Essential gene acquisition destabilizes plasmid inheritance.

Extra-chromosomal genetic elements are important drivers of evolutionary transformations and ecological adaptations in prokaryotes with their evolutionary success often depending on their 'utility' to the host. Examples are plasmids encoding antibiotic resistance genes, which are known to proliferate in the presence of antibiotics. Plasmids carrying an essential host function are recognized as permanent residents in their host. Essential plasmids have been reported in several taxa where they often encode essential metabolic functions; nonetheless, their evolution remains poorly understood. Here we show that essential genes are rarely encoded on plasmids; evolving essential plasmids in Escherichia coli we further find that acquisition of an essential chromosomal gene by a plasmid can lead to plasmid extinction. A comparative genomics analysis of Escherichia isolates reveals few plasmid-encoded essential genes, yet these are often integrated into plasmid-related functions; an example is the GroEL/GroES chaperonin. Experimental evolution of a chaperonin-encoding plasmid shows that the acquisition of an essential gene reduces plasmid fitness regardless of the stability of plasmid inheritance. Our results suggest that essential plasmid emergence leads to a dose effect caused by gene redundancy. The detrimental effect of essential gene acquisition on plasmid inheritance constitutes a barrier for plasmid-mediated lateral gene transfer and supplies a mechanistic understanding for the rarity of essential genes in extra-chromosomal genetic elements.

The role of the cytoskeletal proteins MreB and FtsZ in multicellular cyanobacteria.

Multiseriate and true-branching cyanobacteria are at the peak of prokaryotic morphological complexity. However, little is known about the mechanisms governing multiplanar cell division and morphogenesis. Here, we study the function of the prokaryotic cytoskeletal proteins, MreB and FtsZ in Fischerella muscicola PCC 7414 and Chlorogloeopsis fritschii PCC 6912. Vancomycin and HADA labeling revealed a mixed apical, septal, and lateral trichome growth mode in F. muscicola, whereas C. fritschii exhibits septal growth. In all morphotypes from both species, MreB forms either linear filaments or filamentous strings and can interact with FtsZ. Furthermore, multiplanar cell division in F. muscicola likely depends on FtsZ dosage. Our results lay the groundwork for future studies on cytoskeletal proteins in morphologically complex cyanobacteria.

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach.

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial populations. This is especially the case under rapidly changing environments such as fluctuating antibiotics exposure. We recently showed that plasmids maintain antibiotic resistance genes in Escherichia coli without positive selection for the plasmid presence. Here we describe an experimental system that allows following both the plasmid genotype and phenotype in long-term evolution experiments. We use molecular techniques to design a model plasmid that is subsequently introduced to an experimental evolution batch system approach in an E. coli host. We follow the plasmid frequency over time by applying replica plating of the E. coli populations while quantifying the antibiotic resistance persistence. In addition, we monitor the conformation of plasmids in host cells by analyzing the extent of plasmid multimer formation by plasmid nicking and agarose gel electrophoresis. Such an approach allows us to visualize not only the genome size of evolving plasmids but also their topological conformation-a factor highly important for plasmid inheritance. Our system combines molecular strategies with traditional microbiology approaches and provides a set-up to follow plasmids in bacterial populations over a long time. The presented approach can be applied to study a wide range of mobile genetic elements in the future.