PII,the key regulator of nitrogen metabolism in the cyanobacteria

PII proteins are a protein family important to signal transduction in bacteria and plants. PII plays a critical role in regulation of carbon and nitrogen metabolism in cyanobacteria. Through conformation change and covalent modification, which are regulated by 2-oxoglutarate, PII interacts with different target proteins in response to changes of cellular energy status and carbon and nitrogen sources in cyanobacteria and regulates cellular metabolism. This article reports recent progress in PII research in cyanobacteria and discusses the mechanism of PII regulation of cellular metabolism.     

Molecular properties of the putative nitrogen sensor PII from Arabidopsis thaliana

Although the signal sensing protein PII is well known to play a central role in bacterial nitrogen metabolism, the structure and function of PII in plants remains only partially understood. Comparative modeling was undertaken based on the high degree of amino acid identity between Escherichia coli and Arabidopsis PII. The mature Arabidopsis PII predicted structure superimposes very well onto the E. coli PII structure (Calpha root mean square deviation < 0.4 A). The model of the highly conserved T-loop suggests a molecular mechanism by which the plant PII may regulate putative post-translational modification in response to metabolite binding. Consistent with the presence of key conserved residues necessary for trimer formation, gel filtration showed the oligomeric structure of Arabidopsis thaliana PII to be a homotrimer. We have demonstrated that Arabidopsis PII binds to the small molecules, ATP, ADP, 2KG, and with lesser affinity to OAA, using isothermal titration calorimetry. We have determined the metabolite dissociation constants and compared these with known physiological concentrations of these metabolites in the plant to identify the Arabidopsis PII effector molecules and their possible roles. We predict that the plant PII is likely continually bound by ATP, and its ligand-bound state only varying with respect to the degree of 2KG binding. Based on our in vitro binding studies, the function of plant PII as a 2KG sensor is suggested.     

钝齿棒杆菌GlnK在氮代谢调控及L-精氨酸合成中的功能

【目的】钝齿棒杆菌是重要的氨基酸生产菌株,本研究针对氮代谢PⅡ信号转导蛋白GlnK展开相关功能研究,分析其在钝齿棒杆菌氮代谢调控及L-精氨酸合成中的作用。【方法】以GlnK蛋白为研究对象,通过基因敲除等遗传方法获得过表达、敲除及敲弱glnK的重组钝齿棒杆菌,研究GlnK对NH_4~+吸收的影响,通过RT-qPCR和酶活测定,从转录水平和蛋白水平上揭示GlnK对氮代谢和L-精氨酸合成相关基因表达水平及酶活的影响,通过5-L发酵罐发酵产L-精氨酸研究GlnK对L-精氨酸合成的影响。【结果】过表达glnK能明显促进NH_4~+的吸收,而敲除glnK后则会抑制NH_4~+的摄取;RT-qPCR和酶活测定发现,相比于野生型菌株Cc5-5,glnK过表达菌株Cc-glnK中与铵吸收相关的基因,表达量平均上调约4.58倍,L-精氨酸合成基因簇中基因的表达水平平均上调1.50倍。Cc-glnK中氮代谢相关蛋白的酶活平均提高46.97%;L-精氨酸合成途径上7个关键酶的酶活平均提高30.00%;5-L发酵罐发酵各重组菌株结果表明,Cc-glnK菌株的产量可达49.53 g/L,产率为0.516 g/(L·h),相比于出发菌株Cc5-5,其L-精氨酸产量提高了28.65%。【结论】过表达GlnK能促进NH_4~+的吸收及利用,并通过影响L-精氨酸合成途径上关键基因的表达水平,提高关键酶的酶活,最终提高L-精氨酸的产量。本研究为后续探索钝齿棒杆菌氮代谢调控机制及代谢工程改造钝齿棒杆菌生产L-精氨酸提供了一种新的策略。     

Crystal structures of the apo and ATP bound Mycobacterium tuberculosis nitrogen regulatory PII protein

