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Novel Insights into Fur Regulation in Helicobacter pylori

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Technical Report

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Uniformed Services University Of The Health Sciences Bethesda United States

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Helicobacter pylori is a highly successful pathogen that colonizes the gastric mucosa of 50 of the worlds population. Within this colonization niche the bacteria encounter large fluctuations in nutrient availability. As such, it is critical that this organism regulate expression of key metabolic enzymes so that they are present when environmental conditions are optimal for growth. One such enzyme is the 2-oxoglutarate -ketoglutarate oxidoreductase OOR, which catalyzes the conversion of -ketoglutarate to succinyl-CoA and CO2. Previous studies from our group suggested that the genes that encode for the OOR are activated by iron-bound Fur Fe-Fur microarray analysis showed that expression of oorD, oorA, and oorC were altered in a fur mutant strain of H. pylori. The goal of the present work was to more thoroughly characterize expression of the oor DABC genes in H. pylori as well as to define the role of Fe-Fur in this process. Here in we show that these four genes are co-transcribed as an operon, and that expression of the operon is decreased in a fur mutant strain. Transcriptional start site mapping and promoter analysis revealed the presence of a canonical extended -10element but a poorly conserved -35 upstream of the 1. Additionally, we identified a conserved Fur binding sequence 130 bp upstream of the transcriptional start site. Transcriptional analysis using promoter fusions revealed that this binding sequence was required for Fe-Fur mediated activation. Finally, fluorescence anisotropy assays indicate that Fe-Fur specifically bound this Fur box with a relatively high affinity Kd 200 nM.These findings provide novel insight into the genetic regulation of a key metabolic enzyme and add to our understanding of the diverse roles Fur plays in gene regulation in H. pylori.

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