Cloning, nucleotide sequence and expression in Escherichia coli of a gene (ompM) encoding a 25 kDa major outer-membrane protein (MOMP) of Legionella pneumophila

Abstract

A genomic library derived from a virulent isolate of Legionella pneumophila was constructed in Escherichia culi J M 83 using the cloning vector pUC19. The clones were screened by filter immunoassay using L. pneumuphila rabbit polyclonal antisera and in the absence of in sifu bacterial lysis one such clone, L P 116, expressed L. pneumuphiZa-specific antigens on the surface of E. culi. Restriction endonuclease digest analysis and agarose gel electrophoresis revealed a fragment measuring approximately 750 bp. Southern hybridization confirmed that the fragment was L. pneumophila DNA. Sequencing data showed that the fragment was 810 bp in length with an open reading frame (ORF) of 678 bp. The outer-membrane profiles of the E. c d i parent, the L. pneumuphiZa DNAcontributing strain and clone LP 116 were compared by SDS-PAGE. A protein of 25 kDa was found in outermembrane preparations of both the clone LP 116 and L. pneumuphila but not in E. culi J M 83. This was in agreement with the molecular mass of the deduced peptide of the mature protein. Immunoblots using L. pneumuphila-specific polyclonal antiserum confirmed that this 25 kDa outer-membrane protein (OMP) was a L. pneumuphila polypeptide. Both direct immunofluorescence assay and immunoblots using the commercially produced monoclonal antibody specific for the common antigen of the major outer-membrane protein (MOMP) confirmed that the 25 kDa protein produced by LP 116 was involved with the MOMP complex. The gene encoding this protein has been designated ompM. Furthermore, using the fertile chicken egg virulence assay, clone LP 116 producing the 25 kDa MOMP of L. pneumophila showed an increase in virulence when compared to the E. c d i parent strain.

Identification and serotyping of atypical Legionella pneumophila strains isolated from human and environmental sources

“…The results obtained for the different phenotypic methods are summarized in Table 1. All isolates were positive for the species‐specific epitope of the OmpM (High et al. 1993) using the direct immunofluorescence test MONOFLUO anti‐ Legionella Staining Reagent.…”

“…Two commercially available test kits and one in‐house assay were used for species identification. The MONOFLUO anti‐ Legionella Staining Reagent (Bio‐Rad, Munich, Germany) was used for the recognition of the species‐specific epitope on the outer membrane protein OmpM (High et al. 1993).…”

“…Serological identification of L. pneumophila Two commercially available test kits and one in-house assay were used for species identification. The MONO-FLUO anti-Legionella Staining Reagent (Bio-Rad, Munich, Germany) was used for the recognition of the speciesspecific epitope on the outer membrane protein OmpM (High et al 1993). The Mip protein was detected by immunoblot analysis using the L. pneumophila speciesspecific mAb 20/2 (Helbig et al 1995).…”

Phospholipase PlaB is a new virulence factor of Legionella pneumophila

“…In brief, 10 ml of the fractions were mixed with Laemmli buffer and incubated on ice for 30 min for detection of DotA or boiled at 100 1C for 5 min for detection of MOMP before loading onto 12.5% SDS-polyacrylamide gels. Western blotting was performed using MONOFLUO anti-Legionella staining reagent from the Legionella IFA Test Kit (#32514, Biorad, Munich, Germany) against MOMP (1:100) (Helbig et al, 2007;High et al, 1993) and a DotA antibody (1:1000) kindly provided by Reinhard Marre (University of Ulm, Ulm, Germany). A horseradish peroxidase-conjugated goat anti-mouse antibody was used as the secondary antibody for MOMP (1:5000) and DotA (1:10,000).…”

Diagnostische Bibliothek. Legionellaceae (Legionellose)

“…25-kD-Membranprotein (High et al 1993), Aconitase (major iron containing protein), Heat Shock Protein (HSP 60), Peptidoglycan-assoziiertes 19-kD-Protein, Superoxiddismutase (Übersicht bei Ott 1994).…”