PII constitutes a family of signal transduction proteins that act as nitrogen sensors in microorganisms and plants. Mycobacterium tuberculosis (Mtb) has a single homologue of PII whose precise role has as yet not been explored. We have solved the crystal structures of the Mtb PII protein in its apo and ATP bound forms to 1.4 and 2.4 Å resolutions, respectively. The protein forms a trimeric assembly in the crystal lattice and folds similarly to the other PII family proteins. The Mtb PII:ATP binary complex structure reveals three ATP molecules per trimer, each bound between the base of the T‐loop of one subunit and the C‐loop of the neighboring subunit. In contrast to the apo structure, at least one subunit of the binary complex structure contains a completely ordered T‐loop indicating that ATP binding plays a role in orienting this loop region towards target proteins like the ammonium transporter, AmtB. Arg38 of the T‐loop makes direct contact with the γ‐phosphate of the ATP molecule replacing the Mg²⁺ position seen in the Methanococcus jannaschii GlnK1 structure. The C‐loop of a neighboring subunit encloses the other side of the ATP molecule, placing the GlnK specific C‐terminal 3₁₀ helix in the vicinity. Homology modeling studies with the E. coli GlnK:AmtB complex reveal that Mtb PII could form a complex similar to the complex in E. coli. The structural conservation and operon organization suggests that the Mtb PII gene encodes for a GlnK protein and might play a key role in the nitrogen regulatory pathway.     

Biochemical pathways and mechanisms nitrogen, amino acid, and carbon metabolism

For both nitrogen and carbon metabolism there exist specific regulatory mechanisms to enable cells to assimilate a wide variety of nitrogen and carbon sources. Superimposed are regulatory circuits, the so called nitrogen and carbon catabolite regulation, to allow for selective use of “rich” sources first and “poor” sources later. Evidence points to the importance of specific regulatory mechanisms for short term adaptations, while generalized control circuits are used for long term modulation of nitrogen and carbon metabolism. Similarly a variety of regulatory mechanisms operate in amino acid metabolism. Modulation of enzyme activity and modulation of enzyme levels are the outstanding regulatory mechanisms. In prokaryotes, attenuation and repressor/operator control are predominant, besides a so called “metabolic control” which integrates amino acid metabolism into the overall nutritional status of the cells. In eukaryotic cells compartmentation of amino acid metabolites as well as of part of the pathways becomes an additional regulatory factor; pathway specific controls seem to be rare, but a complex regulatory network, the “general control of amino acid biosynthesis”, coordinates the synthesis of enzymes of a number of amino acid biosynthetic pathways.     

植物的光合作用与光合氮、碳代谢的耦联及调节

概述了光合作用反应与CO2同化和NO^-3/NO^-2还原的耦联关系,提出了应该从氮,碳代谢整合角度讨论作动和光合作用,以便根据生产目的,调节作物的氮,碳代谢,实现农业生产的高产,优质。     

施氮时期对高产夏玉米氮代谢关键酶活性及抗氧化特性的影响

选用玉米品种登海661和郑单958为材料,研究了高产条件下施氮时期对夏玉米产量、氮素利用率、氮代谢相关酶及抗氧化酶活性的影响.结果表明:拔节期一次性施氮不利于夏玉米产量提高和氮素积累,分次施氮且增施花粒肥显著提高了植株和籽粒的吸氮量,并提高了籽粒产量.拔节期、10叶期、花后10d按2∶4∶4施氮,登海661产量最高可达14123.0kg· hm-2;基肥、拔节期、10叶期、花后10 d按1∶2∶5∶2施氮,郑单958产量最高可达14517.1 kg· hm-2,这2种施氮方式较拔节期一次性施氮分别增产14.5％和17.5％.花前分次施氮可以显著提高开花期硝酸还原酶活性;登海661和郑单958在花后0～42 d中,施氮处理的谷氨酰胺合成酶、谷氨酸合成酶、谷氨酸脱氢酶活性分别平均提高了32.6％、47.1％、50.4％和14.5％、61.8％、25.6％,减缓了其下降趋势;超氧化物歧化酶、过氧化氢酶活性提高了22.0％、36.6％和13.4％、62.0％,丙二醛含量显著降低.在高产条件下,分次施氮且适当增加花粒肥施入比例可以提高氮代谢相关酶活性,延缓植株衰老,促进氮素吸收利用,进而提高籽粒产量.     

