Date of Award

Winter 2016

Project Type

Thesis

Program or Major

Genetics

Degree Name

Master of Science

First Advisor

Cheryl A Whistler

Second Advisor

Estelle M Hrabak

Third Advisor

William K Thomas

Abstract

Iron acquisition is well studied in pathogens, and successful virulence is often attributed to iron acquisition by siderophore and heme uptake; however, the role of iron uptake in mutual symbiotic interactions is not as well understood. The mutual symbiosis between Vibrio fischeri and the Hawaiian bobtail squid, Euprymna scolopes, is a well-characterized system in which iron uptake has been implicated as a symbiotic factor. Four studies have implicated iron uptake in the symbiosis: 1) A TnLux reporter assay revealed that siderophore is more highly expressed by V. fischeri in the light organs of juvenile squid compared to V. fischeri in liquid culture; 2) Microarray data showed that genes for siderophore production are upregulated in the light organs of adult squid; 3) A siderophore deficient glnD mutant of V. fischeri had a persistence defect in the light organ that was complemented by addition of iron to the seawater; and 4) A V. fischeri mutant in which the heme uptake locus was deleted had a persistence defect in the squid light organ that was apparent in competition with the ancestor strain V. fischeri ES114. I hypothesize that iron uptake by siderophore is necessary for persistence of V. fischeri in the squid light organ, complementary to heme uptake, and that due to the toxic nature of iron, sequestration by siderophore contributes oxidative stress response.

To assess the role of iron uptake in the interaction between V. fischeri and the Hawaiian bobtail squid we utilized several strategies: 1) I identified itron uptake systems available to V. fischeri by bioinformatically comparing known iron uptake systems against the genome; 2) To reveal potential avenues by which iron uptake is regulated in V. fischeri, we identified genes that influence siderophore biosynthesis by screening a transposon mutant library for siderophore phenotypes; 3) I assessed the physiological role, in growth and oxidative response, of several of the iron uptake genes previously identified; and 4) I directly assessed the symbiotic ability of mutants deficient in iron uptake.

The bioinformatic search revealed several siderophore uptake systems, as well as the previously described heme uptake system; however, only one siderophore biosynthesis system, for aerobactin, was identified. In screening the mutant library, I identified many genes in the flagellar locus and the cellular biosynthesis locus that positively influence siderophore production as well as two quorum sensing genes, AinS and RpoQ, and several cell wall biogenesis/oxidative sensing genes that negatively influence siderophore production. We determined that aerobactin biosynthesis does not contribute to oxidative stress response but does contribute to growth in iron limiting conditions, suggesting a purely nutritional role for siderophore in the symbiosis. When we tested the symbiotic ability of an iucA mutant deficient in siderophore, we could not demonstrate a persistence defect; however, we did find that two siderophore uptake mutants have a competitive defect 24 hours after inoculation, suggesting that siderophore contributes to symbiotic fitness. These findings suggest that regulation of iron uptake in V. fischeri involves more than just response to iron levels, and that iron uptake regulation is intertwined with symbiotically relevant traits. Due to the monospecific nature of the symbiosis, it is unlikely that the non-aerobactin uptake systems contribute to the symbiotic ability of V. fischeri; however, it is clear that aerobactin does contribute to symbiotic ability by conferring a growth advantage over other strains deficient in aerobactin uptake.

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