Anchoring and length regulation of Porphyromonas gingivalis Mfa1 fimbriae by the downstream gene product Mfa2

Porphyromonas gingivalis, a causative agent of periodontitis, has at least two types of thin, single-stranded fimbriae, termed FimA and Mfa1 (according to the names of major subunits), which can be discriminated by filament length and by the size of their major fimbrilin subunits. FimA fimbriae are long filaments that are easily detached from cells, whereas Mfa1 fimbriae are short filaments that are tightly bound to cells. However, a P. gingivalis ATCC 33277-derived mutant deficient in mfa2, a gene downstream of mfa1, produced long filaments (10 times longer than those of the parent), easily detached from the cell surface, similar to FimA fimbriae. Longer Mfa1 fimbriae contributed to stronger autoaggregation of bacterial cells. Complementation of the mutant with the wild-type mfa2 allele in trans restored the parental phenotype. Mfa2 is present in the outer membrane of P. gingivalis, but does not co-purify with the Mfa1 fimbriae. However, co-immunoprecipitation demonstrated that Mfa2 and Mfa1 are associated with each other in whole P. gingivalis cells. Furthermore, immunogold microscopy, including double labelling, confirmed that Mfa2 was located on the cell surface and likely associated with Mfa1 fimbriae. Mfa2 may therefore play a role as an anchor for the Mfa1 fimbriae and also as a regulator of Mfa1 filament length. Two additional downstream genes (pgn0289 and pgn0290) are co-transcribed with mfa1 (pgn0287) and mfa2 (pgn0288), and proteins derived from pgn0289, pgn0290 and pgn0291 appear to be accessory fimbrial components.

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Hypothetical Protein Avin_16040 as the S-Layer Protein of Azotobacter vinelandii and Its Involvement in Plant Root Surface Attachment

Applied and Environmental Microbiology ◽

10.1128/aem.02081-15 ◽

2015 ◽

Vol 81 (21) ◽

pp. 7484-7495 ◽

Cited By ~ 1

Author(s):

Pauline Woan Ying Liew ◽

Bor Chyan Jong ◽

Nazalan Najimudin

Keyword(s):

Cell Surface ◽

Deletion Mutant ◽

Azotobacter Vinelandii ◽

Plant Root ◽

Hypothetical Protein ◽

Surface Hydrophobicity ◽

Root Surface ◽

Bacterial Cells ◽

Wild Type ◽

Content Type

ABSTRACTA proteomic analysis of a soil-dwelling, plant growth-promotingAzotobacter vinelandiistrain showed the presence of a protein encoded by the hypotheticalAvin_16040gene when the bacterial cells were attached to theOryza sativaroot surface. AnAvin_16040deletion mutant demonstrated reduced cellular adherence to the root surface, surface hydrophobicity, and biofilm formation compared to those of the wild type. By atomic force microscopy (AFM) analysis of the cell surface topography, the deletion mutant displayed a cell surface architectural pattern that was different from that of the wild type.Escherichia colitransformed with the wild-typeAvin_16040gene displayed on its cell surface organized motifs which looked like the S-layer monomers ofA. vinelandii. The recombinantE. colialso demonstrated enhanced adhesion to the root surface.

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Lipopolysaccharide biosynthesis-related genes are required for colony pigmentation of Porphyromonas gingivalis

Microbiology ◽

10.1099/mic.0.025163-0 ◽

2009 ◽

Vol 155 (4) ◽

pp. 1282-1293 ◽

Cited By ~ 24

Author(s):

Keiko Sato ◽

Nobuo Kido ◽

Yukitaka Murakami ◽

Charles I. Hoover ◽

Koji Nakayama ◽

...

