在结瘤基因nodA启动子内发现了两个不同功能的结构区域

在豌豆根瘤菌（ＲｈｉｚｏｂｉｕｍＬｅｇｕｍｉｎｏｓａｒｕｍ）结瘤基因ｎｏｄＡ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白ＮｏｄＤ,另一为ＲＩＰ缺失后留下的,我们称为ＲＰ区,只要ＲＰ存在,不需要诱导剂,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。     

Analysis of three nodD genes in Rhizobium leguminosarum biovar phaseoli; nodD 1 is preceded by nolE, a gene whose product is secreted from the cytoplasm

In a strain of Rhizobium leguminosarum biovar phaseoli, three copies of the regulatory nodulation gene nodD were identified on the Sym plasmid and sequenced. Two were closely linked to each other and the third was near, but not adjacent, to the nodABC genes. Each of these nodD genes could correct the Nod- defect of a nodD mutant strain of R. leguminosarum biovar viciae on peas. A truncated form of nodD2 could also correct this mutant, indicating that the C-terminus of NodD2 is not needed for inducing activity. Upstream of nodD1 and in the same operon is a newly described gene, noIE, whose product appears to be exported into the periplasm. Close to nodD2 is another gene, noIP, with no known counterpart in other rhizobia. Both noIP and noIE-nodD1 are preceded by 'nod-box' sequences and, in the former case, there appear to be two tandemly repeated nod-box sequences. Mutations in each of the nodD genes and in the noIE and noIP genes did not abolish nodulation or nitrogen fixation on beans.     

Entry of Rhizobium leguminosarum bv.viciae into root hairs requires minimal Nod factor specificity, but subsequent infection thread growth requires nodO or nodE

Using various mutant strains of Rhizobium leguminosarum bv. viciae, we have investigated the role of nodO in stimulating infection thread development in vetch and pea. Analysis of R. leguminosarum bv. viciae nodE and nodO mutants revealed no significant difference from the wild-type infection phenotype. Conversely, an R. leguminosarum bv. viciae nodE nodO double mutant was severely impaired in its ability to form normal infection threads. This strain displayed a number of novel infection-related events, including intracellular accumulations of bacteria at the base of root hairs, distended and enlarged infection threads, and reversed threads growing up root hairs. Since normal infection was seen in a nodE mutant, nodO must suppress these abnormal infection phenomena A deletion mutant, retaining only the nodD and nodABCIJ genes, also formed intracellular accumulations at the base of root hairs. Addition of R. leguminosarum bv. viciae nodO could alleviate this phenotype and restore some infection thread formation, although these threads appeared to be abnormal. Exogenous application of R. leguminosarum bv. viciae Nod factors could not alleviate the aberrant infection phenotype. Our results show that the most basic Nod factor structure can allow bacterial entry into the root hair, and that nodO can promote subsequent infection thread development.     

Extension of host range of Rhizobium leguminosarum bv. trifolii caused by point mutations in nodD that result in alterations in regulatory function and recognition of inducer molecules

The positive activation of several nodulation genes in strain ANU843 of Rhizobium leguminosarum biovar trifolii is mediated by the product of the nodD gene and by the interaction of NodD with plant-secreted inducer and anti-inducer compounds. We have mutagenized the nodD gene of strain ANU843 with nitrosoguanidine and have found that the ability of the mutated nodD products to interact with inducer and anti-inducer compounds is affected by the amino acid sequence in at least two key regions, including a novel area between amino acids 77 and 123. Several novel classes of mutants were recognized by phenotypic and molecular analysis of the mutant nodD genes. Classes 1 and 4 mutants were able to induce nodA expression independently of the addition of inducer and anti-inducer compounds and were unable to mediate autoregulation of the nodD gene. Classes 2 and 3 mutants retained several properties of the wild-type nodD, including the ability to interact with inducer and anti-inducer compounds and the capacity to autoregulate nodD expression. In addition, class 2 mutants showed an inducer-independent ability to mediate nodA expression to 10-fold higher levels over control strains. The class 3 mutant showed reactivity to compounds that had little or no inducing ability with the wild-type nodD. An alteration in NodD function was demonstrated with classes 2 and 3 mutants, which showed greatly enhanced ability to complement a Tn5-induced mutation in the nodD1 gene of strain NGR234 and to restore nodulation ability on the tropical legume siratro. Mutants of nodD possessing inducer-independent ability to activate nod gene expression (classes 1, 2, and 4) were capable of extending the host range of R. l. bv. trifolii to the nonlegume Parasponia. DNA sequence analysis showed that single base changes were responsible for the altered phenotypic properties of five of six mutants examined. Four of the six mutations affected amino acid residues in a putative receiver domain in the N-terminal end of the nodD protein.     

