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Connecting the Dots in Bacterial Physiology; Methanol and Iron Metabolism

Connecting the Dots in Bacterial Physiology; Methanol and Iron Metabolism. QscR. Amino Acids. Fe uptake. Fald Stress. MeOH. Fur . GSH. Iron. Acid Stress. O 2 stress. Elizabeth Skovran Mary Lidstrom University of Washington. H. H. H. H. OH. C. H. N. C. Purpose. H.

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Connecting the Dots in Bacterial Physiology; Methanol and Iron Metabolism

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  1. Connecting the Dots in Bacterial Physiology; Methanol and Iron Metabolism QscR Amino Acids Fe uptake Fald Stress MeOH Fur GSH Iron Acid Stress O2 stress Elizabeth Skovran Mary Lidstrom University of Washington

  2. H H H H OH C H N C Purpose H H H METHANOL METHYLAMINE Enzyme Activity Proteome Transcriptome Metabolome Growth Analysis Genome Mutagenesis Flux Analysis O- O O O- SUCCINATE

  3. Multiple Metabolic Pathways Affect MeOH Catabolism QscR Amino Acids Fe uptake MeOH Fald Stress Fur Iron GSH Acid Stress O2 stress

  4. Outline • Background:Iron Metabolism Microarrays • Initial Microarray Results -MeOH vs Succinate -Iron -QscR mutant • Is QscR a Regulator of Iron Uptake?

  5. www.ohsu.com/microbiology/Faculty/crosa

  6. Transcriptional control of Fe Uptake High Iron Low Iron Fe FUR Iron Uptake Iron Uptake FUR Fe Fe Fe

  7. Results from one Scan

  8. Comments on Transcriptionl Regulation Not always “on” or “off” -think of as “up” or “down” Microarrays:MeOH vs Succinate Wild-type vs qscR mutant QscR is a regulator of MeOH metabolism

  9. MeOH vs Succ Results Unknown Genes Iron Uptake

  10. QscR-SERINE CYCLE REGULATOR ▪Regulates serine cycle and several C1 transfer genes ▪Arrays were done growing qscR with succinate to see if QscR is a global regulator ▪Numerous iron metabolism genes were differentially expressed

  11. Iron uptake is up-regulated in MeOH and requires QscR

  12. Is the Regulation by QscR Direct or Indirect? To Test: 1. Purify QscR 2. PCR amplify the promoter regions of Iron Uptake Genes 3. Gel Shift Assay Iron Uptake

  13. QscR is Over-expressed and Purified

  14. Gel Shift Assay QscR Fe * * qscR Promoter Region: Ferric anguibactin rec. Lane 1, no protein; lane 2, no protein + 100 M Fe2SO4; Lane 3, 8 g QscR; Lane 4, 8 g QscR + 100 M Fe2SO4

  15. Conclusions Fe Fur Iron Uptake QscR QscR Iron Uptake MeOH metabolism QscR Fe qscR FUR is the global iron regulator Iron regulation by QscR and Fe is a “smart” way to coordinate iron and the iron requirement for methanol metabolism

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