Keyword(s):

Cell Surface ◽

Porphyromonas Gingivalis ◽

Dependent Manner ◽

Wild Type ◽

Gene Encoding ◽

Lipopolysaccharide Biosynthesis ◽

Ladder Pattern ◽

Surface Polysaccharides ◽

Culture Supernatants ◽

Dose Dependent

The periodontopathic bacterium Porphyromonas gingivalis forms pigmented colonies when incubated on blood agar plates as a result of accumulation of μ-oxo haem dimer on the cell surface. Gingipain–adhesin complexes are responsible for production of μ-oxo haem dimer from haemoglobin. Non-pigmented mutants (Tn6-5, Tn7-1, Tn7-3 and Tn10-4) were isolated from P. gingivalis by Tn4351 transposon mutagenesis [Hoover & Yoshimura (1994), FEMS Microbiol Lett 124, 43–48]. In this study, we found that the Tn6-5, Tn7-1 and Tn7-3 mutants carried Tn4351 DNA in a gene homologous to the ugdA gene encoding UDP-glucose 6-dehydrogenase, a gene encoding a putative group 1 family glycosyltransferase and a gene homologous to the rfa gene encoding ADP heptose-LPS heptosyltransferase, respectively. The Tn10-4 mutant carried Tn4351 DNA at the same position as that for Tn7-1. Gingipain activities associated with cells of the Tn7-3 mutant (rfa) were very weak, whereas gingipain activities were detected in the culture supernatants. Immunoblot and mass spectrometry analyses also revealed that gingipains, including their precursor forms, were present in the culture supernatants. A lipopolysaccharide (LPS) fraction of the rfa deletion mutant did not show the ladder pattern that was usually seen for the LPS of the wild-type P. gingivalis. A recombinant chimera gingipain was able to bind to an LPS fraction of the wild-type P. gingivalis in a dose-dependent manner. These results suggest that the rfa gene product is associated with biosynthesis of LPS and/or cell-surface polysaccharides that can function as an anchorage for gingipain–adhesin complexes.

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Kgp and RgpB, but Not RgpA, Are Important for Porphyromonas gingivalis Virulence in the Murine Periodontitis Model

Infection and Immunity ◽

10.1128/iai.01627-06 ◽

2007 ◽

Vol 75 (3) ◽

pp. 1436-1442 ◽

Cited By ~ 66

Author(s):

Rishi D. Pathirana ◽

Neil M. O'Brien-Simpson ◽

Gail C. Brammar ◽

Nada Slakeski ◽

Eric C. Reynolds

Keyword(s):

Bone Loss ◽

Porphyromonas Gingivalis ◽

Alveolar Bone ◽

Alveolar Bone Loss ◽

Enzyme Linked Immunosorbent Assay ◽

Bacterial Cells ◽

Wild Type ◽

Isogenic Mutant ◽

Specific Immunoglobulin ◽

Isogenic Mutants

ABSTRACT The contributions of three proteinase genes (rgpA, rgpB, and kgp) to the virulence of Porphyromonas gingivalis W50 were investigated in the murine periodontitis model. Mice were orally inoculated with eight doses (1 × 1010 cells per dose) of rgpA, rgpB, kgp, rgpA rgpB, or rgpA rgpB kgp isogenic mutants, and the level of alveolar bone loss, immune response induced, and number of bacterial cells per half maxilla were compared with those of animals inoculated with wild-type P. gingivalis. The kgp, rgpB, rgpA rgpB, and rgpA rgpB kgp isogenic mutants induced significantly (P < 0.05) less bone loss than the rgpA isogenic mutant and the wild type did, and the virulence of the rgpA isogenic mutant and the wild type were not significantly different. Mice inoculated with the wild type or the rgpA isogenic mutant exhibited significantly (P < 0.01) more P. gingivalis cells per half maxilla than mice inoculated with rgpB, kgp, rgpA rgpB, and rgpA rgpB kgp isogenic mutants or nonchallenged mice did, as determined using real-time PCR. A significant positive correlation was found between the number of P. gingivalis cells detected per half maxilla and the amount of alveolar bone loss induced. Enzyme-linked immunosorbent assay results showed that each isogenic mutant and the wild type induced a predominant P. gingivalis antigen-specific immunoglobulin G3 (IgG3) response. Furthermore, the kgp and rgpA rgpB kgp isogenic mutants induced significantly (P < 0.05) lower IgG3 antibody responses than the responses induced by the wild type or the rgpA, rgpB, and rgpA rgpB isogenic mutants. The results suggest that the order in which the proteinases contribute to the virulence of P. gingivalis in the murine periodontitis model is Kgp ≥ RgpB ≫ RgpA.

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Cell surface expression of v-fms-coded glycoproteins is required for transformation.

