scholarly journals Recombinant Porphyromonas gingivalis FimA preproprotein expressed in Escherichia coli is lipidated and the mature or processed recombinant FimA protein forms a short filament in vitro

2010 ◽  
Vol 56 (11) ◽  
pp. 959-967 ◽  
Author(s):  
Mikio Shoji ◽  
Atsutoshi Yoshimura ◽  
Hidenobu Yoshioka ◽  
Akemi Takade ◽  
Yasuko Takuma ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Outer Membrane ◽  
Porphyromonas Gingivalis ◽  
Signal Peptide ◽  
Periodontal Diseases ◽  
Microscopic Analysis ◽  
Base Substitution ◽  
Electron Microscopic

The gram-negative anaerobic bacterium Porphyromonas gingivalis is an etiologically important pathogen for chronic periodontal diseases in adults. Our previous study suggested that the major structural components of both Fim and Mfa fimbriae in this organism are secreted through their lipidated precursors. In this study, we constructed Escherichia coli strains expressing various fimA genes with or without the 5′-terminal DNA region encoding the signal peptide, and we determined whether lipidation of recombinant FimA proteins occurred in E. coli. Lipidation occurred for a recombinant protein from the fimA gene with the 5′-terminal DNA region encoding the signal peptide but not for a recombinant protein from the fimA gene without the signal-peptide-encoding region, as revealed by [3H]palmitic acid labeling experiments. A TLR2-dependent signaling response was induced by the recombinant protein from the fimA gene with the signal-peptide-encoding region but not by a recombinant protein from the fimA gene with the signal-peptide-encoding region that had a base substitution causing an amino acid substitution (C19A). Electron microscopic analysis revealed that recombinant FimA (A-47 – W-383) protein was autopolymerized to form filamentous structures of about 80 nm in length in vitro. The results suggest that FimA protein, a major subunit of Fim fimbriae, is transported to the outer membrane by the lipoprotein sorting system, and a mature or processed FimA protein on the outer membrane is autopolymerized to form Fim fimbriae.

Temperature-sensitive processing of outer membrane lipoprotein in an Escherichia coli mutant

1982 ◽  
Vol 152 (3) ◽  
pp. 1163-1168
Author(s):  
H Yamagata ◽  
C Ippolito ◽  
M Inukai ◽  
M Inouye
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Signal Peptide ◽  
Signal Peptidase ◽  
Temperature Sensitive ◽  
Coli Mutant ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein

A mutant of Escherichia coli that accumulated prolipoprotein, a secretory precursor of the outer membrane lipoprotein, was isolated. The prolipoprotein accumulated in this mutant was modified by glyceride, but the in vitro cleavage of the signal peptide of the accumulated prolipoprotein was found to be temperature sensitive. The mutation appears to be located outside the gene for the lipoprotein, thus suggesting that the gene for the signal peptidase for the prolipoprotein was mutated.

scholarly journals Xylanase Attachment to the Cell Wall of the Hyperthermophilic Bacterium Thermotoga maritima

2007 ◽  
Vol 190 (4) ◽  
pp. 1350-1358 ◽  
Author(s):  
Wolfgang Liebl ◽  
Christoph Winterhalter ◽  
Wolfgang Baumeister ◽  
Martin Armbrecht ◽  
Michael Valdez
Keyword(s):  
Outer Membrane ◽  
Signal Peptide ◽  
Cellular Localization ◽  
Culture Supernatant ◽  
Thermotoga Maritima ◽  
Surrounding Medium ◽  
Hydrophobic Core ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Amino Terminal

ABSTRACT The cellular localization and processing of the endo-xylanases (1,4-β-d-xylan-xylanohydrolase; EC 3.2.1.8) of the hyperthermophile Thermotoga maritima were investigated, in particular with respect to the unusual outer membrane (“toga”) of this gram-negative bacterium. XynB (40 kDa) was detected in the periplasmic fraction of T. maritima cells and in the culture supernatant. XynA (120 kDa) was partially released to the surrounding medium, but most XynA remained cell associated. Immunogold labeling of thin sections revealed that cell-bound XynA was localized mainly in the outer membranes of T. maritima cells. Amino-terminal sequencing of purified membrane-bound XynA revealed processing of the signal peptide after the eighth residue, thereby leaving the hydrophobic core of the signal peptide attached to the enzyme. This mode of processing is reminiscent of type IV prepilin signal peptide cleavage. Removal of the entire XynA signal peptide was necessary for release from the cell because enzyme purified from the culture supernatant lacked 44 residues at the N terminus, including the hydrophobic part of the signal peptide. We conclude that toga association of XynA is mediated by residues 9 to 44 of the signal peptide. The biochemical and electron microscopic localization studies together with the amino-terminal processing data indicate that XynA is held at the cell surface of T. maritima via a hydrophobic peptide anchor, which is highly unusual for an outer membrane protein.

Structural features, properties and regulation of the outer-membrane protein W (OmpW) of Vibrio cholerae

Microbiology ◽  
2005 ◽  
Vol 151 (9) ◽  
pp. 2975-2986 ◽  
Author(s):  
Bisweswar Nandi ◽  
Ranjan K. Nandy ◽  
Amit Sarkar ◽  
Asoke C. Ghose
Keyword(s):  
Membrane Protein ◽  
Recombinant Protein ◽  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Outer Membrane Protein ◽  
Secondary Structure Prediction ◽  
Sequence Data ◽  
Insertion Mutagenesis ◽  
Sds Page

The outer-membrane protein OmpW of Vibrio cholerae was studied with respect to its structure, functional properties and regulation of expression. On SDS-PAGE, the membrane-associated form of OmpW protein (solubilized by either 0·1 % or 2 % SDS at 25 °C) migrated as a monomer of 19 kDa that changed to 21 kDa on boiling. The protein was hyperexpressed in Escherichia coli in the histidine-tagged form and the purified His6-OmpW (heated or unheated) migrated as a 23 kDa protein on SDS-PAGE. Circular dichroism and Fourier-transform infrared spectroscopic analyses of the recombinant protein showed the presence of β-structures (∼40 %) with minor amounts (8–15 %) of α-helix. These results were consistent with those obtained by computational analysis of the sequence data of the protein using the secondary structure prediction program Jnet. The recombinant protein did not exhibit any porin-like property in a liposome-swelling assay. An antiserum to the purified protein induced a moderate level (66·6 % and 33·3 % at 1 : 50 and 1 : 100 dilutions, respectively) of passive protection against live vibrio challenge in a suckling mouse model. OmpW-deficient mutants of V. cholerae strains were generated by insertion mutagenesis. In a competitive assay in mice, the intestinal colonization activities of these mutants were found to be either only marginally diminished (for O1 strains) or 10-fold less (for an O139 strain) as compared to those of the corresponding wild-type strains. The OmpW protein was expressed in vivo as well as in vitro in liquid culture medium devoid of glucose. Interestingly, the glucose-dependent regulation of OmpW expression was less prominent in a ToxR− mutant of V. cholerae. Further, the expression of OmpW protein was found to be dependent on in vitro cultural conditions such as temperature, salinity, and availability of nutrients or oxygen. These results suggest that the modulation of OmpW expression by environmental factors may be linked to the adaptive response of the organism under stress conditions.

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