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Olivera Francetic - One of the best experts on this subject based on the ideXlab platform.

  • polar n terminal residues conserved in type 2 secretion pseudopilins determine subunit targeting and membrane extraction steps during fibre assembly
    Journal of Molecular Biology, 2017
    Co-Authors: Javier Santosmoreno, Alexandra East, Ingrid Guilvout, Nathalie Nadeau, Peter J Bond, Guy Tran Van Nhieu, Olivera Francetic
    Abstract:

    Bacterial type 2 secretion systems (T2SS), type 4 pili, and archaeal flagella assemble fibres from initially membrane-embedded pseudopilin and pilin subunits. Fibre subunits are made as precursors with positively charged N-terminal anchors, whose cleavage via the Prepilin Peptidase, essential for pilin membrane extraction and assembly, is followed by N-methylation of the mature (pseudo)pilin N terminus. The conserved Glu residue at position 5 (E5) of mature (pseudo)pilins is essential for assembly. Unlike T4 pilins, where E5 residue substitutions also abolish N-methylation, the E5A variant of T2SS pseudopilin PulG remains N-methylated but is affected in interaction with the T2SS component PulM. Here, biochemical and functional analyses showed that the PulM interaction defect only partly accounts for the PulGE5A assembly defect. First, PulGT2A variant, equally defective in PulM interaction, remained partially functional. Furthermore, pseudopilus assembly defect of pulG(E5A) mutant was stronger than that of the pulM deletion mutant. To understand the dominant effect of E5A mutation, we used molecular dynamics simulations of PulGE5A, methylated PulGWT (MePulGWT), and MePulGE5A variant in a model membrane. These simulations pointed to a key role for an intramolecular interaction between the pseudopilin N-terminal amine and E5 to limit polar interactions with membrane phospholipids. N-methylation of the N-terminal amine further limited its interactions with phospholipid head-groups to facilitate pseudopilin membrane escape. By binding to polar residues in the conserved N-terminal region of PulG, we propose that PulM acts as chaperone to promote pseudopilin recruitment and coordinate its membrane extraction with subsequent steps of the fibre assembly process.

  • recent progress and future directions in studies of the main terminal branch of the general secretory pathway in gram negative bacteria a review
    Gene, 1997
    Co-Authors: Anthony P Pugsley, Olivera Francetic, Odile Possot, Nathalie Sauvonnet, Kim R Hardie
    Abstract:

    Abstract The main terminal branch (MTB) of the general secretory pathway is used by a wide variety of Gram− bacteria to transport exoproteins from the periplasm to the outside milieu. Recent work has led to the identification of the function of two of its 14 (or more) components: an enzyme with type-IV Prepilin Peptidase activity and a chaperone-like protein required for the insertion of another of the MTB components into the outer membrane. Despite these important discoveries, little tangible progress has been made towards identifying MTB components that determine secretion specificity (presumably by binding to cognate exoproteins) or which form the putative channel through which exoproteins are transported across the outer membrane. However, the idea that the single integral outer membrane component of the MTB could line the wall of this channel, and the intriguing possibility that other components of the MTB form a rudimentary type-IV pilus-like structure that might span the periplasm both deserve more careful examination. Although Escherichia coli K-12 does not normally secrete exoproteins, its chromosome contains an apparently complete set of genes coding for MTB components. At least two of these genes code for functional proteins, but the operon in which twelve of the genes are located does not appear to be expressed. We are currently searching for conditions which allow these genes to be expressed with the eventual aim of identifying the protein(s) that E. coli K-12 can secrete.

  • The Cryptic General Secretory Pathway (gsp) Operon of Escherichia coli K-12 Encodes Functional Proteins
    1996
    Co-Authors: A. The Cryptic P. General, Olivera Francetic, P. Pugsley
    Abstract:

