Lüneberg E, Zetzmann N, Alber D, Knirel Y A, Kooistra O, Zähringer U, Frosch M
Institut für Hygiene und Mikrobiologie, Universität Würzburg, Germany.
Int J Med Microbiol. 2000 Mar;290(1):37-49. doi: 10.1016/S1438-4221(00)80104-6.
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.
自发产生的嗜肺军团菌脂多糖（LPS）突变体137不能与LPS特异性单克隆抗体2625结合，用亲本野生型菌株的基因组文库对其进行了互补。筛选转化体以恢复能够结合单克隆抗体2625的野生型LPS表型。通过这种策略，鉴定出一个包含30个开放阅读框（Orfs）的32,661 bp区域。与革兰氏阴性菌LPS或荚膜生物合成所需的编码酶的基因具有显著同源性的Orfs位于该基因座上。菌株137的突变可归因于Orf 8中一个胞嘧啶残基的缺失。Orf 8编码的蛋白质与细菌甲基转移酶具有同源性。嗜肺军团菌LPS基因座包括可能参与LPS核心寡糖生物合成的推导产物的基因（rmlA-D，鼠李糖基转移酶，乙酰转移酶）以及LPS O链生物合成和转运的基因（mnaA，neuB，neuA，wecA，wzt，wzm）。neuA（Orf 25）和neuB（Orf 24）基因产物分别通过荚膜阴性大肠杆菌K1突变体EV5和EV24的互补进行功能表征。通过将嗜肺军团菌neuA基因导入大肠杆菌EV5和neuB基因导入EV24，可以恢复K1多聚唾液酸荚膜的表达。因此，我们得出结论，嗜肺军团菌Sg1 O抗原的结构单元Legionaminic酸的生物合成途径可能与唾液酸的生物合成相似。Southern印迹分析表明整个基因座存在于嗜肺军团菌血清群（Sg）1菌株中，而只有部分DNA片段与Sg2至Sg14菌株的DNA杂交。
