Butyrivibrio hungatei MB2003 Competes Effectively for Soluble Sugars Released by Butyrivibrio proteoclasticus B316T during Growth on Xylan or Pectin

Nikola Palevich; William J. Kelly; Siva Ganesh; Jasna Rakonjac; Graeme T. Attwood

摘要

Rumen bacterial species belonging to the genus Butyrivibrio are important degraders of plant polysaccharides, particularly hemicelluloses (arabinoxylans) and pectin. Currently, four species are recognized; they have very similar substrate utilization profiles, but little is known about how these microorganisms are able to coexist in the rumen. To investigate this question, Butyrivibrio hungatei MB2003 and Butyrivibrio proteoclasticus B316~(T) were grown alone or in coculture on xylan or pectin, and their growth, release of sugars, fermentation end products, and transcriptomes were examined. In monocultures, B316~(T) was able to grow well on xylan and pectin, while MB2003 was unable to utilize either of these insoluble substrates to support significant growth. Cocultures of B316~(T) grown with MB2003 revealed that MB2003 showed growth almost equivalent to that of B316~(T) when either xylan or pectin was supplied as the substrate. The effect of coculture on the transcriptomes of B316~(T) and MB2003 was assessed; B316~(T) transcription was largely unaffected by the presence of MB2003, but MB2003 expressed a wide range of genes encoding proteins for carbohydrate degradation, central metabolism, oligosaccharide transport, and substrate assimilation, in order to compete with B316~(T) for the released sugars. These results suggest that B316~(T) has a role as an initiator of primary solubilization of xylan and pectin, while MB2003 competes effectively for the released soluble sugars to enable its growth and maintenance in the rumen.IMPORTANCE Feeding a future global population of 9 billion people and climate change are the primary challenges facing agriculture today. Ruminant livestock are important food-producing animals, and maximizing their productivity requires an understanding of their digestive systems and the roles played by rumen microbes in plant polysaccharide degradation. Butyrivibrio species are a phylogenetically diverse group of bacteria and are commonly found in the rumen, where they are a substantial source of polysaccharide-degrading enzymes for the depolymerization of lignocellulosic material. Our findings suggest that closely related species of Butyrivibrio have developed unique strategies for the degradation of plant fiber and the subsequent assimilation of carbohydrates in order to coexist in the competitive rumen environment. The identification of genes expressed during these competitive interactions gives further insight into the enzymatic machinery used by these bacteria as they degrade the xylan and pectin components of plant fiber. KEYWORDS: Butyrivibrio , CAZy, rumen, bacteria, genome analysis, pectin, sugar ABC transport system, transcriptome, xylanINTRODUCTIONThe microbial community responsible for degradation of plant fiber in the rumen is diverse (1,–3), but the core rumen bacterial microbiome is composed of seven groups, i.e., Prevotella , Butyrivibrio , and Ruminococcus , as well as unclassified Lachnospiraceae , Ruminococcaceae , Bacteroidales , and Clostridiales (2). Only a few species within these groups possess the enzymatic machinery required to initiate the primary degradation of insoluble plant polysaccharides (4,–10). Cross-feeding interactions are known to occur between the polysaccharide-degrading species and sugar-fermenting microbes, allowing a wider diversity of microorganisms to exist in the rumen (11). Bacterial species belonging to the genus Butyrivibrio are metabolically versatile (2) and efficiently utilize the insoluble complex polysaccharides xylan and pectin (4, 12,–14). At present, there are four recognized Butyrivibrio species (15), but an understanding of the interactions among these species during the process of fiber degradation in the rumen (16,–18) is lacking.Comparisons of the genome sequences of Butyrivibrio hungatei MB2003 (19, 20) and Butyrivibrio proteoclasticus B316~(T) (14), and particularly their carbohydrate-active enzyme (CAZyme) profiles, show that these Butyrivibrio species are functionally similar, and they predict important roles for both of these species in the breakdown of hemicellulose and pectin. B316~(T) contains 342 predicted CAZymes and shows strong growth on both oat spelt xylan and apple pectin (21). However, although MB2003 encodes 225 CAZymes associated with hemicellulose and pectin degradation, phenotypic analysis showed that it cannot grow on these insoluble substrates (19, 20). Therefore, it is hypothesized that MB2003 relies on other rumen organisms with more developed polysaccharide-degrading abilities to initiate the degradation of insoluble substrates and competes for the released oligosaccharides and sugars to enable its growth. To investigate this idea, MB2003 and B316~(T) were grown on oat spelt xylan or apple pectin, separately in monocultures to compare their individual substrate utilization abilities and together in cocultures to investigate their interactions. Growth of the strains was followed with strain-specific