Molecular and Cellular Biology ◽

10.1128/mcb.4.10.1999 ◽

1984 ◽

Vol 4 (10) ◽

pp. 1999-2009 ◽

Cited By ~ 62

Author(s):

M F Roussel ◽

C W Rettenmier ◽

A T Look ◽

C J Sherr

Keyword(s):

Amino Acids ◽

Protein Kinase ◽

Cell Surface ◽

Gene Product ◽

Kinase Activity ◽

Specific Protein ◽

Protein Kinase Activity ◽

Wild Type ◽

Specific Protein Kinase

The viral oncogene v-fms encodes a transforming glycoprotein with in vitro tyrosine-specific protein kinase activity. Although most v-fms-coded molecules remain internally sequestered in transformed cells, a minor population of molecules is transported to the cell surface. An engineered deletion mutant lacking 348 base pairs of the 3.0-kilobase-pair v-fms gene encoded a polypeptide that was 15 kilodaltons smaller than the wild-type v-fms gene product. The in-frame deletion of 116 amino acids was adjacent to the transmembrane anchor peptide located near the middle of the predicted protein sequence and 432 amino acids from the carboxyl terminus. The mutant polypeptide acquired N-linked oligosaccharide chains, was proteolytically processed in a manner similar to the wild-type glycoprotein, and exhibited an associated tyrosine-specific protein kinase activity in vitro. However, the N-linked oligosaccharides of the mutant glycoprotein were not processed to complex carbohydrate chains, and the glycoprotein was not detected at the cell surface. Cells expressing high levels of the mutant glycoprotein did not undergo morphological transformation and did not form colonies in semisolid medium. The transforming activity of the v-fms gene product therefore appears to be mediated through target molecules on the plasma membrane.

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Mutations in the lrpE Gene of Ralstonia solanacearum Affects Hrp Pili Production and Virulence

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-19-0884 ◽

2006 ◽

Vol 19 (8) ◽

pp. 884-895 ◽

Cited By ~ 13

Author(s):

Yukio Murata ◽

Naoyuki Tamura ◽

Kazuhiro Nakaho ◽

Takafumi Mukaihara

Keyword(s):

Cell Surface ◽

Growth Phase ◽

Ralstonia Solanacearum ◽

Vascular System ◽

Exponential Growth Phase ◽

Bacterial Cells ◽

Wild Type ◽

Bacterial Surface ◽

Bacterial Morphology ◽

In The Wild

The Ralstonia solanacearum hrpB-regulated gene lrpE (hpx5/brg24) encodes a PopC-like leucine-rich repeat (LRR) protein that carries 11 tandem LRR in the central region. Defects in the lrpE gene slightly reduced the virulence of R. solanacearum on host plants and changed the bacterial morphology leading to the formation of large aggregates in a minimal medium. The aggregation in the ΔlrpE background required the presence of a functional Hrp type III secretion system. In wild-type R. solanacearum, Hrp pili disappeared from the bacterial surface at the end of the exponential growth phase, when the pili form into long bundles. However, even in the late growth phase, bundled Hrp pili were still observed on the cell surface of the ΔlrpE mutant. Such bundles were entangled and anchored the mutant cells in the aggregates. In contrast to PopC, LrpE accumulated in bacterial cells and did not translocate into plant cells as an effector protein. The expression levels of hrp genes increased three- to fivefold in the ΔlrpE background compared with those in the wild type. We propose that LrpE may negatively regulate the production of Hrp pili on the cell surface of R. solanacearum to disperse bacterial cells from aggregates. In turn, dispersal may contribute to the movement of the pathogen in the plant vascular system and, as a consequence, the pathogenicity of R. solanacearum.

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Role of vimA in cell surface biogenesis in Porphyromonas gingivalis

Microbiology ◽

10.1099/mic.0.038331-0 ◽

2010 ◽

Vol 156 (7) ◽

pp. 2180-2193 ◽

Cited By ~ 13

Author(s):

Devon O. Osbourne ◽

Wilson Aruni ◽

Francis Roy ◽

Christopher Perry ◽

Lawrence Sandberg ◽

...

