La familia de plásmidos pLS20 y la regulación de sus genes de establecimiento

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 25-02-2021Esta tesis tiene embargado el acceso al texto completo hasta el 25-08-2022The spread of antibiotic resistance genes (ARGs) is a major threat to global health. Horizontal gene transfer (HGT) events, and particularly the route of bacterial conjugation, are mainly responsible for the dissemination of ARGs. Conjugation is the process by which a conjugative element is transferred from a donor to a recipient cell through a connecting channel. Conjugative elements are often located on plasmids. Conjugative plasmids are widespread in Gram-positive (Gram+) and Gram-negative (Gram�) bacteria. Most conjugation studies are based on conjugative plasmids from Gram� bacteria though. In our laboratory, we study as a model the conjugative plasmid pLS20 from the Gram+ bacterium Bacillus subtilis. One main objective of this work was to determine the rareness of pLS20; is it a unique plasmid or does it belong to a family of related plasmids. Results obtained in these thesis studies show that pLS20 is the prototype of a new family of conjugative plasmids that is present in several Bacillus species isolated from diverse geographic locations and niches, suggesting that their distribution is worldwide. The pLS20 family of plasmids is characterised by an unusual (i) origin of replication region, that does not encode for a typical replication initiation protein, which is flanked on its left side by (ii) a partitioning system, and on its right side by (iii) a large conjugation operon. Interestingly, the conjugation module of pLS20 is also (partially) present on other Bacillus plasmids as well as on plasmids from other Gram+ bacteria including pathogens such as Listeria, Enterococcus and Clostridium species, indicating that this conjugation module is widely distributed among conjugative plasmids hosted by bacteria of the phylum Firmicutes. Bacteria contain systems to defend themselves against incoming foreign DNA. Establishment of a plasmid in the recipient host after conjugative transfer requires a transient inactivation of these host-defence mechanisms. Many conjugative plasmids contain so-called establishment genes that fulfil this role. In this doctoral thesis, I identified the putative establishment genes present on members of the pLS20 family of plasmids. The establishment genes on these plasmids are organised in a regulon that is composed of several operons. Remarkably, there is a wide variation in the number and kind of establishment genes on the different plasmids. Two fundamentally different regulatory mechanisms control the expression of these regulons. Plasmids pLS20 and p576 are the two prototype plasmids reflecting these alternative mechanisms. In the case of p576, the plasmid encodes a repressor protein (called Reg) that effectively suppresses the establishment promoters. During the conjugation, the DNA but not the repressor is transferred. Consequently, the establishment promoters are transiently active in the recipient cell until sufficient repressor has been synthesised to repress them again. In the case of pLS20, the establishment genes are preceded by a conserved large upstream element (CLUE) that most likely is responsible for transient expression of the establishment genes by a yet unknown mechanis