Worldwide dissemination of multidrug resistance (MDR) within bacterial populations is a major issue in public health. One of the main driving forces of MDR dissemination within bacterial populations is horizontal gene transfer of conjugative plasmids.
Conjugative plasmids encode genes necessary and sufficient for their horizontal propagation via conjugation, but also for their maintenance in a growing population by vertical transfer to the daughter cells during cell division (segregation). Segregation systems locate plasmid copies in the two daughter cells prior to cell division. They are composed of three elements - two genes encoding an NTPase and a DNA binding protein, and a cis-acting centromeric site. However, the extensive sequencing of biological samples has revealed that an increasing number of low copy number plasmids do not encode canonical segregation systems, thus questioning on how they are maintained in a bacterial population. Our studies show that among them, plasmid R388 encodes segregation system of a new type consisting of a single plasmid-encoded DNA binding protein, StbA, and a specific cis-acting site, called stbDR.
Moreover, albeit segregation and conjugation are critical processes for the prevalence of plasmids, they require exclusive subcellular dynamic behaviors of plasmids within the bacterial cell: conjugation requires the recruitment of the plasmid at the cell membrane, while plasmid copies are positioned inside the nucleoid during segregation. Our data hint for the first time a "molecular switch" responsible for alternating these two processes, the Stb system. Indeed, StbA, together with another protein encoded by the stb operon, StbB, control conjugation in an opposite but interdependent fashion. While deletion of the entire stb operon does not affect conjugation, a large increase (50-fold) or complete inhibition of the frequency of conjugation is observed with the deletion of stbA or stbB, respectively. The stb operon is carried by many plasmids, including our R388 conjugative plasmid model, which belongs to the IncW group of broad-host conjugative plasmids found in many bacterial genera, most Proteobacteria (including multidrug-resistant pathogens from the ESKAPE group responsible for the majority of bacterial infections in humans). Our study also shows that a single mutation, for example leading to the inactivation of StbA, can lead to the emergence of plasmids with increased ability to spread ("superspreaders"). An integrated understanding of the molecular mechanisms for conjugation, segregation and their interplay at single cell level is essential for a proper action to fight against MDR dissemination.
We use a multi-scale approach to understand how the Stb system controls plasmid propagation, including biochemistry, genome-wide molecular genetics, crystallography, fluorescence microscopy (classical or coupled to a microfluidic system, and super resolution), etc ...