A genetic screen for suppressors of Escherichia coli Tat signal peptide mutations establishes a critical role for the second arginine within the twin-arginine motif.
The Escherichia coli Tat protein export pathway transports folded proteins synthesized with N-terminal twin-arginine signal peptides. Twin-arginine signal sequences contain a conserved SRRxFLK "twin-arginine" amino acid sequence motif which is required for protein export by the Tat pathway. The E. coli trimethylamine N-oxide reductase (TorA) is a Tat-dependent periplasmic molybdoenzyme that facilitates anaerobic respiration with trimethylamine N-oxide as terminal electron acceptor. Here, we describe mutant strains constructed with modified TorA twin-arginine signal peptides. Substitution of the second arginine residue of the TorA signal peptide twin-arginine motif with either lysine or aspartate, or the simultaneous substitution of both arginines with lysine residues, completely abolished export. In each case, the now cytoplasmically localised TorA retained full enzymatic activity with the artificial electron donor benzyl viologen. However, the mutant strains were incapable of anaerobic growth with trimethylamine N-oxide and the non-fermentable carbon-source glycerol. The growth phenotype of the mutant strains was exploited in a genetic screen with the aim of identifying second-site suppressor mutations that allowed export of the modified TorA precursors.
Buchanan
G
G
Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK.
Sargent
F
F
Berks
B C
BC
Palmer
T
T
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics*; D013489:Suppression, Genetic
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; C408585:twin-arginine translocase complex, E coli; D001120:Arginine; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase