Summer Virtual Research in Miller MEE Laboratory, Yeseo Kwon '21
I resumed my previous research in creating a novel zinc-regulated gene expression system for S. suis to serve as a neutral marker that will distinguish and quantify different strains for experimental evolution experiments in the future.
Benjamin (Yeseo) Kwon
Virtual Summer Research Blog Post
PI: Dr. Eric Miller
Steppingstone for Microbial Experimental Evolution:
Developing a Zinc-Regulated Expression Marker for Streptococcus suis
The field of experimental evolution has led a renaissance in our understanding of ecological diversity and evolution through the use of microbes that are small, easy to maintain, and have short generation times (Bachmann et al., 2017). Fitness is a relative measure of the success of replication of organisms when competing for the same resource, often measured through changes in population ratio over several generations when put in competition with one another (competition assays) (Bachmann et al., 2017). Experimental evolution is characterized by recording and quantifying this fitness across hundreds or thousands of generations, usually under a biotic or abiotic stressor (stressors make it difficult for an organism to survive and reproduce, thus creating a “need” for evolution to take place). Many generations are required for adaptive mutations to occur that increases the bacteria’s fitness. The field has a common goal: to explore how the network of molecular interactions within the organism influences evolutionary change in genes as well as in their expression (Rainey et al., 2017).
In order to measure competition assays throughout an experimental evolution experiment, there must be a way to tell populations apart from each other. In many cases, antibiotics are used to isolate certain populations that have resistances from other populations that do not. However, for bacteria, producing antibiotic resistance come at a fitness cost that may tamper with the process of evolution itself and the impact of dependent variables. Therefore, a control system is needed that will act as a switch to turn off the expression of antibiotic resistance genes during the evolution process and turn on the expression when it is time to measure a competition assay.
The model organism I will be experimenting on is S. suis, an anaerobic, Gram-positive, nonmotile coccus (circular bacteria) that lives in the upper respiratory tract and the tonsils of swine (Merck Veterinary Manual). S. suis is also a zoonotic pathogen that not only is a major cause of bacterial mortality in pigs, but also capable of transmission into humans. The pathogen is known to cause meningitis, septicemia, endocarditis, and often deafness (Feng et al., 2014; Rayanakorn et al., 2018). Beyond its clinical significance, the pathogen is known for its extensive diversity of known and sequenced strains that makes it an optimal model to study the cross-strain differences in evolutionary trajectories (Weinert et al., 2015). Most importantly, the bacterium is known to be naturally transformable, capable of up-taking foreign DNA at significantly higher rates than other common models such as Escherichia coli.
Over the course of last summer, under the guidance of professor Eric Miller, I worked on developing a near neutral, zinc regulated inducible marker for Streptococcus suis. I may have already introduced a couple unfamiliar terms. What do I mean by “near neutral”? Evolutionary biologists rely on proper quantification of fitness levels to ground their findings. If an expression or knockout of a gene has a strong negative effect on the organism’s fitness, the gene is said to have a high fitness cost. However, if tempering with the gene has negligible fitness cost, it is said to be “near neutral”.
This marker operon is adopted from the zinc-efflux sczA+czcD system of Streptococcus pneumoniae, which tightly represses the expression of a zinc-efflux protein in the absence of added zinc (an operon is a set of adjacent genes and regulatory signals that affect transcription of said genes). This operon that we designed, once integrated into S. suis chromosome, will consist of a fluorescent protein unique to a specific strain, and antibiotic resistances (as a redundant screen) that are both expressed only in the presence of zinc. Hence, the gene cassette will act as a near-neutral, latent marker in the absence of zinc.
By creating an efficient and effective method of quantifying and differentiating populations of S. suis in competition assays, I hope to lay a strong groundwork, a “stepping stone,” for future experimental evolution experiments in the Microbial Ecology and Evolution (MEE) laboratory.