Influence of Glycine spp. on Competitiveness of Bradyrhizobium japonicum and Rhizobium fredii

The displacement of indigenous Bradyrhizobium japonicum in soybean nodules with more effective strains offers the possibility of enhanced N₂ fixation in soybean (Glycine max (L.) Merr.). Our objective was to determine whether the wild soybean (G. soja Sieb. & Zucc.) genotype PI 468397 would cause reduced competitiveness of important indigenous B. japonicum strains USDA 31, 76, and 123 and thereby permit nodulation by Rhizobium fredii, the fast-growing microsymbiont of soybean. In an initial experiment, PI 468397 nodulated and fixed moderate amounts of N₂ with USDA 31 and 76 but, despite the formation of nodules, fixed essentially no N₂ with USDA 123. In contrast, PI 468397 formed a highly effective symbiosis with R. fredii strain USDA 193. In two subsequent experiments, Williams soybean and PI 468397 were grown in a pasteurized soil mixture or in soybean rhizobium-free soil and inoculated with both USDA 123 and USDA 193. In each experiment, more than 90% of the nodules of Williams contained USDA 123, while only a maximum of 2% were occupied with USDA 193. In contrast, in the two experiments, 16 and 11%, respectively, of the nodules produced on PI 468397 were occupied by USDA 123, while in both experiments 87% contained USDA 193. Thus, in relation to the cultivar Williams, which is commonly grown and used as a parent in soybean breeding programs in the United States, PI 468397 substantially reduced the competitive ability of B. japonicum strain USDA 123 in relation to R. fredii strain USDA 193.     

Symbiotically defective histidine auxotrophs of Bradyrhizobium japonicum

Four histidine auxotrophs of Bradyrhizobium japonicum strain USDA 122 were isolated by random transposon Tn5 mutagenesis. These mutants arose from different, single transposition events as shown by the comparison of EcoRI and XhoI-generated Tn5 flanking sequences of genomic DNA. The mutants grew on minimal medium supplemented with l-histidine or l-histidinol but failed to grow with l-histidinol phosphate. While two of the muants were symbiotically defective and did not form nodules on Glycine max cvs. Lee and Peking and on Glycine soja, the other two mutants were symbiotically competent. Reversion to prototrophy occurred at a frequency of about 10^-7 on growth medium without added antibiotics, but prototrophs could not be isolated from growth medium containing 200 g/ml kanamycin and streptomycin. The prototrophic revertants formed nodules on all the soybean cultivars examined. When histidine was supplied to the plant growth medium, both nodulation deficient mutants formed effective symbioses. On histidine unamended plants, nodules were observed infrequently. Three classes of bacterial colonies were isolated from such infrequent nodules: class 1 were kanamycin resistant-auxotrophs; class 2 were kanamycin sensitive-prototrophs; and class 3 were kanamycin-sensitive auxotrophs. Our results suggest that two Tn5 insertion mutations in B. japonicum leading to histidine auxotrophy, affect nodulation in some way. These mutations are in regions that show no homology to the Rhizobium meliloti common nodulation genes.     

Bradyrhizobium japonicum mutants defective in root-nodule bacteroid development and nitrogen fixation

The genome of the slow-growing Bradyrhizobium japonicum (strain 110) was mutagenized with transposon Tn5. A total of 1623 kanamycin/streptomycin resistant derivatives were screened in soybean infection tests for nodulation (Nod) and symbiotic nitrogen fixation (Fix). In this report we describe 14 strains possessing a stable, reproducible Nod⁺Fix^- phenotype. These strains were also grown under microaerobic culture conditions to test them for free-living nitrogen fixation activity (Nif). In addition to strains having reduced Fix and Nif activities, there were also strains that had reduced symbiotic Fix activity but were Nif⁺ ex planta.Analysis of the genomic structure revealed that the majority of the strains had a single Tn5 insertion without any further apparent physical alteration. A few strains had additional insertions (by Tn5 or IS50), or a deletion, or had cointegrated part of the vector used for Tn5 mutagenesis. One of the insertions was found in a known nif gene (nifD) whereas all other mutations seem to affect different, hitherto unknown genes or operons.Several mutant strains had an altered nodulation phenotype, inducing numerous, small, widely distributed nodules. Light and electron microscopy revealed that most of these mutants were defective in different stages of bacteroid development and/or bacteroid persistence. The protein patterns of the mutants were inspected by two-dimensional gel electrophoresis after labelling microaerobic cultures with l-(³⁵S)methionine. Of particular interest were mutants lacking a group of proteins the synthesis of which was known to be under oxygen control. Such strains can be regarded as potential regulatory mutants.     