好氧反硝化细菌碳氮代谢特点及途径的研究进展

好氧反硝化作用的发现打破了反硝化只能在严格厌氧条件下进行的传统认知,为生物脱氮提供了一条新的途径,已成为近些年的研究热点。碳源可为好氧反硝化过程提供能量和电子供体,其代谢难易程度直接影响着好氧反硝化细菌的脱氮效率,因此有必要明确碳源在好氧反硝化脱氮过程中的代谢机理。基于此,本文阐述了好氧反硝化细菌的种类及其对硝态氮与亚硝态氮的代谢途径;系统分析了不同好氧反硝化细菌对碳氮源代谢的差异与代谢机理;综合分析了碳代谢差异对好氧反硝化脱氮过程的影响,并对未来的研究方向进行了展望,旨在深入理解好氧反硝化细菌同时去除碳氮的机理,为提高废水生物脱氮除碳效率提供理论依据。     

Micronutrients and nitrogen metabolism

A sand-culture experiment was conducted to study the influence of a deficiency of and an excess of micronutrients on the uptake and assimilation of NH ₄ ⁺ and NO ₃ ⁻ ions by maize. By studying the fate of¹⁵N supplied as¹⁵NH₄NO₃ or NH₄ ¹⁵NO₃, it was demonstrated that in maize plants NH₄−N was absorbed in preference to NO ₃ ⁻ −N. The uptake and distribution of N originating from both NH ₄ ⁺ and NO ₃ ⁻ was considerably modified by deficiency of, or an excess of, micronutrients in the growth medium. The translocation of NH ₄ ⁺ −N from roots to shoots was relatively less than that of NO ₃ ⁻ −N. Deficiency as well as excessive amounts of micronutrients, in the growth medium, substantially reduced the translocation of absorbed N into protein. This effect was more pronounced in the case of N supplied as NO ₃ ⁻ . Amino-N was the predominant non-protein fraction in which N from both NH ₄ ⁺ and NO ₃ ⁻ tended to accumulate. The next important non-protein fractions were NO ₃ ⁻ −N when N was supplied as NO ₃ ⁻ and amide-N when NH ₄ ⁺ was the source. The relative accumulation of¹⁵N into different protein fractions was also a function of imposed micronutrient levels.     

耕作方式及施氮量对砂姜黑土区小麦氮代谢及籽粒产量和蛋白质含量的影响

为明确砂姜黑土区小麦(Triticum aestivum)产量和品质形成的耕作方式及施氮量最优组合, 在大田试验条件下, 以深松、旋耕和常规耕作3种耕作方式为主区, 0、120、225、330 kg·hm^-2 4个施氮量为副区, 研究了不同耕作方式及施氮量组合对小麦拔节后氮代谢、籽粒产量和蛋白质含量的影响。结果表明, 随着生育期的推进, 叶片谷氨酰胺合成酶活性、游离氨基酸含量和可溶性蛋白含量均呈先升后降的趋势, 深松方式配合中高氮处理的峰值在花后10天, 而常规耕作和旋耕的4个施氮处理以及深松的低氮处理峰值多在开花期。与常规耕作和旋耕相比, 深松耕作显著降低了10-40 cm的土壤容重, 提高了土壤总空隙度和根干质量, 有利于中后期根系氮素吸收。耕作方式和施氮量对籽粒产量和蛋白质含量影响显著, 均以深松方式最高。3种耕作方式下小麦产量和蛋白质含量均随施氮量增加而增加, 籽粒产量以深松方式配合330 kg·hm^-2施氮量最高, 而常规耕作和旋耕方式的产量在施氮量为225 kg·hm^-2时达到最大。3种耕作方式下籽粒蛋白质含量均以施氮225 kg·hm^-2最高。因此, 在砂姜黑土区宜采用深松耕作方式配合适宜的施氮量, 以改善土壤条件, 促进根系氮素吸收, 延长叶片功能期, 达到产量与蛋白品质提升之目的。     