    Systematic sequencing of the Escherichia coli K-12 chromosome (GenBank entry U18997) has revealed the presence of an apparently complete operon of genes (the gspC-O operon) similar to genes coding for compo-nents of the main terminal branch of the general secretory pathway (e.g., the Klebsiella oxytoca pulC-O pullulanase secretion operon) and to related genes required for type IV pilus biogenesis. For example, the last gene in the gsp operon, gspO (formerly hopD), encodes a protein which is similar to several type IV Prepilin Peptidases. Expression of gspO from lacZp promotes cleavage of two known Prepilin Peptidase substrates in E. coli K-12: Neisseria gonorrhoeae type IV Prepilin and K. oxytoca prePulG protein. gspO also complements a mutation in the corresponding gene (pulO) of the pullulanase secretion operon when it is expressed from lacZp. Another gene in the gsp operon, gspG (formerly hopG), encodes a protein similar to prePulG, a component of the pullulanase secretion pathway. Expression of gspG from lacZp leads to production of a protein which (i) is recognized by PulG-specific antiserum (and by antiserum against the Pseudomonas aeruginosa PulG homolog XcpG [formerly XcpT]), (ii) is processed in cells expressing gspO, and (iii) restores secretion in cells carrying a pulG mutation. The chromosomal copies of gspG and gspO are apparently not expressed, probably becaus

  • The cryptic general secretory pathway (gsp) operon of Escherichia coli K-12 encodes functional proteins
    1996
    Co-Authors: Olivera Francetic, P. Pugsley
    Abstract:

    Systematic sequencing of the Escherichia coli K-12 chromosome (GenBank entry U18997) has revealed the presence of an apparently complete operon of genes (the gspC-O operon) similar to genes coding for compo-nents of the main terminal branch of the general secretory pathway (e.g., the Klebsiella oxytoca pulC-O pullulanase secretion operon) and to related genes required for type IV pilus biogenesis. For example, the last gene in the gsp operon, gspO (formerly hopD), encodes a protein which is similar to several type IV Prepilin Peptidases. Expression of gspO from lacZp promotes cleavage of two known Prepilin Peptidase substrates in E. coli K-12: Neisseria gonorrhoeae type IV Prepilin and K. oxytoca prePulG protein. gspO also complements a mutation in the corresponding gene (pulO) of the pullulanase secretion operon when it is expressed from lacZp. Another gene in the gsp operon, gspG (formerly hopG), encodes a protein similar to prePulG, a component of the pullulanase secretion pathway. Expression of gspG from lacZp leads to production of a protein which (i) is recognized by PulG-specific antiserum (and by antiserum against the Pseudomonas aeruginosa PulG homolog XcpG [formerly XcpT]), (ii) is processed in cells expressing gspO, and (iii) restores secretion in cells carrying a pulG mutation. The chromosomal copies of gspG and gspO are apparently not expressed, probably because of very weak transcription from the upstream region, as measured by using a chromosomal gspC-lacZ operon fusion. Thus, the gsp operon of E. coli K-12 includes at least two functional genes which, together with the res

Ken F. Jarrell - One of the best experts on this subject based on the ideXlab platform.

  • pilin processing follows a different temporal route than that of archaellins in methanococcus maripaludis
    Life, 2015
    Co-Authors: Divya B. Nair, Ken F. Jarrell
    Abstract:

    Methanococcus maripaludis has two different surface appendages: type IV-like pili and archaella. Both structures are believed to be assembled using a bacterial type IV pilus mechanism. Each structure is composed of multiple subunits, either pilins or archaellins. Both pilins and archaellins are made initially as preproteins with type IV pilin-like signal peptides, which must be removed by a Prepilin Peptidase-like enzyme. This enzyme is FlaK for archaellins and EppA for pilins. In addition, both pilins and archaellins are modified with N-linked glycans. The archaellins possess an N-linked tetrasaccharide while the pilins have a pentasaccharide which consists of the archaellin tetrasaccharide but with an additional sugar, an unidentified hexose, attached to the linking sugar. In this report, we show that archaellins can be processed by FlaK in the absence of N-glycosylation and N-glycosylation can occur on archaellins that still retain their signal peptides. In contrast, pilins are not glycosylated unless they have been acted on by EppA to have the signal peptide removed. However, EppA can still remove signal peptides from non-glycosylated pilins. These findings indicate that there is a difference in the order of the posttranslational modifications of pilins and archaellins even though both are type IV pilin-like proteins.