Transfer and maintenance of IncP and IncW group plasmids into and between extra-slow-growing Bradyrhizobium japonicum strains

Abstract IncP group plasmid pRL180 was conjugally transferred from Agrobacterium tumefaciens LBA928 into extra-slow-growing (ESG) Bradyrhizobium japonicum strains and between ESG strains, RJ17W and RJ12S. pRL180 was integrated into the chromosome of RJ12S, RJ17W and RJ19FY. ESG strains efficiently transferred pRL180 into Escherichia coli at about a 3 × 10⁻⁵ frequency. IncW group plasmid pTY97 was transferred in intergeneric matings from E. coli into ESG strains at a high frequency of 2.5 × 10⁻³; between RJ17W and RJ12S transfer was about 5.6 × 10⁻⁴. pTY97 was maintained as an R' plasmid in RJ12S. The R' plasmid was resolved upon transfer into E. coli C where only pTY97 was autonomously replicated.     

以转座子Tn5作弗氏中华根瘤菌的可识别生态学标记的研究──Tn5的水平转移及其对R．fredii Tn5突变株运动的影响

将一株弗氏中华根瘤菌（Ｒ．ｆｒｅｄｉｉ）ＱＢ１１３０的Ｔｎ５插入突变株ＯＮ－２用于生态学研究,以评估Ｔｎ５在自然环境中的水平转移以及各种水势下Ｔｎ５对突变株ＯＮ－２在土壤中运动的影响．试验表明,在自然潮湿的土壤中,Ｔｎ５本身的水平转移频率很低,且与Ｔｎ５插入相关的突变株卡那霉素抗性表型标记在非选择性平板上连续传４０代后仍然稳定．突变株ＯＮ－２与相对应的野生型菌株ＱＢ１１３０在各种相同水势的土壤中的运动无明显差异（Ｐ＝０．０１）,表明Ｔｎ５的插入不影响突变株的运动．因此,Ｔｎ５可作为研究Ｒ．ｆｒｅｄｉｉ基因工程菌大回应用的一个稳定有效的生态学标记．     

The regulatory status of the fixL- and fixJ-like genes in Bradyrhizobium japonicum may be different from that in Rhizobium meliloti

Summary The cloning, sequencing and mutational analysis of the Bradyrhizobium japonicum symbiotic nitrogen fixation genes fixL and fixJ are reported here. The two genes were adjacent and probably formed an operon, fixLJ. The predicted FixL and FixJ proteins, members of the two-component sensor/regulator family, were homologous over almost their entire lengths to the corresponding Rhizobium meliloti proteins (approx. 50% identity). Downstream of the B. japonicum fixJ gene was found an open reading frame with 138 codons (ORF138) whose product shared 36% homology with the N-terminal part of FixJ. Deletion and insertion mutations within fixL and fixJ led to a loss of approximately 90% wildtype symbiotic nitrogen fixation (Fix) activity, whereas an ORF138 mutant was Fix⁺. In fixL, fixJ and ORF138 mutant backgrounds, the aerobic expression of the fixR-nifA operon was not affected. NifA itself did not regulate the expression of the fixJ gene. Thus, the B. japonicum FixL and FixJ proteins were neither involved in the regulation of aerobic nifA gene expression nor in the anaerobic NifA-dependent autoregulation of the fixRnifA operon; rather they appeared to control symbiotically important genes other than those whose expression was dependent on the NifA protein. The fixL and fixJ mutant strains were unable to grow anaerobically with nitrate as the terminal electron acceptor. Therefore, some of the FixJ-dependent genes in B. japonicum may be concerned with anaerobic respiration.     

