Identification and characterization of Haloferax vocanii hypermotile mutants

Michiyah examining a sample.




Professor of Biology

Project Summary

This summer, I had the great opportunity to carry out research in the Pohlschroder Lab under Mechthild Pohlschroder’s supervision. Dr. Pohlschroder’s lab studies Archaea, prokaryotic microorganisms that are likely the closest relatives to eukaryotes, yet are significantly less studied than organisms from the second prokaryotic domain of life, the Bacteria. In particular, the lab uses Haloferax volcanii as the model organism to get a better understanding of archaeal cell surface biosynthesis and function. For my project, I tried to identify mutants that are unable to change from rod- to disk-shaped cells, a morphology change required for H. volcanii to form effective biofilms. Disk-formation mutants had previously been identified in a screen for hypermotile transposon (Tn) mutants. Hence, I set out to design an effective screen for hypermotile Tn mutants by streaking the Tn library in the center of a motility plate and characterizing those mutants that moved fastest to the edges of the plates. The screen allowed me to successfully isolate 30 hypermotile tn mutants in less than a month. Streaking the mutants onto 3.5% agar plates also revealed that the vast majority of these mutants made smaller and darker colonies compared to the wild-type. Using DIC microscopy, I was also able to show that 20 tn-mutants lack the ability to make disks and appear to be partially defective in disk-formation. Interestingly, some mutants also regained the ability to make disks, suggesting that the switching of shapes is critical for H. volcanii, resulting in the accumulation of suppressor mutations. The mutants are currently being characterized for their ability to form biofilms using the Air-liquid-interface assay routinely carried out in the Pohlschroder lab. Moreover, I am purifying the DNA of these strains to be submitted for genome sequencing to identify the genes disrupted by the Tn.

From this project, I learned how to conduct research for more information related to my subject area and how to create a project plan that contains set goals, deadlines for my project, and when to review my research. I created a database that documents all different hypermotiles identified with their colony color and size and cell shape. This research project also taught me the importance of being organized and detail-oriented. I learned the importance of patience because some experiments did not work at all due to unforeseen obstacles – for example, at some point none of the motility plates in the lab worked, likely due to some difference in the water source. I also learned that some unexpected results, such as the suppressor mutants, can open a new opportunity to obtaining a better understanding of a system. Rather than ignoring the suppressors, we are now eager to identify the SNPs in those suppressors that may provide a better understanding of the proteins involved in cell-shape switching. This project also contributed to my educational experience by learning the molecular, cell biology and microscopy techniques, which will be invaluable as a Molecular and Cellular Biology Major.