The Impact of Iron Deprivation on the Metabolism of \u3cem\u3eEscherichia coli\u3c/em\u3e as Measured by Targeted HPLC-MS/MS Based Metabolomics

Iron is a necessity for all living organisms, and bacteria typically need an internal concentration of iron in the micromolar range.1 This becomes quite problematic, especially in pathogenic bacteria, because the concentration of free ions within the human body is only 10-24 Molar due to iron binding proteins, like transferrin.1 In order to circumvent this problem, bacteria produce small iron chelating molecules called siderophores.1 The bacteria utilized for this study, a K-12 derivative of Escherichia coli, produce only one siderophore, enterobactin. Not only is the transcription of genes associated with iron acquisition initiated in response to the availability of iron in the surrounding environment, but other genes seemingly unrelated to iron acquisition are expressed as well. In many species of bacteria, the availability or absence of iron in the surrounding area often results in the transcription or repression of virulence associated genes as well as those for biofilm formation.2-3 Various analytical techniques have been used to measure the metabolome of an organism including: nuclear magnetic resonance, infrared spectroscopy, gas and liquid chromatography, and mass spectrometry.4-7 Various types of mass spectrometry have been performed, each only targeting a subset of metabolites. The analytical technique presented here was developed by Dr. Rabinowitz of Princeton University.8-9 Using this method of detection, high performance liquid chromatography is utilized in tandem with a triple quadrupole mass spectrometer to identify various iron containing enzymes quantitatively by monitoring the fold changes of their substrate and/or product compounds. Depending upon the cell line and growth conditions, various metabolites demonstrated a fold change. Some of these compounds, such as xanthine and hypoxanthine, were easily traced back to an enzyme with an iron cofactor or an iron sulfur cluster by referencing various pathway maps. Others proved to be more difficult, and a direct connection could not be established based upon published studies and the available pathway maps. Four pathways which were shown experimentally to be affected by iron are central metabolic pathways within the cell: the Pentose Phosphate Pathway, Glycolysis, TCA cycle, and the Urea cycle. The metabolic pathway for the synthesis of Enterobactin was also affected