Cloning and functional characterization of a 30 kb gene locus required for lipopolysaccharide biosynthesis in Legionella pneumophila

• Published in: International journal of medical microbiology Vol. 290; no. 1; pp. 37 - 49
• Main Authors: Lüneberg, Edeltraud, Zetzmann, Nicole, Alber, Dirk, Knirel, Yuriy A., Kooistra, Oliver, Zähringer, Ulrich, Frosch, Matthias
• Format: Journal Article
• Language: English
• Published: Jena Elsevier GmbH 01.03.2000; Elsevier
• Subjects: Bacteriology; Biological and medical sciences; Blotting, Western; cloning; Cloning, Molecular; core oligosaccharide; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; gene locus; Genes, Bacterial; Genetic Complementation Test; Genetics; Humans; Legionella pneumophila; Legionella pneumophila - genetics; Legionella pneumophila - metabolism; Legionella pneumophila - pathogenicity; lipopolysaccharide biosynthesis; Lipopolysaccharides - biosynthesis; Microbiology; monoclonal antibody; Mutation; NeuA; NeuB; O-antigen; Open Reading Frames - genetics; Sequence Analysis, DNA; Structure-Activity Relationship; Virulence; gene locus; NeuB; lipopolysaccharide biosynthesis; NeuA; O-antigen; monoclonal antibody; core oligosaccharide; cloning; Legionellaceae; O antigen; Nucleotide sequence; Molecular assembly; Legionaminic acid; Lipopolysaccharide; Bacteria; Homology; Legionella pneumophila; Gene organization; Biosynthesis; Aminoacid sequence
• DOI: 10.1016/S1438-4221(00)80104-6
• ISSN: 1438-4221

The spontaneous Legionella pneumophila lipopolysaccharide (LPS) mutant 137, which did not bind the LPS-specific mAb 2625, was complemented with a genomic library from the parental wild-type strain. Transformants were screened for reconstitution of the wild-type LPS phenotype, able to bind mAb 2625. By this strategy, a 32,661 bp region comprising 30 open reading frames (Orfs) was identified. Orfs with significant homologies to genes encoding enzymes required for LPS or capsule biosynthesis of Gram-negative bacteria were located on the gene locus. The mutation of strain 137 could be assigned to a deletion of a cytosine residue in Orf 8. The protein encoded by Orf 8 exhibited homology to bacterial methyl-transferases. The L. pneumophila LPS gene locus included genes with deduced products likely to be involved in LPS core oligosaccharide biosynthesis ( rmlA-D, rhamnosyl-transferases, acetyl-transferase) as well as LPS O-chain biosynthesis and translocation ( mnaA, neuB, neuA, wecA, wzt, wzm). The neuA (Orf 25) and neuB (Orf 24) gene products were functionally characterized by complementation of the capsule negative E. coli K1 mutants EV5 and EV24, respectively. By introduction of the L. pneumophila neuA gene into E. coli EV5 and the neuB gene into EV24, expression of the K1 polysialic acid capsule could be restored. We, therefore, conclude that the biosynthesis pathway of legionaminic acid, the structural unit of the L. pneumophila Sg1 O-antigen, might be similar to the biosynthesis of sialic acid. Southern blot analysis indicated the entire gene locus to be present in L. pneumophila serogroup (Sg)1 strains, whereas only parts of the DNA stretch hybridized to DNA from Sg2 to Sg14 strains.