Date of Award

12-11-2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Dr. George E. Pierce

Second Advisor

Dr. Nicole Lopanik

Third Advisor

Dr. Eric Gilbert

Fourth Advisor

Dr. Sidney Crow

Abstract

The marine bryozoan Bugula neritina is the source of the bioactive polyketide compounds, the bryostatins. The biosynthesis of the bryostatins is putatively prescribed by the Type I modular polyketide synthase (PKS), bry, from the uncultured bacterial symbiont “Candidatus Endobugula sertula”. The bry gene cluster has a non-canonical, discrete acyltransferase (AT) enzymatic domain upstream of the PKS gene cluster. The AT is hypothesized to add the polyketide extender units onto an acyl carrier protein (ACP) in the bry PKS and, as such, is termed a trans-AT. In addition, some trans-ATs have been shown to have more extender unit substrate flexibility than cis-ATs, which are usually very substrate-specific. The ability of trans-ATs to discriminate between the modular ACPs and load them with the correct extender unit to form the desired polyketide product during biosynthesis is unclear. To examine how trans-AT’s discriminate between modules, protein-protein interactions between the BryP trans-AT and interdomain regions between ketosynthase (KS) and AT regions of PKS modules were assessed using surface plasmon resonance (SPR) to compare binding events via dissociation data. On average, BryPAT1 had a higher affinity for BryBM4 KS-AT interdomain region as compared to the EryAIII M5 KS-AT interdomain region.

Bryostatins are versatile compounds that are ecologically relevant for the survival of Bugula larvae. The mechanism for this activity could be due to activation of protein kinase C (PKC) via high affinity for the PKC C1b domain. As the symbiont-produced bryostatins are potent activators of a eukaryotic cellular target, the question of how the bryozoan host has adapted to their presence arises. Interestingly, there is variation in symbiont and bryostatin status within the genus Bugula, with some species possessing a symbiont that produces bryostatins, some species possessing a closely-related symbiont that does not produce bryostatins, and some species with no symbiont. Using SPR, on average, bryostatin has a higher affinity for bryostatin-producing B. neritina C1b domain as compared to non-bryostatin containing Bugula pacifica and Rattus norvegicus C1b domains. Understanding aspects of bryostatin biosynthesis and symbiont effects on the host will provide deeper insight into the vital role that they play in the interaction between B. neritina and E. sertula.

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