Bacterial communities are composed of a variety of species, which have the ability to exchange genetic material through horizontal gene transfer (HGT), thus accelerating their diversification, adaptation and genetic evolution. The major gene transfer mechanism is bacterial conjugation, where DNA is transferred from a donor to a recipient cell by direct contact. The importance of conjugation has first become obvious after the Second World War, with the global spread of multidrug resistance. Nowadays, analysis of drug-resistant commensal, environmental and pathogen strains has revealed a vast collection of conjugative plasmids carrying one or multiple resistance genes to most, if not all classes of antibiotics currently used in clinical treatments. Conjugation-mediated drug-resistance dissemination is consequently a major obstacle to successful treatment of infections and is now recognized as one of the biggest threats in public health. In natural and clinical environments, bacterial species predominantly live in spatially structured communities called biofilms, in which a self-produced extracellular matrix holds cells together. Biofilms offer protection against potentially toxic compounds such as antimicrobial drugs, thus intrinsically limiting the efficiency of conventional antimicrobial therapy. Biofilms also protect the bacterial community against bacteriophage infection, rendering phage-based therapeutic approaches unsuitable. Importantly, it has been reported that the biofilm mode of life facilitates horizontal genes transfer by conjugation. Therefore, biofilms not only shelter bacteria against external hazards, but could also offer a niche that facilitates the dissemination of drug-resistance determinants. This possibility highlights the need to invest research effort into the accumulation of basic knowledge about the dynamics of gene transfer by DNA conjugation within bacterial biofilm communities. The central goal of TARGET-Biofilms research proposal is to explore the fundamental and practical aspect of DNA conjugation within bacterial biofilms. The first objective is to provide a comprehensive understanding of the mechanisms, extent, and impact of bacterial conjugation within biofilms. To do so, we will use a genetic system to monitor intra- and interspecies cell-to-cell DNA transfer in live-cells, in combination with an experimental set-up allowing direct imaging of bacterial biofilms in 3D at cellular and sub-cellular resolution. We will address the influence of the biofilm structure and strain composition on the efficiency of DNA transfer and investigate the dissemination of drug-resistance within the community. The second objective is to test the possibility to combine conjugation and CRISPR systems to perform in situ biofilm manipulation, including attempts to disassemble biofilms by killing the resident cells or by suppressing genes required for biofilm formation and stability. Combining DNA transfer functions of conjugation systems with CRISPR/Cas9 genes designed to have a specific effect in targeted bacterial species is an unexplored, yet promising approach, which deserves high interest. The research program will benefit from the consortium’s expertise in live-cell microscopy approaches dedicated to the study of DNA transfer and biofilms, as well as molecular microbiology. TARGET-Biofilms research program will provide outcomes beneficial to public health, life stock, crops, and the environment.

Multiple bacterial natural products including the pepteridine virulence factors and the therapeutically-relevant antibiotic virginiamycin M, are biosynthesized at the intersection between primary and specialized metabolism. In these cases, primary metabolic α-ketoacid dehydrogenase complexes (KADHs) provide essential acyl building blocks to multienzyme complexes of specialized metabolism, including modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). More remarkably, in certain pathways, the KADH components are fully integrated into the PKS/NRPS megaenzymes. At present, nothing is known about the sequence, mechanistic and architectural adaptations that were required relative to the ancestral KADHs to afford such chimaeras – information which is necessary for creating novel types of hybrids in the laboratory by genetic engineering. In this context, the present German-French collaborative project aims to investigate this type of system in detail. Specifically, we will: (i) generate an exhaustive catalog of specialized metabolic pathways that incorporate KADH machinery using genome mining; (ii) structurally characterize the products of newly-identified systems by heterologous expression; (iii) use ancestral protein reconstruction to propose a reasonable evolutionary trajectory to present day KADH enzymes; (iv) deploy an integrative structural biology approach to elucidate key architectural features of multienzyme-integrated KADHs (i.e. oligomerization state, stoichiometry of KADH component binding, and interactions with partner domains within the multienzymes); and (v) exploit the obtained fundamental insights to genetically engineer biosynthetic systems, towards the goal of generating bespoke natural product analogs bearing KADH-derived moieties. The proposed project follows on from previous successful collaboration between three of the partner laboratories, and is fully anchored in all groups’ strong, highly-complementary expertise.