Mutant analysis and cellular localization of the AlgI, AlgJ, and AlgF proteins required for O acetylation of alginate in Pseudomonas aeruginosa
Mutant analysis and cellular localization of the AlgI, AlgJ, and AlgF proteins required for O acetylation of alginate in Pseudomonas aeruginosa
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DOI：
10.1128/jb.184.11.3000-3007.2002
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发表时间：
2002-06-01
影响因子：
3.2
通讯作者：
Ohman, DE
中科院分区：
文献类型：
--
作者：
Franklin, MJ;Ohman, DE
藻酸盐是由铜绿假单胞菌的粘液样菌株产生的细胞外多糖，其通常从慢性感染的囊性纤维化患者的肺道分离。藻酸盐是D-甘露糖醛酸盐和L-古洛糖醛酸盐的线性聚合物，在甘露糖醛酸盐残基的O-2和/或O-3位上具有O-乙酰酯键。O-乙酰基的存在在聚合物作为毒力因子的能力中起着重要作用，并且已知algF、algJ和algI基因对于将O-乙酰基添加到藻酸盐中是必不可少的。为了更好地理解藻酸盐0乙酰化的机制，确定了AlgI、AlgJ和AlgF蛋白的细胞位置。对于这些研究，定义的非极性algI，algJ，和algF缺失突变体的铜绿假单胞菌菌株FRD 1的构建，每个突变体产生藻酸盐缺乏O-乙酰基。algI，algJ，或algF在相应的突变体中的反式表达补充了每个0乙酰化缺陷。随机phoA（碱性磷酸酶[AP]基因）融合algF，algJ和algI构建。所有与algF和algJ的框内融合都具有AP活性，表明AlgF和AlgJ都被输出到周质。原生质球和周质组分的免疫印迹分析表明，AlgF与周质内容物释放，但AlgJ仍然与原生质球组分。AlgJ的N-末端序列分析表明，其推定的AlgJ信号肽没有被切割，这表明AlgJ通过其未切割的信号肽锚定到细胞质膜。AP基因融合也被用来映射的膜拓扑结构的AlgI，结果表明，它是一个完整的膜蛋白与7个跨膜结构域。这些结果表明，AlgI-AlgJ-AlgF可能在膜中形成复合物，该复合物是藻酸盐0乙酰化的反应中心。
Alginate is an extracellular polysaccharide produced by mucoid strains of Pseudomonas aeruginosa that are typically isolated from the pulmonary tracts of chronically infected cystic fibrosis patients. Alginate is a linear polymer Of D-mannuronate and L-guluronate with O-acetyl ester linkages on the O-2 and/or O-3 position of the mannuronate residues. The presence of O-acetyl groups plays an important role in the ability of the polymer to act as a virulence factor, and the algF, algJ, and algI genes are known to be essential for the addition of O-acetyl groups to alginate. To better understand the mechanism of 0 acetylation of alginate, the cellular locations of the AlgI, AlgJ, and AlgF proteins were determined. For these studies, defined nonpolar algI, algJ, and algF deletion mutants of A aeruginosa strain FRD1 were constructed, and each mutant produced alginate lacking O-acetyl groups. Expression of algI, algJ, or algF in trans in the corresponding mutant complemented each 0 acetylation defect. Random phoA (alkaline phosphatase [AP] gene) fusions to algF, algJ, and algI were constructed. All in-frame fusions to algF and algJ had AP activity, indicating that both AlgF and AlgJ were exported to the periplasm. Immunoblot analysis of spheroplasts and periplasmic fractions showed that AlgF was released with the periplasmic contents but that AlgJ remained with the spheroplast fraction. An N-terminal sequence analysis of AlgJ showed that its putative AlgJ signal peptide was not cleaved, suggesting that AlgJ is anchored to the cytoplasmic membrane by its uncleaved signal peptide. AP gene fusions were also used to map the membrane topology of AlgI, and the results suggest that it is an integral membrane protein with seven transmembrane domains. These results suggest that AlgI-AlgJ-AlgF may form a complex in the membrane that is the reaction center for 0 acetylation of alginate.