Characterisation of the antiviral defence system DISARM in the Bacillus cereus group

Details

Supervisors

Gillis, Annika

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

As the evolutionary arms race between bacteria and bacteriophages (phages) continues to progress, researchers constantly discover new bacterial defence mechanisms to evade phage attacks. Understanding these defences is vital for formulating phage-based innovative strategies to combat bacterial infections. This master's dissertation focuses on characterising a novel antiphage defence system known as DISARM, Defence Islands System Associated with Restriction-Modification, within the Bacillus cereus (B. cereus) group. This system is categorised into two classes, with the present research concentrating on Class 2, which is naturally present in Bacillus thuringiensis serovar sotto str. T04001 and is composed of four genes from the drmMII, drmC, drmB and drmA, and two ancillary genes, g1 and g2. The goal of this work is to elucidate the role of DISARM in conferring resistance against viral predation in the B. cereus group through a comprehensive examination of DISARM, including its gene cassette organisation, prediction of protein functional domains and by observing the interactions of DISARM-coding strains with B. cereus s.l.-infecting phages. To do so, the activity of g1g2 compared to B. thuringiensis GSX002 and B. thuringiensis GSX002 (pHT304-18Z) strains (used as controls in this work) was investigated in solid and liquid media. First, bioinformatic analysis reveals predicted sequence homologies of DrmMII to a cytosine-specific methyltransferase, DrmC to a protein containing a phospholipase D-like domain, DrmB to a protein containing a DUF1998 domain along with a MrfA Zn-binding domain, and DrmA to a helicase. Additionally, G1 is found to be associated with a histidine kinase, while G2 is associated with a response regulator. After the successful cloning of g1g2 into the B. thuringiensis naïve host GSX002, the presence of g1g2 reveals variable effects depending on the nine Bacillus phages tested (Vp4, Vp2, RX1, RX2, Deep-Blue, GIL01 lytic mutant, GIL16 lytic mutant, Deep-Purple, and FR(B)), in solid media. Both B. thuringiensis GSX002 (pHT304-18Z) and B. thuringiensis GSX002 (pHT304-18Z::g1g2) display in general lower sensitivity to phages compared to the naïve bacteria, leading to a protective effect of the vector and g1g2. However, experiments in liquid media carried out with phage Vp2 showed a supplementary defensive effect of g1g2 that could be involved in anti-phage defence. Different conditions were tested such as phage-to-bacteria ratio and antibiotics pressure. Finally, a knock out experiment of DISARM in the original host has started in order to compare it to the activity of the original host tested against the nine phages. Overall, this work indicates that genes g1g2 of DISARM seems to have an effect as a defence by reducing the phage infection rate in B. thuringiensis strains in certain conditions and offer surprising insight on a potential anti-phage effect of pHT304-18Z expression vector against Bacillus phages.

Citations

• APA

Alfieri, L. (2024). Characterisation of the antiviral defence system DISARM in the Bacillus cereus group.