紫云英根瘤菌Ra159的巨大质粒上存在有nod和nif基因的证明

在紫云英根瘤菌(Rhizobium astragali)Ra159中存在有两个分子量分别约为300Md(pRal59a)及大于300Md(pRal59b)的巨大质粒。以肺炎克氏杆菌的固氮酶结构基因nifHDK片段和苜蓿根瘤菌共同结瘤基因nod ABCD片段作探针进行的杂交试验证明了紫云英根瘤菌的大质粒pRal59b上存在有nod基因和nif基因。将这些大质粒转移到nod—nif基因缺失的苜蓿根瘤菌突变株Rm627—1,只有带pRal59b的转移接合子能在紫云英植物上形成根瘤,但这些根瘤均不能还原乙炔。     

在结瘤基因nodA启动子内发现了两个不同功能的结构区域

豌豆根瘤菌(Rhizobium Leguminosarum)结瘤基因nod A的启动子内发现了具有两个不同功能的结构区域：其一我们称为Rip在nodA诱导表达中起着关键作用,可能识别经诱导剂作用而发生构象变化的调控蛋自NodD;另一为Rip缺失后留下的,我们称为Rp区。只要Rp存在,不需要诱导荆,NodD蛋白即能导致结瘤基因nodA的表达。因此该区可能识别原始构象的调控蛋白NodD。     

紫云英根瘤菌基因文库的构建及含完整结瘤基因的重组质粒pRaZ15的分离

以紫云英根瘤菌菌株7653R为材料,制备总DNA,经EcoRⅠ限制酶部分酶解,通过10—50％蔗糖梯度离心,分离到20一30 kb的DNA片段。利用能在革兰氏阴性菌中转移和复制的广谱寄主载体——pLAFRl质粒,构建了紫云英根瘤菌基因文库。通过与苜蓿根瘤菌102l菌株中8．7kb的共同结瘤基因(作探针DNA)杂交,从基因文库中分离到紫云英根瘤菌共同结瘤基因片段。以紫云英根瘤菌不结瘤突变株7653R+1(7653R消除共生质粒)为受体、构建的7653R基因文库(E．Coli C600)为供体,通过协助转移质粒pRK2013(LE392)进行三亲交配,在含四环素的根瘤菌台成培养基(sM)上选择接合转移子。将得到的所有接台转移子混合在一起接种植物,通过植物结瘤试验,分离到含紫云英根瘤菌结瘤基因的重组质粒pRaz15。将该质粒用EcoRⅠ完全酶切,得到25kb左右的外源DNA片段,该片段携带完整的结瘤基因簇。     

紫云英根瘤菌基因组中存在有类似IS因子的DNA重复顺序

在紫云英根瘤菌(Rhizobium astragali)的基因组中存在有DNA重复顺序(RSRa)。它在Ra159的基因组中重复4～5次,其中一个拷贝位于nifH基因的上游。以1.25kbPvul片段作探针,在其他紫云英根瘤菌菌株及豌豆根瘤菌RI PRE中也都检测到与RSRa同源的DNA片段。序列测定的结果表明RSRa其结构类似于IS因子,具有原核插入顺序的一些特点。RSRa全长1468bp,在RSRa的两个末端具有反向重复顺序,RSRa中有一个大的开放阅读框架(ORF)。由ORF推定的蛋白与大肠杆菌插入顺序IS903推定的转座酶有较高的同源性。     

氮源对紫云英根瘤菌nod基因表达的作用

高浓度的硫酸铵阻碍了紫云英根瘤菌诱导紫云英根毛发生典型的根毛变形并明显抑制了紫云英结瘤能力。通过对融合子的β-半乳精苷酶活性的测定进一步表明高浓度的硫酸铵对紫云英的结瘤调节基因nodDZ、共同结瘤基因nodA及nodBC的表达有抑制作用而对结瘤调节基因nodD1的表达无抑制作用。     

费氏中华根瘤菌042BS结瘤调节基因的克隆及功能检测

费氏中华根瘤菌 (Sinorhizobiumfredii) 0 4 2BS可以在大豆和苜蓿上结瘤。用费氏中华根瘤菌USDA2 5 7的nodD1和nodD2基因分别作为探针 ,与 0 4 2BS总DNA进行Southern杂交 ,发现其DNA经EcoRI酶切后分别在 3 0kb和 6 0kb处各有一条阳性带。回收这两条阳性带附近的DNA片段 ,建立部分基因文库 ,克隆到带有nodD1基因的 3 0kb片段 ,以及带有nodD2基因的 6 0kb片段。对nodD1和nodD2进行序列分析 ,结果表明 0 4 2BS的nodD1与费氏中华根瘤菌根瘤菌USDA2 5 7和USDA1 91的同源性高达 99% ,而nodD2与USDA2 5 7的同源性为1 0 0 %。再将nodD1的片段克隆到pBBRIMCS 5载体上 ,导入豌豆根瘤菌蚕豆生物变种 (Rhi zobiumleguminosarumbv.viciae)LPR5 0 5 4中进行功能检测 ,显示 0 4 2BS的nodD1均可被大豆分泌的类黄酮物质染料木黄酮以及苜蓿分泌的类黄酮物质毛地黄黄酮所诱导     

