Unraveling a novel mechanism for cellulose decomposition in the bacterial phylum Fibrobacteres.

纤维素是地球上最丰富的有机多糖，是植物细胞壁的主要结构成分。因此，木质纤维素植物生物量在很大程度上难以被微生物分解，并且降解和利用纤维素多糖的能力仅限于少数细菌和真菌群。在自然界中，细菌和真菌利用两种不同的酶机制分解纤维素；需氧真菌和细菌分泌大量的细胞外酶，而厌氧细菌和真菌具有细胞表面结合酶复合物（纤维素体）。反刍食草动物，如家畜，依靠共生肠道微生物消化植物物质。琥珀酸纤维杆菌是纤维杆菌属的模式种，最初是从牛瘤胃中分离出来的，它是植物生物量最多产的细菌降解者。最近的证据表明，在纤维杆菌属中，纤维素降解的“第三种”机制已经进化出来，这可能解释了瘤胃中纤维素降解的优越效率。F.琥珀酸基因不符合纤维素分解的经典模型，纤维杆菌中纤维素降解的一种可能机制涉及去除单个纤维素纤维并随后通过外膜运输，在那里它们被纤维素酶切割。此外，分子方法已经成功地鉴定了纤维素降解的非肠道环境（垃圾填埋场和淡水湖）中纤维杆菌属的成员，这表明纤维杆菌的多样性比以前认为的要大。在这里，我们的方法是对几种纤维杆菌菌株的基因组进行测序，这些菌株代表了目前在该属内检测到的生态和分类多样性的广度。这些分析将包括我们最近从垃圾填埋场分离出的一些新型琥珀酸梭菌菌株，这是该物种首次从非肠道环境中分离出来。此外，这些菌株可以利用纤维素作为唯一的碳来源进行生长。我们的比较基因组分析将使我们能够研究纤维杆菌属中不同菌株和物种的进化相关性，特别强调纤维素降解机制，我们怀疑纤维素降解机制在纤维杆菌属的所有成员中都是保守的，并且是限制该群体的关键生理属性。然后，我们将重点关注该属成员的表型特征，通过观察每个菌株对多糖及其衍生物的降解和利用，获得生长速率和酶活性的定量数据。这些数据将提供关于每种菌株的水解能力和底物特异性的重要信息，目前这些信息缺乏。最后，我们将再次使用高通量测序技术，但这一次将重点放在基因表达谱（mRNA）上，使用转录组学方法，了解在一系列多糖及其衍生物的生长过程中功能基因的差异表达。我们将提供从单糖到复杂木质纤维素植物生物量的生长基质，并比较基因表达谱，使我们能够在特定的降解过程（如纤维素附着或单糖的运输）中暗示特定的基因。这些数据将为我们对纤维细菌降解纤维素的机制的理解提供一个步骤的改变。很明显，纤维细菌是纤维素的多产降解者，因此它们的酶可能在改善经济上重要的反刍动物的营养和提炼用于生产生物乙醇的植物生物量方面找到生物技术应用。

Cellulose is the most abundant organic polysaccharide on Earth and represents a major structural component of plant cell walls. Consequently, lignocellulosic plant biomass is largely recalcitrant to decomposition by microorganisms, and the ability to degrade and utilise cellulosic polysaccharides is limited to only a few bacterial and fungal groups. In nature, two different enzyme mechanisms for cellulose decomposition are utilised by bacteria and fungi; aerobic fungi and bacteria secrete high quantities of extracellular enzymes, whereas anaerobic bacteria and fungi possess cell-surface bound enzyme complexes (cellulosomes). Ruminant herbivores such as domestic cattle rely on symbiotic gut microorganisms for the digestion of plant material. Fibrobacter succinogenes is the type species of the genus Fibrobacter and was first isolated from the bovine rumen where it is established as the most prolific bacterial degrader of plant biomass. This superior efficiency to degrade cellulose in the rumen may be explained by recent evidence that within the genus Fibrobacter, a 'third' mechanism for the degradation of cellulose has evolved. F. succinogenes does not conform to the classical models of cellulose decomposition, and one possible mechanism for cellulose degradation in Fibrobacter involves the removal of individual cellulose fibres and subsequent transport through the outer membrane where they are cleaved by cellulases. Furthermore, molecular approaches have successfully identified members of the genus Fibrobacter in non-gut environments where cellulose is degraded (landfill sites and freshwater lakes), suggesting a greater diversity of fibrobacters than previously thought. Here, our approach is to sequence the genomes of several Fibrobacter strains that represent the breadth of ecological and taxonomic diversity currently detected within the genus. These analyses will include some novel strains of F. succinogenes that we have recently isolated from landfill sites and this is the first isolation of this species from a non-gut environment. Furthermore, these strains can utilise cellulose as the sole source of carbon for growth. Our comparative genomic analyses will enable us to investigate the evolutionary relatedness of the different strains and species within the Fibrobacter genus, with particular emphasis on the mechanism of cellulose degradation that we suspect is conserved across all members of the Fibrobacter genus and is the key physiological attribute that circumscribes the group. We will then focus on phenotypic characterisation of members of the genus, by observing the degradation and utilisation of polysaccharides and their derivatives by each strain, obtaining quantitative data on growth rates and enzyme activities. These data will provide important information on the hydrolytic abilities and substrate specificity of each strain, for which there is a currently a paucity of information. Finally, we will again use high throughput sequencing techniques, but this time focussing on gene expression profiles (mRNA) using transcriptomic approaches that inform on the differential expression of functional genes in response to growth on a range of polysaccharides and their derivatives. We will provide growth substrates that range from simple sugars to complex lignocellulosic plant biomass and compare gene expression profiles to enable us to implicate specific genes in particular degradative processes such as cellulose attachment or the transport of simple sugars. These data will provide a step change in our understanding of the mechanism for cellulose degradation employed by fibrobacters. It is clear that fibrobacters are prolific degraders of cellulose, and their enzymes may therefore find biotechnological application in improving the nutrition of economically important ruminant animals and in the refining of plant biomass for the production of bioethanol.