  • identification of an additional minor pilin essential for piliation in the archaeon methanococcus maripaludis
    PLOS ONE, 2013
    Co-Authors: Divya B. Nair, Daniel K C Chung, James Schneider, Kaoru Uchida, Shin-ichi Aizawa, Ken F. Jarrell
    Abstract:

    Methanococcus maripaludis is an archaeon with two studied surface appendages, archaella and type IV-like pili. Previously, the major structural pilin was identified as MMP1685 and three additional proteins were designated as minor pilins (EpdA, EpdB and EpdC). All of the proteins are likely processed by the pilin-specific Prepilin Peptidase EppA. Six other genes were identified earlier as likely encoding pilin proteins processed also by EppA. In this study, each of the six genes (mmp0528, mmp0600, mmp0601, mmp0709, mmp0903 and mmp1283) was deleted and the mutants examined by electron microscopy to determine their essentiality for pili formation. While mRNA transcripts of all genes were detected by RT-PCR, only the deletion of mmp1283 led to nonpiliated cells. This strain could be complemented back to a piliated state by supplying a wildtype copy of the mmp1283 gene in trans. This study adds to the complexity of the type IV pili system in M. maripaludis and raises questions about the functions of the remaining five pilin-like genes and whether M. maripaludis under other growth conditions may be able to assemble additional pili-like structures.

  • Identification of an Additional Minor Pilin Essential for Piliation in the Archaeon Methanococcus maripaludis
    2013
    Co-Authors: Divya B. Nair, Daniel K C Chung, James Schneider, Kaoru Uchida, Shin-ichi Aizawa, Ken F. Jarrell
    Abstract:

    Methanococcus maripaludis is an archaeon with two studied surface appendages, archaella and type IV-like pili. Previously, the major structural pilin was identified as MMP1685 and three additional proteins were designated as minor pilins (EpdA, EpdB and EpdC). All of the proteins are likely processed by the pilin-specific Prepilin Peptidase EppA. Six other genes were identified earlier as likely encoding pilin proteins processed also by EppA. In this study, each of the six genes (mmp0528, mmp0600, mmp0601, mmp0709, mmp0903 and mmp1283) was deleted and the mutants examined by electron microscopy to determine their essentiality for pili formation. While mRNA transcripts of all genes were detected by RT-PCR, only the deletion of mmp1283 led to nonpiliated cells. This strain could be complemented back to a piliated state by supplying a wildtype copy of the mmp1283 gene in trans. This study adds to the complexity of the type IV pili system in M. maripaludis and raises questions about the functions of the remaining five pilin-like genes and whether M. maripaludis under othe

  • Archaeal signal Peptidases.
    Microbiology, 2007
    Co-Authors: Bonnie Chaban, David J. Vandyke, Ken F. Jarrell
    Abstract:

    Signal Peptidases are vital enzymes in the protein secretion pathway. In Archaea, type I signal Peptidase, responsible for the cleavage of secretory signal peptides from the majority of secreted proteins, and Prepilin Peptidase-like signal Peptidase, responsible for processing signal peptides from Prepilin-like proteins like the preflagellins and various sugar-binding proteins, have been identified. In addition, the archaeal signal peptide Peptidase, responsible for degradation of signal peptides after their removal from precursor proteins, has been characterized. These enzymes seem to have a mosaic of eukaryal and bacterial characteristics, and also possess unique archaeal traits. In this review, the most current knowledge with regard to these enzymes is summarized, including their cellular function, catalytic mechanism and distribution and conservation among archaeal species. Comparisons are drawn of these enzymes to their bacterial and eukaryal counterparts, and unique archaeal features highlighted.

Mechthild Pohlschroder - One of the best experts on this subject based on the ideXlab platform.

  • identification of diverse archaeal proteins with class iii signal peptides cleaved by distinct archaeal Prepilin Peptidases
    Journal of Bacteriology, 2007
    Co-Authors: Zalan Szabo, Adriana Oliveira Stahl, Sonja V Albers, Jessica C Kissinger, Arnold J M Driessen, Mechthild Pohlschroder
    Abstract:

