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Author ORCID Identifier

https://orcid.org/0000-0002-3427-4478

Document Type

Campus-Only Access for One (1) Year

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Food Science

Year Degree Awarded

2019

Month Degree Awarded

September

First Advisor

David A. Sela

Subject Categories

Biotechnology | Food Microbiology

Abstract

In the first study, we investigated the utilization of a human milk nitrogen source, urea, by Bifidobacterium. Urea accounts for ~15% in human milk, which is an abundant non-protein nitrogen (NPN). Some bifidobacteria are found to harbor urease gene clusters that potentially enable their hydrolysis of the human milk urea. However, the underlying mechanisms are still unclear. To incisively link the urease gene cluster with bifidobacterial urea utilization, chemical mutagenesis (i.e. ethyl methanesulfonate) was performed on the urease-positive Bifidobacterium longum subsp. suis UMA399. Mutants were selected on differential media and genetic lesions were identified using whole genome sequencing. A mutant that did not exhibit urease activity, or utilize urea as a primary nitrogen source, was selected for further characterization. We found that a single-point mutation was located on the urease catalytic subunit ureC gene to prompt a substitution at residue 343 from glutamic acid to lysine (E343K). Recombinantly expressed and purified mutant UreC exhibits the loss of urease function. The mutation was complemented by expressing the wild-type UreC in the mutated strain. The restoration of urease activity and urea utilization approached levels exhibited by the wild-type strain. Thus, UreC is essential for the bifidobacterial urea utilization phenotype.

In the ongoing research, we are exploring the ability of Bifidobacterium to utilize cysteine, a sulfur-containing proteinogenic amino acid. Previous studies have shown most Bifidobacterium cannot grow without cysteine (cysteine auxotrophic). It will be interesting to clarify why bifidobacteria cannot synthesize cysteine and how they assimilate cysteine from the gut environment as a necessity for propagation. Thus, we first evaluated bifidobacterial strains on their ability to grow on different sole nitrogen sources as well as sulfur sources. We found that only B. boum LMG10736 was able to grow in methionine as a sole nitrogen source, the rest of the strains are all cysteine auxotroph. However, B. boum LMG10736 was not able to utilize sulfate and sulfide for its growth. We therefore proposed that the methionine degradation pathway may be silenced under the transcriptional or translational regulations.

Available for download on Tuesday, September 01, 2020

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