Title

Quantification of factors influencing the sporulation phenotype in Myxococcus xanthus

Date of Award

August 2016

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

Advisor(s)

Roy Welch

Subject Categories

Life Sciences

Abstract

When starved, Myxococcus xanthus initiates a multifaceted developmental program that culminates in the formation of spore-filled structures called fruiting bodies. When characterizing a strain, a threshold sporulation efficiency frequently serves as the defining metric for developmental competence. While the sporulation assay does provide a quantitative measure of an important phenotypic trait, it displays a high degree of variability even between experimental replicates. This variability means that, while mutant strains displaying strong sporulation defect are easy to detect, ones with more subtle defects are impossible to differentiate from wild-type.

The first aim of this work was to optimize the M. xanthus sporulation assay by determining the source of error associated with each variable, thereby establishing a more comprehensive understanding of how each variable influences spore recovery. These variables, such as incubation temperature, sonication time and intensity, plating dilutions, and germination time, were all tested individually. After maximizing spore recovery and minimizing the variability between independent replicates of wild-type, we characterized a small subset of mutant strains with known sporulation phenotypes to demonstrate that these optimized conditions improve the reproducibility of the spore assay for both mutant and wild-type strains.

The first aim examined the influence of known variables on the sporulation assay; the second aim examined an unknown variable that may also have influence on sporulation. Specifically, we tested how the distribution of cells affected sporulation by altering the surface-to-volume ratio of a starving population. We were able to show a significant change in the spore recovery of wild-type, indicating that the cell population size is an important new variable. We also demonstrated that spore recovery was influenced by the spacing and arrangement of separate populations too far apart to physically interact. This new variable, which we have deemed the “1-3-5 effect”, can be altered through mutation, and requires populations to be placed on the same agar surface. Our current hypothesis is that the 1-3-5 effect is caused by overlapping gradients of a diffusible signal within the agar substrate.

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