Bacteria have evolved a highly conserved chromosomally encoded recombination machinery, Xer, to resolve chromosome dimers. With the exception of a few species, Xer is composed of two tyrosine recombinases, XerC and XerD, which act on a specific chromosome site, dif. Diverse mobile elements harness XerC and XerD for their own benefit. Indeed, Xer was initially discovered because of its role in multicopy plasmid dimer resolution. Since then, numerous Integrative Mobile Elements exploiting Xer (IMEX) have been described. Phages and genetic islands harness Xer recombination to integrate into the dif site of one of their host chromosomes, while small genetic cassettes flanked by pseudo dif (pdif) sites harness Xer to disseminate on plasmids. IMEXs are generally associated with the evolution of pathogenic bacteria, including (in)famous human pathogens (Vibrio cholerae, Yersinia pestis, Neisseria meningitidis and gonorrhea, Acinetobacter baumannii), animal pathogens (a large panel of Vibrios) and plant pathogens (Xanthomonas campestris, Xylella fastidiosa). In particular, cholera toxin, which is responsible for the deadly pandemic diarrhea associated with the disease of the same name, is harbored in the genome of a V. cholerae IMEX, phage CTXΦ. Ecological interactions of CTXΦ with at least two other families of V. cholerae IMEX phage, VGJΦ and TLCΦ, are responsible for the constant rapid emergence of V. cholerae strains harboring new forms of the cholera toxin. The recent appearance of multidrug resistant strains of A. baumannii, a human opportunist pathogen that is responsible for nosocomial diseases, is linked the dissemination of carbapenem resistance genes by a fourth category of IMEXs, the pdif-modules. Thus, understanding how IMEXs exploit Xer recombination for their own benefit and unravelling their ecological interactions is crucial to prepare against new or re-emerging infectious diseases. Nevertheless, reports on IMEXs rarely go beyond the stage of the description of their genetic content and genomic context, due to the methodological challenges involved in probing the system from both a molecular-genetic and mechanistic viewpoint. XerC and XerD proteins cluster in two narrow closely-related phylogenetic groups and act on highly conserved dif sites, which denotes high evolutionary constraints. Remarkably, the core dimer resolution sites of multicopy plasmids exploiting Xer and the attachment sites of IMEXs significantly deviate from the target host dif site. Our preliminary data suggest that IMEXs rely on Integrative Xer recombination complex Stabilization (InXS) factors. The aim of this research program is to search for InXS factors and unravel the complex Xer recombination reactions that they promote. To this end, four teams combining a deep expertise in Xer recombination, bioinformatics, molecular-genetics, genomics, single-molecule biophysics, and structural biology join their forces to study the Xer exploitation strategies of the V. cholerae CTXΦ, VGJΦ and TLCΦ IMEXs and the A. baumannii pdif-modules, which represent the four categories of IMEXs so far described. The results of the InXS project will interest academic research scientists studying protein-DNA transactions and the microbiologist community in general. In addition, the results will be made available in an online database, thereby providing a new tool for the surveillance of pathogenic bacteria. The acquired knowledge could provide opportunities to take advantage and/or control of these 'natural genetic engineers'. In particular, we foresee the development of methodologies to enforce the excision of IMEXs that encode antibiotic resistance elements and/or toxins, which could serve as the basis of new prophylactic medical treatments.

Gram-negative bacteria represent a major public health concern due to their high resistance to antibiotics resulting in millions of human deaths world-wide each year. Their multilayered envelope contains an outer membrane (OM) that forms an effective permeability barrier shielding against noxious molecules, including several antibiotics. Being exposed to the cell surface, the OM represents a promising target for the development of new antimicrobials that can act from the exterior of the cell. The design of new antimicrobial stategies urges a better understanding of the molecular pathways of OM biogenesis. Integral OM proteins are crucial for envelope homeostasis. The beta-barrel assembly machinery (BAM) plays an essential role in OM protein assembly. The activity of BAM is regulated in space and time ensuring the constant supply of protein components to active sites of OM biogenesis. Many questions remain unresolved concerning the protein folding reaction mediated by BAM and the regulation of its activity throughout the OM. Motivated by the need to better understand the biogenesis of the bacterial OM, we have discovered that in the enterobacterium Escherichia coli, a member of gamma-proteobacteria, the lipoprotein DolP associates with the BAM complex and plays a critical role in OM homeostasis and integrity. DolP is widely conserved in gamma-, beta- and some alpha-proteobacteria contributing to the virulence of several pathogens, as well as to their ability to survive in the presence of some antibiotics. Our preliminary data reveal that DolP directly interacts with BamA, the catalytic subunit of BAM, promoting BamA folding and function. Inactivation of DolP phenocopies BamA depletion and makes cells sensitive to antibiotics that are normally excluded by Gram-negative bacteria. DolP localizes at active sites of OM biogenesis, ideally positioned to support BAM activity. The molecular mechanisms by which DolP contributes to OM assembly by the BAM complex and ensures OM integrity remain to be established. Our project uses an interdisciplinary approach to determine how DolP interacts with the BAM complex, influences its organization with partner complexes and regulatory factors, and supports its OM protein assembly activity. By employing a multiscale experimental strategy, we are investigating the molecular processes mediated by DolP i) at the cell envelope-wide scale, ii) in a chemically defined in vitro system, and iii) at the structural level. We will conduct these studies in the enterobacterial model organism E. coli and test our results in other pathogens of the gamma- and beta-proteobacterial classes. Our results will be important for the research of new antibacterial compounds that can interfere with OM integrity in Gram-negative bacterial pathogens.

