茎瘤固氮根瘤菌（Azorhizobium caulinodans）ORS571产生的植物激素

茎瘤固氮根瘤基ＯＲＳ５７１菌株在离体培养条件下，能利用色氨酸合成吲哚乙酸（ＩＡＡ）。随着菌龄的老化，合成的ＩＡＡ量也增加。除ＩＡＡ外，该菌株还产生类ＧＡ物质。本研究未检出细胞分裂素（Ｃｙｔｏｋｉｎｉｎ）类物质。     

茎瘤固氮根瘤菌（Azorhizobium caulinodans）ORS 571产生的植物激素

茎瘤固氮根瘤菌（Ａｚｏｒｈｉｚｏｂｉｕｍｃａｕｌｉｎｏｄａｎｓ）ＯＲＳ５７１菌株在离体培养条件下,能利用色氨酸合成吲哚乙酸（ＩＡＡ）。随着菌龄的老化,合成的ＩＡＡ量也增加。除ＩＡＡ外,该菌株还产生类ＧＡ物质。本研究未检出细胞分裂素（Ｃｙｔｏｋｉｎｉｎ）类物质。     

茎瘤固氮根瘤菌ORS571 GGDEF和EAL结构域蛋白的预测及功能研究

[目的]环二鸟苷酸c-di-GMP是细菌中广泛存在的第二信使,能够调控多种细胞功能。c-di-GMP的合成与水解分别由含有GGDEF结构域和EAL结构域的蛋白催化。本研究针对茎瘤固氮根瘤菌ORS571的GGDEF和EAL结构域相关蛋白进行基因组学分析,并对三个同时含有GGDEF和EAL结构域的蛋白（AZC_3085、AZC_3226和AZC_4658）进行功能研究。[方法]利用SMART数据库对含有GGDEF和EAL结构域的蛋白进行结构域预测。利用CLUSTALW程序对蛋白序列进行比较分析。通过同源重组的方法构建突变株,并对突变株的细胞运动能力、胞外多糖合成、生物膜形成及与豆科宿主的结瘤等表型进行测定。[结果]茎瘤固氮根瘤菌ORS571中一共存在37个GGDEF和EAL结构域蛋白。突变株△4658的运动能力较野生型有下降,但是其胞外多糖合成能力、生物膜形成能力和竞争性结瘤能力较野生型有提高。此外,实验结果表明突变株△4658的胞内c-di-GMP水平高于野生型。突变株△3085和△3226的各种表型与野生型相比没有明显差异。[结论]茎瘤固氮根瘤菌ORS571编码如此大数量的GGDEF和EAL结构域蛋白,表明c-di-GMP可能在其信号转导过程中起到非常重要的作用。同时具有GGDEF和EAL结构域的蛋白AZC_4658对茎瘤固氮根瘤菌ORS571的运动能力、胞外多糖合成、生物膜形成及与宿主的结瘤起到一定的调节作用。     

茎瘤固氮根瘤菌Azorhizobium caulinodans ORS571基因组分泌蛋白的生物信息学分析

为了获取茎瘤固氮根瘤菌(Azorhizobium caulinodans ORS571)的分泌蛋白,以便更深入地了解该菌的共生固氮作用,本研究采用SignalP、TMHMM、PSORTb、TargetP、LipoP、TatP和SecretomeP软件对该菌全部4717个蛋白序列进行分析预测。结果共识别了653个分泌蛋白,其中具有分泌型信号肽的蛋白54个,具有RR-motif型信号肽的蛋白1个,具有脂蛋白信号肽的蛋白2个和非经典分泌蛋白596个。该菌含信号肽分泌蛋白仅占全部蛋白的1.2%,低于其它固氮菌。在分泌蛋白中识别了核酸内切酶和核糖核酸酶等6个核酸酶。它们可能参与宿主植物遗传物质的降解,干扰宿主遗传代谢,进一步在宿主植物侵染过程中起到重要作用。此外还识别了超氧化物歧化酶、过氧化氢酶和谷胱甘肽S-转移酶等4个抗氧化酶。它们可能参与活性氧的清除以保护固氮酶,是该菌固氮过程的重要参与者。     

