Bacterial Cellulose Synthase Modification and Export

许多细菌形成高度耐药的生物膜，由胞外多糖组成，如纤维素，促进其在各种生物和非生物表面的黏附。生物膜基质还为细菌提供保护，使其免受免疫系统组件、抗菌剂、洗涤剂和恶劣环境因素的影响。细菌纤维素也具有重要的工业意义，因为它具有独特的物理/化学性质，与藻类或植物来源的纤维素相比具有显著的优势(例如，纳米级、柔韧性和耐用性)。尽管细菌纤维素的生物学和工业意义正在扩大，但细菌对这种聚合物的合成、修饰和输出仍在阐明中。 BcsABZC基因包括细菌纤维素合成酶的核心基因，然而，也注意到重要的邻近基因座具有适当的聚合物产生、修饰和释放。例如，bcsEFG和wssFGHI分别涉及将磷乙醇胺(PETN)或乙酰基(Ac)安装到新合成的纤维素链上。尽管越来越多的细菌被鉴定出具有不同的纤维素合成酶基因，但对产生这些独特修饰聚合物的固有酶的准确活性缺乏生化验证。增加这一模糊性的是，相关的纤维素酶被预测从细胞释放纤维素链进入生物膜基质是来自不同的糖基水解酶(GH)家族。革兰氏阴性菌中的BcsZ酶属于GH-8，BglX为GH-3，而CcsZ革兰氏阳性同源物为GH-5酶。造成这种偏差的生物学原因以及这些酶如何具体识别它们各自修饰的纤维素聚合物尚不清楚。 这项研究计划旨在通过基因到分子的方法来解决这些缺陷。我们将对导致纤维素生物膜形成的环境条件进行表型分析，并将其与含有新型合成酶复合体的一组生物的特定基因的表达联系起来。这些合成酶中独特的纤维素酶(Ac-/PETN-转移酶和GH酶)将被平行地进行生化表征，以揭示负责聚合物识别、修饰和周转的分子决定因素。这些综合结果将解决关于革兰氏阴性菌和阳性菌不同合成酶中酶的基本要求/协同作用的模棱两可的问题，并将这与特定聚合物对细菌定植和持久性的重要性有关。我们工具箱(菌种、酶、底物和分析)的开发也将用于开发生物催化替代品，用于安装各种生物应用的纤维素聚合物所需的功能和物理化学性质。因此，这项联合研究具有巨大的基础发现潜力，以及加强进一步应用研究的途径。

Many bacteria form highly resistant biofilms composed of extracellular polysaccharides, like cellulose, that promote the adherence to various biotic and abiotic surfaces. The biofilm matrix also affords bacteria protection from immune system components, antimicrobial agents, detergents and harsh environmental factors. Bacterial cellulose is also gaining industrial significance, as it has unique physical/chemical properties that provide significant advantages when compared to algal- or plant-derived cellulose (eg., nano-scale, pliable and durable). Even though the biological and industrial implications of bacterial cellulose are expanding, the synthesis, modification and export of this polymer by bacteria is still being elucidated. The bcsABZC genes comprise the core bacterial cellulose synthase genes, however, important neighboring loci have also been noted for proper polymer production, modification and release. For example, bcsEFG and wssFGHI have been implicated in the installation of phosphoethanolamine (pEtN) or acetyl (Ac) groups onto newly synthesized cellulose chains, respectively. Despite the growing number of bacteria identified with these genes for distinct cellulose synthases, biochemical verification of the precise activities of the inherent enzymes that produce these uniquely modified polymers is lacking. Adding to this ambiguity, the associated cellulases that are predicted to release cellulose chains from the cell into the biofilm matrix are from diverse glycosyl hydrolase (GH) families. BcsZ enzymes in Gram negatives belong to GH-8 and BglX is a GH-3, but the CcsZ Gram positive homologs in Clostridia are GH-5 enzymes. The biological reason for this deviation and how these enzymes specifically recognize their respectively decorated cellulose polymers is unknown. This research proposal aims to address these deficiencies by a gene-to-molecule approach. We will phenotypically analyse environmental conditions that lead to cellulose biofilm formation and link this to expression of specific genes for our panel of organisms containing novel synthase complexes. The distinct cellulose-active enzymes from these synthases (Ac-/pEtN-transferases and GH enzymes) will be biochemically characterized in parallel to uncover molecular determinants responsible for polymer recognition, modification and turnover. These combined results will solve the ambiguity regarding fundamental requirements/synergies of enzymes in the distinct synthases of Gram negatives and positives and relate this to the importance of specific polymers to bacterial colonization and persistence. Exploitation of our toolkit (strains, enzymes, substrates and assays) will also be used to develop biocatalytic alternatives for the installment of desirable functionalities and physicochemical properties to cellulose polymers for various bioapplications. Thus, this combined research has immense potential for basic discoveries, as well as avenues that enhance further applied research.