Insights into the Mechanism of Oxygen-mediated Growth Arrest of Bacteroides fragilis.
Abstract: The goal of
this work was to define the factors that inhibit growth of Bacteroides fragilisat oxygen
levels > 0.05%. One important aspect of this growth arrest is the accumulation of
endogenously-generated reactive oxygen species (ROS) like hydrogen peroxide (H2O2). We
show here that H2O2 scavenging rates were reduced to 20% of the wild-type in a strain
missing alkylhydroperoxide ... read morereductase (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/OD600=0.1 under these conditions. However, deletion of
fumarate reductase gave rise to a strain that accumulated ~19nM H2O2/min//OD600=0.1,
indicating that this enzyme accounts for ~47% of the ROS generated by aerated B.
fragilis. Deleting frdC increased the aerotolerance of a Δsod 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 O2-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 Δoxe strain was characterized by several amino acid
auxotrophies. Though Oxe is not a major source of ROS under room air, a Δoxe
strain was found to scavenge micromolar amounts of H2O2 at a rate approximately triple
that of the wild-type. 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 Δoxe 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
Thesis (Ph.D.)--Tufts University, 2011.
Submitted to the Dept. of Molecular Microbiology.
Advisor: Michael Malamy.
Committee: Andrew Camilli, Andrew Wright, and Ralph Isberg.
Keyword: Microbiology.read less