Induction of conjugative transfer

Bacterial conjugation results in the transfer of genetic material from one bacterial cell (the donor) to another (recipient) cell. The genetic information required for conjugation in Gram-negative bacteria is encoded by a variety of plasmids. In addition to transfer genes conjugative plasmids frequently carry other genes, such as virulence and antibiotic resistance (AR) determinants, which contribute to the burden of infectious disease. Plasmid conjugation functions include the ability to assemble protein filaments on the surface of donor bacteria that are essential for mechanical contacts between cells and the physical transmission of DNA. Elaboration of these plasmid pili is also intricately involved in the ability of bacterial hosts to colonize surfaces. The robust biofilm that develops poses a tremendous clinical challenge to control or remove. Fertility inhibition, or the repression of expression of conjugation genes, is common in natural plasmids. Nonetheless, repressed plasmids exhibit the capacity to override fertility inhibition and promote their rapid dissemination in heterogeneous bacterial communities. Induction and maintenance of fertility derepression is poorly understood. Plasmid transfer is controlled at the initiation stage through intercellular contact, regulation of gene expression, and the enzymatic preparation of DNA for its physical transmission to a new cell. The proposed work aims to elucidate mechanisms, control and consequences of conjugation with the paradigm IncF and IncI classes of conjugative plasmids. We will investigate at the population level the nature of cell-surface and cell-cell interactions that overcome the basal repression of fertility and alter the pattern of plasmid gene expression. Molecular reporter genes fused to plasmid promoters detect variation in gene activity. When present in a population of bacterial hosts, under conditions permissive for conjugation, we expect to reveal the ordered steps leading to derepression. We will investigate in detail the molecular mechanisms and regulation of DNA processing enzymes. These biochemical studies use models of nucleoprotein intermediates of the strand transfer process. We aim to identify the nature of protein-DNA and protein-protein interactions that govern the key step committing the initiation process to plasmid export. Substitution of wild type with mutant and truncated forms of conjugation proteins in a range of enzyme activity assays in vitro reveal insights to the function and regulation of their catalytic activity in this context. The outcome of initiation events is to stimulate the frequency of plasmid gene spread and, in surface attached communities of bacteria, to drive the intercellular interactions that substantially strengthen and expand the bacterial biofilm. Increased molecular understanding of bacterial conjugation may provide insights to inactivate or control these machineries as part of preventive or therapeutic measures.

Bacteria have small genomes compared to other organisms. To compensate for their modest number of genes, bacteria are able to share DNA provided by other cells. Widespread gene transfer occurs between closely related cells and more rarely between distant species. The genetic material is typically organized in mobile, functionally coordinated units such as plasmids, phages and transposons. Evolution has equipped mobile genetic elements with a whole armory of genes that encode useful survival properties for the bacteria. Antibiotic resistance and virulence factors are examples, and gene mobility among bacteria is the driving force behind the massive global increase in cellular resistance to antibiotic therapies. A direct cell to cell injection of the shared genes occurs during bacterial conjugation. In this process specialized protein machinery is synthesized by the donor bacterium to cross the cellular membranes and physically link a pair of cells. The protein channel then actively transports DNA from the donor to the recipient bacterium. This protein machinery has also been adopted over evolution to enable bacteria to inject specific bacterial proteins directly into human, animal and plant cells, typically as part of a successful infection strategy. The medical and economic significance of the secretion process worldwide is enormous. Accordingly an important public health aim is to understand and control the mechanisms of transmission. We study the molecular basis of protein and DNA secretion. Our research applies biochemistry, genetics, structural biology and bioinformatic analyses to reveal the mechanisms involved, with a particular focus on the early steps occurring in donor or infecting bacteria. For both conjugative DNA transfer and virulence related protein transfer, the early steps controlling the start of transfer require a protein-based recognition process that identifies specific protein and DNA complexes for export. The partner receptor protein, which is additionally responsible for controlling access of proteins to the membrane-spanning channel, and for providing the energy for moving these macromolecules, is called the coupling protein. Project P18607 made fantastic progress by identifying the molecular features of a protein that enable its recognition by the export machinery. We have published the first detailed description of structure - function relationships within a recognition feature and its broad conservation in many unrelated bacterial secretion proteins. This knowledge was combined with extensive biochemical reconstitution of several control reactions occurring at transfer start that alter biochemically the coupling protein receptor. Ultimately these changes control how the membrane spanning export machinery is activated. The regulatory interactions discovered provide novel understanding of secretion initiation and present new options for controlled intervention of protein transfer and gene sharing by bacterial conjugation.