Structural Features of the Pseudomonas fluorescens Biofilm Adhesin LapA Required for LapG-Dependent Cleavage, Biofilm Formation, and Cell Surface Localization
Structural Features of the Pseudomonas fluorescens Biofilm Adhesin LapA Required for LapG-Dependent Cleavage, Biofilm Formation, and Cell Surface Localization
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DOI：
10.1128/jb.01629-14
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发表时间：
2014-08-01
影响因子：
3.2
通讯作者：
O'Toole, George A.
中科院分区：
文献类型：
--
作者：
Boyd, Chelsea D.;Smith, T. Jarrod;O'Toole, George A.
LapA蛋白在细胞表面的定位是荧光假单胞菌Pf0-1不可逆附着于表面并形成生物膜的关键步骤。LapA是预测细菌粘附素的一个不同家族的成员，尽管缺乏高度的序列相似性，但家族成员确实具有共同的预测结构域。在这里，通过突变分析，我们确定了LapA的每个结构域特征在其输出和定位到细胞表面以及在生物膜形成中的功能方面的重要性。我们之前的工作表明，在不利于生物膜形成的条件下，LapA的N端是被质周半胱氨酸蛋白酶LapG切割和从细胞表面释放粘附素所必需的。我们定义了LapA蛋白水解所需的N端额外的关键区域。此外，我们的研究结果表明，除了I型分泌信号是LapA输出和细胞表面定位所必需的外，LapA C端内的结构域并不是生物膜形成、输出或定位到细胞表面所绝对需要的。相反，LapA的中心重复区（由100个氨基酸的37个重复组成）的缺失导致无法形成生物膜。我们还使用单分子原子力显微镜进一步表征了这些结构域在疏水和亲水表面生物膜形成中的作用。这些研究首次详细分析了生物膜粘附蛋白LapA家族的结构域。
The localization of the LapA protein to the cell surface is a key step required by Pseudomonas fluorescens Pf0-1 to irreversibly attach to a surface and form a biofilm. LapA is a member of a diverse family of predicted bacterial adhesins, and although lacking a high degree of sequence similarity, family members do share common predicted domains. Here, using mutational analysis, we determine the significance of each domain feature of LapA in relation to its export and localization to the cell surface and function in biofilm formation. Our previous work showed that the N terminus of LapA is required for cleavage by the periplasmic cysteine protease LapG and release of the adhesin from the cell surface under conditions unfavorable for biofilm formation. We define an additional critical region of the N terminus of LapA required for LapG proteolysis. Furthermore, our results suggest that the domains within the C terminus of LapA are not absolutely required for biofilm formation, export, or localization to the cell surface, with the exception of the type I secretion signal, which is required for LapA export and cell surface localization. In contrast, deletion of the central repetitive region of LapA, consisting of 37 repeats of 100 amino acids, results in an inability to form a biofilm. We also used single-molecule atomic force microscopy to further characterize the role of these domains in biofilm formation on hydrophobic and hydrophilic surfaces. These studies represent the first detailed analysis of the domains of the LapA family of biofilm adhesin proteins.