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
| Autor: | M.L. Kalmokoff, Terry D. Cyr, John W. Austin, L. B. Selinger, Ronald M. Teather, Mary Alice Hefford |
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| Rok vydání: | 2009 |
| Předmět: |
Sequence analysis
Molecular Sequence Data Porins Veillonellaceae Biology Microbiology Open Reading Frames chemistry.chemical_compound Protein structure Microscopy Electron Transmission Sequence Analysis Protein Gene Order Cloning Molecular Membrane Glycoproteins Mitsuokella multacida Molecular mass Cryoelectron Microscopy Protein Structure Tertiary Molecular Weight Infectious Diseases Biochemistry chemistry Porin Electrophoresis Polyacrylamide Gel Peptidoglycan Bacterial outer membrane S-layer Bacterial Outer Membrane Proteins |
| Zdroj: | Anaerobe. 15:74-81 |
| ISSN: | 1075-9964 |
| Popis: | 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 beta-barrel and appears to be related to the unusual N-terminal SLH-domain-containing beta-barrel-porins previously described in the cyanobacterium Synechococcus PCC6301. |
| Databáze: | OpenAIRE |
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