Abstract
The outer membrane (OM) protects Gram-negative bacteria against a hostile environment. The proteins embedded in the OM fulfil a number of tasks that are crucial to the bacterial cell. In this study, we identified and characterised a major outer membrane protein (WP_009059494) from Methylacidiphilum fumariolicum SolV. PRED-TMBB and AlphaFold2 predicted this protein to form a porin with a β-barrel structure consisting of ten antiparallel β-sheets and with a small amphipathic N-terminal α-helix in the periplasm. We purified soluble recombinant protein WP_009059494 from E. coli using Tris–HCl buffer with SDS. Antibodies were raised against two peptides in the two large extracellular loops of protein WP_009059494 and immunogold localisation showed this protein to be mainly present in the OM of strain SolV. In addition, this protein is tightly associated with the OM, and is resistant to extraction. Only a small amount can be isolated from the cell envelope using harsh conditions (SDS and boiling). Despite this resistance to extraction, WP_009059494 most likely is an outer membrane protein. A regular lattice could not be detected by negative staining TEM of strain SolV and isolated protein WP_009059494. Considering the specific ecological niche of strain SolV living in a geothermal environment with low pH and high temperatures, this major protein WP_009059494 may act as barrier to resist the extreme conditions found in its natural environment. In addition, we found an absence of the BamB, BamC and BamE proteins of the canonical BAM complex, in Methylacidiphilum and Methylacidimicrobium species. This suggests that these bacteria use a simple BAM complex for folding and transport of OM proteins.