导入dctABD和nifA基因对费氏中华根瘤菌共生固氮的影响研究

以pTR102为载体构建重组质粒pHN307,其上克隆有来自昔蓿中华根瘤菌（Sinorthizobium meliloti）的四碳二羧酸转移酶基因dctABD、来自肺炎克氏杆菌(Klebsiella pneumoniae）的nifA基因和来自pDB30所含的发光酶基因lux-AB。经三亲本接合转移,将pHN307导入费氏中华根瘤菌（S.fredii)NH01、YC4和GR3,并考察了转移接合子中pHN307在传代培养和共生条件下的稳定性。与出发菌相比较的植物盆栽试验结果表明,在与大豆黑农33共生时,导入pHN307后的转移接合子均可显著提高结瘤植株的瘤重、地上部分干重和地上部分总氮量。在与大豆川早一号共生时,转移接合子HN01（pHN307）可显著提高结瘤植株的瘤数和瘤重;GR3（pHN307）可显著提高结瘤植株的瘤数、瘤重、地上部分干重和地上部分总氮量;导入pHN307的YC4却呈现出负作用。本研究表明,导入dctABD可提高固氮效率     

Functional specialization of one copy of glutamine phosphoribosyl pyrophosphate amidotransferase in ureide production from symbiotically fixed nitrogen in Phaseolus vulgaris

Purines are essential molecules formed in a highly regulated pathway in all organisms. In tropical legumes, the nitrogen fixed in the nodules is used to generate ureides through the oxidation of de novo synthesized purines. Glutamine phosphoribosyl pyrophosphate amidotransferase (PRAT) catalyses the first committed step of de novo purine synthesis. In Phaseolus vulgaris there are three genes coding for PRAT. The three full‐length sequences, which are intron‐less genes, were cloned, and their expression levels were determined under conditions that affect the synthesis of purines. One of the three genes, PvPRAT3, is highly expressed in nodules and protein amount and enzymatic activity in these tissues correlate with nitrogen fixation activity. Inhibition of PvPRAT3 gene expression by RNAi‐silencing and subsequent metabolomic analysis of the transformed roots shows that PvPRAT3 is essential for the synthesis of ureides in P. vulgaris nodules.     

Thiamine limitation determines the transition from aerobic to fermentative-like metabolism in Rhizobium etli CE3

Both thiamine and biotin when added to minimal medium subcultures reversed the fermentative-like metabolism exhibited by Rhizobium etli CE3. Thiamine auxotrophs lacking thiCOGE genes were used to investigate the role of thiamine in this medium. A thiC1169∷ miniTn 5lacZ1 thiamine auxotroph subjected to the above subcultures resulted in growth arrest, reduced pyruvate-dehydrogenase activity, and a smaller amount of poly-β-hydroxybutyrate compared with the CE3 strain. Moreover, thiC and thiEb genes were overexpressed as result of thiamine limitation. The absence of classical thi genes suggests that thiamine is synthesized with low efficiency by an alternative pathway. Low levels of thiamine cause the CE3 strain to exhibit a fermentative-like metabolism.     

Control of autogenous activation of Herbaspirillum seropedicae nifA promoter by the IHF protein

Analysis of the expression of the Herbaspirillum seropedicae nifA promoter in Escherichia coli and Herbaspirillum seropedicae, showed that nifA expression is primarily dependent on NtrC but also required NifA for maximal expression under nitrogen-fixing conditions. Deletion of the IHF (integration host factor)-binding site produced a promoter with two-fold higher activity than the native promoter in the H. seropedicae wild-type strain but not in a nifA strain, indicating that IHF controls NifA auto-activation. IHF is apparently required to prevent overexpression of the NifA protein via auto-activation under nitrogen-fixing conditions in H. seropedicae.     

Time course of N2 fixation in common bean (Phaseolus vulgaris L.)

Field and greenhouse experiments were conducted to assess the nitrogen fixation rates of four cultivars of common bean (Phaseolus vulgaris L.) at different growth stages. The ¹⁵N isotope dilution technique was used to quantify biological nitrogen fixation. In the greenhouse, cultivars M4403 and Kallmet accumulated 301 and 189 mg N plant^–1, respectively, up to 63 days after planting (DAP) of which 57 and 43% was derived from atmosphere. Under field conditions, cultivars Bayocel and Flor de Mayo RMC accumulated in 77 DAP, 147 and 135 kg N ha^–1, respectively, of which approximately one-half was derived from the atmosphere. The rates of N₂ fixation determined at different growth stages increased as the plants developed, and reached a maximum during the reproductive stage both under field and greenhouse conditions. Differences in translocation of N were observed between the cultivars tested, particularly under field conditions. Thus, the fixed N harvest index was 93 and 60 for cultivars Flor de Mayo and Bayocel, respectively. In early stages of growth, the total content of ureides in the plants correlated with the N fixation rates. The findings reported in the present paper can be used to build a strategy for enhancing biological N₂ fixation in common bean.     

