title: Targeted protein degradation of outer membrane decaheme cytochrome MtrC metal reductase in Shewanella oneidensis MR-1 measured using biarsenical probe CrAsH-EDT(2).
abstract: 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 (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 h(-1) that is insensitive to O(2) 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 h(-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.
authors: Biological Sciences Division, Fundamental Sciences Directorate, Pacific Northwest National Laboratory, Richland, Washington 99352, United States.
authors: Yijia
authors: Y
authors: Xiong
authors: Baowei
authors: B
authors: Chen
authors: Liang
authors: L
authors: Shi
authors: James K
authors: JK
authors: Fredrickson
authors: Diana J
authors: DJ
authors: Bigelow
authors: Thomas C
authors: TC
authors: Squier
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001674:Bioelectric Energy Sources / Q000382:microbiology; D003574:Cytochrome c Group / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004579:Electron Transport; D005452:Fluoresceins; D005816:Genetic Complementation Test; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009942:Organometallic Compounds; D015202:Protein Engineering; D055550:Protein Stability; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020592:Shewanella / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: C113276:4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein; D001425:Bacterial Outer Membrane Proteins; C518706:CrAsH compound; D003574:Cytochrome c Group; D005452:Fluoresceins; D009942:Organometallic Compounds; D011994:Recombinant Proteins; C433527:MtrC protein, Shewanella