The initial steps of the pLS20 conjugation process: insights into the mechanisms of recipient cell selection and attachment by donor cells

Abstract

Conjugation is the process by which a DNA element is transferred from a donor to a recipient cell through a connecting channel. Conjugation is the principal horizontal gene transfer route that is responsible for the spread of antibiotic resistance, toxin and virulence genes, which is a major global health threat. A better understanding of the conjugation process is required to design strategies or drugs that can impede conjugation-mediated spread of these pernicious genes. Although conjugation occurs in Gram-positive and Gram-negative bacteria, most conjugation studies are based on a limited number of conjugative elements of Gram-negative bacteria. The conjugation process can be divided in different stages that include selection of a plasmid-free recipient cell, attachment of a donor to a recipient cell known as mating pair formation, generation of a connecting channel, and processing and transfer of the DNA from the donor to the recipient cell. Some of these stages appear to be conserved between Gram- negative and -positive bacteria. However, due to the fundamental differences in bacterial cell walls, particularly the initial stages of recipient selection and mating pair formation may be different. Most, if not all, conjugative elements of Gram-negative bacteria produce appendages, named conjugative pili, whose main function is to form mating pairs. Very little is known about mating pair formation by conjugative elements of Gram-positive bacteria. Typical pili associated with conjugation in Gram-negative bacteria have not been observed though for conjugation systems in Gram-positive bacteria suggesting that mating pair formation occurs in a different way(s). The transfer of a conjugative element from one donor to another donor cell affects the efficiency of conjugation. For conjugative elements of Gram-negative bacteria it is has been shown that so-called exclusion systems have evolved that inhibit redundant transfer of conjugative elements between donor cells. Conjugative elements of Gram-positive bacteria are likely to encode also exclusion systems, but these have been studied very little. In this thesis, the initial stages of the conjugation process being mating pair formation and exclusion were studied using the conjugative plasmids pLS20 of the Gram-positive bacterium Bacillus subtilis. Regarding mating pair formation, results were obtained showing that pLS20 contains an exclusion system that is based on a single gene, which we named ses. Ses is the second gene in the large conjugation operon of pLS20 and encodes a surface-located protein. Intriguingly, the level of exclusion is correlated to the amount of Ses protein, and ses is expressed at much higher levels in donor cells having activated the conjugation process than in non-primed donor cells. Due to these differential expression levels, redundant transfer is inhibited very efficiently between two conjugation-primed donor cells, but only moderately between a conjugation primed and non-primed donor cell. Such a differential expression pattern probably occurs also for many or perhaps all other exclusion systems. We propose that that this differential expression serves two purposes: while it favours efficient conjugation, it also allows evolutionary adaptation of conjugative plasmids by not fully blocking redundant transfer. Important progress was also made with respect to mating pair formation of pLS20. It was shown that the seventh gene of the conjugation operon is an adhesion gene containing a thio-ester domain in the N-terminal region of the exported protein. Thio-ester domains are reactive groups permitting the formation of a covalent binding of the protein with another protein via a thio-ester bond. Thio-ester domains have only recently been described to be present in a subgroup of adhesion proteins of Gram-positive bacteria, where they play important roles in adhesion of commensal bacteria to host tissue and in infection o pathogenic bacteria. This is the first time in which an adhesion protein containing a thioester domain has been shown to be crucial for efficient conjugation