    Most secreted archaeal proteins are targeted to the membrane via a tripartite signal composed of a charged N terminus and a hydrophobic domain, followed by a signal Peptidase-processing site. Signal peptides of archaeal flagellins, similar to class III signal peptides of bacterial type IV pilins, are distinct in that their processing sites precede the hydrophobic domain, which is crucial for assembly of these extracytoplasmic structures. To identify the complement of archaeal proteins with class III signal sequences, a PERL program (FlaFind) was written. A diverse set of proteins was identified, and many of these FlaFind positives were encoded by genes that were cotranscribed with homologs of pilus assembly genes. Moreover, structural conservation of primary sequences between many FlaFind positives and subunits of bacterial pilus-like structures, which have been shown to be critical for pilin assembly, have been observed. A subset of pilin-like FlaFind positives contained a conserved domain of unknown function (DUF361) within the signal peptide. Many of the genes encoding these proteins were in operons that contained a gene encoding a novel euryarchaeal Prepilin-Peptidase, EppA, homolog. Heterologous analysis revealed that Methanococcus maripaludis DUF361-containing proteins were specifically processed by the EppA homolog of this archaeon. Conversely, M. maripaludis preflagellins were cleaved only by the archaeal preflagellin Peptidase FlaK. Together, the results reveal a diverse set of archaeal proteins with class III signal peptides that might be subunits of as-yet-undescribed cell surface structures, such as archaeal pili.

Rachida Bouhenni - One of the best experts on this subject based on the ideXlab platform.

  • the role of shewanella oneidensis mr 1 outer surface structures in extracellular electron transfer
    Electroanalysis, 2010
    Co-Authors: Rachida Bouhenni, Gary J Vora, Justin C Biffinger, Sheetal Shirodkar, Kenneth L Brockman, Peter K Wu, Brandy J Johnson, Eulandria M Biddle, Matthew J Marshall
    Abstract:

    The ability of the metal reducer Shewanella oneidensis MR-1 to generate electricity in microbial fuel cells (MFCs) depends on the activity of a predicted type IV Prepilin Peptidase; PilD. Analysis of an S. oneidensis MR-1 pilD mutant indicated that it was deficient in pili production (Msh and type IV) and type II secretion (T2S). The requirement for T2S in metal reduction has been previously identified, but the role of pili remains largely unexplored. To define the role of type IV or Msh pili in electron transfer, mutants that lack one or both pilus biogenesis systems were generated and analyzed; a mutant that lacked flagella was also constructed and tested. All mutants were able to reduce insoluble Fe(III) and to generate current in MFCs, in contrast to the T2S mutant that is deficient in both processes. Our results show that loss of metal reduction in a PilD mutant is due to a T2S deficiency, and therefore the absence of c cytochromes from the outer surface of MR-1 cells, and not the loss of pili or flagella. Furthermore, MR-1 mutants deficient in type IV pili or flagella generated more current than the wild type, even though extracellular riboflavin levels were similar in all strains. This enhanced current generating ability is in contrast to a mutant that lacks the outer membrane c cytochromes, MtrC and OmcA. This mutant generated significantly less current than the wild type in an MFC and was unable to reduce Fe(III). These results indicated that although nanofilaments and soluble mediators may play a role in electron transfer, surface exposure of outer membrane c cytochromes was the determining factor in extracellular electron transfer in S. oneidensis MR-1.

Vladimir Pelicic - One of the best experts on this subject based on the ideXlab platform.

  • cloning independent markerless gene editing in streptococcus sanguinis novel insights in type iv pilus biology
    Nucleic Acids Research, 2017
    Co-Authors: Ishwori Gurung, Jamielee Berry, Alexander M J Hall, Vladimir Pelicic
    Abstract:

    : Streptococcus sanguinis, a naturally competent opportunistic human pathogen, is a Gram-positive workhorse for genomics. It has recently emerged as a model for the study of type IV pili (Tfp)-exceptionally widespread and important prokaryotic filaments. To enhance genetic manipulation of Streptococcus sanguinis, we have developed a cloning-independent methodology, which uses a counterselectable marker and allows sophisticated markerless gene editing in situ. We illustrate the utility of this methodology by answering several questions regarding Tfp biology by (i) deleting single or mutiple genes, (ii) altering specific bases in genes of interest, and (iii) engineering genes to encode proteins with appended affinity tags. We show that (i) the last six genes in the pil locus harbouring all the genes dedicated to Tfp biology play no role in piliation or Tfp-mediated motility, (ii) two highly conserved Asp residues are crucial for enzymatic activity of the Prepilin Peptidase PilD and (iii) that pilin subunits with a C-terminally appended hexa-histidine (6His) tag are still assembled into functional Tfp. The methodology for genetic manipulation we describe here should be broadly applicable.