Identification and Characterization of a Large Protein Essential for Degradation of the Crystalline Region of Cellulose by Cytophaga hutchinsonii
Identification and Characterization of a Large Protein Essential for Degradation of the Crystalline Region of Cellulose by Cytophaga hutchinsonii
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哈钦森噬细胞菌降解纤维素结晶区所必需的大蛋白的鉴定和表征
DOI：
10.1128/aem.02270-16
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发表时间：
2016-10
影响因子：
4.4
通讯作者：
Lu Xuemei
中科院分区：
文献类型：
--
作者：
Wang Sen;Zhao Dong;Bai Xinfeng;Zhang Weican;Lu Xuemei
hutchinsoni细胞噬菌是一种革兰氏阴性细菌，它可以通过一种不同于游离纤维素酶或纤维素体的独特机制有效地降解结晶纤维素。在本研究中，通过插入突变和基因缺失，发现编码假设蛋白chu_3220 （205 kDa）的chu_3220是C. hutchinsonii降解纤维素结晶区而非无定形区所必需的第一个基因。chu_3220缺失突变体在降解结晶纤维素方面存在缺陷，提高了Avicel PH101的结晶度，但仍能完全降解无定形纤维素。发现CHU_3220位于外膜的外表面，可以与纤维素结合。它包含15个phbh1结构域和一个c -末端结构域（CHU_C），该结构域被证明对CHU_3220在细胞表面的定位和CHU_3220在结晶纤维素降解中的功能至关重要。此外，晶体纤维素的降解是完全依赖于细胞的，并且被NaN3抑制。进一步的研究表明，chu_3220是由纤维素诱导的，没有chu_3220，细胞表面的内切葡聚糖酶活性显著降低。Real-time PCR结果显示，chu_3220缺失突变体中大部分位于细胞表面的内切葡聚糖酶编码基因转录减少，表明chu_3220也可能对某些内切葡聚糖酶的表达起调控作用。重要意义：哈钦索细胞噬菌可以在不需要纤维素体和游离纤维素酶的情况下，以独特的机制高效降解结晶纤维素。它缺乏被认为在破坏纤维素晶体区域中起重要作用的蛋白质，包括外葡聚糖酶、水解多糖单加氧酶、膨胀蛋白、膨胀蛋白样蛋白或膨胀蛋白，而且它的大多数内切葡聚糖酶缺乏碳水化合物结合模块。结晶纤维素降解的机理尚不清楚。在本研究中，chu_3220被确定为降解纤维素结晶区而非无定形区所必需的第一个基因。CHU_3220是一种位于外膜外表面的高分子量蛋白，可以与纤维素结合。我们提出CHU_3220可能是细胞表面负责结晶纤维素脱晶的蛋白质复合物的重要组成部分。哈金梭菌对结晶纤维素的降解不仅依赖于完整的细胞，而且需要细胞提供能量。这明显不同于其他已知的纤维素解聚体系。我们的研究揭示了哈金梭菌降解结晶纤维素的新策略。
ABSTRACT Cytophaga hutchinsonii is a Gram-negative bacterium that can efficiently degrade crystalline cellulose by a unique mechanism different from the free cellulase or cellulosome strategy. In this study, chu_3220, encoding the hypothetical protein CHU_3220 (205 kDa), was identified by insertional mutation and gene deletion as the first gene essential for degradation of the crystalline region but not the amorphous region of cellulose by C. hutchinsonii. A chu_3220 deletion mutant was defective in the degradation of crystalline cellulose and increased the degree of crystallinity of Avicel PH101 but could still degrade amorphous cellulose completely. CHU_3220 was found to be located on the outer surface of the outer membrane and could bind to cellulose. It contains 15 PbH1 domains and a C-terminal domain (CHU_C) that was proved to be critical for the localization of CHU_3220 on the cell surface and the function of CHU_3220 in crystalline cellulose degradation. Moreover, the degradation of crystalline cellulose was intact-cell dependent and inhibited by NaN3. Further study showed that chu_3220 was induced by cellulose and that the endoglucanase activity on the cell surface was significantly reduced without chu_3220. Real-time PCR revealed that the transcription of most genes encoding endoglucanases located on the cell surface was decreased in the chu_3220 deletion mutant, indicating that chu_3220 might also play a role in the regulation of the expression of some endoglucanases. IMPORTANCE Cytophaga hutchinsonii could efficiently degrade crystalline cellulose with a unique mechanism without cellulosomes and free cellulases. It lacks proteins that are thought to play important roles in disruption of the crystalline region of cellulose, including exoglucanases, lytic polysaccharide monooxygenases, expansins, expansin-like proteins, or swollenins, and most of its endoglucanases lack carbohydrate binding modules. The mechanism of the degradation of crystalline cellulose is still unknown. In this study, chu_3220 was identified as the first gene essential for the degradation of the crystalline region but not the amorphous region of cellulose. CHU_3220 is a high-molecular-weight protein located on the outer surface of the outer membrane and could bind to cellulose. We proposed that CHU_3220 might be an essential component of a protein complex on the cell surface in charge of the decrystallization of crystalline cellulose. The degradation of crystalline cellulose by C. hutchinsonii was not only dependent on intact cells but also required the energy supplied by the cells. This was obviously different from other known cellulose depolymerization system. Our study has shed more light on the novel strategy of crystalline cellulose degradation by C. hutchinsonii.
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