Bacterial toxin-antitoxin systems (TAs) are ubiquitous genetic elements found on chromosomes and mobile genetic elements. TAs consist of an operon encoding a toxin that inhibits cell growth and an antitoxin that counteracts the toxin. Typically, the toxins disrupt essential functions of the host bacterium, including translation, replication or peptidoglycan synthesis. Several biological roles associated with TAs have been demonstrated, including stabilising mobile genetic elements that confer resistance to antibiotics, providing bacterial immunity against phages and contributing to the virulence and persistence of pathogenic bacteria. Toxins use a variety of strategies and mechanisms to control bacterial growth, offering interesting alternatives to traditional antibiotics. However, the development of inovative antibacterial strategies requires a thorough understanding of the mechanisms of inhibition, expression and regulatory control of TAs. The Patho-TOX project focuses on the conserved Rosmer TA family (RmrTA), which has recently been identified as a potential defence mechanism against phages. RmrA antitoxins are paired with different RmrT toxins that generally show no detectable similarity to other protein families. RmrA proteins combine a DNA-binding HTH domain with a metallopeptidase-like ImmA domain, the function of which has not yet been elucidated. The fine-tuning of RmrT activity is critical for bacterial survival. Remarkably, some RmrT toxins have been shown to be responsible for the essentiality of ClpXP in bacteria such as Streptococcus pneumoniae and Escherichia coli, thus suggesting that RmrT toxins are kept inactive by the ClpXP AAA+ protease. The aim of this project is to reveal the cellular functions and activation mechanisms of chromosomal RmrTA systems in three major human pathogens, namely E. coli, S. pneumoniae, and Staphylococcus aureus. The project combines genetic, biochemical, and structural approaches to understand (i) the molecular mechanisms that cause bacterial growth inhibition by RmrT toxins, (ii) the roles of the RmrTA components in the transcriptional regulation of the rmrTA operon and in bacterial physiology, and (iii) how the AAA+ ClpXP protease control the activation of RmrT toxins, both in vitro and in vivo in response to relevant stress. This project will provide an in-depth analysis of novel toxin-mediated growth inhibition mechanisms and TA activation networks, which could be used to develop new strategies to combat bacterial pathogens and antibiotic resistance.

Transition metals are required by all life forms to carry out central functions necessary for survival. Moreover, they are at the heart of the host-pathogen battle as the host immune system sequesters essential metals from pathogens. In order to acquire metals and maintain their metal homeostasis, microbial pathogens have developed vital mechanisms involving a range of proteins that constitute their metalloproteome. Bacteria of the Mycoplasma genus have amongst the smallest genomes known for an autonomously replicating cell. Through evolution, genomes of mycoplasmas underwent drastic reduction to select a minimal set of essential genes. As such, the metalloproteome of mycoplasmas might represent a minimal metalloproteome required to perform some of the most fundamental functions supporting life. Many mycoplasmas are pathogens of vertebrates that are alarming in the raise of antimicrobial resistance, e.g., the avian pathogen Mycoplasma gallisepticum and the human pathogen Mycoplasma pneumoniae causing respiratory diseases. An attractive way to design drugs against these pathogens is to target essential components of their metalloproteome. However, microbial metalloproteomes are largely uncharacterized, especially in mycoplasmas. The MIMESIS project aims to decipher the mechanisms of metal acquisition and utilization in the minimalist mycoplasma organisms through a multidisciplinary approach combining biochemistry, structural biology, biophysics and bioinformatics. The project will unravel new biochemical mechanisms and answer fundamental questions regarding the metal homeostasis and metalloenzyme chemistry essential to minimal microbial pathogens. Ultimately, the project will identify key components in the survival of mycoplasmas, thereby uncovering putative new drug targets in order to combat antimicrobial resistance.

Chromosome segregation is a fundamental yet poorly understood cell cycle process allowing cells to transmit genetic material to their progeny. In all organisms it is tightly regulated and highly accurate to avoid the loss of genetic information (aneuploidy, chromosome breaks). In spite of an apparent diversity, in particular in the bacterial world, sister chromosome segregation follows conserved rules. In this project, we will use the E. coli model to study the mechanisms underlying one of these rules in bacteria: the pairing of newly replicated sister chromatids and the controlled release of this pairing. We will build up on our recent observations suggesting that sister chromatid pairing involves the establishment of specific chromatin, particular dynamics of paired loci and their segregated neighbors, and transient restructuring of the whole genome. Our working hypothesis is that these features reflect the dynamics of sister chromatid linkages, mainly topological linkages called precatenates and catenates that entangle sister chromatids, and the mechanisms responsible for their release. Based on these results, we will characterize the pairing and segregation mechanisms (WP1), the functioning of the terminal region of the chromosome as a hub dedicated to the control of decatenation (WP2) and we will develop in silico models allowing a quantitative interpretation of the experimental data (WP3). Technically, SISTERS proposes to develop complementary assays to characterize the paired state, of which the monitoring of chromosome loci and the dynamics of sister chromatids by fluorescence microscopy as well as population-based genomic studies and the physical characterization of catenated states. SISTERS also relies on the development of new polymer physics models adapted to the study of braided molecules. This dialogue between modeling and experiments will allow us to evaluate the relevance of our working hypotheses and to feed new research routes. The experiments and models planned during SISTERS will establish the mechanistic basis of the control of sister chromatid pairing and will pave the way to understanding the integration of this process in the bacterial cell cycle.