RNA landscape of glp operon The role of glycerol metabolism in the Lyme disease agent Bethany Crouse, University of Montana Abstract Borrelia burgdorferi, the bacterium that causes Lyme disease, is maintained in nature through an enzootic cycle, transiting between a mammalian host and tick vector. Following acquisition by a tick, B. burgdorferi uses the sugar glucose from the blood meal to survive. When this source of carbon runs out, the bacterium undergoes a state of nutrient stress. During this time, it uses the sugar glycerol as an alternate carbon and energy source. The glp operon is composed of three annotated genes that enable B. burgdorferi to import and metabolize glycerol. glpD, the last gene in the operon, encodes the enzyme glycerol-3-phosphate dehydrogenase that shuttles glycerol into glycolysis, the main pathway in the bacterium for extracting energy. The glpD gene is regulated differently than the other genes in the glp operon, which respond to nutrient stress, so I hypothesize that glpD is required for B. burgdorferi to use glycerol in both the tick and the mammal. Therefore, I have taken a genetic approach to test the role of glpD in survival of the bacterium and have constructed a mutant of B. burgdorferi lacking the glpD gene. My preliminary results suggest that the glpD mutant does not utilize glycerol as well as wild-type B. burgdorferi. I am currently generating a complement of this mutant to use as a control, and I will assay the phenotype of both the mutant and the complement for their ability to grow in glucose and glycerol. Eventually, I will test the mutant for its ability to survive in ticks and mice using the tick-murine model of Lyme disease. GlpD Structure RNA landscape of glp operon Structure of GlpD protein in Escherichia coli. The membrane associated protein has two conformational states, and while not yet confirmed in B. burgdorferi, the amino acid sequence is 49% similar, implying possible similar structure. In its truncated form, the CAP Domain is missing from the protein. From Yeh et. al., 2008 (3). RNA-seq data of the glp operon. The frequency of the glpD mRNA sequence is the same in both wild-type and a rel mutant, which does not have the gene that responds to nutrient stress. This demonstrates that glpD is regulated independently from the rest of the operon. Adapted from Drecktrah et al., 2015 (4). Construction of truncated glpD mutant a) Methods I have taken a genetic approach to assay the carbon utilization of B. burgdorferi, which requires the construction of a mutant with the glpD gene removed. A complement to use as a control and a truncated glpD mutant were also created. Two DNA sequences on either side of the gene to be removed were amplified and ligated together in a vector An antibiotic resistant cassette was inserted into the vector The vector was linearized and mixed with competent B. burgdorferi cells The bacteria were plated in a 96-well plate with antibiotics to select for transformants Putative mutants were tested for proper gene sequence Enzootic Cycle b) Enzootic cycle of B. burgdorferi. Bacterium is acquired by a larval tick that feeds on an infected mammal. After the tick molts into a nymph, B. burgdorferi will transit into the next mammalian host. From Brisson et al., 2012 (1). a) Construct of truncated glpD mutant. b) Depiction of the mRNA of the full length and truncated glpD transcripts. Conclusions A null glpD mutant, a truncated glpD mutant, and a complemented strain of B. burgdorferi have been constructed The next step will be to assay the survival of these mutants in vitro in glycerol and glucose Further experiments will be done to assess bacterial survival in both ticks and mice Construction of glpD mutant and complement Glycerol Utilization a) wild-type glpD gene Glycerol utilization in B. burgdorferi. glpD, which encodes glycerol-3-phosphate dehydrogenase, plays a crucial role in the fate of glycerol in the bacterium, deciding whether it is used for membrane biosynthesis or shuttled into glycolysis to be used as an energy source. Adapted from Corona and Schwartz, 2015 (2). References Brisson D, Drecktrah D, Eggers CH, Samuels DS. 2012. Genetics of Borrelia burgdorferi. Annu. Rev. Genet. 46:515-536. Corona A, Schwartz I. 2015. Borrelia burgdorferi: Carbon metabolism and the tick-mammal enzootic cycle. Microbiol. Spectr. 3:MBP-0011-2014. Yeh JI, Chinte U, Du S. 2008. Structure of glycerol-3-phosphate dehydrogenase, an essential monotopic membrane enzyme involved in respiration and metabolism. PNAS. 105: 3280–3285. Drecktrah D, Lybecker M, Popitsch N, Rescheneder P, Hall LS, Samuels DS. 2015. The Borrelia burgdorferi RelA/SpoT homolog and stringent response regulate survival in the tick vector and global gene expression during starvation. PLoS Pathog. 11:e1005160. b) Null mutant c) glpD complement Acknowledgements Special thanks to Scott Samuels, Laura Hall and Dan Drecktrah for all of their help and support in completing this research and the making of this poster.