Abstract
dc:description.abstractIndole is a heterocyclic signaling molecule, synthesized by a wide array of bacterial species, but most studied in *Escherichia coli*. Indole is formed by the degradation of L-tryptophan through the action of the enzyme tryptophanase (TnaA). The *tnaA* gene is part of the tryptophanase (*tna*) operon, which is regulated by catabolite repression and tryptophan-induced transcription antitermination. Recent studies have unveiled two distinct kinetic modes of indole signalling in *E. coli*: a long-lasting but low-level (persistent) signal, and a transient, high-level (pulse) signal. The underlying mechanisms that regulate the indole pulse phenotype have not been fully characterised. Pulse signalling is thought to be linked to the increased expression of tryptophanase during the transition from exponential to stationary phase. Nonetheless, empirical data from the literature indicates the presence of tryptophanase during early to mid-exponential phase when indole synthesis is low or non-existent. This contradictory evidence indicates the existence of additional regulatory mechanisms controlling tryptophanase activity, and the indole pulse. This work has combined conventional shake-flask culture approaches with single-cell analysis to investigate tryptophanase expression and activity across all *E. coli* growth phases. From these studies a hypothesis has emerged that the indole pulse is triggered, at least in part, by post-translational activation of tryptophanase that results in the surge of indole production. Initial experiments focused on characterising the indole pulse. Significant variability was observed in the timing of the indole pulse in shake-flask culture, its timing varying within a window of about 40 min and its duration ranging from 30 to 45 minutes. The inability experimentally to regulate the timing of the pulse by mutation of the *tna* operon or by plasmid-based tryptophanase expression indicated a post-translational regulatory mechanism might be acting on tryptophanase. Tryptophanase expression measured throughout growth showed tryptophanase protein levels *per* cell are similar in exponential and early stationary phase. This suggests the pulse is triggered by both *de novo* synthesis and activation of pre-existing tryptophanase enzyme. Tryptophanase activity assays on enzyme harvested at different growth phases were consistent with this idea. An indication of the mechanism of post-translational regulation came from the use of single-cell microfluidic studies. Within individual cells, tryptophanase was either concentrated at a polar focus or dispersed throughout the cell. The dispersal of polar foci appears to be critical for the triggering of the indole pulse. As a result of the COVID-pandemic, laboratory access was restricted for a substantial portion of this study, prompting the use of alternative research methods. Consequently, an exploration of tryptophanase distribution and potential evolutionary history was undertaken in *E. coli* and other gut microbial species. The results indicate TnaA is maintained in *E. coli*, and there is evidence that it is a component of the core genome. TnaA extends beyond *E. coli*, being identified in 10% of the bacterial species sequenced in the microbiome. It is present across various phyla of the gut microbiome, including Proteobacteria, Firmicutes, and Bacteroidota.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2024
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Kelly, Ellis
- Advisor dc:contributor.advisor
-
- Summers, David
Subjects
dc:subject × 2Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.112440
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/374345