title: Molecular characterization of the 28- and 31-kilodalton subunits of the Legionella pneumophila major outer membrane protein.
abstract: The major outer membrane protein of Legionella pneumophila exhibits an apparent molecular mass of 100 kDa. Previous studies revealed the oligomer to be composed of 28- and 31-kDa subunits; the latter subunit is covalently bound to peptidoglycan. These proteins exhibit cross-reactivity with polyclonal anti-31-kDa protein serum. In this study, we present evidence to confirm that the 31-kDa subunit is a 28-kDa subunit containing a bound fragment of peptidoglycan. Peptide maps of purified proteins were generated following cyanogen bromide cleavage or proteolysis with staphylococcal V8 protease. A comparison of the banding patterns resulting from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a common pattern. Selected peptide fragments were sequenced on a gas phase microsequencer, and the sequence was compared with the sequence obtained for the 28-kDa protein. While the amino terminus of the 31-kDa protein was blocked, peptide fragments generated by cyanogen bromide treatment exhibited a sequence identical to that of the amino terminus of the 28-kDa protein, but beginning at amino acid four (glycine), which is preceded by methionine at the third position. This sequence, (Gly-Thr-Met)-Gly-Pro-Val-Trp-Thr-Pro-Gly-Asn ... , confirms that these proteins have a common amino terminus. An oligonucleotide synthesized from the codons of the common N-terminal amino acid sequence was used to establish by Southern and Northern (RNA) blot analyses that a single gene coded for both proteins. With regard to the putative porin structure, we have identified two major bands at 70 kDa and at approximately 120 kDa by nonreducing SDS-PAGE. The former may represent the typical trimeric motif, while the latter may represent either a double trimer or an aggregate. Analysis of these two forms by two-dimensional SDS-PAGE (first dimensions, nonreducing; second dimensions, reducing) established that both were composed of 31- and 28-kDa subunits cross-linked via interchain disulfide bonds. These studies confirm that the novel L. pneumophila major outer protein is covalently bound to peptidoglycan via a modified 28-kDa subunit (31-kDa anchor protein) and cross-linked to other 28-kDa subunits via interchain disulfide bonds.
authors.affiliation: Department of Microbiology, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: P S
authors.initials: PS
authors.lastname: Hoffman
authors.forename: J H
authors.initials: JH
authors.lastname: Seyer
authors.forename: C A
authors.initials: CA
authors.lastname: Butler
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D016952:Legionella pneumophila / Q000737:chemistry*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010446:Peptide Fragments / Q000737:chemistry*; D010449:Peptide Mapping; D010457:Peptidoglycan / Q000737:chemistry; D018272:Porins
chemical_list: D001425:Bacterial Outer Membrane Proteins; D010446:Peptide Fragments; D010457:Peptidoglycan; D018272:Porins