Keyword(s):

Cell Surface ◽

Outer Membrane ◽

Porphyromonas Gingivalis ◽

Type Strain ◽

Wild Type Strain ◽

Phenotypic Expression ◽

Surface Proteins ◽

Wild Type ◽

In The Wild

The Porphyromonas gingivalis vimA gene has been previously shown to play a significant role in the biogenesis of gingipains. Further, in P. gingivalis FLL92, a vimA-defective mutant, there was increased auto-aggregation, suggesting alteration in membrane surface proteins. In order to determine the role of the VimA protein in cell surface biogenesis, the surface morphology of P. gingivalis FLL92 was further characterized. Transmission electron microscopy demonstrated abundant fimbrial appendages and a less well defined and irregular capsule in FLL92 compared with the wild-type. In addition, atomic force microscopy showed that the wild-type had a smoother surface compared with FLL92. Western blot analysis using anti-FimA antibodies showed a 41 kDa immunoreactive protein band in P. gingivalis FLL92 which was missing in the wild-type P. gingivalis W83 strain. There was increased sensitivity to globomycin and vancomycin in FLL92 compared with the wild-type. Outer membrane fractions from FLL92 had a modified lectin-binding profile. Furthermore, in contrast with the wild-type strain, nine proteins were missing from the outer membrane fraction of FLL92, while 20 proteins present in that fraction from FLL92 were missing in the wild-type strain. Taken together, these results suggest that the VimA protein affects capsular synthesis and fimbrial phenotypic expression, and plays a role in the glycosylation and anchorage of several surface proteins.

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Cell surface expression of v-fms-coded glycoproteins is required for transformation

Molecular and Cellular Biology ◽

10.1128/mcb.4.10.1999-2009.1984 ◽

1984 ◽

Vol 4 (10) ◽

pp. 1999-2009

Author(s):

M F Roussel ◽

C W Rettenmier ◽

A T Look ◽

C J Sherr

Keyword(s):

Amino Acids ◽

Protein Kinase ◽

Cell Surface ◽

Gene Product ◽

Kinase Activity ◽

Specific Protein ◽

Protein Kinase Activity ◽

Wild Type ◽

Specific Protein Kinase

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Involvement of the Type IX Secretion System in Capnocytophaga ochracea Gliding Motility and Biofilm Formation

Applied and Environmental Microbiology ◽

10.1128/aem.03452-15 ◽

2016 ◽

Vol 82 (6) ◽

pp. 1756-1766 ◽

Cited By ~ 21

Author(s):

Daichi Kita ◽

Satoshi Shibata ◽

Yuichiro Kikuchi ◽

Eitoyo Kokubu ◽

Koji Nakayama ◽

...

Keyword(s):

Cell Surface ◽

Biofilm Formation ◽

Laser Scanning ◽

Secretion System ◽

Gliding Motility ◽

Confocal Laser ◽

Bacterial Cells ◽

Wild Type ◽

Content Type ◽

Type Ix Secretion System

ABSTRACTCapnocytophaga ochraceais a Gram-negative, rod-shaped bacterium that demonstrates gliding motility when cultured on solid agar surfaces.C. ochraceapossesses the ability to form biofilms; however, factors involved in biofilm formation by this bacterium are unclear. A type IX secretion system (T9SS) inFlavobacterium johnsoniaewas shown to be involved in the transport of proteins (e.g., several adhesins) to the cell surface. Genes orthologous to those encoding T9SS proteins inF. johnsoniaehave been identified in the genome ofC. ochracea; therefore, the T9SS may be involved in biofilm formation byC. ochracea. Here we constructed three ortholog-deficientC. ochraceamutants lackingsprB(which encodes a gliding motility adhesin) orgldKorsprT(which encode T9SS proteins inF. johnsoniae). Gliding motility was lost in each mutant, suggesting that, inC. ochracea, the proteins encoded bysprB,gldK, andsprTare necessary for gliding motility, and SprB is transported to the cell surface by the T9SS. For the ΔgldK, ΔsprT, and ΔsprBstrains, the amounts of crystal violet-associated biofilm, relative to wild-type values, were 49%, 34%, and 65%, respectively, at 48 h. Confocal laser scanning and scanning electron microscopy revealed that the biofilms formed by wild-typeC. ochraceawere denser and bacterial cells were closer together than in those formed by the mutant strains. Together, these results indicate that proteins exported by the T9SS are key elements of the gliding motility and biofilm formation ofC. ochracea.

