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。     

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。     

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。     

Isolation and characterization of Tn5-induced NADPH-glutamate synthase (GOGAT-) mutants of Azorhizobium sesbaniae ORS571 and cloning of the corresponding glt locus

Summary Random Tn5 mutagenesis was used to isolate two independent Azorhizobium sesbaniae ORS571 mutants disturbed in ammonium assimilation (Asm^-). Both Asm^- mutant strains were shown to lack NADPH-glutamate synthase (NADPH-GOGAT) activity and to carry Tn5 insertions ca. 1.5 kb apart in the ORS571 chromosome. The Tn5-containing region of one of the GOGAT^- mutant strains was cloned in pACYC184 and used to identify the wild-type glt (GOGAT) locus in a phage clone bank of ORS571. The cloned region was shown to have DNA homology with the Escherichia coli glt locus and to complement the Asm^- phenotype of E. coli and ORS571 GOGAT^- strains. The ORS571 GOGAT^- mutations were found to interfere with free-living as well as symbiotic nitrogen fixation. Expression of ORS571 NADPH-GOGAT activity was shown to be independent of the nitrogen regulation (ntr) system.     

Comparative genomic and protein sequence analyses of the chemotaxis system of Azorhizobium caulinodans北大核心CSCD

［Objective］ Azorhizobium caulinodans ORS571 can fix nitrogen not only as a free-living organism and an associative-symbiotic bacterium by colonizing the root surface of non-leguminous plants, but also as a symbiotic bacterium by interacting with leguminous plant Sesbania rostrata.Due to its ability to grow and fix nitrogen under three conditions, A.caulinodans uses sophisticated chemotaxis signal transduction systems to transform environmental cues into corresponding behavioral responses.Chemotaxis appears crucial for the growth of A.caulinodansin complicated environment and the construction of associative relationship with the plant.However, little is known about the chemotactic pathway of A.caulinodans.Thus, our study aimed to compare the chemotaxis-like genes of A.caulinodans with those of well-studied species.［Methods］ NCBI protein BLAST was used for searching sequence similarity with default parameter values against the genomes of A.caulinodans.HMMER3, based on Pfam database, was used for comparative analyses of methyl-accepting chemotaxis protein （MCP）.［Results］ There was a major chemotaxis cluster in A.caulinodans and the CheR methylated MCPs independently of pentapeptide motif.There were 43 MCP homologs containing diverse signal-sensing architectures in A.caulinodans.In addition,cytoplasmic domains of these MCPs were all composed of 38 heptad repeats.［Conclusion］ Despite the extremely high homology presented between the chemotactic system of A.caulinodans and those of well-studied species, A.caulinodans shows its own unique characteristics.The classification of these chemotactic pathways by comparative genomics enables us to better understand how A.caulinodansresponds to changes in environment via exquisite signal transductions in chemotaxis system.     

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.     

Cloning and characterization of nifA and ntrC genes of the stem nodulating bacterium ORS571, the nitrogen fixing symbiont of Sesbania rostrata: Regulation of nitrogen fixation (nif) genes in the free living versus symbiotic state

Summary A cosmid bank of ORS571, a diazotrophic bacterium capable of inducing aerial stem and root nodules on Sesbania rostrata, was constructed in the vector pLAFR1. A DNA probe carrying the Klebsiella pneumoniae nifA gene was used to identify nifA-and ntrC-like regions of ORS571 in the cosmid bank by colony hybridization. Cosmids carrying these regions were mapped by restriction endonuclease analysis, Southern blotting and transposon Tn5 mutagenesis. Selected Tn5 insertion mutations in the nifA/ntrC homologous regions were used for gene-replacement experiments and the resulting ORS571 mutants were examined for Nif, Fix and Ntr phenotypes. Two clearly distinct regulatory loci were thus identified and named nifA and ntrC. Plasmids carrying gene fusions of the ORS571 nifH and nifD genes to lacZ were constructed and the regulation of the ORS571 nifHDK promoter, and of the Rhizobium meliloti nifHDK promoter, was studied under varying physiological conditions in ORS571, ORS571 nifA::Tn5 and ORS571 nitrC::Tn5 strains. A model for the role of nifA and ntrC in the regulation of ORS571 nif and other nitrogen assimilation genes is proposed.     

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.     

Common nodABC genes in Nod locus 1 of Azorhizobium caulinodans: Nucleotide sequence and plant-inducible expression

Summary Azorhizobium caulinodans strain ORS571 induces nitrogen-fixing nodules on roots and stem-located root primordia of Sesbania rostrata. Two essential Nod loci have been previously identified in the bacterial genome, one of which (Nod locus 1) shows weak homology with the common nodC gene of Rhizobium mehloti. Here we present the nucleotide sequence of this region and show that it contains three contiguous open reading frames (ORFA, ORFB and ORFC) that are related to the nodABC genes of Rhizobium and Bradyrhizobium species. ORFC is followed by a fourth (ORF4) and probably a fifth (ORF5) open reading frame. ORF4 may be analogous to the nod[ gene of R. leguminosarum, whereas ORF5 could be similar to the rhizobial nodF genes. Coordinated expression of this set of five genes seems likely from the sequence organization. There is no typical nod promoter consensus sequence (nod box) in the region upstream of the first gene (ORFA) and there is no nodD-like gene. LacZ fusions constructed with ORFA, ORFB, ORFC, and ORF4 showed inducible -galactosidase expression in the presence of S. rostrata seedlings as well as around stem-located root primordia. Among a series of phenolic compounds tested, the flavanone naringenin was the most efficient inducer of the expression of this ORS571 nod gene cluster.     

