Abstract
Members of the genus Mycobacterium contain species that are adapted to thrive under a wide variety of environmental conditions. A key factor in the life cycle of mycobacteria is their ability to persist in the absence of growth triggered by either low oxygen levels or nutrient limitation. During these transitions from growth to non-growth, mycobacteria are faced with the challenge of redox homeostasis and balancing electron flux to meet maintenance energy requirements. Mycobacteria are able to synthesize a large number of flavoproteins and have biosynthetic pathways for a number of flavins (e.g. FMN, FAD and F420) that vary in redox potential. In this study, I have investigated the molecular function of two flavoproteins in the fast-growing saprophyte Mycobacterium smegmatis: a flavin-sequestering protein (Fsq) and malate menaquinone oxidoreductase (Mqo). Analysis of Fsq showed it was a member of the diverse superfamily of flavin and deazaflavin-dependent oxidoreductases (FDORs) and was widely distributed in mycobacterial species. I created a markerless deletion mutant of fsq and demonstrated that fsq is required for cell survival during hypoxia. Using fsq deletion and overexpression, I found that fsq enhances cellular resistance to hydrogen peroxide treatment. We solved the X-ray crystal structure of Fsq to 2.7 Å revealing a homodimeric organization with FAD bound noncovalently. The Fsq structure also uncovered no potential substrate- binding cavities, as the FAD is fully enclosed, and electrochemical studies indicated that the Fsq:FAD complex is relatively inert and does not share common properties with electron- transfer proteins. Taken together, our results suggest that Fsq modulates the formation of reactive oxygen species (ROS) by sequestering free FAD during recovery from hypoxia, thereby protecting the cofactor from undergoing autoxidation to produce ROS. This finding represents a new paradigm in mycobacterial adaptation to hypoxia.
The physiological role of Mqo in mycobacteria is unknown despite its high conservation throughout the mycobacterial genera. Mqo serves the role of oxidizing malate to oxaloacetate and is a highly conserved member of the tricarboxylic acid (TCA) cycle within mycobacteria. Genetic deletion of mqo revealed that mqo was essential for growth on non-fermentable carbon sources, and delayed growth was observed on fermentable carbon sources. Biochemical analyses showed that deletion of mqo resulted in significant alteration in the activity of other enzymes in the TCA cycle and electron transport chain. Further, the oxygen consumption of the Dmqo mutant was reduced and displayed an inability to increase oxygen consumption upon addition of an uncoupler. Growth of the Dmqo mutant on fermentable carbon sources resulted in significantly lower pH levels in the growth medium, which was partially caused by the secretion of malate. Taken together these data suggest that the deletion of mqo in M. smegmatis causes the operation of an incomplete TCA cycle that can be bypassed on fermentable carbon sources.
The results of this thesis provide insight in M. smegmatis flavoprotein function and advance the current understanding of Fsq and Mqo in mycobacterial adaptation and metabolism.