Date of Award

2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

Abstract

Nitric oxide (NO) is an important signal secreted by the squid, Euprymna scolopes, and is thought to limit symbiotic biofilm formation, the initial step in colonization by the bacterium, Vibrio fischeri. Previous work demonstrated that a mutant defective for NO-sensor HnoX (heme nitric oxide/oxygen binding) had a competitive advantage over wild-type V. fischeri, but the mechanism by which this occurred remains unknown. HnoX is encoded upstream of hahK, a recently identified positive regulator of biofilm formation. I thus hypothesized that HnoX inhibits colonization by controlling HahK-induced biofilm formation during the initiation of colonization.

I assessed the impact of an hnoX deletion on biofilm formation in various genetic backgrounds by evaluating in vitro read-outs for biofilm formation, namely: wrinkled colony development, pellicle formation, and cell clumping. Deletion of hnoX resulted in early biofilm formation dependent on active alleles of hahK. I also generated an increased activity allele of hnoX that severely delayed wrinkled colony formation. Finally, I found that the addition of a NO-generator abrogated pellicle formation and cell clumping. NO-dependent inhibition of biofilm formation required hnoX and occurred at the level of transcription.

These results suggest NO-bound HnoX inhibits the activity of HahK, thus inhibiting biofilm formation. This work has uncovered the first host-relevant signal controlling biofilms and a mechanism for inhibition of biofilm formation by V. fischeri. The study of HnoX permits

us to understand not only the mechanism for control of biofilm formation in the context of a host, but also the function of HnoX domain proteins as regulators of important bacterial processes.

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Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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