title: Sequence analysis, expression, and binding activity of recombinant major outer sheath protein (Msp) of Treponema denticola.
abstract: The gene encoding the major outer sheath protein (Msp) of the oral spirochete Treponema denticola ATCC 35405 was cloned, sequenced, and expressed in Escherichia coli. Preliminary sequence analysis showed that the 5' end of the msp gene was not present on the 5.5-kb cloned fragment described in a recent study (M. Haapasalo, K. H. Müller, V. J. Uitto, W. K. Leung, and B. C. McBride, Infect. Immun. 60:2058-2065,1992). The 5' end of msp was obtained by PCR amplification from a T. denticola genomic library, and an open reading frame of 1,629 bp was identified as the coding region for Msp by combining overlapping sequences. The deduced peptide consisted of 543 amino acids and had a molecular mass of 58,233 Da. The peptide had a typical prokaryotic signal sequence with a potential cleavage site for signal peptidase 1. Northern (RNA) blot analysis showing the msp transcript to be approximately 1.7 kb was consistent with the identification of a promoter consensus sequence located optimally upstream of msp and a transcription termination signal found downstream of the stop codon. The entire msp sequence was amplified from T. denticola genomic DNA and cloned in E. coli by using a tightly regulated T7 RNA polymerase vector system. Expression of Msp was toxic to E. coli when the entire msp gene was present. High levels of Msp were produced as inclusion bodies when the putative signal peptide sequence was deleted and replaced by a vector-encoded T7 peptide sequence. Recombinant Msp purified to homogeneity from a clone containing the full-length msp gene adhered to immobilized laminin and fibronectin but not to bovine serum albumin. Attachment of recombinant Msp was decreased in the presence of soluble substrate. Attachment of T. denticola to immobilized laminin and fibronectin was increased by pretreatment of the substrate with recombinant Msp. These studies lend further support to the hypothesis that Msp mediates the extracellular matrix binding activity of T. denticola.
authors.affiliation: Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.
authors.forename: J C
authors.initials: JC
authors.lastname: Fenno
authors.forename: K H
authors.initials: KH
authors.lastname: Müller
authors.forename: B C
authors.initials: BC
authors.lastname: McBride
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005353:Fibronectins / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial / Q000235:genetics; D007797:Laminin / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018272:Porins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins; D017422:Sequence Analysis, DNA; D014208:Treponema / Q000235:genetics*; D014764:Viral Proteins
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D005353:Fibronectins; D007797:Laminin; D018272:Porins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C099361:major outer sheath protein, Treponema; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases