Delaunay, C. | bioRxiv Science

bioRxiv2024

Within-host environmental heterogeneity is associated with phenotypic but not genomic diversity in Wolbachia endosymbionts

The mechanisms underlying within-host diversification in parasite populations are poorly understood. Yet, phenotypic and genotypic variation in parasites can shape their evolutionary trajectories and have important epidemiological consequences. Our aim was to determine whether the constraints associated with infecting different host tissues lead to the emergence and coexistence of multiple parasite sub-populations with distinct phenotypes. We tested this hypothesis using the most widespread bacterial endosymbiont, Wolbachia. We injected Wolbachia bacteria isolated from three tissues of the common pill-bug (Armadillidium vulgare) into uninfected individuals and monitored the growth rate and virulence of each bacterial sub-population in the new hosts. Our results highlight that within-host tissue heterogeneity leads to diverse Wolbachia phenotypes. High-depth genome re-resequencing of Wolbachia sub-populations revealed that this polymorphism was not due to genomic variation but was more likely a result of phenotypic plasticity. Indeed, we found no recurrent tissue-specific genomic variation among infected individuals. Our single nucleotide polymorphism (SNP) filtration pipeline, developed to ensure SNP validity, detected only one substitution. This Wolbachia variant, observed in one female, was present in all three bacterial sub-populations, with frequencies ranging from 24% to 58% depending on the tissue. Overall, our results support the stability of the Wolbachia genome with respect to the rarity of point mutations, in agreement with reports from other symbiotic systems. From a methodological perspective, our study highlights the need for considerable caution when detecting variants in endosymbiont populations, as our conservative approach led us to exclude more than 99.5% of the initially called variants.

evolutionary biology↗

bioRxiv2021

A uniform benchmark for testing ssrA-derived degrons in the Escherichia coli ClpXP pathway

The ssrA degron is commonly used in fusion proteins to control protein stability in bacteria or as an interaction module. These applications often rely on the modular activities of the ssrA tag in binding to the SspB adaptor and in engaging the ClpXP protease. However, a comparison of these activities for a substantial standard set of degron variants has not been conducted previously, which may hinder developments of new variants optimized exclusively for one application. Here, we strive to establish a benchmark that will facilitate the comparison of ssrA variants under uniform conditions. In our workflow, we included methods for expression and purification of ClpX, ClpP, SspB and eGFP-degrons, assays of ClpX ATPase activity, of eGFP-degron binding to SspB and for measuring eGFP-degron degradation in vitro and in vivo. Using uniform, precise and sensitive methods under the same conditions on a range of eGFP-degrons allowed us to determine subtle differences in their properties that can affect their potential applications. Our findings can serve as a reference and a resource for developing targeted protein degradation approaches. SUMMARYThis work lays standards for assays used to compare engagement of SspB and ClpXP by a set of ssrA-derived degrons that can be used to fine-tune tools for protein stability control.

biochemistry↗