非共生固氮菌及其固氮作用

生物固氮作用是生态系统中重要的氮素来源,参与固氮作用的微生物对植物的生长发育至关重要。与共生固氮微生物相比,非共生固氮微生物地域分布更广泛、种类更多,对全球生态系统中氮素循环有着重要意义。本文总结了非共生固氮菌的分类及系统发育,非共生固氮菌的群落构建过程和机制;归纳了不同生态系统(如草原、森林、海洋、农田等)、植物不同部位(如林冠、叶际、根际、根内、凋落物等)和土壤中非共生固氮菌的群落组成及固氮潜力的差异,以及影响非共生固氮菌群落组成和固氮潜力的主要因素(如气候因素、土壤理化性质、人为措施等);并整理了常用的研究非共生固氮菌及其固氮潜力的检测方法。     

植物内生固氮菌

植物内生固氮菌的发现和研究显现了一个新的固氮系统,深化了根际联合共生固氮的研究。文章综述了10年来植物内生固氮菌的研究进展,并探讨了内生固氮菌进入植物体内的方式和行使固氮功能的可能性。     

落地生根内生固氮菌多样性和促生特性

【背景】内生固氮菌可以定殖于植物体内为植物提供营养物质,还能通过代谢促进植物生长,目前对于落地生根内生菌的研究鲜见报道。【目的】研究落地生根中内生固氮菌多样性。【方法】从表面消毒的植物组织中分离纯化内生菌,并通过乙炔还原法测定菌株的固氮酶活性。采用SDS-PAGE全细胞蛋白电泳和IS指纹图谱对菌株聚类,各类群代表菌株进行16S rRNA基因系统发育分析和生理生化鉴定。测定菌株固氮、分泌生长素和ACC脱氨酶、产铁载体、溶磷和解钾等促生特性。【结果】从落地生根中分离纯化出26株内生固氮菌,聚为5个类群,隶属于4个属的5个菌种,且各类群代表菌株具有多种促生功能。【结论】从落地生根中分离获得的内生菌具有丰富的遗传多样性和促生特性,并且存在新的微生物资源,有待开发利用。     

泓森槐可培养内生固氮细菌多样性与潜在促生长特性评价

【背景】泓森槐为豆科刺槐属的速生型落叶乔木,生长快,营养需求多。目前对于泓森槐内生固氮细菌的研究鲜有报道。【目的】研究泓森槐可培养内生固氮细菌的多样性及潜在促生长特性。【方法】从表面消毒的泓森槐根、茎、叶及根瘤中进行内生固氮细菌的分离纯化,采用IS-PCR指纹图谱技术将分离得到的菌株进行聚类,对各类群代表菌株进行16SrRNA基因系统发育分析、生理生化试验、固氮酶活性测定、促生长特性试验。【结果】从泓森槐中分离出56株细菌,聚为10个类群,分别为Paenibacillus sabinae、Klebsiella michiganensis、Kosakonia radicincitans、Kosakonia pseudosacchari、Mesorhizobium erdmanii、Mesorhizobium huakuii、Mesorhizobium silamurunense、Pseudomonas geniculata、Burkholderia territorii和Devosia riboflavina,其中5个类群为联合固氮菌、3个类群为共生固氮菌、2个类群为非固氮菌,表明泓森槐组织内生固氮细菌具有丰富的多样性。促生特性试验表明7个类群具溶磷能力、6个类群具解钾能力、6个类群具有产生长素能力、6个类群具产铁载体能力、2个类群具有产蛋白酶能力。【结论】泓森槐组织内生固氮细菌具有遗传多样性与促生长特性,其在农业微生物菌肥开发利用方面具有潜在价值。     

弗兰克氏菌在构建新的共生固氮物种研究中的优越性

弗兰克氏菌在构建新的共生固氮物种研究中的优越性解放军石家庄医学高等专科学院０５００８１曲东明范浩南四川师范大学细胞研究室６１００６８韩善华弗兰克氏菌（Ｆｒａｎｋｉａ）能与多种木本被子植物建立共生固氮关系，它形成的根瘤具有较强的固氮能力，有些超过大豆根...     

植物内生固氮菌及其固氮机理研究进展

氮素是植物生长必不可少的元素,植物内生固氮菌不仅能够在植物体内产生氮素以供植物利用,而且在自然界氮素循环过程中发挥积极作用,对农业可持续发展具有重要意义。近年来,植物内生固氮菌逐渐成为研究热点。由植物内生固氮菌的发现、作物共生、侵入途径、固氮机理、促生作用机制等方面系统地综述了植物内生固氮菌的研究进展,探讨了植物内生固氮菌新的研究思路以及一些尚未解决的问题,以期为植物内生固氮菌及生物固氮研究提供参考。     

