Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Microbiology and Molecular Genetics

First Advisor

Aimee Shen

Second Advisor

John Barlow


Clostridium difficile is a Gram-positive spore-forming strict anaerobe that can cause severe colitis in humans. C. difficile is best known as the leading cause of nosocomial-acquired diarrhea, particularly in people undergoing antibiotic therapies, since it is naturally resistant to most antibiotics. A clinical feature that makes C. difficile infection, or CDI, particularly difficult to treat is the organism's inherent ability to resist antibiotic therapies while in its spore form. Since oxygen is toxic to C. difficile, spores are the major transmissive form; they are also resilient to most disinfectants, which makes them extremely difficult to eliminate to prevent additional infections.

While over fifty years of studies on the spore-forming model organism Bacillus subtilis laid the foundation of how sporulation and germination occurs, little was known about how C. difficile regulates spore formation and/or what proteins are necessary for sporulation and germination processes. The work presented in this dissertation addresses how C. difficile regulates sporulation, identifies genes that are regulated during sporulation, and characterizes some key proteins that are required for either sporulation or germination.

During the developmental process of sporulation, a cell divides into two asymmetrical compartments. In each compartment, specific transcriptional programs controlled by sporulation-specific sigma factors, drive the cell through a series of morphological events, culminating in the formation of a spore. Using genetic and cell biological techniques, we show that mutations in the genes encoding the master transcriptional regulator Spo0A and the sporulation-specific sigma factors σF, σE, σG, and σK block sporulation at various stages. Analysis of the mutants and wild type C. difficile strain using RNA-Sequencing identified genes regulated by a given sigma factor and revealed that the sigma factors control sporulation in a manner that differs from B. subtilis. Whereas the sporulation-specific sigma factor activity is regulated in a sequential manner involving cross talk between the different compartments in B. subtilis, C. difficile regulates these factors in a bifurcated manner, with less cross-compartment regulation.

Guided by our RNA-Sequencing results, we constructed targeted gene mutations in spoIIQ and spoIIIA-H, which are important for forming a channel known as the 'feeding tube' in B. subtilis. We demonstrated that these proteins are necessary for maintaining forespore integrity, tethering the coat to the forespore, and engulfment. Using metabolic labeling, we show that while spoIIQ and spoIIIA mutants cannot finish the phagocytic-like process of engulfment, they are capable of transforming peptidoglycan, which is a necessary step for engulfment to occur.

We also constructed a targeted gene mutation in a gene that is highly transcribed during sporulation, now known as gerS. We show that a gerS mutant cannot degrade cortex during germination and is required for SleC-mediated cortex hydrolysis, making GerS a novel regulator of C. difficile spore germination.

Altogether, this research provides a framework for understanding how the pathogen C. difficile undergoes sporulation and is therefore capable of infecting humans. Further, our studies reveal important factors that mediate the essential process of engulfment during sporulation and an important factor that mediates cortex hydrolysis during germination. This work has demonstrated that C. difficile regulates sporulation and germination differently than what has previously been described in other Firmicutes.



Number of Pages

351 p.

Included in

Microbiology Commons