Molecular analysis of an outer membrane protein, MopB, of Methylococcus capsulatus (Bath) and structural comparisons wit
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© Springer-Verlag 2000
O R I G I N A L PA P E R
Anne Fjellbirkeland · Vahid Bemanian · Ian R. McDonald · J. Colin Murrell · Harald B. Jensen
Molecular analysis of an outer membrane protein, MopB, of Methylococcus capsulatus (Bath) and structural comparisons with proteins of the OmpA family Received: 5 November 1999 / Revised: 26 January 2000 / Accepted: 28 January 2000 / Published online: 26 February 2000 © Springer-Verlag 2000
Abstract The gene encoding a major outer membrane protein (MopB) of the methanotroph Methylococcus capsulatus (Bath) was cloned and sequenced. The cloned DNA contained an open reading frame of 1044 bp coding for a 348-amino-acid polypeptide with a 21-amino-acid leader peptide. Comparative sequence analysis of the predicted amino acid sequence revealed that the C-terminal part of MopB possessed sequences that are conserved in the OmpA family of proteins. The N-terminal half of the protein had no significant sequence similarity to other proteins in the databases, but the predicted secondary structure showed stretches of amphipathic β-strands typical of transmembrane segments of outer membrane proteins. A region with four cysteines similar to the cysteineencompassing region of the OprF of Pseudomonas aeruginosa was found toward the C-terminal part of MopB. Results from whole-cell labeling with the fluorescent thiol-reacting reagent 5-iodoacetamidofluorescein indicated a surface-exposed location for these cysteines. A probe consisting of the 3′-end of the mopB gene hybridized to the type I methanotroph Methylomonas methanica S1 in Southern blots containing DNA from nine methanotrophic strains representing six different genera. Key words Methanotroph · Methylococcus capsulatus · Outer membrane protein · OprF protein · OmpA protein Abbreviation OMP Outer membrane protein
A. Fjellbirkeland (✉) · V. Bemanian · H.B. Jensen Department of Molecular Biology, University of Bergen, HIB, 5020 Bergen, Norway e-mail: [email protected], Tel.: +47-55-584500, Fax: +47-55-589683 I.R. McDonald · J.C. Murrell Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
Introduction The methanotrophs are characterized by their ability to use methane as a sole source of carbon and energy. They are gram-negative bacteria and are phylogenetically grouped within the α- or γ-subdivision of the Proteobacteria (Bowman et al. 1993). They are also separated into type I or type II, based on their route of carbon assimilation and the ultrastructure of their characteristic intracytoplasmic membranes. In nature they appear to be ubiquitous and have been studied because of their contribution to global methane cycling and their potential commercial application (Lidstrom and Stirling 1990; Hanson and Hanson 1996). Methylococcus capsulatus (Bath) is one of the most extensively studied methanotrophs. It is similar to the type I methanotrophs in that it clusters with the γ-Proteobacteria and has its inner membranes arranged in bundles of vesicular disks. The routes of formaldehyde assimilation, howe
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