Date of Completion

Spring 5-10-2009

Thesis Advisor(s)

Joerg Graf

Honors Major

Cell Biology


Cell Biology | Molecular Biology


The digestive tract symbiosis of the medicinal leech, Hirudo verbana, is a model system for studying the genes required for microbial colonization of digestive tracts, as H. verbana has only two species of bacteria that dominate the crop microbiota, Aeromonas veronii and a Rikenella-like bacterium. Signature-tagged mutagenesis (STM) of the A.

veronii strain, HM21R, revealed genes required for the colonization of the digestive tract. One of these mutants, JG573, has an interrupted gene that is predicted to encode a hypothetical protein. The region flanking the transposon insertion of this mutant was sequenced by primer walking.

Comparison of the flanking DNA to the databases revealed similarity to the rhs gene, whose function is not known. PCR was performed to confirm the presence of a transposon insertion by detecting a size difference between

JG573 and the parent strain. The wild type rhs gene was isolated from the fosmid library DNA, and a Southern blot was performed on fosmids obtained from the wild type and mutant DNA. To identify in vitro phenotypes of the mutant, the following experiments were performed: an SDS sensitivity assay, a growth curve, and a screen for two hundred-forty possible phenotypes using Biolog plates. The ability of the mutant to colonize the leech digestive tract was assessed in a competition assay, which revealed that this mutant had no colonization defect at 18 h after feeding but had a dramatic defect by 24 h. These data suggest that an important physiological change occurs during this time that affects the viability of this mutant inside the leech.