Physiological Role and Phylogenetic Diversity of Termite Gut Symbionts

IBN-0114505：白蚁肠道共生的生理作用和系统发育多样性主要调查者：密歇根州立大学约翰·A·布雷兹纳克白蚁是地球主要生物质形式--木质纤维植物材料及其衍生的残留物，如腐殖质的重要分解者。为了在这种相对耐火、缺氮的食物资源上茁壮成长，白蚁得到了肠道微生物共生体的多样化群落的帮助。肠道微生物的性质，以及它们在白蚁营养和活力中的作用，多年来一直是PI实验室持续研究的重点。目前奖项支持的研究在很大程度上是一项突破的副产品，这项突破导致了前一个资助期的两项重大发现。突破是在纯培养中分离出了白蚁肠道微生物群落的一个长期公认的、主要的和形态上独特的、但迄今难以捉摸的组成部分，即螺旋体。这些发现是：(A)认识到它们是能够从包括H2+CO2在内的各种底物中产生醋酸酯的密螺旋体的新物种；以及(B)在这些(随后在自由生活的)螺旋体中展示了固氮酶结构基因(NifH)和固氮作用。由于微生物产生的醋酸盐提供了白蚁每日所需能量的100%，肠道微生物固定氮气可以提供白蚁生长所需氮素的60%，螺旋体在白蚁营养中的重要性变得越来越明显。此外，由于以前从未在螺旋体中发现过H2/CO2-乙酰化或固氮作用，这些发现也为这些令人着迷的细菌的代谢多样性提供了新的见解。现在将结合生理、生化和分子生物学方法来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，并评估螺旋体在H2/CO2-乙酰化和原位氮气固定中的重要性。还将研究螺旋体与肠道微生物区系其他成员之间的合作和拮抗作用，包括：利用螺旋体产物的其他肠道微生物(如醋酸盐氧化剂)提供叶酸化合物(白蚁肠道螺旋体生长所必需的)；以及产氢/二氧化碳的螺旋体和消耗氢的甲烷菌之间的拮抗作用--两组微生物竞争相同的能源。还将进行研究，以检验这一假设，即许多(非螺旋体)肠道微生物定居在上皮附近的后肠微氧区，是以前在纯培养中逃脱隔离的微需氧微生物，因为它们不会在空气或缺氧条件下生长。据推测，这种微嗜氧菌是醋酸盐氧化剂，消耗向内扩散的氧气，从而使肠道的管腔区域缺氧，以便(由螺旋体和原生动物)发酵产生更多的醋酸盐。后一项努力可能会揭示有关低氧下生命的新原理，低氧是一种普遍存在但研究很少的情况，我们生物圈中的许多生物都经历过这种情况。这项研究项目将涉及研究生和博士后，因此，将扩大受过培训的科学家基础，以探索微生物世界最显著的两个特征--其巨大但鲜为人知且基本上未被开发的多样性，以及它对地球上大型生物生命的重要性。

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.