Date of Completion

8-27-2014

Embargo Period

8-26-2014

Major Advisor

Kenneth M. Noll

Associate Advisor

Daniel J. Gage

Associate Advisor

J. Peter Gogarten

Field of Study

Genetics and Genomics

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

The ATP-binding cassette (ABC) transporters play an important role in the uptake of nutrients in bacterial and archaeal cells. The bacterium Thermotoga maritima has an unusually high number of ABC transporters. However, only a few of them have been characterized due to the variety of substrates they can transport and the time consuming efforts needed to thoroughly characterize each ABC transporter. In this work, a technique has been optimized to determine the sugars that interact with the substrate-binding proteins (SBP) of the ABC transporter complexes in Thermotoga maritima. Using this high throughput assay, the ligands of trehalose-binding protein (TreE) and xylose-binding protein (XylE2) in Thermotoga maritima as well as representatives of the mannoside-binding proteins (Man) in the Thermotogaspecies were identified. The phylogeny of the mannose-binding proteins is interesting because of the presence of two paralogous proteins in this family (ManD and ManE). Ten representatives of the mannoside-binding proteins (ManD and ManE) encoded in the Thermotogales were characterized. At 37°C and 60°C, the ManD and ManE homologs bind cellobiose, cellotriose, cellotetraose, β-mannotriose, and β-mannotetraose. However, the ManE homologs have additional function, as they are able to bind β-mannobiose, laminaribiose, laminaritriose and sophorose. An examination of the selective pressure that acted on manD and manE and the identification of the residues located in the binding-site of their encoded proteins suggest that early in the evolution of manD and manE, these genes had different codon sites under positive selection which encode important regions of their binding site. Taken together, these analysis suggest that these paralogs likely evolved under different evolutionary constrains.

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