O-acetylation of alginate produced by the opportunistic human pathogen Pseudomonas aeruginosa significantly contributes to its pathogenesis. Three proteins, AlgI, AlgJ and AlgF have been implicated to form a complex and act together with AlgX for O-acetylation of alginate. AlgI was proposed to transfer the acetyl group across the cytoplasmic membrane, while periplasmic AlgJ was hypothesised to transfer the acetyl group to AlgX that acetylates alginate. To elucidate the proposed O-acetylation multiprotein complex, isogenic knockout mutants of algI, algJ and algF genes were generated in the constitutively alginate overproducing P. aeruginosa PDO300 to enable mutual stability studies. All knockout mutants were O-acetylation negative and complementation with the respective genes in cis or trans restored O-acetylation of alginate. Interestingly, only the AlgF deletion impaired alginate production suggesting a link to the alginate polymerisation/secretion multiprotein complex. Mutual stability experiments indicated that AlgI and AlgF interact independent of AlgJ as well as impact on stability of the alginate polymerisation/secretion multiprotein complex. Deletion of AlgJ did not destabilise AlgX and vice versa. When the alginate polymerase, Alg8, was absent, then AlgI and AlgF stability was strongly impaired supporting a link of the O-acetylation machinery with alginate polymerisation. Pull-down experiments suggested that AlgI interacts with AlgJ, while AlgF interacts with AlgJ and AlgI. Overall, these results suggested that AlgI-AlgJ-AlgF form a multiprotein complex linked via Alg8 to the envelope-spanning alginate polymerisation/secretion multiprotein complex to mediate O-acetylation of nascent alginate. Here, we provide the first insight on how the O-acetylation machinery is associated with alginate production.

中文翻译：

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铜绿假单胞菌中藻酸盐的O-乙酰化机制分析。

机会性人类病原体铜绿假单胞菌产生的藻酸盐的O-乙酰化极大地促进了其发病机理。已经暗示了三种蛋白质AlgI，AlgJ和AlgF形成复合物，并与AlgX一起作用于藻酸盐的O-乙酰化。提出了AlgI将乙酰基转移到细胞质膜上，而假设周质的AlgJ将乙酰基转移到使藻酸盐乙酰化的AlgX上。为了阐明拟议的O-乙酰化多蛋白复合物，在组成型海藻酸盐生产铜绿假单胞菌PDO300中生成了algI，algJ和algF基因的同基因敲除突变体，以实现相互稳定性研究。所有敲除突变体均为O-乙酰化阴性，并与藻酸盐的顺式或反式恢复的O-乙酰化中的相应基因互补。有趣的是 只有AlgF缺失会破坏藻酸盐的产生，这暗示了与藻酸盐聚合/分泌多蛋白复合物的联系。相互稳定性实验表明，AlgI和AlgF相互作用独立于AlgJ，并且对藻酸盐聚合/分泌多蛋白复合物的稳定性有影响。删除AlgJ不会破坏AlgX的稳定性，反之亦然。当缺少藻酸盐聚合酶Alg8时，会严重削弱AlgI和AlgF的稳定性，从而支持O-乙酰化机制与藻酸盐聚合的联系。下拉实验表明，AlgI与AlgJ相互作用，而AlgF与AlgJ和AlgI相互作用。总体，这些结果表明，AlgI-AlgJ-AlgF形成了一种多蛋白复合物，通过Alg8与跨膜的藻酸盐聚合/分泌多蛋白复合物相连，以介导新生藻酸盐的O-乙酰化。在这里，我们提供了关于O-乙酰化机制与藻酸盐生产有关的第一见解。