一氧化氮对豆科植物结瘤及固氮的影响机制

豆科植物-根瘤菌共生过程受双方基因复杂且精细的调控, 能够产生特异的根瘤结构并可将大气中的惰性氮气(N₂)转化为可被植物直接利用的氨态氮。结瘤与固氮受多种因素影响, 其中, 一氧化氮(NO)作为一种自由基反应性气体信号分子, 可参与调节植物的许多生长发育过程, 如植物的呼吸、光形态建成、种子萌发、组织和器官发育、衰老以及响应各种生物及非生物胁迫。在豆科植物中, NO不仅影响寄主与菌共生关系的建立, 还参与调控根瘤菌对氮气的固定并提高植株氮素营养利用效率。该文主要从豆科植物及共生菌内NO的产生、降解及其对结瘤、共生固氮的影响和对环境胁迫的响应, 阐述了NO调控豆科植物共生体系中根瘤形成和共生固氮过程的作用机制, 展望了NO信号分子在豆科植物共生固氮体系中的研究前景。     

耐氨固氮菌分泌物对水稻秧苗生长的影响*

研究了催娩克氏菌(Klebsiella oxytoca)的不同菌株(耐氨固氮型、野生型、不固氮型)对杯栽水稻秧苗生长的影响,发现耐氨固氮菌可分泌某种植物生长促进物质。此类物质可被阳离子树脂吸附,在80℃下活性不受影响。在适宜浓度下,可使水稻秧苗的根重和苗重增加30％。     

耐氨固氮菌分泌物对水稻秧苗生长的影响

研究了催娩克氏菌（Klebsiella oxytoca)的不同菌株（耐氨固氮型,野生型,不固氮型）对杯栽水稻秧苗生长的影响,发现耐氨固氮菌可分泌某种植物生长促进物质,此类物质可被阳离子树脂吸附,在80℃下活性不受影响,在适宜浓度下,可使水稻秧苗的根重和苗重增加30%。     

植物联合固氮菌分子生态学的研究方法和应用

联合固氮菌定殖于植物根际或体内 ,它们与植物联合没有像根瘤菌和豆科植物共生形成根瘤那样形成特化的结构。由于二者的联合缺少明显的表型变化 ,以致研究这种植物和微生物间相互作用的工作进展缓慢。最近 ,免疫学技术、寡核苷酸探针杂交技术和监测标记 /报告基因等分子生物学技术在这一领域中得到了广泛的应用 ,不起才有了许多细致的联合固氮菌分子生态学研究。如研究与植物联合的固氮菌在何处表达固氮功能时 ,就应用了抗固氮酶铁蛋白抗体专一识别铁蛋白、寡核苷酸探针与ｎｉｆＨ基因 (编码固氮酶铁蛋白 )的ｍＲＮＡ杂交或ｎｉｆＨ启动子与…     

Sulfate is transported at significant rates through the symbiosome membrane and is crucial for nitrogenase biosynthesis

Legume–rhizobia symbioses play a major role in food production for an ever growing human population. In this symbiosis, dinitrogen is reduced (“fixed”) to ammonia by the rhizobial nitrogenase enzyme complex and is secreted to the plant host cells, whereas dicarboxylic acids derived from photosynthetically produced sucrose are transported into the symbiosomes and serve as respiratory substrates for the bacteroids. The symbiosome membrane contains high levels of SST1 protein, a sulfate transporter. Sulfate is an essential nutrient for all living organisms, but its importance for symbiotic nitrogen fixation and nodule metabolism has long been underestimated. Using chemical imaging, we demonstrate that the bacteroids take up 20‐fold more sulfate than the nodule host cells. Furthermore, we show that nitrogenase biosynthesis relies on high levels of imported sulfate, making sulfur as essential as carbon for the regulation and functioning of symbiotic nitrogen fixation. Our findings thus establish the importance of sulfate and its active transport for the plant–microbe interaction that is most relevant for agriculture and soil fertility.     

Hydrogen metabolism and energy costs of nitrogen fixation

Abstract The high energy costs of biological nitrogen fixation are partly caused by hydrogen production during the reduction of dinitrogen to ammonia. Some nitrogen-fixing organisms can recycle the evolved hydrogen via a membrane-bound uptake hydrogenase. The energetic aspects of hydrogen metabolism and nitrogen fixation are discussed.
Studies on both isolated nitrogenase proteins and nitrogen-fixing chemostat cultures show that energy limitation will result in a high hydrogen production by nitrogenase. In plant- Rhizobium symbiosis, the supply of oxygen or photosynthetate is the limiting factor for nitrogen fixation. In both cases, nitrogen fixation is energy-limited, and it is concluded that a large amount of hydrogen is produced during nitrogen fixation in these symbioses.
Hydrogen reoxidation yields less energy than the oxidation of endogenous substrates, and therefore expression of hydrogenase under oxygen-limited conditions is energetically unfavourable. Moreover, hydrogen reoxidation can never completely regain the energy invested during hydrogen production. The controversial reports of the effect of hydrogen reoxidation on the efficiency of nitrogen fixation are being discussed.
The determination of the energy costs of nitrogen fixation (expressed as the amount of ATP needed to fix 1 mol of N₂) using chemostat cultures is described. Calculations show that the nitrogenase-catalysed hydrogen production has more influence on the efficiency of nitrogen fixation than the absence or presence of a hydrogen uptake system.     

