pXO16 from Bacillus thuringiensis : a proteomic vista on this unique conjugative plasmid

Details

Supervisors

Mahillon, Jacques

Faculty

Faculté des bioingénieurs

Degree label

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

Abstract

pXO16, a giant conjugative plasmid from Bacillus thuringiensis sv. israelensis, encodes a unique conjugative apparatus that allows the seemingly non-specific transfer, mobilization and retro-mobilization of a variety of DNA fragments within or between species in the B. cereus group. Besides this potential to promote gene exchange between the group members, not much is known about pXO16 or its conjugative apparatus. Plasmids typically interfere with the way their host interacts with their environment, and pXO16 is likely no exception. With a focus on exported proteins, this Master’s thesis aimed at tackling the study of pXO16 lifestyle, from gene expression and localization to host-plasmid interaction, as preliminary work towards a better understanding of pXO16 ecology and impact on its host. In this work, bioinformatic analysis predicted that 17.5% of the 434 CDSs of pXO16 encode surface-exposed or secreted proteins, reaffirming the potential of pXO16 to alter the cell surface and exported signals of the host cell. A putative exported proteins from the tip locus, TipD, was tagged with a GFP, but expression of TipD-GFP could not be observed with the fluorescent microscope. The tagged version of pXO16 seemed to exhibit slightly reduced conjugative capacity compared to wild-type pXO16. This may be due to the disruption of TipD folding and/or activity, or to a polar effect in the predicted tipC-D-E operon. Further work is necessary to understand this effect and assess the importance of TipC, TipD and TipE in pXO16 conjugative machinery. Next, in an effort to evaluate the impact of pXO16 on its host, a preliminary proteomic experiment was carried out to compare the proteome of two isogenic Bacillus thuringiensis sv. israelensis strains, with and without pXO16. Although the exploratory nature of the experiment limited the interpretation of the results, this work showed that pXO16 does impact its host and expresses at least some proteins in the exponential growth phase. This experiment also led to the identification of a potential region of interest. This region, upstream of the tip locus, contains several proteins with predicted signal peptides and transmembrane domains and thus may be involved in interaction with environmental factors. The overall abundance of potentially exported proteins encoded by pX016 bears witness to a tremendous potential in alteration of host surface and secreted signals. This supports that pXO16 is involved in interactions between the host and its environment. Of course, much experimental work is needed to confirm this and understand exactly how pXO16 impacts its host.