Export of Thermus thermophilus cytoplasmic beta-glycosidase via the E. coli Tat pathway.
The Tat pathway is distinct from the Sec machinery given its unusual capacity to export folded proteins, which contain a twin-arginine (RR) signal peptide, across the plasma membrane. The functionality of the Tat pathway has been demonstrated for several Gram-negative and Gram-positive mesophilic bacteria. To assess the specificity of the Tat system, and to analyze the capacity of a mesophilic bacterial Tat system to translocate cytoplasmic proteins from hyperthermophilic bacteria, we fused the Thermus thermophilus beta-glycosidase (Glc) to the twin-arginine signal peptide of the E. coli TorA protein. When expressed in E. coli, the thermophilic RR-Glc chimera was successfully synthesized and efficiently translocated into the periplasm of the wild type strain. In contrast, the beta-glycosidase accumulated within the cytoplasm of all the tat mutants analyzed. The beta-glycosidase synthesized in these strains exhibited thermophilic properties. These results demonstrated, for the first time, the capacity of the E. coli Tat system to export cytoplasmic hyperthermophilic protein, implying an important potential of the Tat system for the production of thermostable enzymes used in bioprocessing applications.
Gérard
Fabien
F
UPR9043, Institut de Biologie Structurale et Microbiologie, CNRS, Marseille, France.
Angelini
Sandra
S
Wu
Long-Fei
LF
D001709:Biotechnology / Q000379:methods; D003470:Culture Media; D003593:Cytoplasm / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019897:Periplasm / Q000201:enzymology; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
D003470:Culture Media; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; C408585:twin-arginine translocase complex, E coli; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase; D006026:Glycoside Hydrolases