Protoporphyrin (PPIX) efflux by the MacAB-TolC pump in Escherichia coli.
Bottom Line: Escherichia coli regulates heme biosynthesis by a feedback loop involving heme-induced proteolytic cleavage of HemA, glutamyl-tRNA reductase, which is the first enzyme in the heme biosynthetic pathway.We also show that it is disrupted by iron chelation, which reduces the intracellular iron concentration necessary for loading iron into protoporphyrin IX (PPIX, the immediate heme precursor).We propose that PPIX is an endogenous substrate of the MacAB-TolC pump in E. coli and S. typhimurium and that this compound is produced inside bacteria when natural heme homeostasis is disrupted by iron shortage, as happens when bacteria invade the mammalian host.
Affiliation: Unité des Membranes Bactériennes, Département de Microbiologie, Institut Pasteur, 75724, Paris Cedex 15, France.Show MeSH
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Mentions: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot were carried out according to standard protocols. Cultures were grown to an OD600 of 1. Cells were harvested by centrifugation for 20 min at 5000g at 4°C. The cell pellet was resuspended in 50 mmol/L Tris-HCl pH 8.0, 0.3 mol/L NaCl to OD600 of 50/mL and disrupted by sonication, then centrifuged at 20,000g for 60 min at 4°C to remove cellular debris. Protein concentration in cell extracts was determined by Bradford protein assay and is indicated in the Figure 1. Ten microliters of aliquots were loaded on SDS PAGE. Bound antibodies were detected with secondary anti-rabbit antibodies coupled with horseradish peroxidase and revealed by chemiluminescence.
Affiliation: Unité des Membranes Bactériennes, Département de Microbiologie, Institut Pasteur, 75724, Paris Cedex 15, France.