琥珀酸弧菌L-天门冬酰胺酶基因的初级克隆和表达

本文以表达型噬菌体λgtll为载体,以及¹²⁵I标记的放射免疫抗体为探针,从EcDR I酶切的琥珀酸弧菌(Vyibrto succinogenes)染色体DNA片段中克隆得到携带天门冬酰胺酶基因的目的片段,在宿主菌E．Coil Y 1090 中得到表达。经酶解和凝胶电泳分析表明该插入DNA片段的分子量为5．8kb．重组DNA感染另一宿主菌E．ColiYl089后所产生的酶蛋白具有L-天门冬酰胺酶活力。用重组DNA(λgt11-AS8)为探针进行southern DNA杂交,琥珀酸弧菌染色体DNA的Ec0R I酶切片段中,出现一条位置在5．8kb处的杂交带,证明我们克隆到的携带L-天门冬酰胺酶基因的目的片段来自琥珀酸弧菌。     

转座子Tn5诱变对三叶草根瘤菌早期侵染和结瘤的影响

对三叶草根瘤菌Rhizobium trifolii野生型菌株ANU843及其转座子Tn5诱导的突变株nod258和nod261的侵染和结瘤能力进行了比较。Ⅲ医结瘤基因nodFE的突变林(nod258)能在地三叶草(Subterranean clover)上正常侵染和结瘤,但瘤数比ANU843所诱导的略有减少。而Ⅱ区结瘤基因nodIJ的突变株(nod261)却表现侵染力减弱,诱导无效瘤和瘤数大大减少。显微镜强察和超微结构研究表明菌株ANU843在接种后24h侵染已经从植物根毛开始,48h侵染线,(infeetioa thread)发展到表皮细胞并开始分支,接种后72h侵染线进一步发展,深入皮层细胞,这时皮层细胞已经大量分裂增生,形成瘤的分生组织。成熟的瘤细胞内充满了类菌体。而nod261突变株侵染植物比ANU843推迟了24h,侵染线的发展受阻碍,接种后72h侵染线仍然停留在根毛细胞中。Nod26l突变株所诱导的瘤细胞内没有或仅有个别类菌体,是无效瘤。这表明结瘤基因nodIJ与侵染线的正常形成和发展密切相关。     

分离自杭子梢等宿主根瘤的30株菌的结瘤特性的研究

对分离自杭子梢、菜豆和决明等宿主根瘤、处于Agrobacterium系统发育分支、DNA-DNA杂交与A.rubi的相似性达到100%的30株土壤杆菌,分属于Agrobacterium、Bradyrhizobium、Mesorhizobium、Rhizobium和Sinorhizobium 5个属的12个参比菌株。nodA PCR的结果表明,30株供试菌中扩增不出nodA,即没有结瘤性。以Sinorhizobium meliloti USDA1002T的nodA做探针对所提取的细菌总DNA进行斑点杂交,在65℃-68℃严谨洗膜条件下,该探针只能与同种的根瘤菌进行杂交,不能与其它属的根瘤菌或土壤杆菌杂交,初步推测共同结瘤基因nodA探针只能对种内根瘤菌的结瘤性进行鉴定。     

紫云英根瘤菌结瘤基因的定位研究

以根瘤菌nod ABC基因的结构同源性为依据,对紫云英根瘸菌结瘤基因(nod)进行了定位。结果表明,紫云英根瘤菌菌株7653R的nod基因同时存在于小质粒pR37653Ra和大质粒pRa7653Rb上;S52的nod基因位于小质粒pRa52a上;SR72的nod基因单拷贝位于小质粒pRa72a上,含有部分nod ABC基因的BamHI、HiadIII和Pstl酶切片段分别为22.4kb、11．9kb和2．2kb,EcoRl酶切片段为4．6kb和3．0kb;HR104的nod基因也是单拷贝,仅存在于小质粒PRal04a中,含有部分nod ABC基因的 BaraHI、EcoRL和Pstl酶切片段分别为 22．4kb、9．1kb和2．2kb。     

