Analyses of Thermotogales secretomes
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The aim of this study was to analyse the secretomes of the deeply branching order Thermotogales, using the species of Thermosipho africanus, Petrotoga mobilis, and Kosmotoga olearia, which have all been isolated from hot and deep North Sea oil reservoirs, as experimental systems. These strains are thermophilic and anaerobic heterotrophs and were grown in mineral media supplemented with yeast extract and carbohydrates. This is the first study of the exoproteomes in this bacterial order. Proteins from culture supernatants, which were concentrated by ultrafiltration, were both analysed by SDS-PAGE and proteomics-based methods. A large number (60-90), extracellular proteins with signal peptides, including solute-binding proteins, various classes of hydrolytic enzymes, as well as previously uncharacterised (hypothetical) proteins, were identified in spent media of P. mobilis and K. olearia. T. africanus culture supernatants contained almost only typical intracellular proteins, and almost no proteins with signal peptides, possibly due to extensive leakage of cytoplasmic proteins from the cells or a high degree of cellular lysis during growth. P. mobilis grown on maltodextrin as a carbon source, a specific alpha amylase accumulated in the medium, demonstrating a substrate-controlled regulation. For K. olearia, a particular large number (33) of uncharacterised proteins with signal peptides were identified, a few of which were among the most abundant extracellular proteins. Some of these proteins are shared with other Thermotogales representatives, while other are unique to K. olearia. This organism is thus a rich source of new extracellular functions. K. olearia also secreted 19 different extracellular solute-binding proteins, belonging to carbohydrate-, oligo/dipeptide-, amino acid-binding families, and 12 different hydrolases, including amylases, peptidases and nucleotidases. This species appears to be an efficient scavenger with a large arsenal of enzymes and proteins for digestion and uptake of various types of nutrients. A smaller number of the same types of the extracellular proteins, were identified in the P. mobilis secretome. A large number of proteins without signal peptides were also detected in the exoproteomes. For T. africanus and P. mobilis, these are possibly due to unspecific leakage or partial cell lysis, but for K. olearia, the fraction with extracellular proteins without signal peptides (160) appeared to be limited and specific. These proteins may be secreted by a hitherto unknown mechanism often referred to as non-classically protein secretion. They are also sometimes termed moon-lighting" proteins, indicating that they may have dual functions, one known intracellular function and one unknown extracellular function.