Effects of pH and Osmotic Stress on Cellular Polyamine Contents in the Soybean Rhizobia Rhizobium fredii P220 and Bradyrhizobium japonicum A1017

Homospermidine is a polyamine present in its highest concentrations in root nodule bacteria. By using the soybean rhizobia Rhizobium fredii P220 and Bradyrhizobium japonicum A1017, the effects of the pH and osmolarity of the medium on rhizobial growth and cellular polyamine contents were investigated. Elevation of medium pH repressed the growth of slowly growing B. japonicum A1017 and resulted in a slight increase in cellular putrescine, while homospermidine content was not significantly affected. In contrast, in fast-growing R. fredii P220, which showed good growth over a wide range of the medium pHs from 4.0 to 9.5, homospermidine content increased with the lowering of the medium pH. Under the acid-stressed conditions, cellular Mg²⁺ content in strain P220 also increased. Strain P220 was able to grow in NaCl concentrations up to 0.4 M, while strain A1017 did not grow in media containing 0.15 M NaCl. Glutamic acid and K⁺ contents of salt-tolerant P220 cells increased in response to NaCl concentrations, but homospermidine and Mg²⁺ contents were inversely related to the NaCl concentrations. External salinity had no effect on the contents of other polyamines in P220 cells. On the basis of osmotic strength, NaCl, KCl, sucrose, or glycerol induced similar decreases in cellular homospermidine content. These results suggested that the cellular levels of homospermidine in strain P220 may be regulated by mechanisms related to their pH and osmotic tolerance.     

Mutants of Rhizobium japonicum defective in nodulation

Several mutants defective in nodulation were isolated from Rhizobium japonicum strains 3I1b110 and 61A 76. Mutants of class I do not form nodules after incubation with soybean [Glycine max (L.) Merrill] for 17 days, but will do so by 28 days. When host plants other than G. max are infected with several of these strains, there is no detectable difference in the time of nodulation or size of nodules as compared to the wild type. Two mutants of class I (i. e., SM1 and SM2) have been shown previously to be altered in the lipopolysaccharide portion of their cell wall. Mutants of class II are not slow to nodulate but form fewer nodules than the wild type on all the host plants tested. Mutants of class III are unable to form nodules. Some bacteriophage-resistant mutants, altered in cell surface structure, fall into this class. Two mutants of class III do not bind to soybean roots.     

The formation of R-prime deletion mutants and the identification of the symbiotic genes in Rhizobium fredii strain USDA191

Summary R-prime plasmids were formed between the plasmid of Rhizobium fredii strain USDA191 containing nodulation and nitrogen-fixation genes, pRjaUSDA191c, and pRL180, and RP1 derivative. R. fredii USDA191 contains four HindIII fragments that hybridize with an 8.7 kb EcoRI fragment that contains nodulation genes from R. meliloti. These four fragments are on pRjaUSDA191c and are 15.5 kb, 12.5 kb, 6.8 kb, and 5.2 kb in size. A series of R-primes generated in E. coli of pRjaUSDA191c were transferred into a Nod^- Nif^- derivative of strain USDA191 to determine which nodulation region is necessary for nodule formation. Transconjugants containing the 12.5 kb and the 6.8 kb HindIII fragments on segments of pRjaUSDA191c produced nodules on soybean plants. However, transconjugants containing the 12.5 kb HindIII fragment alone were unable to form nodules, suggesting that the 6.8 kb HindIII fragment or the 6.8 kb and the 12.5 kb HindIII fragments together were needed for nodule formation. The 6.8 kb HindIII fragment was subcloned into the vector pVK102 and transferred into transconjugants containing no sequences homologous to R. meliloti nodulation DNA or to transconjugants containing only the 12.5 kb HindIII fragment. Nodules were formed on soybeans only when both the 12.5 kb and the 6.8 kb HindIII fragments were present in R. frediistrain USDA191.     

Isolation and characterization of an R-prime plasmid from Rhizobium meliloti.

Using a simple enrichment procedure, we isolated an R-prime derivative of plasmid R68.45 carrying a 17.8-megadalton segment of the Rhizobium meliloti 41 chromosome. The chromosomal segment carried on this plasmid (pGY1) includes the markers cys-24+, cys-46+, and att16-3. Plasmid pGY1 mobilized the chromosome in a polarized way starting from the region of homology, but cannot promote chromosome transfer from other sites. The att16-3 site on pGY1 allowed the integration of phage 16-3 into pGY1, and a composite plasmid of 91.8 megadaltons was formed. This vector (pGY2) is suitable for the introduction of Rhizobium bacteriophage 16-3 into other gram-negative bacteria.     

