Rocchetta H L, Lam J S
Department of Microbiology and Canadian Bacterial Diseases Network, University of Guelph, Ontario.
J Bacteriol. 1997 Aug;179(15):4713-24. doi: 10.1128/jb.179.15.4713-4724.1997.
Pseudomonas aeruginosa coexpresses two distinct lipopolysaccharide (LPS) molecules known as A band and B band. B band is the serospecific LPS, while A band is the common LPS antigen composed of a D-rhamnose O-polysaccharide region. An operon containing eight genes responsible for A-band polysaccharide biosynthesis and export has recently been identified and characterized (H. L. Rocchetta, L. L. Burrows, J. C. Pacan, and J. S. Lam, unpublished data; H. L. Rocchetta, J. C. Pacan, and J. S. Lam, unpublished data). In this study, we report the characterization of two genes within the cluster, designated wzm and wzt. The Wzm and Wzt proteins have predicted sizes of 29.5 and 47.2 kDa, respectively, and are homologous to a number of proteins that comprise ABC (ATP-binding cassette) transport systems. Wzm is an integral membrane protein with six potential membrane-spanning domains, while Wzt is an ATP-binding protein containing a highly conserved ATP-binding motif. Chromosomal wzm and wzt mutants were generated by using a gene replacement strategy in P. aeruginosa PAO1 (serotype 05). Western blot analysis and immunoelectron microscopy using A-band- and B-band-specific monoclonal antibodies demonstrated that the wzm and wzt mutants were able to synthesize A-band polysaccharide, although transport of the polymer to the cell surface was inhibited. The inability of the polymer to cross the inner membrane resulted in the accumulation of cytoplasmic A-band polysaccharide. This A-band polysaccharide is likely linked to a carrier lipid molecule with a phenol-labile linkage. Chromosomal mutations in wzm and wzt were found to have no effect on B-band LPS synthesis. Rather, immunoelectron microscopy revealed that the presence of A-band LPS may influence the arrangement of B-band LPS on the cell surface. These results demonstrate that A-band and B-band O-antigen assembly processes follow two distinct pathways, with the former requiring an ABC transport system for cell surface expression.
铜绿假单胞菌共表达两种不同的脂多糖（LPS）分子，即A带和B带。B带是血清特异性LPS，而A带是由D - 鼠李糖O - 多糖区域组成的常见LPS抗原。最近已鉴定并表征了一个包含八个负责A带多糖生物合成和输出的基因的操纵子（H. L. Rocchetta、L. L. Burrows、J. C. Pacan和J. S. Lam，未发表数据；H. L. Rocchetta、J. C. Pacan和J. S. Lam，未发表数据）。在本研究中，我们报告了该基因簇内两个基因（命名为wzm和wzt）的表征。Wzm和Wzt蛋白的预测大小分别为29.5 kDa和47.2 kDa，并且与许多构成ABC（ATP结合盒）转运系统的蛋白同源。Wzm是一种具有六个潜在跨膜结构域的整合膜蛋白，而Wzt是一种含有高度保守ATP结合基序的ATP结合蛋白。通过在铜绿假单胞菌PAO1（血清型05）中使用基因替换策略产生了染色体wzm和wzt突变体。使用A带和B带特异性单克隆抗体的蛋白质印迹分析和免疫电子显微镜表明，wzm和wzt突变体能够合成A带多糖，尽管聚合物向细胞表面的转运受到抑制。聚合物无法穿过内膜导致细胞质A带多糖积累。这种A带多糖可能通过酚不稳定键与载体脂质分子相连。发现wzm和wzt中的染色体突变对B带LPS合成没有影响。相反，免疫电子显微镜显示A带LPS的存在可能会影响B带LPS在细胞表面的排列。这些结果表明，A带和B带O抗原组装过程遵循两条不同的途径，前者需要ABC转运系统进行细胞表面表达。
