Connecting the Dots in Bacterial Physiology; Methanol and

Connecting the Dots in Bacterial Physiology; Methanol and

Connecting the Dots in Bacterial Physiology; Methanol and Iron Metabolism QscR Amino Acids Fe uptake MeOH Fald Stress Fur GSH

Iron Acid Stress O2 stress Elizabeth Skovran Mary Lidstrom University of Washington H H Purpose

C OH H H C H METHANOL H H N H

METHYLAMINE Enzyme Activity Proteome Transcriptome Metabolome Growth Analysis Mutagenesis O-

O O O- SUCCINATE Genome Flux Analysis Multiple Metabolic Pathways Affect MeOH Catabolism QscR Amino Acids

Fe uptake MeOH Fald Stress Fur GSH Iron Acid Stress O2

stress Outline Background: Iron Metabolism Microarrays Initial Microarray Results -MeOH vs Succinate -Iron -QscR mutant Is QscR a Regulator of Iron Uptake? www.ohsu.com/microbiology/Faculty/crosa Transcriptional control of Fe Uptake

High Iron Low Iron Fe FUR Iron Uptake Iron Uptake FUR Fe Fe

Fe Results from one Scan 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 MeOH vs Succ Results

Unknown Genes Iron Uptake 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 Iron uptake is up-regulated in MeOH and requires QscR Gene Predicted Function Fold Induction

by C1 Substrates RMQ10957 Siderophore biosynthesis protein 4 10 RMQ06259 Ferric anguibactin receptor 3

6 RMQ08929 Iron(III)-transport system permease NI 3 RMQ08574 Ferrichrome-iron receptor

5 3 RMQ09337 Ferrioxamine receptor 5 2 RMQ08930 Iron(III)-binding protein

NI 2 RMQ10956 Siderophore biosynthesis protein 2 2 RMQ06568 Ferrichrysobactin receptor

NI 2 RMQ00643 Iron(III) dicitrate transport NI 2 RMQ04738 Chorismate mutase

NI 2 Fold Repression in qscR mutant 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

QscR is Over-expressed and Purified Gel Shift Assay Fe QscR * * Promoter Region: Ferric anguibactin rec.

qscR Lane 1, no protein; lane 2, no protein + 100 M Fe2SO4; Lane 3, 8 g QscR; Lane 4, 8 g QscR + 100 M Fe2SO4 Conclusions Fe Fur Iron Uptake QscR QscR

Iron Uptake MeOH metabolism Fe QscR 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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