title: Identification and cloning of the gene encoding BmpC: an outer-membrane lipoprotein associated with Brachyspira pilosicoli membrane vesicles.
abstract: The intestinal spirochaete Brachyspira pilosicoli causes colitis in a wide variety of host species. Little is known about the structure or protein constituents of the B. pilosicoli outer membrane (OM). To identify surface-exposed proteins in this species, membrane vesicles were isolated from B. pilosicoli strain 95-1000 cells by osmotic lysis in dH(2)O followed by isopycnic centrifugation in sucrose density gradients. The membrane vesicles were separated into a high-density fraction (HDMV; rho=1.18 g cm(-3)) and a low-density fraction (LDMV; rho=1.12 g cm(-3)). Both fractions were free of flagella and soluble protein contamination. LDMV contained predominantly OM markers (lipo-oligosaccharide and a 29 kDa B. pilosicoli OM protein) and was used as a source of antigens to produce mAbs. Five B. pilosicoli-specific mAbs reacting with proteins with molecular masses of 23, 24, 35, 61 and 79 kDa were characterized. The 23 kDa protein was only partially soluble in Triton X-114, whereas the 24 and 35 kDa proteins were enriched in the detergent phase, implying that they were integral membrane proteins or lipoproteins. All three proteins were localized to the B. pilosicoli OM by immunogold labelling using specific mAbs. The gene encoding the abundant, surface-exposed 23 kDa protein was identified by screening a B. pilosicoli 95-1000 genome library with the mAb and was expressed in Escherichia coli. Sequence analysis showed that it encoded a unique lipoprotein, designated BmpC. Recombinant BmpC partitioned predominantly in the OM fraction of E. coli strain SOLR. The mAb to BmpC was used to screen a collection of 13 genetically heterogeneous strains of B. pilosicoli isolated from five different host species. Interestingly, only strain 95-1000 was reactive with the mAb, indicating that either the surface-exposed epitope on BmpC is variable between strains or that the protein is restricted in its distribution within B. pilosicoli.
authors.affiliation: School of Veterinary Science, The University of Queensland, St Lucia, Queensland, Australia.
authors.forename: Darren J
authors.initials: DJ
authors.lastname: Trott
authors.affiliation: Bacterial Diseases of Livestock Research, National Animal Disease Center, Ames, IA, USA.
authors.forename: David P
authors.initials: DP
authors.lastname: Alt
authors.affiliation: Bacterial Diseases of Livestock Research, National Animal Disease Center, Ames, IA, USA.
authors.forename: Richard L
authors.initials: RL
authors.lastname: Zuerner
authors.affiliation: Bacterial Pathogenesis Research Group, Department of Microbiology, Monash University, Victoria, Australia.
authors.forename: Dieter M
authors.initials: DM
authors.lastname: Bulach
authors.affiliation: Veterinary Medical Research Institute, Iowa State University, Ames, IA, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Wannemuehler
authors.affiliation: Microscopy Services, National Animal Disease Center, Ames, IA, USA.
authors.forename: Judi
authors.initials: J
authors.lastname: Stasko
authors.affiliation: School of Veterinary Science, The University of Queensland, St Lucia, Queensland, Australia.
authors.forename: Kirsty M
authors.initials: KM
authors.lastname: Townsend
authors.affiliation: Pre-Harvest Food Safety Research, National Animal Disease Center, Ames, IA, USA.
authors.forename: Thaddeus B
authors.initials: TB
authors.lastname: Stanton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D001433:Bacteriolysis; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D021961:Cell Membrane Structures; D002500:Centrifugation, Isopycnic; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009997:Osmotic Pressure; D017422:Sequence Analysis, DNA; D013144:Spirochaetales / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D008074:Lipoproteins