Abstract
Salmonella enterica serovar Typhimurium is a Gram-negative bacterium with a flexible respiratory capability. Under anaerobic conditions, S. enterica can utilize a range of terminal electron acceptors, including selenate, to sustain respiratory electron transport. The S. enterica selenate reductase is a membrane-bound enzyme encoded by the ynfEFGH-dmsD operon. The active enzyme is predicted to comprise at least three subunits where YnfE is a molybdenum-containing catalytic subunit. The YnfE protein is synthesized with an N-terminal twin-arginine signal peptide and biosynthesis of the enzyme is coordinated by a signal peptide binding chaperone called DmsD. In this work, the interaction between S. enterica DmsD and the YnfE signal peptide has been studied by chemical crosslinking. These experiments were complemented by genetic approaches, which identified the DmsD binding epitope within the YnfE signal peptide. YnfE signal peptide residues L24 and A28 were shown to be important for assembly of an active selenate reductase. Conversely, a random genetic screen identified the DmsD V16 residue as being important for signal peptide recognition and selenate reductase assembly.
Figures
![Fig. 4. DSS or formaldehyde, but not EDC, can crosslink a peptide ligand to DmsD. Purified DmsD (2 µM) and synthetic peptide 2 (Table 2; 100 µM) were incubated together in a final volume of 100 µl prior to the addition of chemical crosslinkers [1 mM DSS; 1 mM EDC; 1% (v/v) formaldehyde, final concentrations]. Reactions were stopped after 30min with 50 mM Tris/ HCl (pH 8.0) before 10 µl samples were analysed by SDS-PAGE [17% (w/v) acrylamide gel] and Western immunoblotting. Antibodies used were anti-E. coli DmsD (1 : 20 000) with a horseradish peroxidase-conjugated anti-rabbit IgG secondary (1 : 10 000). The asterisk marks the position of peptide crosslinked DmsD.](https://s3-eu-west-1.amazonaws.com/com.mendeley.prod.article-extracted-content/images/ad844be8-e560-3f24-9377-b0d2ae0e74d9/thumbnail-fa38aa8b-a728-43a2-8b76-837aedb890f9-5.png)
![Fig. 5. DSS-mediated crosslinking of signal peptides to DmsD prevents homo-dimerization of the chaperone. purified DmsD (2 µM) and synthetic peptides 1 and 2 (Table 2; 100 µM) were incubated together with 1 mM DSS (final concentration) in a final volume of 100 µl. The reaction was stopped after 30min with the addition of 50 mM Tris/HCl (pH 8) before 10 µl samples were analysed by SDS-PAGE [17% (w/v) acrylamide gel] and Western immunoblotting. Antibodies used were anti-DmsD (1 : 20 000) and an anti-rabbit IgG secondary (1 : 10 000). The positions of the DmsD monomer (DmsD), dimer (2 DmsD) and peptide crosslinked forms (*) are indicated.](https://s3-eu-west-1.amazonaws.com/com.mendeley.prod.article-extracted-content/images/ad844be8-e560-3f24-9377-b0d2ae0e74d9/thumbnail-8822535e-99dd-459c-9ed2-c3a587dc92c7-6.png)
![Fig. 6. A synthetic peptide with a single internal lysine within the Tat motif can only be weakly crosslinked to DmsD. DSS crosslinking was induced between purified DmsD and synthetic peptide 3 (Table 2), which is a 28-mer carrying a single lysine within the twin-arginine motif. Varying molar ratios of DmsD (1 µM) to peptide (as indicated) were incubated for 1 h with 1 mM DSS. The sample marked ( ) contains no peptide and no crosslinker. Reactions were stopped by the addition of 50 mM Tris/HCl (pH 8), before 10 µl samples were analysed by SDS-PAGE [17% (w/v) acrylamide gel] and Western immunoblot (anti-DmsD, 1 : 20 000; anti-rabbit IgG secondary, 1 : 10 000). The asterisk marks the position of peptide crosslinked DmsD.](https://s3-eu-west-1.amazonaws.com/com.mendeley.prod.article-extracted-content/images/ad844be8-e560-3f24-9377-b0d2ae0e74d9/thumbnail-8eea167d-6cc5-4cc0-aee6-c65f318624d9-7.png)
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Connelly, K. R. S., Stevenson, C., Kneuper, H., & Sargent, F. (2016). Biosynthesis of selenate reductase in Salmonella enterica: Critical roles for the signal peptide and DmsD. Microbiology (United Kingdom), 162(12), 2136–2146. https://doi.org/10.1099/mic.0.000381
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