Nitrogen-Fixing Activity in Peat Soils from a Raised Bog

The nitrogenase (acetylene reductase) activity in monolithic and minced peat samples was found to be low, no more than 0.014–0.022 mg N/(kg h). Incorporation of the ¹⁵N₂ isotope into organic compounds of peat soil was 2.71–8.13 mg N/kg over 15 days. The nitrogen-fixing activity was the highest in a 10- to 20-cm layer of soil and much lower in the upper (under green moss) and deeper (20- to 30-cm) layers. The addition of glucose to soil samples stimulated nitrogen fixation considerably after 18–26 h. The maximum nitrogenase activity (3.5–3.8 mg N/(kg h)), observed after 60–70 h, coincided with the peak of respiratory activity. A repeated addition of glucose after its exhaustion increased nitrogenase activity, without a lag period, to 8.5 mg N/(kg h). Investigation of the effect of environmental factors (temperature, pH, aeration, and light intensity) on potential nitrogen-fixing activity in peat samples revealed that nitrogen fixation could proceed in a wide range of pH values (from 3.0 to 7.5) and temperatures (from 5 to 35°C). The nitrogen-fixing bacteria belonging to different trophic groups were enumerated by using nitrogen-free media with pH values and mineralization levels close to those in situ. In samples of peat soil, diazotrophic methanol-utilizing bacteria prevailed (2.0–2.5 × 10⁶ cells/g); the second largest group was facultatively anaerobic bacteria of the family Enterobacteriaceae.     

Nitrogen fixation in eukaryotes – New models for symbiosis

Background  

Nitrogen, a component of many bio-molecules, is essential for growth and development of all organisms. Most nitrogen exists in the atmosphere, and utilisation of this source is important as a means of avoiding nitrogen starvation. However, the ability to fix atmospheric nitrogen via the nitrogenase enzyme complex is restricted to some bacteria. Eukaryotic organisms are only able to obtain fixed nitrogen through their symbiotic interactions with nitrogen-fixing prokaryotes. These symbioses involve a variety of host organisms, including animals, plants, fungi and protists.     

联合固氮菌的合成生物学研究进展

氮素是作物生长过程中最重要的元素,氮素缺乏将会严重影响作物生长。随着人类对粮食的需求量增加,化学氮肥的施用量越来越多。生物固氮在全球氮素循环中有着重要的作用,60%的氮来源于生物固氮。因此,生物固氮,尤其是能够在作物中定殖的联合固氮菌,最有可能代替氮肥成为粮食作物的主要氮源。长期以来,如何提高生物固氮效率以及在作物中实现生物固氮是生物学家的重要研究方向。合成生物学的出现和发展为能够生物固氮的研究带了新的机遇,有望缓解粮食作物对化学氮肥的大量需求。本文概述了固氮菌的种类、联合固氮菌中固氮基因岛的组成以及转录调控机理,阐述了合成生物学在生物固氮领域中的研究现状,对未来的联合固氮菌合成生物学的发展方向作出了展望。     

Induction of nitrogen-fixing nodules on clover requires only 32 kilobase pairs of DNA from the Rhizobium trifolii symbiosis plasmid.

Overlapping subclones from the Rhizobium trifolii symbiosis plasmid pRt843a were generated by using in vivo and in vitro methods. Subclones were assayed for symbiotic phenotype by introducing them into a derivative of R. trifolii ANU843 cured of its symbiosis plasmid and testing the transconjugant strains for the ability to induce nitrogen-fixing nodules on clover. One subclone spanning 32 kilobase pairs (kb) of DNA from pRt843a was found to restore nitrogen fixation ability. This subclone included all known nodulation genes of R. trifolii ANU843 and the nitrogenase structural genes nifHDK. In addition, regions homologous to fixABC, nifA, nifB, nifE, and nifN genes of other nitrogen-fixing bacteria were identified in this 32-kb subclone by DNA-DNA hybridization. Transposon mutagenesis of this subclone confirmed that regions containing these nif and fix genes were required for induction of nitrogen-fixing nodules on clover. In addition, a region located 5 kb downstream of the nifK gene was found to be required for induction of nitrogen-fixing nodules. No homology to known nif and fix genes could be detected in this latter region.     

非共生生物固氮微生物分子生态学研究进展

氮是限制生态系统生产力的主要元素,生物固氮是自然生态系统中氮的主要来源.生物固氮包括共生、联合和自生固氮3种类型,其中联合固氮和自生固氮统称为非共生固氮.相对于共生固氮而言,非共生固氮速率虽然较低,但其不需要与其他生物形成共生体系就可以生存并进行固氮,在时空分布上更加广泛,因此对生态系统氮循环特别是素输入具有重要贡献.本文对近年有关非共生固氮微生物的多样性、土壤和叶际固氮微生物的分布特征及影响因素等研究进展进行了综述,并在此基础上阐述了现有研究中存在的问题和发展前景.