1. Dr. Kimberly Murphy, Mackenzie Ryan, Francesca Scribano
Effects of Evolution on Laboratory Sublines of Myxococcus xanthus DK1622
Dr. Kimberly Murphy, Mackenzie Ryan, Francesca Scribano
Photo courtesy of L. Ritchie

2. Background/Relevance
Bacterial characteristics
Rod-shaped
Gram negative
Cytoplasmic and outer membrane
Multicellular phenotypes
Fruiting body formation, rippling, motility
Why is M. xanthus a good model organism
Social Behavior/Motility
Reproduction Rate
Non-pathogenic
Cellular patterns of M, xanthus cells with 1mm scale bars. © 2009 Federation of European Microbiological Societies

3. A motility Adventurous Hard, dry surfaces
Higher agar concentration
1.5%
Movement of single cells or small groups of cells
Unknown propulsion mechanism; slime extrusion
Thinner flare edge
Swarming colonies micrograph; adventurous motility. Microbiology: Shi and Zusman. Proc. Natl. Acad. Sci.

4. S motility Social Softer surfaces Movement of cells in groups
Lower agar concentration
0.4%
Movement of cells in groups
Achieved through the attachments and retraction on type IV pili
Thicker flare at the colony edge
Swarming colonies micrograph; adventurous motility. Microbiology: Shi and Zusman. Proc. Natl. Acad. Sci.

5. Original Wild Type
Subline
Subline

6. Our Research Question
Will disrupting the same gene in three wild-type DK1622 sublines result in the same phenotype?

8. Methods Plasmid Miniprep Restriction Digest Plasmid Dialysis
Isolate and purify plasmid DNA from E.coli
Restriction Digest
Confirm the presence of gene fragment of interest
Plasmid Dialysis
Reduce salt concentration before electroporation
Electroporation
Introduce plasmid into M. xanthus
Motility Assays
Measure swarm diameter size on different CTTYE-Kan agar plates (A vs S motility). Compare swarm sizes between sublines

9. Complications and Limitations
Population
During each step of the experiment, there is the possibility for a reduction in cell count
Time constraints
Scheduling conflicts
Requirement of extended time frames

10. Results: A motility
1.5% agar
Averages (in cm)
WT Shimkets
1.5
1.4
1.6
1.7
S2020
M2020
WT Kirby
1.8
1.9 2
No significant difference in swarm size between WT Shimkets and Shimkets 2020

11. Results: S Motility
0.4% agar
Averages (in cm)
WT Shimkets
2.2
2.5
2.3
2.4
2.8
2.6
S2020
2.1
2.7 2
1.9
M2020
3.6
3.4
3.3
2.9
WT Kirby 3
No significant difference in swarm size between WT Shimkets and Shimkets 2020

12. Conclusions
No significant differences observed between wild type and mutant sublines
A and S motility
Morphological differences were noted
Larger transparent edge (WT Kirby)
Indicates some varied response to gene disruption
Could be true of other laboratory bacteria

13. Future Directions Compare WT and mutant sublines in Murphy and Kirby
Restreak Murphy WT
Remake Kirby and Shimkets 1548 strains
Grow remaining freezer stocks
Fruiting body development
Sporulation assays

14. Acknowledgements Funding: NSF, Augustana College Charlie Bentley
Linnea Ritchie
Roy Welch lab
Research partners: Marie Skuby & Eliot Kmiec

15. References
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