Title

Characterization of Novel Streptomyces Developmental Genes Identified by Transposon Mutagenesis

Date Written

Spring 4-6-2017

Document Type

Distinction Paper

Degree Name

Biochemistry and Molecular Biology-BS

Department

Biochemistry and Molecular Biology

Advisor

J. Bennett

First Committee Member

J. Bennett

Second Committee Member

J. Lehman

Third Committee Member

D. Robertson

Keywords

Streptomyces, Microbial Development, Transposon Mutagenesis, Sporulation

Subject Categories

Microbiology

Abstract

Streptomyces coelicolor is a bacterium with a high GC content that is predominately found in soil and decomposing vegetation. The linear chromosome is the largest bacterial genome to be sequenced to date. The genus synthesizes over two-thirds of available antibiotics. Transposon mutagenesis was employed to identify novel developmental genes involved in the complex life cycle. First, a vegetative mycelium forms, consisting primarily of hyphal elongation and DNA replication without cellular division. Aerial hyphae growth follows and then sporogenic cells are developed. There are many developmental genes still believed to be undiscovered and they are thought to play key roles in this complex life cycle. Random transposon mutagenesis of S. coelicolor was performed. The wild-type strain was then compared to mutants that exhibited potential developmental defects based on visual screens followed by phase-contrast and fluorescence microscopy. The transposon insertion site was identified by chromosomal DNA isolation, restriction enzyme digestion, ligation, and inverse PCR followed by sequencing. This short sequence of mutant DNA was then subjected to a nucleotide BLAST to locate the insertion point of the mini-Tn5 transposon. Once the insertion site was identified for each mutant strain, the strains were characterized, using a variety of methods including phase-contrast, propidium iodide staining of DNA, and wheat germ agglutinin staining of peptidoglycan in the cell wall. One of the genes was also deleted using λ Red recombination. This research partially characterizes two developmental genes of interest in Streptomyces.

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