Characterization of Pullulanase (glgX/pulA) in Francisella novicida U112 Growth, Gene Expression, and Biofilm Formation

Abstract

Francisella (F.) tularensis is a Gram-negative bacterium that causes the highly infectious human disease, tularemia. Within Francisella, like many other bacteria, polysaccharides play pivotal roles in many different pathways, whether they be intracellular polysaccharides or extracellular. One extracellular polysaccharide is pullulan; a water-soluble polysaccharide consisting of α(1,4) and α(1,6) glycosidic linkages and is cleaved by the enzyme pullulanase. Pullulan can be found everywhere in nature and is used in commercial food production. Interestingly, we noticed that Francisella encodes a gene for pullulanase. Within F. tularensis, pullulanase can be found within all four strains. In F. tularensis subsp. novicida (or F. novicida U112), the pullulanase gene is called glgX (Glycogen debranching enzyme), pulA and pulB depending on the annotation. Pullulanase (pul) is an enzyme that hydrolyze α(1,4) and α(1,6) glycosidic linkages. Within Francisella, the same genes annotated as type I and type II pullulanase can both be found. After a comparative analysis of the sequences, I conclude that Francisella pulA and pulB genes encode the same protein. My thesis focuses on characterizing the role of pullulanase in Francisella physiology and virulence. For my first aim, I identified the role pullulanase plays in Francisella in vitro growth stages (lag phase, exponential, and stationary phase) by characterizing the effect that a glgX mutant has under various conditions. Under this aim, I compared glgX mutants with WT F. novicida through various pH ranges, temperature ranges, and nutrient variations. I predicted that glgX will be a positive regulator for in vitro growth. Based on research conducted in K. oxytoca, E. coli, and Francisella on glgX-connected genes and biofilm regulators, I characterized the role pullulanase plays in biofilm formation in the second aim. Comparing all four strains of Francisella, F. novicida has the strongest evidence supporting the premise that biofilms are important for its environmental and stress stability. I compared WT F. novicida against glgX mutants in terms of biofilm morphology. I predicted that WT glgX is a positive regulator on biofilm production and mutating it will result in a negative regulation. A paper written by Uda et al demonstrated that in F. tularensis SCHU S9, pullulanase is an important factor in intracellular replication in macrophages but not in disease progression in mice. On the contrary, other literature reporting on the effect of mutants in glgX/pulA/pulB in Francisella suggests that pullulanase is not needed for intracellular replication. Overall, I expect to demonstrate that pullulanase (glgX) is important for in vitro growth, is a positive regulator in biofilm formation, and will have no effect on the intracellular replication of Francisella novicida U112.