Physical and genetic characterization of an outer-membrane protein (OmpM1) containing an N-terminal S-layer-like homology domain from the phylogenetically Gram-positive gut anaerobe Mitsuokella multacida

Thin sectioning and freeze-fracture-etch of the ovine ruminal isolate Mitsuokella multacida strain 46/5(2) revealed a Gram-negative envelope ultra-structure consisting of a peptidoglycan wall overlaid by an outer membrane. Sodium-dodecyl-sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis...

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Bibliographic Details
Published in:Anaerobe 2009-06, Vol.15 (3), p.74-81
Main Authors: Kalmokoff, M.L., Austin, J.W., Cyr, T.D., Hefford, M.A., Teather, R.M., Selinger, L.B.
Format: Article
Language:English
Subjects:
Bacterial Outer Membrane Proteins - chemistry
Bacterial Outer Membrane Proteins - genetics
Bacterial Outer Membrane Proteins - isolation & purification
Cloning, Molecular
Cryoelectron Microscopy
Cyanobacteria
Electrophoresis, Polyacrylamide Gel
Gene Order
Gram-negative
Membrane Glycoproteins - genetics
Microscopy, Electron, Transmission
Molecular Sequence Data
Molecular Weight
Open Reading Frames
Outer membrane
Outer-membrane protein
Porin
Porins - chemistry
Porins - genetics
Porins - isolation & purification
Protein Structure, Tertiary
Sequence Analysis, Protein
Surface-layer homology (SLH) domain
Synechococcus
Veillonellaceae - chemistry
Veillonellaceae - genetics
Veillonellaceae - ultrastructure
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Summary:Thin sectioning and freeze-fracture-etch of the ovine ruminal isolate Mitsuokella multacida strain 46/5(2) revealed a Gram-negative envelope ultra-structure consisting of a peptidoglycan wall overlaid by an outer membrane. Sodium-dodecyl-sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of whole cells, cell envelopes and Triton X-100 extracted envelopes in combination with thin-section and N-terminal sequence analyses demonstrated that the outer membrane contained two major proteins (45 and 43 kDa) sharing identical N-termini (A-A-N-P-F-S-D-V-P-A-D-H-W-A-Y-D). A gene encoding a protein with a predicted N-terminus identical to those of the 43 and 45 kDa outer-membrane proteins was cloned. The 1290 bp open reading frame encoded a 430 amino acid polypeptide with a predicted molecular mass of 47,492 Da. Cleavage of a predicted 23 amino acid leader sequence would yield a protein with a molecular mass of 45,232 Da. Mass spectroscopic analysis confirmed that the cloned gene ( ompM1) encoded the 45 kDa outer-membrane protein. The N-terminus of the mature OmpM1 protein (residues 24–70) shared homology with surface-layer homology (SLH) domains found in a wide variety of regularly structured surface-layers (S-layers). However, the outer-membrane locale, resistance to denaturation by SDS and high temperatures and the finding that the C-terminal residue was a phenylalanine suggested that ompM1 encoded a porin. Threading analysis in combination with the identification of membrane spanning domains indicated that the C-terminal region of OmpM1 (residues 250–430) likely forms a 16-strand β-barrel and appears to be related to the unusual N-terminal SLH-domain-containing β-barrel-porins previously described in the cyanobacterium Synechococcus PCC6301.
ISSN:1075-9964
1095-8274
DOI:10.1016/j.anaerobe.2009.01.001