A genetic analysis of in vivo selenate reduction by Salmonella enterica serovar Typhimurium LT2 and Escherichia coli K12
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ORIGINAL PAPER
A genetic analysis of in vivo selenate reduction by Salmonella enterica serovar Typhimurium LT2 and Escherichia coli K12 David Guymer · Julien Maillard · Frank Sargent
Received: 3 February 2009 / Revised: 1 April 2009 / Accepted: 6 April 2009 / Published online: 5 May 2009 © Springer-Verlag 2009
Abstract The twin-arginine transport (Tat) system is dedicated to the translocation of folded proteins across the bacterial cytoplasmic membrane. Proteins are targeted to the Tat system by signal peptides containing a twin-arginine motif. In Salmonella enterica serovar Typhimurium and Escherichia coli many Tat substrates are known or predicted to bind a molybdenum cofactor in the cytoplasm prior to export. In the case of N- and S-oxide reductases, coordination of molybdenum cofactor insertion with protein export involves a ‘Tat proofreading’ process where chaperones of the TorD family bind the signal peptides, thus preventing premature export. Here, a genetic approach was taken to determine factors required for selenate reductase activity in Salmonella and E. coli. It is reported for both biological systems that an active Tat translocase and a TorD-like chaperone (DmsD) are required for complete in vivo reduction of selenate to elemental red selenium. Further mutagenesis and in vitro biophysical experiments implicate the Salmonella ynfE gene product, and the E. coli YnfE and YnfF proteins, as putative Tat-targeted selenate reductases.
Communicated by John Helmann. D. Guymer · F. Sargent (&) Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK e-mail: [email protected] J. Maillard ENAC-ISTE/Laboratoire de Biotechnologie Environnementale (LBE), EPF Lausanne, Bâtiment Chimie-B Ecublens, CH-1015 Lausanne, Switzerland
Keywords Enteric bacteria · Bacterial respiration · Twin-arginine translocation pathway · Molybdo-enzymes · Selenate reductase · Molecular chaperone · Mutagenesis · Isothermal titration calorimetry Abbreviations TMAO Trimethylamine N-oxide Tat Twin-arginine translocation
Introduction The twin-arginine translocation (Tat) apparatus is a protein translocation system found in the cytoplasmic membranes of many bacteria and archaea (Berks et al. 2003). Proteins routed through the Tat pathway are synthesised as precursors with N-terminal signal peptides bearing a conserved SRRxFLK ‘twin-arginine’ amino acid motif (Berks 1996) and transported in a fully folded conformation (DeLisa et al. 2003). Salmonella enterica serovar Typhimurium (hereafter ‘Salmonella’) and Escherichia coli are genetically related enteric bacteria and members of the -Proteobacteria. In these biological systems the majority of known, or predicted, Tat-targeted proteins are complex redox enzymes that must acquire their prosthetic groups in the cytoplasm prior to the export event (Sargent 2007). Furthermore, a high proportion of these are ‘molybdo-enzymes’ that are known, or predicted, to contain a version of the molybdenum cofactor at their active sites (Kisker et al.
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