Microbes Breathe Iron: Characterization of Dissimilatory Iron Reduction by Shewanella oneidensis MR-1

Open Access
Author:
Qian, Yufeng
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
July 06, 2011
Committee Members:
  • Ming Tien, Dissertation Advisor
  • Ming Tien, Committee Chair
  • Carsten Krebs, Committee Member
  • Squire J Booker, Committee Member
  • James David Kubicki, Committee Member
  • B Tracy Nixon, Committee Member
Keywords:
  • Shewanella
  • Iron reduction
Abstract:
This dissertation concerns the biological process of dissimilatory iron reduction (DIR) by a model iron-reducing microorganism: Shewanella oneidensis MR-1. Biochemical studies were performed to probe the iron binding site on a small tetraheme cytochrome and to elucidate the electron transfer network in the periplasm. An outer membrane (OM)-localized TonB-dependent receptor was identified as a putative iron transport protein involved in DIR. A periplasm-localized small tetraheme cytochrome (STC) was heterologously expressed and purified from Escherichia coli. Kinetic studies have been performed to probe the electron flow on STC. Chemical modifications followed by site-directed mutagenesis along with isothermal titration calorimetry (ITC) and stopped flow measurements were performed to identify the iron binding site on STC. Chemical modification of STC revealed that carboxyl groups are involved in iron binding. Scanning mutagenesis on Asp and Glu was carried out to probe the putative iron binding site on STC. Two STC mutants (D21N; D80N) showed approximately 70% decrease in observed electron transfer rate constant with EDTA-Fe3+ from transient-state kinetic measurements. The impaired reactivity of STC (D80N/D21N) with EDTA-Fe3+ was further confirmed by a significant decrease (>10 fold) in iron binding affinity. Metal-catalyzed protein oxidation was used to identify membrane proteins which bind iron, acting either as an iron transporter or as a terminal iron reductase. This oxidation system catalyzed the modification of amino acids in close proximity to the iron binding site. The carbonyl groups formed from this oxidation can then be labeled with fluoresceinamine (FLNH2). The peptide containing the FLNH2 can then be proteolytically digested, purified by HPLC and then identified by MALDI-TOF tandem MS. A predominant peptide was identified to be part of SO2907 that encodes a putative TonB dependent receptor. Compared to wild type (wt), the so2097 gene deletion (∆SO2907) mutant has impaired ability to reduce soluble Fe(III). In vitro iron binding assay demonstrated that a truncated form of heterologous-expressed SO2907 that contains the Fe(III) binding site, is capable of binding soluble Fe(III) forms with a Kd of approximate 50 μM. Biochemical studies were carried out to investigate the electron transfer network in the periplasm. In vitro protein-protein interactions were conducted between three important electron transfer components: cytoplamic-membrane-localized CymA, soluble periplamsic-localized STC and OM associated MtrA in S. oneidensis MR-1. The specific interaction was found between CymA and MtrA while no apparent interaction was observed between CymA and STC. Through the in vitro membrane-catalyzed (formate-dehydrogenase-dependent) MtrA reduction, the direct electron transfer was demonstrated between CymA with MtrA. A proposed electron transfer route in the periplasm is presented.