Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
mBio. 2013 Jan 22;4(1):e00631-12. doi: 10.1128/mBio.00631-12.
Common polysaccharide antigen (CPA) is a conserved cell surface polysaccharide produced by Pseudomonas aeruginosa. It contains a rhamnan homopolymer and is one of the two forms of O polysaccharide attached to P. aeruginosa lipopolysaccharide (LPS). Our laboratory has previously characterized an eight-gene cluster (pa5447-pa5454 in P. aeruginosa PAO1) required for biosynthesis of CPA. Here we demonstrate that an adjacent five-gene cluster pa5455-pa5459 is also involved. Using reverse transcriptase PCR (RT-PCR), we showed that the original eight-gene cluster and the new five-gene cluster are both organized as operons. We have analyzed the LPS phenotypes of in-frame deletion mutants made in each of the five genes, and the results verified that these five genes are indeed required for CPA biosynthesis, extending the CPA biosynthesis locus to contain 13 contiguous genes. By performing overexpression experiments of different sets of these biosynthesis genes, we were able to obtain information about their possible functions in CPA biosynthesis.
Lipopolysaccharide (LPS) is an important cell surface structure of Gram-negative bacteria. The human opportunistic pathogen Pseudomonas aeruginosa simultaneously produces an O-antigen-specific (OSA) form and a common polysaccharide antigen (CPA) form of LPS. CPA, the focus of this study, is composed of α-1-2, α1-3-linked d-rhamnose sugars and has been shown to be important for attachment of the bacteria to human airway epithelial cells. Genome sequencing of this species revealed a new five-gene cluster that we predicted to be involved in CPA biosynthesis and modification. In this study, we have generated chromosomal knockouts by performing in-frame deletions and allelic replacements. Characterizing the function of each of the five genes is important for us to better understand CPA biosynthesis and the mechanisms of chain length termination and regulation of this unique D-rhamnan polysaccharide.
常见多糖抗原（CPA）是铜绿假单胞菌产生的一种保守的细胞表面多糖。它含有一个鼠李聚糖均聚物，是连接到铜绿假单胞菌脂多糖（LPS）的两种 O 多糖形式之一。我们的实验室以前已经对参与 CPA 生物合成的八个基因簇（铜绿假单胞菌 PAO1 中的 pa5447-pa5454）进行了特征描述。在这里，我们证明了相邻的五个基因簇 pa5455-pa5459 也参与其中。通过逆转录 PCR（RT-PCR），我们表明原始的八个基因簇和新的五个基因簇都组织为操纵子。我们分析了每个基因缺失突变体的 LPS 表型，结果证实这五个基因确实是 CPA 生物合成所必需的，将 CPA 生物合成基因座扩展到包含 13 个连续基因。通过对这些生物合成基因的不同组合进行过表达实验，我们能够获得有关它们在 CPA 生物合成中可能的功能的信息。
脂多糖（LPS）是革兰氏阴性细菌的重要细胞表面结构。人类机会性病原体铜绿假单胞菌同时产生 O-抗原特异性（OSA）和常见多糖抗原（CPA）形式的 LPS。CPA 是本研究的重点，由α-1-2、α1-3 连接的 d-鼠李糖组成，已被证明对细菌与人呼吸道上皮细胞的附着很重要。对该物种的基因组测序揭示了一个新的五个基因簇，我们预测该基因簇参与 CPA 生物合成和修饰。在这项研究中，我们通过进行框内缺失和等位基因替换生成了染色体敲除。对五个基因中的每一个的功能进行表征对于我们更好地理解 CPA 生物合成以及这种独特的 D-鼠李聚糖的链长终止和调控机制非常重要。
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