title: Functional analysis of the sortase YhcS in Bacillus subtilis.
abstract: Sortases of Gram-positive bacteria catalyze the covalent C-terminal anchoring of proteins to the cell wall. Bacillus subtilis, a well-known host organism for protein production, contains two putative sortases named YhcS and YwpE. The present studies were aimed at investigating the possible sortase function of these proteins in B. subtilis. Proteomics analyses revealed that sortase-mutant cells released elevated levels of the putative sortase substrate YfkN into the culture medium upon phosphate starvation. The results indicate that YfkN required sortase activity of YhcS for retention in the cell wall. To analyze sortase function in more detail, we focused attention on the potential sortase substrate YhcR, which is co-expressed with the sortase YhcS. Our results showed that the sortase recognition and cell-wall-anchoring motif of YhcR is functional when fused to the Bacillus pumilus chitinase ChiS, a readily detectable reporter protein that is normally secreted. The ChiS fusion protein is displayed at the cell wall surface when YhcS is co-expressed. In the absence of YhcS, or when no cell-wall-anchoring motif is fused to ChiS, the ChiS accumulates predominately in the culture medium. Taken together, these novel findings show that B. subtilis has a functional sortase for anchoring proteins to the cell wall.
authors.affiliation: Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
authors.forename: Hamidreza
authors.initials: H
authors.lastname: Fasehee
authors.forename: Helga
authors.initials: H
authors.lastname: Westers
authors.forename: Albert
authors.initials: A
authors.lastname: Bolhuis
authors.forename: Haike
authors.initials: H
authors.lastname: Antelmann
authors.forename: Michael
authors.initials: M
authors.lastname: Hecker
authors.forename: Wim J
authors.initials: WJ
authors.lastname: Quax
authors.forename: Agha F
authors.initials: AF
authors.lastname: Mirlohi
authors.forename: Jan Maareten
authors.initials: JM
authors.lastname: van Dijl
authors.forename: Gholamreza
authors.initials: G
authors.lastname: Ahmadian
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002473:Cell Wall / Q000201:enzymology / Q000378:metabolism*; D002688:Chitinases / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010710:Phosphates / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D010710:Phosphates; D019881:Aminoacyltransferases; D002688:Chitinases; D003546:Cysteine Endopeptidases