Sodium transport in filamentous nitrogen fixing cyanobacteria

Two filamentous, nitrogen fixing cyanobacteria were examined for their salt tolerance and sodium (Na⁺) transport.Anabaena torulosa, a saline form, grew efficiently and fixed nitrogen even at 150 mM salt (NaCl) concentration while,Anabaena L-31, a fresh water cyanobacterium, failed to grow beyond 35 mM NaCl.Anabaena torulosa showed a rapidly saturating kinetics of Na⁺ transport with a high affinity for Na⁺ (K _m, 0.3 mM).Anabaena L-31 had a much lower affinity for Na⁺ (K_m, 2.8 mM) thanAnabaena torulosa and the pattern of uptake was somewhat different. BothAnabaena spp. exhibited an active Na⁺ extrusion which seems to be mediated by a Na⁺-K⁺ ATPase and aided by oxidative phosphorylation.Anabaena L-31 was found to retain much more intracellular Na⁺ thanAnabaena torulosa. The results suggest that the saline form tolerates high Na⁺ concentrations by curtailing its influx and also by an efficient Na⁺ extrusion, although these alone may not entirely account for its success in saline environment.     

Appearance and establishment of diazotrophic cyanobacteria in Lake Kinneret,Israel

1. We propose that the appearance and establishment of Nostocales (cyanobacteria) species of the genera Aphanizomenon and Cylindrospermopsis in the warm subtropical Lake Kinneret (Sea of Galilee, Israel) from 1994 was linked to changes in climate conditions and summer nitrogen (N) availability. 2. From 1994 to 2009, an increase in frequency of events of elevated water temperature (>29 °C) in summer, and to some extent a greater frequency of lower summer wind speed events, affected water turbulence and water column stratification, thus providing better physical conditions for the establishment of these populations. 3. In recent years, N‐depleted conditions in Lake Kinneret in early summer have promoted the development and domination of Nostocales that could gain an ecological advantage owing to their N₂‐fixing capability. 4. Nitrogen fixation rates coincided both with heterocyst abundance and with Nostocales biomass. The N supplied to the lake via nitrogen fixation ranged from negligible quantities when Nostocales represented only a minor component of the phytoplankton community to 123 tonnes when Cylindrospermopsis bloomed in 2005. This high N₂ fixation rate equals the average summer dissolved inorganic nitrogen load to the lake via the Jordan River.     

外源葡萄糖对山定子生长及根系氮素代谢的影响

以有机质含量仅为0.65%的低碳冲积沙土为栽培基质,以当年生山定子幼苗为试材,分别添加与土壤本体微生物生物量碳(MBC)等量的碳量(2 g·kg^-1)、5倍MBC碳量(10g·kg^-1)的葡萄糖,以不添加葡萄糖为对照,处理后0~30 d内定期采集根系样品,研究外源葡萄糖对低碳土壤中山定子幼苗生长、根系构型及氮素代谢的影响.结果表明:5倍MBC碳源处理后山定子幼苗的株高、总生物量、总根长和根表面积分别显著增加12.3%、26.4%、23.2%和14.6%,而茎粗、根体积和平均直径无显著变化.等量及5倍MBC碳源处理均显著提高了山定子的根系活力,分别在第3和15天达到峰值,高于对照119.1%和75.7%.在整个处理期间,等量及5倍MBC碳源处理显著增加了根中NO_3^-、NO_2^-和NH_4^+含量;整体上,等量及5倍MBC碳源处理均显著增强根系中硝酸还原酶、谷氨酰胺合酶、谷氨酸脱氢酶、谷氨酸合酶、谷草转氨酶和谷丙转氨酶的活性,其中5倍MBC处理的作用最显著.5倍MBC的外源葡萄糖浓度更有利于促进低碳土壤中山定子根系中氮素的吸收代谢过程,诱导植株生长、干物质积累和根系构型改变.     