茎瘤固氮根瘤菌环二鸟苷酸代谢相关基因的功能鉴定

【目的】考察茎瘤固氮根瘤菌ORS571中c-di-GMP合成酶AZC-2412的编码基因缺失的突变表型,初步探究其功能机理。【方法】本实验构建基于cre-loxp重组酶系统的根瘤菌基因敲除系统,以及采用三亲接合技术构建突变株。测定野生型和突变株的生长速率、趋化能力、胞外多糖产量、生物膜形成等表型。【结果】突变株与野生型生长速率几乎相同。与野生型相比突变株由于细胞内c-di-GMP水平降低,胞外多糖、生物膜产量等均有所下降。【结论】实验表明,环二鸟苷酸合成酶AZC-2412缺失,使得c-di-GMP水平降低,对胞外多糖生成、细菌的运动能力、生物膜的形成、细胞絮凝、与植物的互作等均有调控作用。     

茎瘤固氮根瘤菌ORS571中motA基因的功能分析

[目的] MotA是细菌的鞭毛马达蛋白,是跨膜质子通道的重要组成结构之一,在调控鞭毛运动中具有至关重要的作用。本研究探究了Azorhizobium caulinodans ORS571中鞭毛马达基因motA对菌株表型和植物互作的影响。[方法] 通过同源重组原理和三亲接合转移方法构建突变菌株∆motA,测定野生型与突变体在菌体生长、运动、固氮、胞外多糖合成、生物膜形成及根系定殖能力的差异。[结果] 与野生型相比,突变体菌体生长没有明显差异,但其运动能力完全丧失,固氮、胞外多糖合成、生物膜形成及根系定殖能力减弱。[结论] MotA鞭毛马达蛋白对A.caulinodans ORS571的运动、固氮、胞外多糖合成、生物膜形成及根系定殖能力均有调控作用。     

田菁茎瘤固氮根瘤菌对小麦叶组织的促生作用研究

利用GFP标记的田菁茎瘤固氮根瘤菌(Azorhizobium caulinodans ORS 571)侵染露白24h的小麦种子,分别在侵染后0、6、12、24、48、72和96h采样,利用实时荧光定量PCR方法检测小麦体内6条与促生作用相关miRNAs(miR156、miR159、miR160、miR167、miR168和miR403)的表达模式,检测其中3条miRNAs(miR159、miR167和miR168)的靶基因表达模式;以接菌8d的小麦样品做切片,利用激光共聚焦显微镜检测小麦叶部田菁茎瘤固氮根瘤菌的分布,并测定小麦的生理指标。结果显示:(1)田菁茎瘤固氮根瘤菌侵染小麦后能够在叶片边缘部位定殖。(2)小麦叶片中与促生作用相关的6条miRNAs出现了不同程度变化,在12~24h到达其峰值,随后逐渐下降,其中miR159在峰值时的表达量为初始表达量的2.88倍。(3)3条miRNAs的靶基因表达模式与相应miRNA表达模式相对应,但并不严格。(4)生理指标测定结果显示,接种田菁茎瘤固氮根瘤菌对小麦叶片产生明显的促生作用,其中叶鲜重在96h的变化与对照差异极显著。研究表明,接种的田菁茎瘤固氮根瘤菌能够到达小麦叶组织,对小麦叶片的生长产生明显的促生作用,其中miRNAs在促生过程中发挥重要作用。     

茎瘤固氮根瘤菌ORS571鞭毛马达基因fliN与fliM的功能分析

【目的】考察茎瘤固氮根瘤菌ORS571中鞭毛马达蛋白FliN、FliM的编码基因分别缺失的突变体表型,初步探究其功能机理。【方法】本研究采用同源重组和三亲本接合转移的方法构建突变体,测定野生型及突变株的生长曲线、趋化性、胞外多糖的分泌、生物膜的形成及细胞絮凝等表型。【结果】三种菌株的生长速率基本无差,与野生型菌株相比突变株鞭毛结构丧失,趋化能力、分泌的胞外多糖和生物膜形成能力均下降,但相同时间内细胞絮凝程度比野生型明显。【结论】实验表明,鞭毛基因fliN、fliM对茎瘤固氮根瘤菌ORS571鞭毛的形成、趋化运动、胞外多糖的分泌、生物膜的形成及细胞絮凝能力等均有调控作用。     

