Fall Fitness Challenge

I have been thinking about fitness recently. Like how to improve my pitiful 9.5 minute mile time (ba-dum-che). Kidding! I have been thinking about how (evolutionary) fitness relates to selection and adaptation and my Streptomyces. I am interested in the evolutionary processes driving diversification between two recently diverged, but distinct, Streptomyces populations. If I can identity different fitness patterns in my isolates under laboratory growth conditions, I hope to identify possible advantages this provides in their natural habitat. Then I can extrapolate and infer potential ecological adaptations driving (or consequence of) the divergence between these populations.

Fitness is a fundamental concept in evolution. In population biology, fitness (often noted ‘w’) is the reproductive success of a genotype in a given environment. In other words, fitness refers to the genetic contribution of an individual to subsequent generations. There are generally two measurements of fitness, absolute fitness and relative fitness. Absolute fitness is the ratio of a given genotype (number of progeny or alleles) in the subsequent generation under different environmental conditions. Relative fitness is a means to normalize between different genotypes and is the number of progeny (or alleles) of one genotype with relation to another genotype in the subsequent generation under the same environmental conditions. The more fit genotype outcompetes the less fit genotype, thus contributing more genetic material to future generations: differential fitness between genotypes is necessary for natural selection to occur.

The reproductive success of an organism can be difficult to quantify, so biologists often use components of fitness as a proxy measurement (e.g. pollen production or size). Microbial systems can be ideal for exploring selection and fitness dynamics since population sizes are so vast, generation times so short, and it’s so easy manipulate growth conditions. For microbes, maximum growth rate of a pure culture is often used as a proxy for absolute fitness. Relative fitness can be assessed by competing two microbial genotypes against each other and measuring the relative contribution of each genotype to the next generation. The latter approach is exemplified by Richard Lenski’s E. coli Long-term Experimental Evolution (LTEE) project. This is brilliant work and should be required reading for both microbiologists and evolutionary biologists.

Streptomyces, life cycle involves the formation of branching mycelia and aerial hyphae.
The life cycle of Streptomyces involves the formation of branching mycelia and aerial hyphae containing spores.                           

Maximum growth rate in microbes (like E. coli) is usually determined by measuring optical density (OD) of liquid cultures. The problem for me is Streptomyces don’t like to grow in liquid media. The cells clump into a flocculent culture. This is partially because Streptomyces don’t follow the canonical bacterial life style, and instead practice a filamentous life style forming branch-like substrate mycelia that elongate from the tips. While there is a substantial body of literature detailing different techniques for measuring growth rate of Streptomyces (like this), I’m not actually interested in growth rate so much as using growth as a proxy for fitness. Wait, what about counting spores? Have you ever tried to count Streptomyces spores?!?!? Instead of going down this painful rabbit hole, I have  explored two approaches for quantifying absolute fitness proxies for Strepotmyces 1) biomass production and 2) growth rate on solid media.

Filters + Biomass #prettyinpurple
Filters + Biomass #prettyinpurple

Biomass Production For this approach, spores are plated onto solid media overlaid with a 0.22 µM filter (made of material Streptomyces can’t eat). Cultures are grown under the desired
condition (e.g. antibiotics, nutrient limitation, iron starvation) and after incubation, filters are oven dried for a few days. Biomass can be determined by subtracting the weight of the dry filter+biomass minus the weight of the. Absolute fitness is inferred from the ratio of an isolate’s biomass production under a control to experimental condition.

Streptomyces cultures at the end of the experiment.
Streptomyces cultures at the end of the experiment.

Growth Rate  For this approach , spores are inoculated into culture plate wells containing solid media. Cultures are again grown under the desired condition. Growth on solid media is measured with a plate reader (ABS 450 nm). Despite heterogeneity in pigmentation and colony morphology, rate of growth seems to fit a linear approximation. Absolute fitness is inferred from the ratio of an isolate’s growth rate under a control to experimental condition.

Each color represents the growth curve of a single Stireptomyces isolate. Lines are linear fit.
Each color represents the growth curve of a single Streptomyces isolate. Lines are linear fit~growth rate.

I think both of these approaches are practical, informative, and provide insight into a complex and extremely important parameter in population biology. #thinkingaboutfitness

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