Rocchetta H L, Burrows L L, Pacan J C, Lam J S
Department of Microbiology and Canadian Bacterial Diseases Network, University of Guelph, ON, Canada.
Mol Microbiol. 1998 Jun;28(6):1103-19. doi: 10.1046/j.1365-2958.1998.00871.x.
The Pseudomonas aeruginosa A-band lipopolysaccharide (LPS) molecule has an O-polysaccharide region composed of trisaccharide repeat units of alpha1-->2, alpha1-->3, alpha1-->3 linked D-rhamnose (Rha). The A-band polysaccharide is assembled by the alpha-D-rhamnosyltransferases, WbpX, WbpY and WbpZ. WbpZ probably transfers the first Rha residue onto the A-band accepting molecule, while WbpY and WbpX subsequently transfer two alpha1-->3 linked Rha residues and one alpha1-->2 linked Rha respectively. The last two transferases are predicted to be processive, alternating in their activities to complete the A-band polymer. The genes coding for these transferases were identified at the 3' end of the A-band biosynthetic cluster. Two additional genes, psecoA and uvrD, border the 3' end of the cluster and are predicted to encode a coenzyme A transferase and a DNA helicase II enzyme respectively. Chromosomal wbpX, wbpY and wbpZ mutants were generated, and Western immunoblot analysis demonstrates that these mutants are unable to synthesize A-band LPS, while B-band synthesis is unaffected. WbpL, a transferase encoded within the B-band biosynthetic cluster, was previously proposed to initiate B-band biosynthesis through the addition of Fuc2NAc (2-acetamido-2,6-dideoxy-D-galactose) to undecaprenol phosphate (Und-P). In this study, chromosomal wbpL mutants were generated that did not express A band or B band, indicating that WbpL initiates the synthesis of both LPS molecules. Cross-complementation experiments using WbpL and its homologue, Escherichia coli WecA, demonstrates that WbpL is bifunctional, initiating B-band synthesis with a Fuc2NAc residue and A-band synthesis with either a GlcNAc (N-acetylglucosamine) or GalNAc (N-acetylgalactosamine) residue. These data indicate that A-band polysaccharide assembly requires four glycosyltransferases, one of which is necessary for initiating both A-band and B-band LPS synthesis.
铜绿假单胞菌A带脂多糖（LPS）分子具有一个O-多糖区域，该区域由α1→2、α1→3、α1→3连接的D-鼠李糖（Rha）的三糖重复单元组成。A带多糖由α-D-鼠李糖基转移酶WbpX、WbpY和WbpZ组装而成。WbpZ可能将第一个Rha残基转移到A带接受分子上，而WbpY和WbpX随后分别转移两个α1→3连接的Rha残基和一个α1→2连接的Rha残基。预计最后两个转移酶是持续性的，它们的活性交替进行以完成A带聚合物。编码这些转移酶的基因在A带生物合成簇的3'端被鉴定出来。另外两个基因psecoA和uvrD位于该簇的3'端边界，预计分别编码一种辅酶A转移酶和一种DNA解旋酶II。构建了染色体wbpX、wbpY和wbpZ突变体，蛋白质免疫印迹分析表明这些突变体无法合成A带LPS，而B带合成不受影响。WbpL是一种在B带生物合成簇内编码的转移酶，先前有人提出它通过将Fuc2NAc（2-乙酰氨基-2,6-二脱氧-D-半乳糖）添加到十一异戊烯磷酸（Und-P）上来启动B带生物合成。在本研究中，构建了不表达A带或B带的染色体wbpL突变体，这表明WbpL启动了两种LPS分子的合成。使用WbpL及其同源物大肠杆菌WecA进行的交叉互补实验表明，WbpL具有双功能，用一个Fuc2NAc残基启动B带合成，用一个GlcNAc（N-乙酰葡糖胺）或GalNAc（N-乙酰半乳糖胺）残基启动A带合成。这些数据表明，A带多糖组装需要四种糖基转移酶，其中一种对于启动A带和B带LPS合成都是必需的。