广东省豆科植物结瘤固氮及根瘤菌资源的初步研究

于2000～2001年在广东省境内54个县（市、区）进行了豆科植物结瘤固氮资源的调查及根瘤菌的采集工作。共采集到豆科植物根瘤样品484份,隶属于37属78种;根瘤的形状以圆形、椭圆形、珊瑚状和姜状居多,大小一般在1～10 mm之间,颜色多为淡红色或黄色。用乙炔还原法对24种93份根瘤样品进行了固氮酶活性测定,结果表明,大多数样品的固氮酶活性在1～10 μmol C₂H₄·g^-1 fresh nodule·h^-1之间。从采集到的根瘤中分离纯化出410株根瘤菌,对其中312株进行了回接试验,回接成功率为93.3 %。调查发现广东省现有栽培豆科作物种类与《广州植物志》的记载相似,所以本研究在豆科栽培作物方面有一定的代表性。在调查采集到根瘤的豆科植物中,三尖叶猪屎豆（Crotalaria micans）、毛排钱草（Phyllodium elegans）、细长柄山蚂蝗（Podorcarpium leptopus）等在本研究之前未见到有结瘤固氮的报道。     

田菁根瘤菌YIC4027可溶性趋化受体Tlp1的功能鉴定

【目的】初步探究田菁根瘤菌Sinorhizobium alkalisoli YIC4027中唯一含有PAS结构域可溶性趋化受体Tlp1的功能机理。【方法】本研究基于Red重组系统以及三亲接合技术进行缺失突变株的构建。对野生型和突变株的生长情况、趋化能力、趋氧性、细胞凝结、生物膜的形成、胞外多糖产量、在宿主根表的定殖及竞争性结瘤等表型进行了测定。【结果】与野生型相比,突变株的生长不受影响,趋化和趋氧能力降低,在宿主根表的定殖及竞争性结瘤能力降低,而细胞凝结能力、生物膜形成以及胞外多糖产生能力等均有所提高【。结论】本研究首次证实了S. alkalisoli YIC4027中可溶性趋化受体Tlp1影响细胞的趋化运动。     

毛萼田菁茎瘤菌被种子浸提物诱导表达基因的筛选及其功能

摘要：【目的】选择毛萼田菁茎瘤菌ORS571为研究对象,筛选毛萼田菁茎瘤菌在菌植互作早期被宿主植物种子浸提物诱导表达的基因。【方法】采用新颖的基于抗性的活体表达筛选技术,以种子浸提物为诱导物,筛选获得能够稳定被种子浸提物诱导的突变株。通过中间片断融合报告基因的方法研究信号分子对相关基因的诱导情况。【结果】随机引物PCR产物测序及网上BLAST结果表明,被诱导表达的基因为LysE家族的成员。而且,对另外3个属根瘤菌中lysE基因的研究发现,它们都可以被相应的宿主植物种子浸提物诱导表达。此外,这四株根瘤菌的lysE突变株都表现对刀豆氨酸极为敏感。本文首次报道了lysE家族在根瘤菌中的生理功能。【结论】我们推测：LysE家族蛋白可能广泛存在于根瘤菌中,并在根瘤菌与宿主植物的早期相互作用中发挥重要作用。     

Responses of Azorhizobium caulinodans to cadmium stress

The symbiosis of Azorhizobium caulinodans and an annul legume Sesbania rostrata was recently found to be tolerant to cadmium pollution by an unknown mechanism. In this study, A. caulinodans ORS571 and ZY-20 showed much stronger tolerance to cadmium than a mutant ORS571-X15 and a common Rhizobium sp., with minimum inhibitory concentration values as high as 4 and 5 mM (versus 1 and 0.1 mM) on yeast extract mannitol agar medium, respectively. Although Cd uptake by all three strains of A. caulinodans were mostly from absorption rather than binding (both loosely or tightly) on cell surface, in resistant strains a higher portion of extractable Cd was bound on the cell surface vs. absorbed (about 1:2.5 ratio) compared to the sensitive mutant (about 1:35.1 ratio). These results suggest that certain level of metal exclusion by a permeability barrier was involved in the mechanism of resistance to Cd by A. caulinodans ORS571 and ZY-20. Over the 12-h period of cultivation in yeast extract mannitol agar medium with Cd addition, the Cd concentrations in the outer membrane and periplasm and spheroplast were the highest at the first 3 h, and declined steadily over time. The fact that Cd concentrations in spheroplast of all three strains were many folds higher than those in outer membrane and periplasm, suggests that extracellular sequestration was not the only mechanism of Cd tolerance in A. caulinodans. The decline of Cd concentrations was significantly faster and started earlier in strains ORS571 and ZY-20 than in ORS571-X15. This suggests a second, probably more substantial, mechanism involves active transport of the metal from the cell, e.g., some efflux system for maintaining homeostasis under cadmium stress.     

