ECF σ因子在哈氏噬纤维菌纤维素降解新策略中的调控作用机制研究

•.Cytophaga hutchinsonii is a common cellulolytic soil bacterium belonging to the phylum Bacteroidetes. Cells of C. hutchinsonii are capable of actively digesting crystalline cellulose using a third but poorly understood strategy to digest cellulose. The precise aspects of the digesting process as well as how the process is controlled is largely unknown. The pattern of gene expression dedicated by the extracytoplasmic function (ECF) σ factor has been thought to constitute the thrid pillar of bacteria signal transduction in response to changes in environmental cues. Our previous study identified an ECF σ encoding gene whose insterted mutation resulted in the inability of C. hutchinsonii to digest filter paper. Immediately adjacent to this gene locus is a gene coding for a protein predicted to be a cognate anti-σ factor. The present project thus intends to fully characterize the ECF σ/anti-σ pair, trying to elucidate the mechanism by which this ECF σ system participates in controlling the cellulose degradation in Cytophaga hutchinsonii. The project will focus on the structure and function of the ECF σ and anti-σ, the pattern of their interactions and how this interaction pattern changes under different conditions, the relationship between this ECF σ/anti-σ pair and a PDZ protease whose mutation also leads to cellulolytic defect but can be complemented by ECF σ, and the effect of the ECF σ may inflict on the responsive netwrok of C. hutchinsonii to carbon sources. The above results are anticipated to help elucidating the precise mechanism by which the cellulose responsive ECF σ factor is activated, and thus how the process of cellulose assimilation by C. hutchinsonii is regulated. The results will also provide the theoretical basis for the establishment and development of new strategies for the efficient conversion of cellulose by the cell-bound noncomplexed enzymatic hydrolytic system, which will enrich and develop the theoretical system of microbial degradation of cellulose.

哈氏噬纤维菌（Cytophaga hutchinsonii）具有极强的结晶纤维素降解能力，但其纤维素降解过程及过程其调控机理尚未得到阐明。胞质外功能（ECF）σ因子所介导的基因表达模式改变是原核生物细胞响应环境信号变化的重要途径之一。我们在前期研究中发现了一个影响哈氏噬纤维菌降解利用纤维素的ECF σ 因子基因簇。本项研究拟对该因子在纤维素利用过程中的作用机制进行系统研究，主要包括对基因簇中ECF σ 因子(SigC)和anti-σ因子(SigAC)的结构功能、两者间相互作用模式及其动态变化、SigC所控制的碳源响应基因网络进行研究，深入阐述SigC的活性控制机制及其在哈氏噬纤维菌纤维素利用过程中的调控机制, 从而丰富和发展纤维素降解的理论体系, 为建立和发展纤维素转化利用的新策略提供理论基础。

纤维素降解微生物在生物质的资源转化中发挥着重要的作用。哈氏噬纤维菌（Cytophaga hutchinsonii）具有高效和独特的纤维素降解降解策略，可迅速降解利用结晶纤维素。本研究表明，哈氏噬纤维菌中存在两种胞质外功能σ因子系统即SigE/anti-SigE（CHU_3097/CHU_3096）和SigF/anti-SigF（CHU_3562/CHU_3563）参与该菌对纤维素的降解。除anti-SigF外，其它三组分的缺失均导致纤维素利用缺陷。分析显示SigE的缺失不影响菌体纤维素酶的产生能力，但导致菌体部分外膜蛋白组分缺失和菌体运动能力丧失。通过细菌双杂交方式证明，SigE 和anti-SigE 之间存在直接的相互作用。细胞定位分析发现，anti-SigE以三聚体的形式镶嵌于细胞膜上，SigE在非诱导条件下与anti-SigE结合于细胞膜上，在纤维素存在的诱导条件下部分被释放至细胞质中。利用转录组测序技术比较了SigE缺失突变株与野生型之间的基因表达差异。在葡萄糖、无碳源、和微晶纤维素三种碳源条件下，与野生型相比突变株都存在较多的差异表达上调和差异下调的基因，其中372个基因（129个未知功能，243个为已知基因）是在Avicel诱导条件下特异性下调的。在本研究中对发现的ECF σ因子调节蛋白ChSR（CHU_0195）也进行了研究。综合分析显示，SigE主要影响菌体内多糖利用位点（PLUs）SUS-like（CHU_1276-1280）的组装和细胞内蛋白物质输送的 Ⅱ 型分泌系统。ECF σ因子SigE的缺失，使得菌体细胞膜与菌体细胞外膜功能性蛋白复合体的完整性受到破坏，进而导致胞内产生的纤维素酶难以输送至细胞外膜或在细胞外膜上难以正确定位，从而导致菌体纤维素利用缺陷。由此可以说明，ECFσ/anti-σ系统在哈氏噬纤维菌中对于纤维素的降解具有中心性的调节功能。