Comparative analysis of extremely thermophilic Caldicellulosiruptor species reveals common and differentiating cellular strategies for plant biomass utilization. Comparative analysis of extremely thermophilic Caldicellulosiruptor species reveals common and differentiating cellular strategies for plant biomass utilization

Microbiological, genomic and transcriptomic analyses were used to examine three species from the bacterial genus Caldicellulosiruptor with respect to their capacity to convert the carbohydrate content of lignocellulosic biomass at 70°C to simple sugars, acetate, lactate, CO2 and H2. C. bescii, C. kronotskyensis and C. saccharolyticus solubilized 38%, 36% and 29% (by weight) of unpretreated switchgrass (5 g/l), repectively, which was about half of the concentration of crystalline cellulose (Avicel, 5 g/l) that was solubilized under the same conditions. The lower yields with C. saccharolyticus were unexpected, given that its genome encodes the same GH9-GH48 multi-domain cellulase (CelA) found in the other two species. However, the genome of C. saccharolyticus lacks two other cellulases with GH48 domains, which could be responsible for its lower levels of solubilization. Transcriptomes for growth of each species comparing Cellulose to switchgrass showed that many carbohydrate ABC transporters and multi-domain extracellular glycoside hydrolases were differentially regulated, reflecting the heterogeneity of lignocellulose. However, significant differences in transcription levels for conserved genes among the three species were noted, indicating unexpectedly diverse regulatory strategies for deconstruction for these closely related bacteria. Genes encoding the Che-type chemotaxis system and flagella biosynthesis were up-regulated in C. kronotskyensis and C. bescii during growth on cellulose, implicating motility in substrate utilization. The results here show that capacity for plant biomass deconstruction varies across Caldicellulosiruptor species and depends in a complex way on GH genome inventory, substrate composition, and gene regulation. Overall design: A dye swap was completed with each of three Caldicellulosiruptor species: C. bescii, C. kronotskyensis and C. saccharolyticus growing on cellulose and switchgrass. Half of the RNA sample for one condition was labeled with Cy3 and the other half Cy5. The two differentially labeled samples were run on two different slides and analyzed to investigate differences in transcription during growth on cellulose and switchgrass.

本研究采用微生物学、基因组学与转录组学分析方法，针对热纤梭菌属（Caldicellulosiruptor）的3个物种开展研究，以解析其在70℃条件下将木质纤维素生物质中的碳水化合物转化为单糖、乙酸、乳酸、二氧化碳及氢气的能力。
其中，贝氏热纤梭菌（C. bescii）、克氏热纤梭菌（C. kronotskyensis）与解糖热纤梭菌（C. saccharolyticus）对未预处理柳枝稷（浓度5g/L）的碳水化合物溶出率分别为38%、36%与29%（按重量计），仅为相同培养条件下微晶纤维素（Avicel，5g/L）溶出率的约一半。
出乎意料的是，解糖热纤梭菌的溶出率较低，尽管其基因组编码了另外两个物种共有的GH9-GH48多结构域纤维素酶（CelA）。然而，解糖热纤梭菌的基因组缺少另外两种带有GH48结构域的纤维素酶，这可能是其溶出水平较低的原因。
对各物种在纤维素与柳枝稷上生长的转录组进行比较分析后发现，大量碳水化合物ATP结合盒（ATP-binding cassette, ABC）转运蛋白与多结构域胞外糖苷水解酶（glycoside hydrolase，GH）的表达存在显著差异，这反映了木质纤维素底物的异质性。不过，三个物种间保守基因的转录水平存在显著差异，表明这些亲缘关系紧密的细菌在生物质解构过程中采用了出人意料的多样化调控策略。
在纤维素培养基上生长时，克氏热纤梭菌与贝氏热纤梭菌中编码Che型趋化系统（chemotaxis system）及鞭毛生物合成的基因表达上调，提示运动性参与了底物利用过程。
本研究结果表明，不同热纤梭菌属物种的植物生物质解构能力存在差异，且其能力与糖苷水解酶家族（GH）基因组编码谱、底物组成及基因调控之间存在复杂的关联。
实验总体设计：针对3个热纤梭菌属物种——贝氏热纤梭菌（C. bescii）、克氏热纤梭菌（C. kronotskyensis）与解糖热纤梭菌（C. saccharolyticus）在纤维素与柳枝稷上的生长样本，完成了染料互换（dye swap）实验。将某一培养条件下的RNA样品均分为两份，分别用Cy3与Cy5进行荧光标记；将两份差异标记的样品分别在两张不同的芯片玻片上进行杂交检测，以分析纤维素与柳枝稷培养条件下的转录差异。