The common nodulation genes of Astragalus sinicus rhizobia are conserved despite chromosomal diversity

The nodulation genes of Mesorhizobium sp. (Astragalus sinicus) strain 7653R were cloned by functional complementation of Sinorhizobium meliloti nod mutants. The common nod genes, nodD, nodA, and nodBC, were identified by heterologous hybridization and sequence analysis. The nodA gene was found to be separated from nodBC by approximately 22 kb and was divergently transcribed. The 2. 0-kb nodDBC region was amplified by PCR from 24 rhizobial strains nodulating A. sinicus, which represented different chromosomal genotypes and geographic origins. No polymorphism was found in the size of PCR products, suggesting that the separation of nodA from nodBC is a common feature of A. sinicus rhizobia. Sequence analysis of the PCR-amplified nodA gene indicated that seven strains representing different 16S and 23S ribosomal DNA genotypes had identical nodA sequences. These data indicate that, whereas microsymbionts of A. sinicus exhibit chromosomal diversity, their nodulation genes are conserved, supporting the hypothesis of horizontal transfer of nod genes among diverse recipient bacteria.     

Nucleotide sequence of Rhizobium meliloti RCR2011 genes involved in host specificity of nodulation.

A 6 kb DNA segment of the R. meliloti 2011 pSym megaplasmid, which contains genes controlling host specificity of root hair infection and of nodulation, was cloned and sequenced. The DNA sequence analysis, in conjunction with previous genetic data, allowed identification of four nod genes designated as E, F, G and H. nodH is divergently transcribed with respect to nodFE and nodG. A conserved nucleotide sequence was found around 200 bp upstream of the translation start of nodF, nodH and nodA. This sequence is also present upstream of common nodA and species specific nodF genes of other Rhizobium species. The predicted protein products of nodF and nodG show homology with acyl carrier protein and ribitol dehydrogenase, respectively. The nodH product contains a rare sequence of four contiguous proline residues. Comparison with the nod gene products of R. leguminosarum shows that species specific nodFE products are as well conserved as those of common nodABC and nodD genes.     

Molecular cloning and expression of Rhizobium fredii USDA 193 nodulation genes: extension of host range for nodulation.

DNA hybridization with the cloned nodulation region of Rhizobium meliloti as a probe revealed DNA homology with four HindIII fragments, 12.5, 6.8, 5.2, and 0.3 kilobases (kb) in size, of the symbiotic plasmid pRjaUSDA193. Both hybridization and complementation studies suggest that the common nodulation genes nodABC and nodD of R. fredii USDA 193 are present on the 5.2-kb HindIII and 2.8-kb EcoRI fragments, respectively, of the Sym plasmid. Both fragments together could confer nodulation ability on soybeans when present in Sym plasmid-cured (Sym-) and wild-type (Sym+) Rhizobium strains or in a Ti plasmid-cured Agrobacterium tumefaciens strain. Furthermore, the 2.8-kb EcoRI fragment alone was able to form nodulelike structures on Glycine max L. cv. "Peking" (soybean). Microscopic examination of these nodules revealed bacterial invasion of the cells, probably via root hair penetration. Bacterial strains harboring plasmids carrying the 5.2- and 2.8-kb nod fragments elicited root-hair-curling responses on infection. These data suggest that the genes responsible for host range determination and some of the early events of nodulation may be coded for by the 5.2-kb HindIII and 2.8-kb EcoRI fragments.     

Identification and structure of the Rhizobium galegae common nodulation genes: evidence for horizontal gene transfer

Rhizobia are soil bacteria able to fix atmospheric nitrogen in symbiosis with leguminous plants. In response to a signal cascade coded by genes of both symbiotic partners, a specific plant organ, the nodule, is formed. Rhizobial nodulation (nod) genes trigger nodule formation through the synthesis of Nod factors, a family of chitolipooligosaccharides that are specifically recognized by the host plant at the first stages of the nodulation process. Here, we present the organization and sequence of the common nod genes from Rhizobium galegae, a symbiotic member of the RHIZOBIACEAE: This species has an intriguing phylogenetic position, being symbiotic among pathogenic agrobacteria, which induce tumors instead of nodules in plant shoots or roots. This apparent incongruence raises special interest in the origin of the symbiotic apparatus of R. galegae. Our analysis of DNA sequence data indicated that the organization of the common nod gene region of R. galegae was similar to that of Sinorhizobium meliloti and Rhizobium leguminosarum, with nodIJ downstream of nodABC and the regulatory nodD gene closely linked to the common nod operon. Moreover, phylogenetic analyses of the nod gene sequences showed a close relationship especially between the common nodA sequences of R. galegae, S. meliloti, and R. leguminosarum biovars viciae and trifolii. This relationship in structure and sequence contrasts with the phylogeny based on 16S rRNA, which groups R. galegae close to agrobacteria and separate from most other rhizobia. The topology of the nodA tree was similar to that of the corresponding host plant tree. Taken together, these observations indicate that lateral nod gene transfer occurred from fast-growing rhizobia toward agrobacteria, after which the symbiotic apparatus evolved under host plant constraint.     

