Targeted Protein Degradation of Outer Membrane Decaheme Cytochrome MtrC Metal Reductase in Shewanella oneidensis MR-1 Measured Using Biarsenical Probe CrAsH-EDT2

Journal Article · Fri Oct 14 04:00:00 EDT 2011 · Biochemistry (Eaton)

DOI:https://doi.org/10.1021/bi200602f· OSTI ID:1031434

Xiong, Yijia; Chen, Baowei; Shi, Liang; Fredrickson, Jim K; Bigelow, Diana J; Squier, Thomas C

Development of efficient microbial biofuel cells requires an ability to exploit interfacial electron transfer reactions to external electron acceptors, such as metal oxides; such reactions occur in the facultative anaerobic gram-negative bacterium Shewanella oneidensis MR-1 through the catalytic activity of the outer membrane decaheme c-type cytochrome MtrC. Central to the utility of this pathway to synthetic biology is an understanding of cellular mechanisms that maintain optimal MtrC function, cellular localization, and renewal by degradation and resynthesis. In order to monitor trafficking to the outer membrane, and the environmental sensitivity of MtrC, we have engineered a tetracysteine tag (i.e., CCPGCC) at its C-terminus that permits labeling by the cell impermeable biarsenical fluorophore, carboxy-FlAsH (CrAsH) of MtrC at the surface of living Shewanella oneidensis MR-1 cells. In comparison, the cell permeable reagent FlAsH permits labeling of the entire population of MtrC, including proteolytic fragments resulting from incorrect maturation. We demonstrate specific labeling by CrAsH of engineered MtrC which is dependent on the presence of a functional type-2 secretion system (T2S), as evidenced by T2S system gspD or gspG deletion mutants which are incapable of CrAsH labeling. Under these latter conditions, MtrC undergoes proteolytic degradation to form a large 35-38 kDa fragment; this degradation product is also resolved during normal turnover of the CrAsH-labeled MtrC protein. No MtrC protein is released into the medium during turnover, suggesting the presence of cellular turnover systems involving MtrC reuptake and degradation. The mature MtrC localized on the outer membrane is a long-lived protein, with a turnover rate of 0.043 hr-1 that is insensitive to O2 concentration. Maturation of MtrC is relatively inefficient, with substantial rates of turnover of the immature protein prior to export to the outer membrane (i.e., 0.028 hr-1) that are consistent with the inherent complexity associated with correct heme insertion and acylation of MtrC that occurs in the periplasm prior to its targeting to the outer membrane. These latter results suggest that MtrC protein trafficking to the outer membrane and its subsequent degradation are tightly regulated, which is consistent with cellular processing pathways that target MtrC to extracellular structures and their possible role in promoting electron transfer from Shewanella to extracellular acceptors.

• Research Organization: Pacific Northwest National Laboratory (PNNL), Richland, WA (US)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-76RL01830
• OSTI ID: 1031434
• Report Number(s): PNNL-SA-79477; KP1601010
• Journal Information: Biochemistry (Eaton), Journal Name: Biochemistry (Eaton) Journal Issue: 45 Vol. 50; ISSN 0006-2960; ISSN BICHAW
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
ACYLATION
AFFINITY
BACTERIAL NANOWIRES
BINDING ENERGY
BIOFUELS
BIOLOGY
C-TYPE CYTOCHROMES
CYTOCHROMES
ELECTRON CONDUIT
ELECTRON TRANSFER
ELECTRONS
EXPORTS
FUNCTIONALS
HEME
II SECRETION
IN-VIVO
LIVING CELLS
MEMBRANES
METHIONINE OXIDATION
MONITORS
MUTANTS
OMCA
OXIDES
OXIDOREDUCTASES
PROBES
PROTEINS
SECRETION
STRAIN MR-1
TARGETS
VALENCE