Effects of elevated ozone and nitrogen addition on leaf nitrogen metabolism in poplar

臭氧和氮添加对杨树叶片氮代谢的影响臭氧(O₃)污染和氮(N)沉降/施肥都能同时影响植物的生长。然而,几乎没有研究探究O₃和N添加对植物叶片N代谢过程的复合影响。本研究在开顶式气室(OTC)中对杨树进行了为期95 d的熏蒸实验,包括两个O₃水平(NF,环境O₃水平;NF60,NF + 60 ppb O₃)和4个N处理(N0,没有N添加;N50,N0 + 50 kg N ha⁻¹ yr⁻¹;N100,N0 + 100 kg N ha⁻¹ yr⁻¹;N200,N0 + 200 kg N ha⁻¹ yr⁻¹)。测定了与叶片N代谢相关的一些指标,包括叶片N代谢酶的活性、总叶片N浓度、NO₃⁻-N浓度、NH₄⁺-N浓度、总氨基酸浓度(TAA)、总可溶性糖的浓度(TSP)。研究结果表明,相对于NF,在8月份NF60处理显著刺激了硝酸还原酶(NR)的活性,使其升高了47.2%。当平均所有的N处理和两次取样时间时,NF60处理下谷氨酰胺酶(GS)的活性比NF处理下的高57.3%。但是O₃处理并没有显著影响TSP浓度,并且在8月也没有降低TAA的浓度。相对N0,高的N添加处理(N200)显著增加了杨树叶片的饱和光合速率(A_sat) 24%,并且分 别在8和9月增加了总叶片N浓度70.3%和43.3%。但是在8月份,N200处理下光合N利用效率比N0的低26.1%。这表明N添加导致的A_sat和叶片总的N浓度的升高是不匹配的,高N处理下,叶片中一些剩余的N没有被用于优化植物碳的同化。同时,也发现高N添加显著刺激了叶片N代谢过程,叶片中的NO₃⁻-N浓度、NH₄⁺-N浓度、TAA浓度、NR和GS活性都显著升高。然而,O₃和N添加对杨树叶片所有N代谢相关的指标都没有交互影响。这些结果将有助于更好地了解在高O₃污染和N沉降/施肥下植物的N代谢过程以及生物地球化学循环过程。     

The effects of different light–dark cycles on the metabolism of the diazotrophic,unicellular cyanobacteria Cyanothece sp. ATCC 51142, and Cyanothecesp. PCC 7822

The diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142 demonstrates circadian patterns in nitrogenase activity, H₂ production and glycogen storage when grown under nitrogen‐fixing, 12:12 light:dark (L:D) conditions. In this study, we grew Cyanothece sp. ATCC 51142, and another strain in this genus, Cyanothece sp. PCC 7822, under long‐day (16:8 L:D) and short‐day (8:16 L:D) nitrogen‐fixing conditions to determine if they continued to display circadian rhythms. Both strains demonstrated similar circadian patterns for all three metabolic parameters when grown under long‐day conditions. However, the strains responded differently to short‐day growth conditions. Cyanothece sp. ATCC 51142 retained reasonable circadian patterns under 8:16 L:D conditions, whereas Cyanothece sp. PCC 7822 had quite damped patterns without a clear circadian pattern. In particular, glycogen storage changed very little throughout the day and we ascribe this to the difference in the type of glycogen granules in Cyanothece sp. PCC 7822 which has small β‐granules, compared to the large, starch‐like granules in Cyanothece sp. ATCC 51142. The results suggested that both mechanistic and regulatory processes play a role in establishing the basis for these metabolic oscillations.