Increased copy number couples the evolution of plasmid horizontal transmission and plasmid-encoded antibiotic resistance

Conjugative plasmids are mobile elements that spread horizontally between bacterial hosts and often confer adaptive phenotypes, including antimicrobial resistance (AMR). Theory suggests that opportunities for horizontal transmission favor plasmids with higher transfer rates, whereas selection for plasmid carriage favors less-mobile plasmids. However, little is known about the mechanisms leading to variation in transmission rates in natural plasmids or the resultant effects on their bacterial host. We investigated the evolution of AMR plasmids confronted with different immigration rates of susceptible hosts. Plasmid RP4 did not evolve in response to the manipulations, but plasmid R1 rapidly evolved up to 1,000-fold increased transfer rates in the presence of susceptible hosts. Most evolved plasmids also conferred on their hosts the ability to grow at high concentrations of antibiotics. This was because plasmids evolved greater copy numbers as a function of mutations in the copA gene controlling plasmid replication, causing both higher transfer rates and AMR. Reciprocally, plasmids with increased conjugation rates also evolved when selecting for high levels of AMR, despite the absence of susceptible hosts. Such correlated selection between plasmid transfer and AMR could increase the spread of AMR within populations and communities.

Increased copy number couples the evolution of plasmid horizontal transmission and antibiotic resistance

Antimicrobial resistance (AMR) in bacteria is commonly encoded on conjugative plasmids, mobile elements which can spread horizontally between hosts. Conjugative transfer disseminates AMR in communities but it remains unclear when and how high transfer rates evolve, and with which consequences. Here we studied experimentally the evolution of two antibiotic resistance encoding plasmids when confronted to different immigration rates of susceptible, plasmid-free hosts. While plasmid RP4 did not evolve detectably, plasmid R1 rapidly evolved up to 1000-fold increased transfer rates in the presence of susceptible hosts, at a cost to its host. Unexpectedly, most evolved plasmids also conferred to their hosts the ability to grow at high concentrations of antibiotics. The most common mutations in evolved plasmids were contained within the copA gene which controls plasmid replication and copy number. Evolved copA variants had elevated copy number, leading to both higher transfer rates and AMR. Due to these pleiotropic effects, host availability and antibiotics were each sufficient to select for highly transmissible plasmids conferring high levels of antibiotic resistance.

Cryo-EM reconstruction of AlfA fromBacillus subtilisreveals the structure of a simplified actin-like filament at 3.4-Å resolution

Low copy-number plasmid pLS32 ofBacillus subtilissubsp.nattocontains a partitioning system that ensures segregation of plasmid copies during cell division. The partitioning locus comprises actin-like protein AlfA, adaptor protein AlfB, and the centromeric sequenceparN. Similar to the ParMRC partitioning system fromEscherichia coliplasmid R1, AlfA filaments form actin-like double helical filaments that arrange into an antiparallel bipolar spindle, which attaches its growing ends to sister plasmids through interactions with AlfB andparN. Because, compared with ParM and other actin-like proteins, AlfA is highly diverged in sequence, we determined the atomic structure of nonbundling AlfA filaments to 3.4-Å resolution by cryo-EM. The structure reveals how the deletion of subdomain IIB of the canonical actin fold has been accommodated by unique longitudinal and lateral contacts, while still enabling formation of left-handed, double helical, polar and staggered filaments that are architecturally similar to ParM. Through cryo-EM reconstruction of bundling AlfA filaments, we obtained a pseudoatomic model of AlfA doublets: the assembly of two filaments. The filaments are antiparallel, as required by the segregation mechanism, and exactly antiphasic with near eightfold helical symmetry, to enable efficient doublet formation. The structure of AlfA filaments and doublets shows, in atomic detail, how deletion of an entire domain of the actin fold is compensated by changes to all interfaces so that the required properties of polymerization, nucleotide hydrolysis, and antiparallel doublet formation are retained to fulfill the system’s biological raison d’être.