At least two nodD genes are necessary for efficient nodulation of alfalfa by Rhizobium meliloti

A Rhizobium meliloti DNA region (nodD1) involved in the regulation of other early nodulation genes has been delimited by directed Tn5 mutagenesis and its nucleotide sequence has been determined. The sequence data indicate a large open reading frame with opposite polarity to nodA, -B and -C, coding for a protein of 308 (or 311) amino acid residues. Tn5 insertion within the gene caused a delay in nodulation of Medicago sativa from four to seven days. Hybridization of nodD1 to total DNA of Rhizobium meliloti revealed two additional nodD sequences (nodD2 and nodD3) and both were localized on the megaplasmid pRme41b in the vicinity of the other nod genes. Genetic and DNA hybridization data, combined with nucleotide sequencing showed that nodD2 is a functional gene, while requirement of nodD3 for efficient nodulation of M. sativa could not be detected under our experimental conditions. The nodD2 gene product consists of 310 amino acid residues and shares 86.4% homology with the nodD1 protein. Single nodD2 mutants had the same nodulation phenotype as the nodD1 mutants, while a double nodD1-nodD2 mutant exhibited a more severe delay in nodulation. These results indicate that at least two functional copies of the regulatory gene nodD are necessary for the optimal expression of nodulation genes in R. meliloti.     

Rhizobium meliloti nodulation genes: identification of nodDABC gene products, purification of nodA protein, and expression of nodA in Rhizobium meliloti.

A set of conserved, or common, bacterial nodulation (nod) loci is required for host plant infection by Rhizobium meliloti and other Rhizobium species. Four such genes, nodDABC, have been indicated in R. meliloti 1021 by genetic analysis and DNA sequencing. An essential step toward understanding the function of these genes is to characterize their protein products. We used in vitro and maxicell Escherichia coli expression systems, together with gel electrophoresis and autoradiography, to detect proteins encoded by nodDABC. We facilitated expression of genes on these DNA fragments by inserting them downstream of the Salmonella typhimurium trp promoter, both in colE1 and incP plasmid-based vectors. Use of the incP trp promoter plasmid allowed overexpression of a nodABC gene fragment in R. meliloti. We found that nodA encodes a protein of 21 kilodaltons (kDa), and nodB encodes one of 28 kDa; the nodC product appears as two polypeptide bands at 44 and 45 kDa. Expression of the divergently read nodD yields a single polypeptide of 33 kDa. Whether these represent true Rhizobium gene products must be demonstrated by correlating these proteins with genetically defined Rhizobium loci. We purified the 21-kDa putative nodA protein product by gel electrophoresis, selective precipitation, and ion-exchange chromatography and generated antiserum to the purified gene product. This permitted the immunological demonstration that the 21-kDa protein is present in wild-type cells and in nodB- or nodC-defective strains, but is absent from nodA::Tn5 mutants, which confirms that the product expressed in E. coli is identical to that produced by R. meliloti nodA. Using antisera detection, we found that the level of nodA protein is increased by exposure of R. meliloti cells to plant exudate, indicating regulation of the bacterial nod genes by the plant host.     

Reiteration of genes involved in symbiotic nitrogen fixation by fast-growing Rhizobium japonicum.

By using cloned Rhizobium meliloti nodulation (nod) genes and nitrogen fixation (nif) genes, we found that the genes for both nodulation and nitrogen fixation were on a plasmid present in fast-growing Rhizobium japonicum strains. Two EcoRI restriction fragments from a plasmid of fast-growing R. japonicum hybridized with nif structural genes of R. meliloti, and three EcoRI restriction fragments hybridized with the nod clone of R. meliloti. Cross-hybridization between the hybridizing fragments revealed a reiteration of nod and nif DNA sequences in fast-growing R. japonicum. Both nif structural genes D and H were present on 4.2- and 4.9-kilobase EcoRI fragments, whereas nifK was present only on the 4.2-kilobase EcoR2 fragment. These results suggest that the nif gene organizations in fast-growing and in slow-growing R. japonicum strains are different.