbid plaques in lawns of Bacillus thuringiensis. The lytic variants were used to generate a collection of resistant B. thuringiensis mutants by using an improved method that combined the classical spotting assay (to generate the bacterial mutants) and an inverted spotting assay to evaluate the resistance of the candidate mutants. Twenty fully-resistant mutants to GIL01 and GIL16 were selected mainly because these resistant bacteria showed differences in cell and colony morphotypes and displayed distinct adaptation features (i.e. biofilm formation, sporulation rate, swarming motility, differences in metabolic profiles and antibiotic susceptibilities). To unravel the genetic changes responsible for tectivirus-resistance in B. thuringiensis, a pooled high-throughput whole genome sequencing was used. Potential genes causing the resistant phenotype were identified and several genes associated with cell-wall metabolism and turn-over, as well as cell-surface proteins, have been pinpointed. Since the phage resistant mutants were analysed by library-pooling and tag-pooling combined with high-throughput sequencing (a technique that might had masked potential SNPs), three mutants were selected to be re-sequenced individually (MiSeq, Illumina). We performed a single nucleotide variants (SNVs) analysis calling from the generated NGS data. These analyses revealed different genetic changes (SNPs and INDELs) in several genes in the three B. thuringiensis mutants. These resistant mutants were also characterized using a quantitative shotgun proteomic approach, based on isotope-coded protein labelling (ICPL). Preliminary results suggested that several enzymes involved in peptidoglycan metabolism and turn-over could be responsible for at least part of the resistance phenotypes observed in the bacterial mutants. Currently, the most interesting SNVs are being verified by Sanger sequencing and compared with the levels of protein expression to identify the proteins that are playing an important role in the dynamic process of “arms race” between tectiviruses and their Gram-positive host.
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Gillis, A., & Mahillon, J. (2018). Spontaneous phage-resistant mutants in Bacillus thuringiensis: the tectivirus-bacteria interplay. BSPIT 2018 colloque «Bactéries sporulantes pathogènes ou d’intérêt technologique», Paris, France. https://hdl.handle.net/2078.5/220770