Physiological Role and Phylogenetic Diversity of Termite Gut Symbionts

IBN-0114505：白蚁肠道共生体的生理作用和系统发育多样性首席研究员：John A. Breznak，密歇根州立大学白蚁是地球上主要生物质形式的重要分解者——木质纤维素植物材料及其残留物，如腐殖质。为了在这种相对难处理、缺乏氮的食物资源上茁壮成长，白蚁需要多种肠道微生物共生体的帮助。肠道微生物的性质及其在白蚁营养和活力中的作用，多年来一直是PI实验室研究的重点。目前资助的研究在很大程度上是一项突破的结果，这些突破在上一个资助期内导致了两项重大发现。这一突破是在纯培养中分离出白蚁肠道微生物群落中一个长期以来公认的、主要的、形态上独特的、但迄今为止难以捉摸的组成部分，即螺旋体。这些发现是：(a)确认它们是密螺旋体的新物种，能够从各种底物（包括H2+CO2）中生产乙酸；(b)在这些（随后在自由生活的）螺旋体中展示了氮酶结构基因（nifH）和固氮作用。由于微生物产生的醋酸盐可支持白蚁100%的日常能量需求，而肠道微生物对N2的固定可为白蚁的生长提供高达60%的氮，因此螺旋体在白蚁营养中的重要性越来越明显。此外，由于此前从未发现螺旋体具有H2/ co2 -丙酮生成和N2固定作用，这些发现也为了解这些迷人细菌的代谢多样性提供了新的见解。生理学、生物化学和分子生物学方法的结合现在将用于更详细地探索这些代谢活动。我们试图确定影响其表达和活性的因素，并评估螺旋体对H2/ co2 -丙酮生成和N2原位固定的重要性。还将研究螺旋体与肠道微生物群其他成员的合作和拮抗相互作用，包括：利用螺旋体产物（如醋酸氧化剂）的其他肠道微生物提供叶酸化合物（白蚁肠道螺旋体生长所需）；以及产H2/ co2的螺旋体和消耗H2的产甲烷菌之间的对抗——两组微生物争夺相同的能量来源。还将进行研究来验证这样一种假设，即在靠近上皮的后肠的微氧区定植的许多（非螺藻）肠道微生物是以前在纯培养中逃脱分离的嗜氧微生物，因为它们不能在空气或缺氧条件下生长。据推测，这些微嗜氧菌是醋酸氧化菌，它们消耗向内扩散的O2，从而使肠道内腔区缺氧，以便（由螺旋体和原生动物）发酵产生更多的醋酸。后一种努力可能会揭示缺氧下生命的新原理，缺氧是我们生物圈中许多生物普遍经历的一种但研究很少的条件。这个研究项目将包括研究生和博士后，因此，将扩大科学家的基础，以探索微生物世界的两个最显著的特征-其巨大的，但知之甚少和很大程度上未开发的多样性，以及它对我们星球上的微生物生命的重要性。

IBN-0114505: Physiological Role and Phylogenetic Diversity of Termite Gut SymbiontsPrincipal Investigator: John A. Breznak, Michigan State UniversityTermites are important decomposers of earth's major form of biomass -- lignocellulosic plant material and residues derived from it, e.g. humus. In order to thrive on such relatively refractory, nitrogen-poor food resources, termites are aided by a diverse community of gut microbial symbionts. The nature of the gut microbes, and their role in termite nutrition and vitality, has been an ongoing focus of research in the PI's laboratory for many years. Research supported by the current award is largely an outgrowth of a breakthrough, which led to two major discoveries during the previous funding period. The breakthrough was the isolation in pure culture of a long-recognized, major, and morphologically distinct, but hitherto elusive, component of the termite gut microbial community, i.e. spirochetes. The discoveries were: (a) the recognition of them as novel species of Treponema capable of acetate production from a variety of substrates, including H2+CO2; and (b) the demonstration of nitrogenase structural genes (nifH) and N2 fixation in these (and subsequently in free-living) spirochetes. Inasmuch as microbially-produced acetate supports up to 100% of the daily energy requirement of termites, and N2 fixation by gut microbes can supply up to 60% of the nitrogen for termite growth, the importance of spirochetes in termite nutrition was becoming clearer. Moreover, as neither H2/CO2-acetogenesis nor N2 fixation had ever been recognized in spirochetes before, these discoveries were also providing new insight into the metabolic diversity of these fascinating bacteria.A combination of physiological, biochemical, and molecular biological methods will now be used to explore these metabolic activities in greater detail. We seek to identify factors affecting their expression and activity, and to assess the importance of spirochetes to H2/CO2-acetogenesis and N2 fixation in situ. Cooperative and antagonistic interactions of spirochetes with other members of the gut microbiota will also be examined, including: the provision of folate compounds (required for growth of termite gut spirochetes) by other gut microbes that utilize spirochete products, e.g. acetate oxidizers; and antagonisms between H2/CO2-acetogenic spirochetes and H2-consuming methanogens - two groups of microbes competing for the same energy source. Studies will also be done to test the hypothesis that many of the (non-spirochetal) gut microbes colonizing the microoxic region of hindguts near the epithelium are microaerophiles that have previously escaped isolation in pure culture, because they will not grow in air or under anoxia. It is hypothesized that such microaerophiles are acetate-oxidizers that consume inwardly diffusing O2, thereby rendering the luminal region of the gut anoxic for the fermentative production (by spirochetes and protozoa) of more acetate. This latter effort is likely to reveal new principles about life under hypoxia, a pervasive but poorly studied condition experienced by many organisms in our biosphere. This research project will involve graduate students and a postdoctoral and, hence, will expand the base of scientists trained to explore two of the most salient features of the microbial world - its enormous, but poorly understood and largely untapped diversity, and its importance to macrobial life on our planet.