Phenotype sequencing uncovers mutations in candidate genes that mediate tectivirus-resistance in Bacillus thuringiensis

The family Tectiviridae is a relative rare group that includes tail-less phages having a membrane beneath their icosahedral protein shell, formed of approximately equal amounts of virus-encoded proteins and lipids derived from the host cell plasma membrane. The 15 kb linear dsDNA genomes have long inverted terminal repeat sequences (~100 bp) and are coiled within the lipid membrane. This family contains two groups of phages: the lytic PRD1-like infecting Gram-negative enterobacteria, and the temperate ones preying on Gram-positive bacteria belonging to the Bacillus cereus group. Phages GIL01, GIL16, Bam35, AP50 and Wip1 are the fully-sequenced representatives of the second group. GIL01 and relatives are capable to reside as temperate phages that do not integrate into the host genome upon infection and remain as an autonomous linear plasmid in the cell. Therefore, it is important to understand the selective pressures undergone by the bacteria when facing this type of phages. The aim of this work was to study the primary interaction between these tectiviruses and their host, and the phenotypes of bacterial resistances triggered by the presence of these phages. For this purpose, Bacillus thuringiensis sv. israelensis strain GBJ002 was subjected to a selective pressure after repetitive propagation with clear plaque (CP) mutants of GIL01 and GIL16. The CP mutants showed an elevated efficiency of killing mainly because they propagate exclusively lytically. Twenty completely tectivirus-resistant bacterial mutants were isolated. These resistant bacteria showed differences in colony morphotype and displayed distinct adaptation features, such as biofilm formation, sporulation rate, swarming motility, exopolysaccharide production and some differences in metabolic profiles. These observations indicated that tectiviruses may drive life-trait changes and ecological adaptations in the B. cereus group members, as results of a phage-selective pressure. To unravel the genetic changes responsible for the phage-resistant phenotype, a whole genome sequencing method was approached. Using a pooled high-throughput sequencing analysis of multiple independent mutants, potential genes causing the tectivirus-resistant phenotype in B. thuringiensis were identified. Several genes associated with cell-wall metabolism and turn-over, as well as cell-surface proteins, have been pinpointed. Currently these candidate genes are been studied intensely to confirm their enrolment in the bacteriophage-resistant phenotype. These results have shed new light on the cell wall component(s) that could act as receptor(s) or mediate the interaction between these phages and their hosts. This approach will also permit to gain insights into the different strategies used by bacteria to elude phage infection