Chemotaxis to aromatic and hydroaromatic acids: comparison of Bradyrhizobium japonicum and Rhizobium trifolii

Rhizobia are bacteria well known for their ability to fix nitrogen in symbiosis with leguminous plants. Members of diverse rhizobial species grow at the expense of hydroaromatic and aromatic compounds commonly found in plant cells and plant litter. Using a quantitative capillary assay to measure chemotaxis, we tested the ability of hydroaromatic acids, selected aromatic acids, and their metabolites to serve as chemoattractants for two distantly related rhizobial species, Bradyrhizobium japonicum and Rhizobium trifolii. Slow-growing B. japonicum I-110 demonstrated positive chemotaxis to shikimate, quinate, protocatechuate, and vanillate; threshold concentrations for the compounds were as low as 10(-6) M. The dicarboxylic acids succinate and beta-ketoadipate, metabolites in the catabolism of many aromatic compounds, were positive chemoattractants with low threshold concentrations as well. Taxis to beta-ketoadipate occurred constitutively and, of the tested compounds, beta-ketoadipate gave the strongest peak response. Taxis to shikimate or quinate was induced by growth on either substrate but not by growth on protocatechuate or succinate. In contrast, fast-growing R. trifolii 2066 was only weakly attracted to quinate and other aromatic and dicarboxylic acids that were strong attractants for B. japonicum. The R. trifolii strain exhibited positive chemotaxis to shikimate, but the threshold concentration of shikimate required to elicit a response (10(-4) M) was 2 orders of magnitude higher than that for the B. japonicum strain.     

高温胁迫对根瘤菌Tn5在土壤中的存活及其表型表达的影响

研究了３株弗氏中华根瘤菌（Ｒｈｉｚｏｂｉｕｍｆｒｅｄｉｉ）Ｔｎｓ突变株于适宜温度和高温胁迫两种条件下在土壤中的存活和Ｔｎｓ表型的表达．在适宜温度（２８℃）条件下的灭菌和未灭菌土壤中的存活研究表明生物因素抑制了突变株和野生型的生长．但野生型和突变株的存活种群密度之间无显著差异（Ｐ＝０．０１）．在高温胁迫（４０℃）条件下,土壤中野生型和突变株的种群密度迅速下降,其中部分ＯＮ－２和ＯＮ－３细胞丢失了Ｔｎｓ表型,说明部分细菌的Ｔｎ５表型在高温胁迫条件下不能表达．     

Molecular characterization of a gene region involved in lipopolysaccharide biosynthesis in Bradyrhizobium japonicum: cloning, sequencing and expression of rfaf gene

A 3.0-kb region involved in lipopolysaccharide biosynthesis in Bradyrhizobium japonicum was sequenced. One complete open reading frame was identified which encodes a polypeptide of 354 amino acid residues with a predicted molecular mass of 38 209 Da. Expression of the protein using a T7 gene expression system revealed a band of similar molecular mass after sodium dodecyl sulfate polyacrylamide gel electrophoresis. A database search against known gene sequences revealed a significant sequence similarity to the rfaF gene cloned from several Gram-negative bacteria. The rfaF gene is known to encode heptosyltransferase II that transfers a second heptose to the inner core of lipopolysaccharide. The cloned B. japonicum open reading frame was able to functionally complement a rfaF mutant of Salmonella typhimurium SL3789. Transformation of this mutant with the B. japonicum gene restored production of an intact lipopolysaccharide and resistance to the hydrophobic antibiotic, novobiocin. An additional open reading frame having a significant sequence similarity to the rfaD gene was found to be divergently oriented to the rfaF gene.     

Isolation and characterization of an ndvB locus from Rhizobium fredii

A gene (ndvB) in Rhizobium meliloti that is essential for nodule development in Medicago sativa (alfalfa), specifies synthesis of a large membrane protein. This protein appears to be an intermediate in beta-1,2-glucan synthesis by the microsymbiont. Southern hybridization analysis showed strong homology between an ndvB (chvB) probe and genomic DNA of R. fredii but not from Bradyrhizobium japonicum. A cosmid clone containing the putative ndvB locus was isolated from a Rhizobium fredii gene library. The cosmid clone which complemented R. meliloti ndvB mutants for synthesis of beta-1,2-glucans and effective nodulation of alfalfa was mapped and subcloned. Fragment-specific Tn5 mutagenesis followed by homologous recombination into the R. fredii genome indicated that the region was essential for beta-1,2-glucan synthesis and for formation of an effective symbiosis with Glycine max (soybean).     

