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首页> 外文期刊>Applied Microbiology >Caldicellulosiruptor bescii Adheres to Polysaccharides via a Type IV Pilin-Dependent Mechanism
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Caldicellulosiruptor bescii Adheres to Polysaccharides via a Type IV Pilin-Dependent Mechanism

机译：Caldicellulosiruptor Bescii通过IV型粘蛋白依赖性机制粘附在多糖上

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摘要

Biological hydrolysis of cellulose above 70°C involves microorganisms that secrete free enzymes and deploy separate protein systems to adhere to their substrate. Strongly cellulolytic Caldicellulosiruptor bescii is one such extreme thermophile, which deploys modular, multifunctional carbohydrate-acting enzymes to deconstruct plant biomass. Additionally, C. bescii also encodes noncatalytic carbohydrate binding proteins, which likely evolved as a mechanism to compete against other heterotrophs in carbon-limited biotopes that these bacteria inhabit. Analysis of the Caldicellulosiruptor pangenome identified a type IV pilus (T4P) locus encoded upstream of the tāpirins, that is encoded by all Caldicellulosiruptor species. In this study, we sought to determine if the C. bescii T4P plays a role in attachment to plant polysaccharides. The major C. bescii pilin (CbPilA) was identified by the presence of pilin-like protein domains, paired with transcriptomics and proteomics data. Using immuno-dot blots, we determined that the plant polysaccharide xylan induced production of CbPilA 10- to 14-fold higher than glucomannan or xylose. Furthermore, we are able to demonstrate that recombinant CbPilA directly interacts with xylan and cellulose at elevated temperatures. Localization of CbPilA at the cell surface was confirmed by immunofluorescence microscopy. Lastly, a direct role for CbPilA in cell adhesion was demonstrated using recombinant CbPilA or anti-CbPilA antibodies to reduce C. bescii cell adhesion to xylan and crystalline cellulose up to 4.5- and 2-fold, respectively. Based on these observations, we propose that CbPilA and, by extension, the T4P play a role in Caldicellulosiruptor cell attachment to plant biomass.IMPORTANCE Most microorganisms are capable of attaching to surfaces in order to persist in their environment. Type IV (T4) pili produced by certain mesophilic Firmicutes promote adherence; however, a role for T4 pili encoded by thermophilic members of this phylum has yet to be demonstrated. Prior comparative genomics analyses identified a T4 pilus locus possessed by an extremely thermophilic genus within the Firmicutes . Here, we demonstrate that attachment to plant biomass-related carbohydrates by strongly cellulolytic Caldicellulosiruptor bescii is mediated by T4 pilins. Surprisingly, xylan but not cellulose induced expression of the major T4 pilin. Regardless, the C. bescii T4 pilin interacts with both polysaccharides at high temperatures and is located to the cell surface, where it is directly involved in C. bescii attachment. Adherence to polysaccharides is likely key to survival in environments where carbon sources are limiting, allowing C. bescii to compete against other plant-degrading microorganisms.
机译：70℃以上纤维素的生物水解涉及分泌游离酶的微生物，并展开单独的蛋白质系统粘附在其基材上。强烈的纤维素钙霉素ulosuptor Bescii是一种这种极端嗜热，其部署模块化多功能碳水化合物作用酶以解构植物生物质。另外，C.Bescii还编码了非催化碳水化合物结合蛋白，其可能被发展为与这些细菌栖息的碳限量的其他异位竞争的机制。分析Caldicellulosiruptor pangenome鉴定了由所有Caldicellulosupor型物种编码的静脉内部编码的IV型菌落（T4P）基因座。在这项研究中，我们试图确定C. Bescii T4p是否在植物多糖附着中发挥作用。通过含氏蛋白样蛋白质结构域的存在，将主要的C.Bescii pilin（cbpila）鉴定出与转录组织和蛋白质组学数据配对。使用免疫点污，我们确定植物多糖木聚糖诱导的CBPILA 10-至14倍，高于葡糖胺或木糖。此外，我们能够证明重组CBPILA在升高的温度下直接与木聚糖和纤维素相互作用。通过免疫荧光显微镜证实细胞表面处CBPILA的定位。最后，使用重组CBPILA或抗CBPILA抗体来证明CBPILA在细胞粘附中的直接作用，以将C. Bescii细胞的粘附性分别降低至木聚糖和结晶纤维素至4.5-和2倍的结晶纤维素。基于这些观察结果，我们提出了CBPILA和通过延伸，T4P在Caldicellulosiruptor Cell附着中发挥作用，以植物生物质。大多数微生物能够在其环境中坚持持续到表面。由某种嗜合性的型蛋白化促进依从性产生的IV型（T4）pili;然而，尚未证明由该字段的嗜热成员编码的T4 pili的作用。现有的比较基因组学分析鉴定了在芯片内极其嗜热属的T4菌落轨迹。在这里，我们证明将植物生物质相关的碳水化合物的附着由强纤维分解的Caldicellulosiruptor Bescii介导的T4粘蛋白介导。令人惊讶的是，Xylan但不是纤维素诱导的主要T4毛蛋白的表达。无论如何，C.Bescii T4菌液在高温下与多糖相互作用，位于细胞表面，在那里它直接参与C. Bescii附着。对多糖的粘附可能是在碳源限制的环境中存活的关键，从而允许C.Bescii与其他植物降解的微生物竞争。

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