Date of Award

Spring 4-2022

Document Type

Distinction Paper

Degree Name

Biochemistry and Molecular Biology-BS

Department

Biology & Earth Science

Advisor

Jennifer Bennett

First Committee Member

Jennifer Bennett

Second Committee Member

Robin Grote

Third Committee Member

John Tansey

Keywords

Streptomyces coelicolor, Transposon Mutagenesis, Aerial Mycelium, Sporulation, Medium-Dependent Phenotype, SCO2186

Subject Categories

Bacteriology | Biochemistry | Cell Biology | Genetics | Higher Education | Other Microbiology

Abstract

Streptomyces coelicolor is a soil-dwelling, Gram-positive Actinobacteria. Streptomyces species produce thousands of secondary metabolites which include many antibiotics used in medicine today. In Streptomyces development, as colonies grow, secondary metabolites are produced, and ultimately mature spores are dispersed. The production of each of these secondary metabolites in wild-type Streptomyces is determined by a distinct biosynthetic pathway. In order to identify new genes for antibiotic production and other aspects of development, an engineered Tn5-derived transposon was used to mutagenize wild-type cells in vivo and produce single, stable insertions.

Colonies that were visually distinct from the wild type were selected for further analysis. The transposon insertion site for each mutant was then identified using the following steps. Chromosomal DNA was prepared from the mutants, digested with a restriction endonuclease, and ligated into many circular DNA segments that were amplified using inverse PCR. The PCR products were then subjected to Sanger sequencing.

BLAST analysis revealed that the four mutants contained insertions in genes of unknown function or putative function, including a potential transcriptional regulator. The primary mutant of interest possessed an insertion in a gene encoding a protein of unknown function. When compared to the wild type this mutant was enhanced in aerial mycelium formation when grown on Mannitol Soy Flour Agar. Interestingly, when the same mutant was grown on R2YE medium it was delayed in its production of aerial mycelium and was able to recover when in close proximity to the wild type. This medium-dependent phenotype has been previously described for Streptomyces bald (bld) mutants. This newly identified gene is potentially a novel developmental gene influencing aerial mycelium formation.

Licensing Permission

Copyright, all rights reserved. Fair Use

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