Identification of a NodD repressible gene adjacent to nodM in Rhizobium leguminosarum biovar viciae

The nodFEL and nodMNT operons in Rhizobium leguminosarum biovar viciae are transcribed in the same orientation and induced by NodD in response to flavonoids secreted by legumes. In the narrow intergenic region between nodFEL and nodMNT, we identified a small gene divergently transcribed from nodM to the 3' end of nodL. Unlike the promoters upstream of nodF and nodM, the promoter of this gene is constitutively expressed. It appeared that its promoter might partially overlap with that of nodM and its expression was repressed by nodD. A deletion mutation was made and proteins produced by the mutant were compared with those by wild-type using 2D gel electrophoresis. Several protein differences were identified suggesting that this small gene influences the expression or stability of these proteins. However, the mutant nodulated its host plant (pea) normally.     

Induction of microbial genes for pathogenesis and symbiosis by chemicals from root border cells.

Reporter strains of soil-borne bacteria were used to test the hypothesis that chemicals released by root border cells can influence the expression of bacterial genes required for the establishment of plant-microbe associations. Promoters from genes known to be activated by plant factors included virE, required for Agrobacterium tumefaciens pathogenesis, and common nod genes from Rhizobium leguminosarum bv viciae and Rhizobium meliloti, required for nodulation of pea (Pisum sativum) and alfalfa (Medicago sativum), respectively. Also included was phzB, an autoinducible gene encoding the biosynthesis of antibiotics by Pseudomonas aureofaciens. The virE and nod genes were activated to different degrees, depending on the source of border cells, whereas phzB activity remained unaffected. The homologous interaction between R. leguminosarum bv viciae and its host, pea, was examined in detail. Nod gene induction by border cells was dosage dependent and responsive to environmental signals. The highest levels of gene induction by pea (but not alfalfa) border cells occurred at low temperatures, when little or no bacterial growth was detected. Detached border cells cultured in distilled water exhibited increased nod gene induction (ini) in response to signals from R. leguminosarum bv viciae.     

Distribution of O-acetyl groups in the exopolysaccharide synthesized by Rhizobium leguminosarum strains is not determined by the Sym plasmid

The patterns of O-acetylation of the exopolysaccharide (EPS) from the Sym plasmid-cured derivatives of Rhizobium leguminosarum bv. trifolii strain LPR5, R. leguminosarum bv. trifolii strain ANU843 and R. leguminosarum bv. viciae strain 248 were determined by 1H and 13C NMR spectroscopy. Beside a site indicative of the chromosomal background, these strains have one site of O-acetylation in common, namely residue b of the repeating unit. The O-acetyl esterification pattern of EPS of the Sym plasmid-cured derivatives of strains LPR5, ANU843, and 248 was not altered by the introduction of a R. leguminosarum bv. viciae Sym plasmid or a R. leguminosarum bv. trifolii Sym plasmid. The induction of nod gene expression by growth of the bacteria in the presence of Vicia sativa plants or by the presence of the flavonoid naringenin, produced no significant changes in either amount or sites of O-acetyl substitution. Furthermore, no such changes were found in the EPS from a Rhizobium strain in which the nod genes are constitutively expressed. The substitution pattern of the exopolysaccharide from R. leguminosarum is, therefore, determined by the bacterial genome and is not influenced by genes present on the Sym plasmid. This conclusion is inconsistent with the suggestion of Philip-Hollingsworth et al. (Philip-Hollingsworth, S., Hollingsworth, R. I., Dazzo, F. B., Djordjevic, M. A., and Rolfe, B. G. (1989) J. Biol. Chem. 264, 5710-5714) that nod genes of R. leguminosarum bv. trifolii, by influencing the acetylation pattern of EPS, determine the host specificity of nodulation.     

Regulation of Syrm and Nodd3 in Rhizobium Meliloti

The early steps of symbiotic nodule formation by Rhizobium on plants require coordinate expression of several nod gene operons, which is accomplished by the activating protein NodD. Three different NodD proteins are encoded by Sym plasmid genes in Rhizobium meliloti, the alfalfa symbiont. NodD1 and NodD2 activate nod operons when Rhizobium is exposed to host plant inducers. The third, NodD3, is an inducer-independent activator of nod operons. We previously observed that nodD3 carried on a multicopy plasmid required another closely linked gene, syrM, for constitutive nod operon expression. Here, we show that syrM activates expression of the nodD3 gene, and that nodD3 activates expression of syrM. The two genes constitute a self-amplifying positive regulatory circuit in both cultured Rhizobium and cells within the symbiotic nodule. We find little effect of plant inducers on the circuit or on expression of nodD3 carried on pSyma. This regulatory circuit may be important for regulation of nod genes within the developing nodule.