Nucleotide sequence of the fixABC region of Azorhizobium caulinodans ORS571: similarity of the fixB product with eukaryotic flavoproteins, characterization of fixX, and identification of nifW.

Summary The nucleotide sequence of a 4.1 kb DNA fragment containing the fixABC region of Azorhizobium caulinodans was established. The three gene products were very similar to the corresponding polypeptides of Rhizobium meliloti. The C-terminal domains of both fixB products displayed a high degree of similarity with the -subunits of rat and human electron transfer flavoproteins, suggesting a role for the FixB protein in a redox reaction. Two open reading frames (ORF) were found downstream of fixC. The first ORF was identified as fixX on the basis of sequence homology with fixX from several Rhizobium and Bradyrhizobium strains. The second ORF potentially encoded a 69 amino acid product and was found to be homologous to a DNA region in the Rhodobacter capsulatus nif cluster I. Insertion mutagenesis of the A. caulinodans fixX gene conferred a Nif^– phenotype to bacteria grown in the free-living state and a Fix^– phenotype in symbiotic association with the host plant Sesbania rostrata. A crude extract from the fixX mutant had no nitrogenase activity. Furthermore, data presented in this paper also indicate that the previously identified nifO gene located upstream of fixA was probably a homologue of the nifW gene of Klebsiella pneumoniae and Azotobacter vinelandii.     

Use of oligonucleotide probes to identify members of two-component regulatory systems in Xanthomonas campestris pathovar campestris

Summary Two-component regulatory systems comprising a sensor and a regulator protein, both with highly conserved amino acid domains, and commonly genetically linked, have been described in a range of bacterial species and are involved in sensing environmental stimuli. We used two oligonucleotide probes matching the postulated coding regions for domains of sensor and regulator proteins respectively in Xanthomonas campestris pathovar campestris (Xcc) to identify possible two-component regulatory systems in Xcc. Two different fragments of Xcc DNA with homology to both of these probes were cloned. The DNA sequence of part of one of these fragments encompassed a potential open reading frame (ORF), the predicted amino acid sequence of which had extensive homology with regulator proteins of two-component regulatory systems. Analysis of the predicted amino acid sequence for the 3 end of an adjacent ORF revealed a very high level of homology with the C-terminal end of sensor proteins. Strains of Xcc with Tn5-induced mutations in the regulator gene were affected in extracellular polysaccharide production, and also in resistance to salt and chloramphenicol. No effects of mutation in the second clone were observed.     

A translocation activating the cryptic nitrogen regulation gene areB inactivates a previously unidentified gene involved in glycerol utilisation in Aspergillus nidulans

Summary The chromosome VIII translocation breakpoint of the areB-404 translocation, selected for its ability to activate the cryptic nitrogen metabolism regulatory gene areB, and the mutation glcD-100 both lead to loss of mitochondrial FAD-dependent sn-glycerol-3-phosphate dehydrogenase in Aspergillus nidulans. These two lesions therefore define glcD, a second gene (in addition to glcB) where mutation can result in loss of this enzyme. The glcD gene has been localised to a centromere-proximal region of the right arm of chromosome VIII. Although all six known areB-activating mutations involve chromosomal rearrangements and presumably therefore gene fusions, areB-404 is the first such rearrangement where the gene involved in an areB fusion has been identified.     

Hydrogen oxidation and efficiency of nitrogen fixation in succinate-limited chemostat cultures ofRhizobium ORS 571

Rhizobium ORS 571, isolated from stem nodules of the tropical legumeSesbania rostrata is able to grow in the chemostat with molecular nitrogen as sole nitrogen source at a specific growth rate of 0.1 h^-1. Samples from nitrogenfixing cultures showed high acetylene reduction activities: 1,500 nmol ethylene formed per milligram dry weight per hour. Under nitrogen-fixing conditions an uptake hydrogenase is induced. Ammonia-assimilating cultures, without additional hydrogen, did not induce hydrogenase. The addition of hydrogen to succinate-limited nitrogen-fixing cultures resulted in an increase in the molar growth yield on succinate (Y _succinate) from 27 to 35 and a slight decrease in the molar growth yield on oxygen ( ), showing that hydrogen oxidation is less energy-yielding than the oxidation of endogenous substrates. Respiration-driven proton translocation measured with starved cells indicated the functioning of site 1 and 2 of oxidative phosphorylation. Cytochrome spectra showed that cytochromea ₆₀₀, present at high dissolved oxygen tension (d.o.t.) almost completely disappeared at low d.o.t. In flash-photolysis spectra only thea-type cytochrome could be detected as an oxidase in cells both grown at high and low d.o.t. Growth yields in ammonia-assimilating cultures were higher than those measured in nitrogen-fixing cultures. Assuming two sites of oxidative phosphorylation, a molar growth yield on ATP (Y _ATP) of about 3 and 6 was calculated for respecticely nitrogen-fixing and ammonia-assimilating cultures. TheY _ATP under nitrogen-fixing conditions is dependent on the amount of H₂ formed per mol N₂ fixed (H₂/N₂ ratio). A method has been described to calculate the total amount of ATP use by nitrogenase during the fixation of 1 mol N₂ (ATP/N₂ ratio) and H₂/N₂ ratios in aerobic nitrogen fixing organisms. This calculation yielded that nitrogen fixation inRhizobium ORS 571 is a high ATP-consuming process. The calculated ATP/N₂ and H₂/N₂ ratios were respectively 42 and 7.5.Abbreviations d.o.t. dissolved oxygen tension A preliminary account of this work was presented at the 5th International Symposium on Nitrogen Fixation, September 1983, Noordwijkerhout, The Netherlands