title: A Type I Signal Peptidase Is Required for Pilus Assembly in the Gram-Positive, Biofilm-Forming Bacterium Actinomyces oris.
abstract: The Gram-positive bacterium Actinomyces oris, a key colonizer in the development of oral biofilms, contains 18 LPXTG motif-containing proteins, including fimbrillins that constitute two fimbrial types critical for adherence, biofilm formation, and polymicrobial interactions. Export of these protein precursors, which harbor a signal peptide, is thought to be mediated by the Sec machine and require cleavage of the signal peptide by type I signal peptidases (SPases). Like many Gram-positive bacteria, A. oris expresses two SPases, named LepB1 and LepB2. The latter has been linked to suppression of lethal "glyco-stress," caused by membrane accumulation of the LPXTG motif-containing glycoprotein GspA when the housekeeping sortase srtA is genetically disrupted. Consistent with this finding, we show here that a mutant lacking lepB2 and srtA was unable to produce high levels of glycosylated GspA and hence was viable. However, deletion of neither lepB1 nor lepB2 abrogated the signal peptide cleavage and glycosylation of GspA, indicating redundancy of SPases for GspA. In contrast, the lepB2 deletion mutant failed to assemble the wild-type levels of type 1 and 2 fimbriae, which are built by the shaft fimbrillins FimP and FimA, respectively; this phenotype was attributed to aberrant cleavage of the fimbrillin signal peptides. Furthermore, the lepB2 mutants, including the catalytically inactive S101A and K169A variants, exhibited significant defects in polymicrobial interactions and biofilm formation. Conversely, lepB1 was dispensable for the aforementioned processes. These results support the idea that LepB2 is specifically utilized for processing of fimbrial proteins, thus providing an experimental model with which to study the basis of type I SPase specificity.
  
  
  
  Sec-mediated translocation of bacterial protein precursors across the cytoplasmic membrane involves cleavage of their signal peptide by a signal peptidase (SPase). Like many Gram-positive bacteria, A. oris expresses two SPases, LepB1 and LepB2. The latter is a genetic suppressor of lethal "glyco-stress" caused by membrane accumulation of glycosylated GspA when the housekeeping sortase srtA is genetically disrupted. We show here that LepB1 and LepB2 are capable of processing GspA, whereas only LepB2 is required for cleavage of fimbrial signal peptides. This is the first example of a type I SPase dedicated to LPXTG motif-containing fimbrial proteins. Thus, A. oris provides an experimental model with which to investigate the specificity mechanism of type I SPases.
authors.affiliation: Department of Microbiology & Molecular Genetics, University of Texas Health Science Center, Houston, Texas, USAThe University of Chicago.
authors.forename: Sara D
authors.initials: SD
authors.lastname: Siegel
authors.affiliation: Department of Microbiology & Molecular Genetics, University of Texas Health Science Center, Houston, Texas, USAThe University of Chicago chenggang.wu@uth.tmc.edu ton-that.hung@uth.tmc.edu.
authors.forename: Chenggang
authors.initials: C
authors.lastname: Wu
authors.affiliation: Department of Microbiology & Molecular Genetics, University of Texas Health Science Center, Houston, Texas, USAThe University of Chicago chenggang.wu@uth.tmc.edu ton-that.hung@uth.tmc.edu.
authors.forename: Hung
authors.initials: H
authors.lastname: Ton-That
mesh_terms: D000190:Actinomyces / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018441:Biofilms; D015536:Down-Regulation; D010861:Fimbriae, Bacterial / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D012697:Serine Endopeptidases; C026515:type I signal peptidase