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Decrease in Cell Surface Galactose Residues ofSchizosaccharomyces pombe Enhances Its Coflocculation with Pediococcus damnosus

Applied and Environmental Microbiology ◽

10.1128/aem.67.8.3413-3417.2001 ◽

2001 ◽

Vol 67 (8) ◽

pp. 3413-3417 ◽

Cited By ~ 11

Author(s):

Xuan Peng ◽

Jun Sun ◽

Chris Michiels ◽

Dirk Iserentant ◽

Hubert Verachtert

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Cell Surface ◽

Concanavalin A ◽

Cell Walls ◽

Type Species ◽

Bacterial Cells ◽

Wild Type ◽

Protease Treatment ◽

Type Strains

ABSTRACT Pediococcus damnosus can coflocculate withSaccharomyces cerevisiae and cause beer acidification that may or may not be desired. Similar coflocculations occur with other yeasts except for Schizosaccharomyces pombe which has galactose-rich cell walls. We compared coflocculation rates ofS. pombe wild-type species TP4-1D, having a mannose-to-galactose ratio (Man:Gal) of 5 to 6 in the cell wall, with its glycosylation mutants gms1-1 (Man:Gal = 5:1) and gms1Δ (Man:Gal = 1:0). These mutants coflocculated at a much higher level (30 to 45%) than that of the wild type (5%). Coflocculation of the mutants was inhibited by exogenous mannose but not by galactose. The S. cerevisiae mnn2 mutant, with a mannan content similar to that ofgms1Δ, also showed high coflocculation (35%) and was sensitive to mannose inhibition. Coflocculation of P. damnosus and gms1Δ (or mnn2) also could be inhibited by gms1Δ mannan (with unbranched α-1,6-linked mannose residues), concanavalin A (mannose and glucose specific), or NPA lectin (specific for α-1,6-linked mannosyl units). Protease treatment of the bacterial cells completely abolished coflocculation. From these results we conclude that mannose residues on the cell surface of S. pombe serve as receptors for a P. damnosuslectin but that these receptors are shielded by galactose residues in wild-type strains. Such interactions are important in the production of Belgian acid types of beers in which mixed cultures are used to improve flavor.

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Preferential dipeptide incorporation of Porphyromonas gingivalis mediated by proton-dependent oligopeptide transporter (Pot)

FEMS Microbiology Letters ◽

10.1093/femsle/fnaa204 ◽

2020 ◽

Author(s):

Yuko Ohara-Nemoto ◽

Mohammad Tanvir Sarwar ◽

Yu Shimoyama ◽

Takeshi Kobayakawa ◽

Takayuki K Nemoto

Keyword(s):

Porphyromonas Gingivalis ◽

Systemic Diseases ◽

Bacterial Cells ◽

Wild Type ◽

Oligopeptide Transporter ◽

Potential Therapeutic Target ◽

Metabolic Analysis ◽

Dipeptidyl Peptidases ◽

Dipeptide Uptake ◽

Cellular Components

Abstract Multiple dipeptidyl-peptidases (DPPs) are present in the periplasmic space of Porphyromonas gingivalis, an asaccharolytic periodontopathic bacterium. Dipeptides produced by DPPs are presumed to be transported into the bacterial cells and metabolized to generate energy and cellular components. The present study aimed to identify a transporter responsible for dipeptide uptake in the bacterium. A real-time metabolic analysis demonstrated that P. gingivalis preferentially incorporated Gly-Xaa dipeptides, and then, single amino acids, tripeptides, and longer oligopeptides to lesser extents. Heterologous expression of the P. gingivalis serine/threonine transporter (SstT) (PGN_1460), oligopeptide transporter (Opt) (PGN_1518), and proton-dependent oligopeptide transporter (Pot) (PGN_0135) genes demonstrated that Escherichia coli expressing Pot exclusively incorporated Gly-Gly, while SstT managed Ser uptake and Opt was responsible for Gly-Gly-Gly uptake. Dipeptide uptake was significantly decreased in a P. gingivalis Δpot strain and further suppressed in a Δpot-Δopt double-deficient strain. In addition, the growth of the Δpot strain was markedly attenuated and the Δpot-Δopt strain scarcely grew, whereas the ΔsstT strain grew well almost like wild type. Consequently, these results demonstrate that predominant uptake of dipeptide in P. gingivalis is mostly managed by POT. We thus propose that Pot is a potential therapeutic target of periodontal disease and P. gingivalis-related systemic diseases.

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