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

Citation
E. Luneberg et al., Cloning and functional characterization of a 30 kb gene locus required forlipopolysaccharide biosynthesis in Legionella pneumophila, INT J MED M, 290(1), 2000, pp. 37-49
Citations number
65
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Microbiology
Journal title
INTERNATIONAL JOURNAL OF MEDICAL MICROBIOLOGY
ISSN journal
1438-4221 → ACNP
Volume
290
Issue
1
Year of publication
2000
Pages
37 - 49
Database
ISI
SICI code
1438-4221(200003)290:1<37:CAFCOA>2.0.ZU;2-U
Abstract
The spontaneous Legionella pneumophila lipopolysaccharide (LPS) mutant 137, which did not bind the LPS-specific mAb 2625, was complemented with a geno mic 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 (Orf s) was identified. Orfs with significant homologies to genes encoding enzym es 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 exh ibited homology to bacterial methyl-transferases. The L. pneumophila LPS ge ne locus included genes with deduced products likely to be involved in LPS core oligosaccharide biosynthesis (rmlA-D, rhamnosyl-transferases, acetyl-t ransferase) as well as LPS O-chain biosynthesis and translocation (mnaA, ne uB, neuA, wecA, wzt, wzm). The neuA (Orf 25) and neuB (Orf 24) gene product s were functionally characterized by complementation of the capsule negativ e 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, express ion of the K1 polysialic acid capsule could be restored. We, therefore, con clude that the biosynthesis pathway of legionaminic acid, the structural un it of the L. pneumophila Sg1 O-antigen, might be similar to the biosynthesi s of sialic acid. Southern blot analysis indicated the entire gene locus to be present in L. pneumophila serogroup (Sg)1 strains, whereas only parts o f the DNA stretch hybridized to DNA from Sg2 to Sg14 strains.