Biotin synthesis in Bacillus subtilis

Biotin is a cofactor required by all three domains of life. It is an enzyme cofactor that acts as a “swinging arm” to transfer carboxyl groups in important metabolic pathways involving carboxylation, decarboxylation and transcarboxylation reactions. Despite its importance, the biotin biosynthetic pathway has only been fully characterized in Escherichia coli. Our current understanding of other possible biotin synthetic pathways remains incomplete since various biotin synthesizing organisms have diverse bio genes that are not homologous to those of E. coli. The diversity in pathways lies in the first stage of synthesis of a pimelate thioester moiety. Bacillus subtilis represents a group of microorganisms that follow a different pathway for biotin synthesis. Genetic and biochemical studies identified bioW and bioI as two genes required for pimeloyl-CoA and pimeloyl-ACP synthesis, respectively. The question of the significance of each gene is striking due to the redundancy of pimelate thioester generation. BioW, a structurally unique enzyme, remained to be fully characterized for its importance and enzyme activity. BioW enables B. subtilis to use free pimelic acid as a precursor for biotin synthesis unlike the case in E. coli. However, the source of pimelic acid is unknown in bacterial metabolism. My results show bioW is essential for biotin synthesis whereas bioI is dispensable. I uncovered a unique function of BioW as a proofreading enzyme of noncognate acyl-adenylate substrates other than pimeloyl-adenylate to ensure proper initiation of biotin synthesis. I also report pimeloyl-CoA as the preferred substrate of B. subtilis BioF unlike E. coli BioF. My 13C-NMR studies of labeled biotin elucidated the presence of pimelic acid in the cells and provided direct evidence for generation of pimelate through fatty acid synthesis. Decreased biotin production in presence of fatty acid inhibitors further verified my findings to establish pimelic acid as a link between fatty acid synthesis and biotin synthesis. Hence, in this Thesis, I report my observations that answer long-standing questions about bioW-bioI gene redundancy and pimelate source, to gain further understanding of biotin synthesis in B. subtilis.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Microbiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2018

Author dc:creator

• Manandhar, Miglena

Contributors dc:contributor

• Cronan, John E.
• Gardner, Jeffrey F.
• Imlay, James A.
• Vanderpool, Cari

• Biotin
• Bacillus

Statement dc:rights

• Copyright 2017 Miglena Manandhar

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/99286

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/99286