Department of Biology, Wilfrid Laurier University, Waterloo, Ontario, Canada.
Department of Biology, Wilfrid Laurier University, Waterloo, Ontario, Canada.
J Biol Chem. 2023 Jul;299(7):104849. doi: 10.1016/j.jbc.2023.104849. Epub 2023 May 22.
In microbial biofilms, bacterial cells are encased in a self-produced matrix of polymers (e.g., exopolysaccharides) that enable surface adherence and protect against environmental stressors. For example, the wrinkly spreader phenotype of Pseudomonas fluorescens colonizes food/water sources and human tissue to form robust biofilms that can spread across surfaces. This biofilm largely consists of bacterial cellulose produced by the cellulose synthase proteins encoded by the wss (WS structural) operon, which also occurs in other species, including pathogenic Achromobacter species. Although phenotypic mutant analysis of the wssFGHI genes has previously shown that they are responsible for acetylation of bacterial cellulose, their specific roles remain unknown and distinct from the recently identified cellulose phosphoethanolamine modification found in other species. Here, we have purified the C-terminal soluble form of WssI from P. fluorescens and Achromobacter insuavis and demonstrated acetylesterase activity with chromogenic substrates. The kinetic parameters (k/K values of 13 and 8.0 M s, respectively) indicate that these enzymes are up to four times more catalytically efficient than the closest characterized homolog, AlgJ from the alginate synthase. Unlike AlgJ and its cognate alginate polymer, WssI also demonstrated acetyltransferase activity onto cellulose oligomers (e.g., cellotetraose to cellohexaose) with multiple acetyl donor substrates (p-nitrophenyl acetate, 4-methylumbelliferyl acetate, and acetyl-CoA). Finally, a high-throughput screen identified three low micromolar WssI inhibitors that may be useful for chemically interrogating cellulose acetylation and biofilm formation.
在微生物生物膜中，细菌细胞被包裹在由聚合物（例如胞外多糖）自产生的基质中，这使它们能够附着在表面上并保护免受环境胁迫。例如，荧光假单胞菌的皱缩散布表型定植于食物/水源和人体组织，形成能够在表面上扩散的强大生物膜。这种生物膜主要由纤维素合酶蛋白编码的 wss（WS 结构）操纵子产生的细菌纤维素组成，该操纵子也存在于其他物种中，包括致病性不动杆菌物种。尽管以前对 wssFGHI 基因的表型突变分析表明它们负责细菌纤维素的乙酰化，但它们的具体作用仍不清楚，并且与最近在其他物种中发现的纤维素磷酸乙醇胺修饰不同。在这里，我们从荧光假单胞菌和不动杆菌中纯化了 WssI 的 C 端可溶性形式，并使用显色底物证明了乙酰酯酶活性。动力学参数（k/K 值分别为 13 和 8.0 M s）表明，这些酶的催化效率比最接近的特征化同源物 AlgJ（来自藻酸盐合酶）高 4 倍。与 AlgJ 及其同源藻酸盐聚合物不同，WssI 还表现出对纤维素低聚物（例如纤维四糖至纤维六糖）的乙酰转移酶活性，具有多种乙酰供体底物（对硝基苯乙酸酯、4-甲基伞形酮乙酸酯和乙酰辅酶 A）。最后，高通量筛选鉴定出三种低微摩尔的 WssI 抑制剂，它们可能有助于化学研究纤维素乙酰化和生物膜形成。
