Defense Date

11-5-2021

Graduation Date

Fall 12-17-2021

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Biological Sciences

Committee Chair

Joseph R. McCormick

Committee Member

Jana Patton-Vogt

Committee Member

John F. Stolz

Committee Member

Mihaela Rita Mihailescu

Keywords

Streptomyces, ssdA, Germination, Vegetative growth, Sporulation, Spore maturation, Spore shape, Spore sacculus, HPLC-MS, whiD

Abstract

Streptomycetes are Gram-positive, soil-dwelling bacteria that possess a complex life cycle with the alternation of vegetative mycelium, aerial mycelium, and spores. Streptomyces. coelicolor spore maturation is a complex process that involves spore shape metamorphosis from cylindrical pre-spores into ellipsoid spores, but the details of this process have remained enigmatic. Previously, our lab identified a novel gene ssdA that might play a role in spore shape determination using a transposon-based insertion mutagenesis in S. coelicolor. In this study, I isolated a S. coelicolor ssdA-null mutant that showed increased colony hydrophobicity and misshapen spores in sizes and shapes, confirming the phenotype of the ssdA insertion mutant. In order to further investigate the function of ssdA, I switched the model species to S. venezuelae due to its advantages. Here, I demonstrate that a S. venezuelae ssdA mutant showed delayed morphological differentiation on both solid and liquid media, arising in part from germination and growth defects of the mutant. The ssdA mutant also generated heterogeneously-sized spores, possibly due to sporulation and cell-cell separation defects. Deletion of ssdA also resulted in spore sensitivity to heat, osmotic stress, and cell-wall targeting antibiotics. The spore sacculus was isolated and a preliminary HPLC-MS result demonstrated the accumulation of peptidoglycan muropeptides in the ssdA mutant. SsdA-EGFP localizes in vegetative hyphae, sporulation septa, and the periphery of spores, consistent with its roles in cell wall development. A bacterial two-hybrid assay shows that the central cytoplasmic region of SsdA interacts with a dynamin-like, sporulation-specific protein DynB, explaining the septal localization of SsdA-EGFP. Of interest, ssdA also affects Streptomyces’s exploration, a novel growth mode. whiD is a regulatory gene in Streptomyces that potentially controls ssdA expression. Here I show that the S. venezuelae whiD mutant has a white colony and produces heterogeneously sized spores, indicating a sporulation defect. Also the whiD mutant is drastically sensitive to heat and slightly sensitive to salt, different from the sensitivity profile for the ssdA mutant. Further elucidation the regulatory mechanism of WhiD will provide more insights into the sporulation and spore maturation in this filamentous bacterium.

Language

English

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