Spontaneous phage-resistant mutants in Bacillus thuringiensis defy tectiviral infection through different mechanisms and phenotypes

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. 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 B. 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 were selected and validated to confirm complete resistance to tectiviruses GIL01 and GIL16. 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. Additionally, two phage-resistant mutants were re-sequenced independently and a variant calling mapping, along with differential proteomic analyses, was performed as these mutants displayed striking phenotypic changes that resulted in blocking tectiviral infection. SNPs and indels found in several genes, combined with differential expression of proteins, underlay the cost of resistance in the complicated tectivirus-bacteria interplay