Zhang Weixin, Li Lizhu, Li Tengxin, Li Xin, Wang Xia, Yao Qiang, Lu Xuemei, Chen Guanjun, Liu Weifeng
State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China.
National Center of Technology Innovation for Comprehensive Utilization of Saline-Alkali Land, Dongying, China.
Appl Environ Microbiol. 2025 Apr 23;91(4):e0247124. doi: 10.1128/aem.02471-24. Epub 2025 Mar 25.
The widespread cellulolytic specialist belonging to the phylum Bacteroidetes adopted a unique cellulose utilization strategy that did not conform to the known cellulose-degrading paradigms involving free cellulases or cellulosomes. The strategy used by still remains largely unclear. In this study, we showed that within the gene cluster, which has been previously shown to be important for cellulose utilization by , encodes an outer membrane protein, and its elimination prohibited bacterial growth on cellulose. Structural prediction revealed that CHU_1279 is a surface glycan-binding protein B (SGBP-B)-like protein comprising two putative carbohydrate-binding module (CBM)-like domains. Further analyses verified that recombinant CHU_1279 displayed significant cellulose-binding protein, and its C-terminal domain is predominantly responsible for cellulose binding. Expression of the C-terminal domain but not the N-terminal domain restored cellulose utilization of ∆. Moreover, site-directed mutagenesis analyses identified three aromatic residues important for cellulose binding of the recombinant CHU_1279 protein. The defective cellulose utilization of ∆ cells otherwise could be recovered by CHU_1279 variants with significantly damaged cellulose-binding capability. Sequence analyses revealed that orthologs of CHU_1279 as well as the atypical polysaccharide utilization loci (PUL) constituted by the gene cluster are also present in two other cellulolytic Bacteroidetes bacteria, and , which are closely related to . Our results contribute to unveiling the unique mechanism underlying the efficient cellulose utilization by and similar cellulolytic bacteria.IMPORTANCEMost members of the phylum Bacteroidetes are highly competitive and efficient degraders of complex polysaccharides largely ascribed to their employment of a SusC-like system encoded by a polysaccharide utilization locus (PUL). However, characterization of PULs is limited to those responsible for utilization of (semi)soluble glycans. PULs involved in the utilization of cellulose, the most abundant renewable polymer, have not been identified and functionally characterized yet. We demonstrated that in the cellulolytic specialist encodes an SGBP-B-like protein that is required for cellulose utilization, supporting that the gene cluster in encodes an atypical PUL system dedicated to cellulose assimilation. Further analyses showed that this atypical PUL system is also present in two other cellulolytic Bacteroidetes bacteria. This study not only contributes to unveiling the unusual cellulose utilization strategy adopted by and similar cellulolytic bacteria but also helps expand our understanding of atypical PULs for nutrient acquisition by cellulolytic bacteria.
属于拟杆菌门的广泛存在的纤维素分解专家采用了一种独特的纤维素利用策略，该策略不符合涉及游离纤维素酶或纤维小体的已知纤维素降解模式。其使用的策略在很大程度上仍不清楚。在本研究中，我们表明，在先前已证明对纤维素利用很重要的基因簇中，编码一种外膜蛋白，其缺失会阻止细菌在纤维素上生长。结构预测显示，CHU_1279是一种表面聚糖结合蛋白B（SGBP-B）样蛋白，包含两个假定的碳水化合物结合模块（CBM）样结构域。进一步分析证实，重组CHU_1279表现出显著的纤维素结合蛋白特性，其C端结构域主要负责纤维素结合。C端结构域而非N端结构域的表达恢复了∆的纤维素利用能力。此外，定点诱变分析确定了对重组CHU_1279蛋白的纤维素结合很重要的三个芳香族残基。纤维素结合能力显著受损的CHU_1279变体可以恢复∆细胞有缺陷的纤维素利用能力。序列分析表明，CHU_1279的直系同源物以及由基因簇构成的非典型多糖利用位点（PUL）也存在于另外两种纤维素分解拟杆菌和中，它们与密切相关。我们的结果有助于揭示纤维素分解拟杆菌和类似纤维素分解细菌高效利用纤维素的独特机制。
重要性
拟杆菌门的大多数成员是复杂多糖的高度竞争性和高效降解者，这在很大程度上归因于它们使用由多糖利用位点（PUL）编码的类似SusC的系统。然而，PUL的表征仅限于那些负责利用（半）可溶性聚糖的PUL。参与利用最丰富的可再生聚合物纤维素的PUL尚未被鉴定和进行功能表征。我们证明，纤维素分解专家中的编码一种纤维素利用所需的SGBP-B样蛋白，支持中的基因簇编码一个专门用于纤维素同化的非典型PUL系统。进一步分析表明，这种非典型PUL系统也存在于另外两种纤维素分解拟杆菌中。本研究不仅有助于揭示纤维素分解拟杆菌和类似纤维素分解细菌采用的异常纤维素利用策略，还有助于扩展我们对纤维素分解细菌获取营养的非典型PUL的理解。
