Characterization of protein-protein interactions involved in iron reduction by Shewanella oneidensis MR-1

Daniel E. Ross, Shane S. Ruebush, Susan L. Brantley, Robert S. Hartshorne, Thomas A. Clarke, David J. Richardson, Ming Tien

Research output: Contribution to journalArticle

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Abstract

The interaction of proteins implicated in dissimilatory metal reduction by Shewanella oneidensis MR-1 (outer membrane [OM] proteins OmcA, MtrB, and MtrC; OM-associated protein MtrA; periplasmic protein CctA; and cytoplasmic membrane protein CymA) were characterized by protein purification, analytical ultracentrifugation, and cross-linking methods. Five of these proteins are heme proteins, OmcA (83 kDa), MtrC (75 kDa), MtrA (32 kDa), CctA (19 kDa), and CymA (21 kDa), and can be visualized after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by heme staining. We show for the first time that MtrC, MtrA, and MtrB form a 198-kDa complex with a 1:1:1 stoichiometry. These proteins copurify through anion-exchange chromatography, and the purified complex has the ability to reduce multiple forms of Fe(III) and Mn(IV). Additionally, MtrA fractionates with the OM through sucrose density gradient ultracentrifugation, and MtrA comigrates with MtrB in native gels. Protein cross-linking of whole cells with 1% formaldehyde show new heme bands of 160, 151, 136, and 59 kDa. Using antibodies to detect each protein separately, heme proteins OmcA and MtrC were shown to cross-link, yielding the 160-kDa band. Consistent with copurification results, MtrB cross-links with MtrA, forming high-molecular-mass bands of approximately 151 and 136 kDa.

Original languageEnglish (US)
Pages (from-to)5797-5808
Number of pages12
JournalApplied and environmental microbiology
Volume73
Issue number18
DOIs
StatePublished - Sep 1 2007

Fingerprint

Shewanella oneidensis
Shewanella
protein-protein interactions
Iron
iron
protein
Hemeproteins
heme
Membrane Proteins
Proteins
proteins
Ultracentrifugation
Heme
Periplasmic Proteins
ultracentrifugation
membrane
crosslinking
Sodium Dodecyl Sulfate
Formaldehyde
Anions

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

Cite this

Ross, Daniel E. ; Ruebush, Shane S. ; Brantley, Susan L. ; Hartshorne, Robert S. ; Clarke, Thomas A. ; Richardson, David J. ; Tien, Ming. / Characterization of protein-protein interactions involved in iron reduction by Shewanella oneidensis MR-1. In: Applied and environmental microbiology. 2007 ; Vol. 73, No. 18. pp. 5797-5808.
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abstract = "The interaction of proteins implicated in dissimilatory metal reduction by Shewanella oneidensis MR-1 (outer membrane [OM] proteins OmcA, MtrB, and MtrC; OM-associated protein MtrA; periplasmic protein CctA; and cytoplasmic membrane protein CymA) were characterized by protein purification, analytical ultracentrifugation, and cross-linking methods. Five of these proteins are heme proteins, OmcA (83 kDa), MtrC (75 kDa), MtrA (32 kDa), CctA (19 kDa), and CymA (21 kDa), and can be visualized after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by heme staining. We show for the first time that MtrC, MtrA, and MtrB form a 198-kDa complex with a 1:1:1 stoichiometry. These proteins copurify through anion-exchange chromatography, and the purified complex has the ability to reduce multiple forms of Fe(III) and Mn(IV). Additionally, MtrA fractionates with the OM through sucrose density gradient ultracentrifugation, and MtrA comigrates with MtrB in native gels. Protein cross-linking of whole cells with 1{\%} formaldehyde show new heme bands of 160, 151, 136, and 59 kDa. Using antibodies to detect each protein separately, heme proteins OmcA and MtrC were shown to cross-link, yielding the 160-kDa band. Consistent with copurification results, MtrB cross-links with MtrA, forming high-molecular-mass bands of approximately 151 and 136 kDa.",
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Characterization of protein-protein interactions involved in iron reduction by Shewanella oneidensis MR-1. / Ross, Daniel E.; Ruebush, Shane S.; Brantley, Susan L.; Hartshorne, Robert S.; Clarke, Thomas A.; Richardson, David J.; Tien, Ming.

In: Applied and environmental microbiology, Vol. 73, No. 18, 01.09.2007, p. 5797-5808.

Research output: Contribution to journalArticle

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T1 - Characterization of protein-protein interactions involved in iron reduction by Shewanella oneidensis MR-1

AU - Ross, Daniel E.

AU - Ruebush, Shane S.

AU - Brantley, Susan L.

AU - Hartshorne, Robert S.

AU - Clarke, Thomas A.

AU - Richardson, David J.

AU - Tien, Ming

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