土壤中质粒pLV1016 在快生型大豆根瘤菌间的水平转移

在滤膜、液体培养基和土壤微宇宙3种系统中,研究了接合型质粒pLV1016 由快生型大豆根瘤菌(Rhizobiumfredii)QB1131 向R.frediilux Lux3的水平转移及pLV1016 由QB1131 向土著细菌的转移.接合培养1d后,分别计算供、受体菌的生长速率和质粒转移速率常数(γ).结果表明,相同接种浓度下,滤膜接合时γ值最高,土壤中γ值最低,γ值不受土壤是否灭菌和是否有大豆植株的影响,γ值与初始接种浓度负相关,与供、受体的生长速率正相关.在未灭菌土中检测到pLV1016 可转移到土著细菌,土著接合子分别属于根瘤菌属和假单胞菌属.     

Tn5—Mob转座系统对紫云英根瘤菌SR72的诱变和诱动作用

通过接合作用将携带有转座子 Tn5—Mob 的“自杀”性载体质粒 pSUP5011引入紫云英根瘤菌 SR72,得到卡那霉素抗性(Km~r)菌落的频率为6.99×10~(-6),测得受体菌的 Km~r 自发突变频率<10~(-8)。从对1071个 Km~r 突变体进行的植物砂培结瘤试验中筛选出结瘤不固氮(Nod~ ,Fix~-)突变株17个,不结瘤(Nod~-)突变株4个。另外,还从近3000个 Km~r 突变体中选出腺苷营养缺陷型突变株3个。通过 Tn5探针进行的菌落原位杂交试验证明:这21个共生固氮突变株中均含有 Tn5序列,进一步通过接合作用将协助质粒 RP4—4(Tc~r)引入 Nod~ ,Fix~ 突变株,获得了含有 Tn5—Mob 和 RP4—4的新突变株 SR72ZR(Km~r,Tc~r),但试图通过它们的协同作用将SR72中的大质粒诱动转移到根癌农杆菌 A136的试验未获成功.     

Evidence of Horizontal Transfer of Symbiotic Genes from a Bradyrhizobium japonicum Inoculant Strain to Indigenous Diazotrophs Sinorhizobium (Ensifer) fredii and Bradyrhizobium elkanii in a Brazilian Savannah Soil

The importance of horizontal gene transfer (HGT) in the evolution and speciation of bacteria has been emphasized; however, most studies have focused on genes clustered in pathogenesis and very few on symbiosis islands. Both soybean (Glycine max [L.] Merrill) and compatible Bradyrhizobium japonicum and Bradyrhizobium elkanii strains are exotic to Brazil and have been massively introduced in the country since the early 1960s, occupying today about 45% of the cropped land. For the past 10 years, our group has obtained several isolates showing high diversity in morphological, physiological, genetic, and symbiotic properties in relation to the putative parental inoculant strains. In this study, parental strains and putative natural variants isolated from field-grown soybean nodules were genetically characterized in relation to conserved genes (by repetitive extragenic palindromic PCR using REP and BOX A1R primers, PCR-restriction fragment length polymorphism, and sequencing of the 16SrRNA genes), nodulation, and N₂-fixation genes (PCR-RFLP and sequencing of nodY-nodA, nodC, and nifH genes). Both genetic variability due to adaptation to the stressful environmental conditions of the Brazilian Cerrados and HGT events were confirmed. One strain (S 127) was identified as an indigenous B. elkanii strain that acquired a nodC gene from the inoculant B. japonicum. Another one (CPAC 402) was identified as an indigenous Sinorhizobium (Ensifer) fredii strain that received the whole symbiotic island from the B. japonicum inoculant strain and maintained an extra copy of the original nifH gene. The results highlight the strategies that bacteria may commonly use to obtain ecological advantages, such as the acquisition of genes to establish effective symbioses with an exotic host legume.