Isolation and characterization of hydrogenase-negative mutants of Azorhizobium caulinodans ORS571

Hydrogenase-negative (Hup^-) mutants of Azorhizobium caulinodans ORS571 were isolated by means of Tn5 mutagenesis. The colony test used for screening for Hup^- strains was based on the absence of reduction of triphenyltetrazolium chloride with hydrogen. Suspensions from cultures of the mutant strains grown under derepressing conditions did not use hydrogen with methylene blue or oxygen as the hydrogen acceptor. The mutants were shown to carry single Tn5 insertions at different locations in the A. caulinodans genome. Molar growth yields (corrected for poly--hydroxybutyrate formation) in chemostat cultures of the mutants were similar to those of the wild type. Molar growth yields of the mutants were not increased by passing additional hydrogen through chemostat cultures, which is in agreement with the hydrogenase-negative phenotype of the mutants. H₂/N₂ ratios (mol H₂ formed per mol N₂ fixed) were calculated from the hydrogen content of the effluent gas and the N-content of the bacterial dry weight. Low H₂/N₂ ratios (between 1.2 and 1.9) were found in both energy-limited (oxygen or succinate) cultures and in cultures limited by the supply of an anabolic substrate (Mg²⁺). ATP/2e values (mol ATP used at the transport of 2e to nitrogen or H⁺) were calculated from the H₂/N₂ ratios and the molar growth yields of nitrogen-fixing and ammonia-assimilating cultures. ATP/2e values were between 7 and 11. It was concluded that the calculated ATP/2e values comprise not only 4 mol ATP used at the transport of 2e through nitrogenase but also energy equivalents needed for reversed electron flow from NADH to the low-potential hydrogen donor used by nitrogenase.     

Molecular and functional characterization of the Azorhizobium caulinodans ORS571 hydrogenase gene cluster

In this work, we report the cloning and sequencing of the Azorhizobium caulinodans ORS571 hydrogenase gene cluster. Sequence analysis revealed the presence of 20 open reading frames hupTUVhypFhupSLCDFGHJK hypABhupRhypCDEhupE. The physical and genetic organization of A. caulinodans ORS571 hydrogenase system suggests a close relatedness to that of Rhodobacter capsulatus. In contrast to the latter species, a gene homologous to Rhizobium leguminosarum hupE was identified downstream of the hyp operon. A hupSL mutation drastically reduced the high levels of hydrogenase activity induced by the A. caulinodans ORS571 wild-type strain in symbiosis with Sesbania rostrata plants. However, no significant effects on dry weight and nitrogen content of S. rostrata plants inoculated with the hupSL mutant were observed in plant growth experiments.     

Characterization of the fixABC region of Azorhizobium caulinodans ORS571 and identification of a new nitrogen fixation gene

Summary The fast growing strain, Azorhizobium caulinodans ORS571, isolated from stem nodules of the tropical legume Sesbania rostrata, can grow in the free-living state at the expense of molecular nitrogen. Five point mutants impaired in nitrogen fixation in the free-living state have been complemented by a plasmid containing the cloned fix-ABC region of strain ORS571. Genetic analysis of the mutants showed that one was impaired in fixC, one in fixA and the three others in a new gene, located upstream from fixA and designated nifO. Site-directed Tn5 mutagenesis was performed to obtain Tn5 insertions in fixB and fixC. The four genes are required for nitrogen fixation both in the free-living state and under symbiotic conditions. The nucleotide sequence of nifO was established. The gene is transcribed independently of fixA and does not correspond to fixX, recently identified in Rhizobium meliloti and R. leguminosarum. Biochemical analysis of the five point mutants showed that they synthesized normal amounts of nitrogenase components. It is unlikely that fixA, fixC and nifO are involved in electron transport to nitrogenase. FixC could be required for the formation of a functional nitrogenase component 2.     

The flavonoid naringenin stimulates the intercellular colonization of wheat roots by Azorhizobium caulinodans

We have studied intercellular colonization of wheat roots by Azorhizobium caulinodans and other diazotrophic bacteria, using strains marked with the lacZ reporter gene to facilitate their detection and identification. A. caulinodans was observed by light and electron microscopy to enter the roots of wheat at high frequency at the points of emergence of lateral roots (lateral root cracks). After lateral root crack colonization, bacteria moved into intercellular spaces within the cortical cell layer of roots. The flavonoid naringenin at 10 and 100 mmol m^–3 significantly stimulated root colonization. The roles of the structural nodABC genes and the regulatory nodD gene were also studied; lateral root crack colonization of wheat was shown to be Nod factor- and NodD-independent. Similar frequencies of lateral root crack colonization were observed following inoculation of wheat with Azospirillum brasilense. Colonization by A. brasilense was stimulated by naringenin and also by other flavonoid molecules.