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.
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