机译：属于Butyrivibrio属的瘤胃细菌是植物多糖的重要降解剂，尤其是半纤维素（阿拉伯木聚糖）和果胶。目前，已识别出四种。它们具有非常相似的底物利用率曲线，但对这些微生物如何在瘤胃中共存的了解甚少。为了调查该问题，将霍氏丁酸杆菌MB2003和变形丁酸杆菌B316〜（T）单独或在木聚糖或果胶上共培养，并检查它们的生长，糖的释放，发酵终产物和转录组。在单培养中，B316〜（T）能够在木聚糖和果胶上生长良好，而MB2003无法利用这些不溶性底物中的任何一种来支持显着的生长。与MB2003一起生长的B316〜（T）的共培养物表明，当以木聚糖或果胶作为底物时，MB2003的生长几乎与B316〜（T）的相等。评估了共培养对B316〜（T）和MB2003转录组的影响; B316〜（T）的转录在很大程度上不受MB2003的影响，但是MB2003表达了广泛的基因编码蛋白，这些蛋白编码碳水化合物的降解，中枢代谢，寡糖转运和底物同化，以便与B316〜（T）竞争释放的糖。这些结果表明B316〜（T）作为木聚糖和果胶的主要增溶剂的引发剂，而MB2003有效竞争释放的可溶性糖以使其在瘤胃中生长和维持。重要养活全球9个人口十亿人口和气候变化是当今农业面临的主要挑战。反刍家畜是重要的粮食生产动物，要使其生产力最大化，就需要了解其消化系统以及瘤胃微生物在植物多糖降解中的作用。 Butyrivibrio物种是系统发育上多样的细菌，通常在瘤胃中发现，在瘤胃中，它们是木质纤维素材料解聚的多糖降解酶的重要来源。我们的研究结果表明，紧密相关的Butyrivibrio物种已开发出独特的策略来降解植物纤维并随后吸收碳水化合物，从而在竞争性瘤胃环境中共存。在这些竞争性相互作用过程中表达的基因的鉴定为这些细菌降解植物纤维的木聚糖和果胶成分提供了进一步的了解。关键词：Butyrivibrio，CAZy，瘤胃，细菌，基因组分析，果胶，糖ABC转运系统，转录组，木聚糖诱导瘤胃中植物纤维降解的微生物群落多种多样（1，–3），但瘤胃的核心微生物群是由七个组组成，即普雷沃氏菌，Butyrivibrio和Ruminococcus，以及未分类的Lachnospiraceae，Ruminococcaceae，Bacteroidales和Clostridiales（2）。这些组中只有极少数的物种具有引发不溶性植物多糖初步降解所需的酶促机制（4，-10）。已知在降解多糖的物种和发酵糖的微生物之间会发生交叉进料的相互作用，从而使瘤胃中存在更多种类的微生物（11）。属于Butyrivibrio属的细菌在代谢上具有多方面的优势（2），可有效利用不溶性复合多糖木聚糖和果胶（4、12–14）。目前，有4个公认的Butyrivibrio物种（15），但缺乏对瘤胃中纤维降解过程中这些物种之间相互作用的了解（16，–18）。Butyrivibrio hungatei MB2003基因组序列的比较19，20）和Butyrivibrio proteoclasticus B316〜（T）（14），尤其是它们的碳水化合物活性酶（CAZyme）谱显示，这些Butyrivibrio物种在功能上相似，并且它们预测这两种物种在分解中的重要作用半纤维素和果胶。 B316〜（T）包含342种预测的CAZymes，并且在燕麦拼木聚糖和苹果果胶中均表现出强劲的生长（21）。然而，尽管MB2003编码与半纤维素和果胶降解有关的225种酶，但表型分析表明MB2003不能在这些不溶性底物上生长（19、20）。因此，假设MB2003依赖于具有更发达的多糖降解能力的其他瘤胃生物来引发不溶性底物的降解，并竞争释放的寡糖和糖以使其生长。为了研究这个想法，MB2003和B316〜（T）分别在单种栽培中分别在燕麦拼合木聚糖或苹果果胶上生长，以比较它们各自的底物利用能力，并在共培养中一起生长以研究它们的相互作用。菌株生长后进行菌株特异性

Butyrivibrio hungatei MB2003 Competes Effectively for Soluble Sugars Released by Butyrivibrio proteoclasticus B316T during Growth on Xylan or Pectin

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