Defense Date

10-1-2019

Graduation Date

Fall 12-20-2019

Availability

One-year Embargo

Submission Type

dissertation

Degree Name

PhD

Department

Biological Sciences

Committee Chair

Joseph R. McCormick

Committee Member

Nancy Trun

Committee Member

Jana Patton-Vogt

Committee Member

Michael Cascio

Keywords

Stretpomyces coelicolor, microbial development, spores, WhiJ, BldB, spore-associated proteins

Abstract

Streptomyces coelicolor is a Gram positive, filamentous soil dwelling bacterium that exhibits a complex life cycle including the transition from a vegetative mycelium to a sporulating, aerial mycelium. Early genetic studies identified two classes of genes that resulted in developmental blocks, bald and white. The bald (bld) phenotype occurs when colonies cannot produce aerial hyphae. White (whi) colonies exhibit incomplete sporulation and/or loss of production of the concurrently produced grey pigment. While some of the original mutants have been explored, the developmental functions of many of the identified genes are not well understood. Of particular interest is a multi-gene regulatory system, with multiple paralogs of each gene present in S. coelicolor chromosome, which are well conserved among other streptomycetes and morphologically complex actinomycetes. The three genes encode 1) a predicted helix-turn-helix protein (WhiJR-like proteins), 2) a small, acidic protein of unknown function (WhiJS-like proteins), and 3) an anti-sigma factor-like protein (WhiJA-like proteins). In order to explore the roles of these genes in development, one such gene system involved in the regulation of the spore-associated protein (sap) operon, sapCED, was analyzed. sapR (whiJR-like), sapS (whiJS-like) comprise a divergently transcribed operon from sasA (whiJA-like), which is the first gene in the sapCED operon. In order to investigate the potential roles of this gene system in the developmental regulation of this spore-associated protein operon, null mutants were isolated by recombineering. Single and double null mutants were isolated and the effects on expression were assayed by the extraction of spore-associated proteins and a transcriptional fusion of the sapCEDp to luciferase. These assays indicate that in the absence of sapR and sapS, (either individually or together) there was an increase in expression of the sapCED operon. However, a single deletion of sasA has no observed phenotype. To determine protein-protein interactions, sapR, sapS, and sasA were cloned into a bacterial adenylate cyclase two-hybrid (BACTH) system. Analysis suggests self-interactions of SapR, SapS, and SasA. SapR was shown to interact with SapS; and, SapS was shown to interact with SasA. These interactions, save for SapS interacting with SasA, were recapitulated in a homologous multi-gene system (SCO3421, SCO3423, SCO3424). Using an electrophoretic mobility shift assay, SapR has been shown to interact with the promoter region of sapCED. These results suggest a model for SapR and SapS in repressing the development-associated sapCED operon, while SasA may function as a feedback mechanism through its interactions with SapS. Additionally, nine spore associated-proteins were identified in common laboratory strains of S. coelicolor that may play important roles for nutrient harvesting during germination.

Language

English

Additional Citations

Mintmier, B., (Sallmen) et al. (2018). "Molecular cloning, expression and biochemical characterization of periplasmic nitrate reductase from Campylobacter jejuni." FEMS Microbiol Lett 365(16).

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