Complex O-acetylation in Legionella pneumophila serogroup 1 lipopolysaccharide. Evidence for two genes involved in 8-O-acetylation of legionaminic acid.
A putative gene encoding an O-acetyl transferase, lag-1, is involved in biosynthesis of the O-polysaccharide (polylegionaminic acid) in some Legionella pneumophila serogroup 1 strains. To study the effect of the presence and absence of the gene on the O-polysaccharide O-acetylation, lag-1 from strain Philadelphia 1 was expressed in trans in the naturally lag-1-negative OLDA strain RC1, and immunoblot analysis revealed that the lag-1-encoded O-acetyl transferase is active. O-Polysaccharides of different size were prepared from the lipopolysaccharides of wild-type and transformant strains by mild acid degradation followed by gel-permeation chromatography. Using NMR spectroscopy and MALDI-TOF mass spectrometry, it was found that O-acetylation of the first three legionaminic acid residues next to the core occurs in the short-chain O-polysaccharide (<10 sugars) from both strains. Hence, there is another O-acetyl transferase encoded by a gene different from lag-1. In the longer-chain O-polysaccharide, a legionaminic acid residue proximal to the core is N-methylated and could be further 8-O-acetylated in the lag-1-dependent manner. Only strains expressing a functional lag-1 gene were recognized in Western blot analysis by monoclonal antibody 3/1 requiring 8-O-acetylated polylegionaminic acid for binding. The highly O-acetylated outer core region of the lipopolysaccharide is involved in the epitope of another serogroup 1-specific monoclonal antibody termed LPS-1. The O-acetylation pattern of the L. pneumophila serogroup 1 core oligosaccharide was revised using MALDI-TOF mass spectrometry. lag-1-independent O-acetylation of the core and short-chain O-polysaccharide was found to be a common feature of L. pneumophila serogroup 1 strains. The biological importance of conserved lag-1-independent and variable lag-1-dependent O-acetylation is discussed.
Kooistra
O
O
Forschungszentrum Borstel, Zentrum für Medizin und Biowissenschaften, Parkallee 22, D-23845 Borstel, Germany.
Lüneberg
E
E
Lindner
B
B
Knirel
Y A
YA
Frosch
M
M
Zähringer
U
U
D000107:Acetylation; D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001426:Bacterial Proteins; D015139:Blotting, Southern; D002240:Carbohydrate Sequence; D005591:Chemical Fractionation; D005798:Genes, Bacterial; D016952:Legionella pneumophila / Q000235:genetics* / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D019081:O Antigens / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D012794:Sialic Acids / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020349:Surface Plasmon Resonance; D014169:Transformation, Bacterial
C110570:5,7-diacetamido-8-O-acetyl-3,5,7,9-tetradeoxy-glycero-talo-nonulosonic acid; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D008070:Lipopolysaccharides; D019081:O Antigens; D010455:Peptides; D012794:Sialic Acids; C120065:lag-1 protein, Legionella; D000123:Acetyltransferases