Relaxases, key enzymes in bacterial conjugation

Bacterial conjugation is the primary mechanism of horizontal transmission of genetic information within bacterial communities. It allows for the rapid spread and establishment of genes that are necessary for the survival of bacteria under stress conditions. In the case of pathogenic bacteria, acquisition of multi-resistance plasmids renders antibiotic treatments of infectious bacterial diseases useless. To address the world-wide problem of the spread of multi-resistant plasmids within pathogenic bacterial populations, an EU-consortium was founded to identify specific inhibitors of bacterial conjugation (COINS). This approach to the control of infectious diseases targets the spread of antibiotic resistance rather than the pathogenic bacteria themselves. In preparation and in support of the European effort we propose to carry out a comprehensive structural and biochemical characterization of key enzymes of bacterial conjugation. Plasmid-encoded relaxases and their auxiliary DNA binding proteins, which are integral components of the initiation stage of plasmid transfer, were selected. 1. The primary objective of this project is the structural analysis through X-ray diffraction. 2. In addition to macromolecular crystallography, biophysical techniques such as circular dichroism (CD), fluorescence and biocalorimetry (DSC and ITC) will be used in this study to verify the integrity of the purified proteins, and to determine their biophysical characteristics and DNA-binding behavior. We will focus on relaxosomes of self-transmissible IncF and IncP plasmids and family 2 of rolling circle replicating (RCR) mobilizable plasmids. These systems are well studied. They encompass a variety of host range specificities including Gram-negative and Gram-positive bacteria and are expected to exhibit some mechanistic diversity. The structure determination of relaxosomal components will help to elucidate the mechanisms that initiate and terminate the plasmid DNA strand transfer process. Furthermore the structure of relaxases and accessory conjugation proteins can provide the basis for a rational design of conjugation inhibitors (COINS) and for improvement of relaxase-specific lead compounds discovered by the COINS project.

Bacterial conjugation is the primary mechanism of horizontal transmission of genetic information within bacterial communities. It allows for the rapid spread and estab-lishment of genes that are necessary for the survival of bacteria under stress condi-tions. In the case of pathogenic bacteria, the acquisition of multiresistance plasmids renders antibiotic treatments of infectious bacterial diseases useless. Relaxases are the key enzymes of bacterial conjugation. Together with a number of accessory proteins they form a complex (the relaxosome) which is responsible for key steps in the bacterial conjugation (site specific cleavage of the plasmid DNA, transfer of the DNA from the donor to the acceptor cell, termination of the transfer). In this project we focussed on the biophysical and structural characterization of re-laxases of the self transmissible IncF and IncP plasmids, and the relaxases of plas-mids derived from gram-positive bacteria, MobM of plasmid pMV158 and TraA of plasmid pIP501. Constructs for the expression of the full length relaxases as well as truncation mutants were provided by our collaborators. The expression systems were established in our laboratory and expression and purification schemes on a prepara-tive scale were developed. The purified proteins were investigated with biophysical methods (CD spectroscopy, fluorescence spectroscopy, etc.) in order to establish the solubility and the correct folding of the proteins. DNA binding studies were performed with TraA (pIP501), TraI (RP4), and TraI (plasmid R1). Intensive crystallization trials were performed with relaxases as well as relaxase DNA complexes. During the last year of this project, crystallization trials of an S-layer protein, SbsC of Geobacillus stearothermophilus, were successfully performed. Crystals of two trunca-tion mutants of this S-layer protein could be obtained and data for it`s structure de-termination were collected.