Insights into the Mechanism of Oxygen-mediated Growth Arrest of Bacteroides fragilis A Dissertation submitted by Brian Michael Meehan In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular Biology and Microbiology TUFTS UNIVERSITY Sackler School of Graduate Biomedical Sciences February 2011 Advisor: Michael Malamy Abstract The goal of this work was to define the factors that inhibit growth of Bacteroides fragilis at oxygen levels > 0.05%. One important aspect of this growth arrest is the accumulation of endogenously-generated reactive oxygen species (ROS) like hydrogen peroxide (H 2O2). We show here that H 2O2 scavenging rates were reduced to 20% of the wild-type in a strain missing alkylhydroperoxide reductase ( ahpC ), catalase, and thioredoxin- dependent peroxidase ( tpx ). This strain was hypersensitive to room air and was found to generate ROS at a rate of ~35nM/min/OD 600 =0.1 under these conditions. However, deletion of fumarate reductase gave rise to a strain that accumulated ~19nM H2O2/min//OD 600 =0.1, indicating that this enzyme accounts for ~47% of the ROS generated by aerated B. fragilis . Deleting frdC increased the aerotolerance of a ∆sod strain by ~100-fold during 9 hours of exposure to room air. Spontaneous mutants of B. fragilis capable of growth under 1% oxygen arise at a frequency of ~10 -6. These strains carried mutations in an orf designated oxe . Deletion of oxe established the O 2-enabled phenotype, and this mutant could be re-sensitized to oxygen by providing a wild-type copy of oxe in trans . Microaerobic growth of the ∆oxe strain was characterized by several amino acid auxotrophies. Though Oxe is not a major source of ROS under room air, a ∆oxe strain was found to scavenge micromolar amounts of H 2O2 at a rate approximately triple that of the wild-type. 2 Fecal strains of B. fragilis did not grow under ~1% oxygen. Ten clinical strains plated microaerobically with efficiencies greater than 10%, 14 plated with frequencies similar to the fecal strains, and 7 had an intermediate phenotype. The oxe locus was sequenced for 7 clinical strains that plated with high efficiencies microaerobically and four were found to encode a wild-type Oxe, while 3 were predicted to contain amino acid substitutions in Oxe. Oxe may contribute to the utilization of amino sugars by B. fragilis , as a ∆oxe strain bearing a mutant allele of nagB could not grow on N-acetylglucosamine or glucosamine. However, the role of Oxe in the anaerobic physiology of this organism is still under investigation. 3 Table of Contents Abstract .............................................................................................................................. 2 Table of Contents .............................................................................................................. 4 List of Tables ..................................................................................................................... 8 List of Figures .................................................................................................................. 10 Introduction ..................................................................................................................... 14 Iron Sulfur Clusters ....................................................................................................... 15 The Birth of Oxygen ..................................................................................................... 16 How Reactive Oxygen Species Form ........................................................................... 19 ROS damage ................................................................................................................. 20 Anaerobe vs. aerobe ...................................................................................................... 22 Bacteroides fragilis ....................................................................................................... 25 The B. fragilis Response to Oxygen ............................................................................. 27 Cytochrome bd oxidase ........................................................................................................ 28 OxyR ..................................................................................................................................... 29 Superoxide Dismutase .......................................................................................................... 31 Iron Sequestration ................................................................................................................. 33 Catalase ................................................................................................................................. 33 Alkylhydroperoxide reductase (AhpC) ................................................................................. 35 Tpx and thioredoxins ............................................................................................................ 36 Other Peroxidases ................................................................................................................. 37 Other oxygen-responsive components .................................................................................. 39 B. fragilis metabolism ................................................................................................... 40 4 Endogenous sources of ROS ......................................................................................... 43 Why can't B. fragilis grow in room air? ........................................................................ 45 References ..................................................................................................................... 46 CHAPTER 1 .................................................................................................................... 61 Identification of Fumarate Reductase as a Major Source of Reactive Oxygen Species in Bacteroides fragilis ...................................................................................................... 61 Abstract ......................................................................................................................... 62 Introduction ................................................................................................................... 64 Materials and Methods .................................................................................................. 67 Results ........................................................................................................................... 76 Tpx and AhpC are major scavengers of low concentrations of hydrogen peroxide ............. 76 Deletions of ahpC and tpx do not impair detoxification of high concentrations of H 2O2.... 77 AhpC and Tpx are important components of aerotolerance ................................................. 77 Fumarate reductase is a major source of ROS when B. fragilis is exposed to oxygen ......... 78 Deleting frdC from a superoxide dismutase mutant partially restores aerotolerance ........... 80 Discussion ..................................................................................................................... 81 Acknowledgments......................................................................................................... 84 References ..................................................................................................................... 91 CHAPTER 2 .................................................................................................................... 97 Characterization of Bacteroides fragilis Mutants That Grow Under Increased Oxygen ............................................................................................................................. 97 Abstract ......................................................................................................................... 98 Introduction ................................................................................................................. 100 5 Materials and Methods ................................................................................................ 102 Results ......................................................................................................................... 111 Isolation of oxygen-enabled mutants .................................................................................. 111 A ∆oxe strain plates with the same efficiency under anaerobic and microaerobic conditions ............................................................................................................................................ 113 A ∆oxe strain grows in liquid medium under microaerobic conditions but is auxotrophic for certain amino acids ............................................................................................................. 114 ∆oxe retains greater viability under room air than wild-type ............................................. 115 A strain lacking oxe accumulates H 2O2 at a slightly faster rate than wild-type .................. 115 ∆oxe strain scavenges H 2O2 more efficiently than wild-type ............................................. 116 Spontaneous oxygen-enabled mutants arise in B. thetaiotaomicron .................................. 116 Discussion ................................................................................................................... 118 References ..................................................................................................................