Biotin synthesis in Bacillus subtilis

Manandhar, Miglena

Permalink

https://hdl.handle.net/2142/99286 Copy

Description

Title

Biotin synthesis in Bacillus subtilis

Author(s)

Manandhar, Miglena

Issue Date

2017-09-07

Director of Research (if dissertation) or Advisor (if thesis)

Cronan, John E.

Doctoral Committee Chair(s)

Cronan, John E.

Committee Member(s)

• Gardner, Jeffrey F.
• Imlay, James A.
• Vanderpool, Cari

Department of Study

Microbiology

Discipline

Microbiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Biotin
• Bacillus

Abstract

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.

Graduation Semester

2017-12

Type of Resource

text

Permalink

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

Copyright and License Information

Copyright 2017 Miglena Manandhar

Owning Collections