title: The canonical twin-arginine translocase components are not required for secretion of folded green fluorescent protein from the ancestral strain of Bacillus subtilis.
abstract: Cellular processes, such as the digestion of macromolecules, phosphate acquisition, and cell motility, require bacterial secretion systems. In Bacillus subtilis, the predominant protein export pathways are Sec (generalized secretory pathway) and Tat (twin-arginine translocase). Unlike Sec, which secretes unfolded proteins, the Tat machinery secretes fully folded proteins across the plasma membrane and into the medium. Proteins are directed for Tat-dependent export by N-terminal signal peptides that contain a conserved twin-arginine motif. Thus, utilizing the Tat secretion system by fusing a Tat signal peptide is an attractive strategy for the production and export of heterologous proteins. As a proof of concept, we expressed green fluorescent protein (GFP) fused to the PhoD Tat signal peptide in the laboratory and ancestral strains of B. subtilis. Secretion of the Tat-GFP construct, as well as secretion of proteins in general, was substantially increased in the ancestral strain. Furthermore, our results show that secreted, fluorescent GFP could be purified directly from the extracellular medium. Nonetheless, export was not dependent on the known Tat secretion components or the signal peptide twin-arginine motif. We propose that the ancestral strain contains additional Tat components and/or secretion regulators that were abrogated following domestication.
authors.affiliation: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Snyder
authors.forename: Sampriti
authors.initials: S
authors.lastname: Mukherjee
authors.forename: J Kyle
authors.initials: JK
authors.lastname: Glass
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Kearns
authors.forename: Suchetana
authors.initials: S
authors.lastname: Mukhopadhyay
mesh_terms: D001120:Arginine / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058947:Bacterial Secretion Systems; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D021382:Protein Sorting Signals; D021381:Protein Transport; D014166:Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; D021382:Protein Sorting Signals; D049452:Green Fluorescent Proteins; D001120:Arginine; D014166:Transferases