Characterization of the promoter of the Rhizobium etli recA gene.

The promoter of the Rhizobium etli recA gene has been identified by primer extension and by making deletions affecting several regions located upstream of its coding region. A gel mobility shift assay carried out with crude extracts of cells of R. etli has been used to show that a DNA-protein complex is formed in the R. etli recA promoter region in vitro. Analysis of the minimal region of the recA promoter giving rise to this DNA-protein complex revealed the presence of an imperfect palindrome corresponding to the sequence TTGN11CAA. Site-directed mutation of both halves of this palindrome indicated that both motifs, TTG and CAA, are necessary for both normal DNA-protein complex formation in vitro and full DNA damage-mediated inducibility of the recA gene in vivo. However, the TTG motif seems to be more dispensable than the CAA one. The presence of this same palindrome upstream of the recA genes of Rhizobium meliloti and Agrobacterium tumefaciens, whose expression is also regulated in R. etli cells, suggests that this TTGN11CAA sequence may be the SOS box of at least these three members of the Rhizobiaceae.     

厚荚相思树根瘤菌HJ06菌株的16S rDNA全序列和nifA基因片段分析

对厚荚相思(Acaciacrassicarpa)根瘤菌HJ06菌株的16SrDNA全序列和nifA基因片段进行了测定。结果表明,HJ06菌株在以16SrDNA序列构建的系统发育树状图中位于根瘤菌属(Rhizobium)分支中,与根瘤菌属各个种的相似性达95%以上;从HJ06菌株克隆出的585bpnifA基因片段与Klebsiellapneumoniae的同源性达到99.3%,与Klebsiellaoxytoca的NifF,NifL,NifA,NifB蛋白基因的同源性为97.8%。     

Inactivation of the nodH gene in Sinorhizobium sp. BR816 enhances symbiosis with Phaseolus vulgaris L

Sulfate modification on Rhizobium Nod factor signaling molecules is not a prerequisite for successful symbiosis with the common bean (Phaseolus vulgaris L.). However, many bean-nodulating rhizobia, including the broad host strain Sinorhizobium sp. BR816, produce sulfated Nod factors. Here, we show that the nodH gene, encoding a sulfotransferase, is responsible for the transfer of sulfate to the Nod factor backbone in Sinorhizobium sp. BR816, as was shown for other rhizobia. Interestingly, inactivation of nodH enables inoculated bean plants to fix significantly more nitrogen under different experimental setups. Our studies show that nodH in the wild-type strain is still expressed during the later stages of symbiosis. This is the first report on enhanced nitrogen fixation by blocking Nod factor sulfation.     

Rhizobium etli taxonomy revised with novel genomic data and analyses

The taxonomic position of Phaseolus vulgaris rhizobial strains with available sequenced genomes was examined. Phylogenetic analyses with concatenated conserved genomic fragments accounting for over half of each genome showed that Rhizobium strains CIAT 652, Ch24-10 (newly reported genome) and CNPAF 512 constituted a well-supported group independent from Rhizobium etli CFN 42(T). DNA-DNA hybridization results indicated that CIAT 652, Ch24-10 and CNPAF 512 could correspond to R. etli, although the hybridization values were at the borderline that distinguishes different species. In contrast, experimental hybridization results were higher (over 80%) with Rhizobium phaseoli type strain ATCC 14482(T) in congruence to phylogenetic and ANIm analyses. The latter criterion allowed the reclassification of R. etli strains 8C-3 and Brasil5 as R. phaseoli. It was therefore concluded, based on all the evidence, that the CIAT 652, Ch24-10, and CNPAF 512 strains should be reclassified as R. phaseoli in spite of several common features linking them to R. etli. The R. phaseoli and R. etli speciation process seems to be a more recent event than the speciation that has occurred among other sister species, such as R. leguminosarum-R. etli or R. rhizogenes-R. tropici.     

Cultivar and Rhizobium strain effects on the symbiotic performance of pea (Pisum sativum)

The symbiotic performance of four pea ( Pisum sativum L.) cultivars in combination with each of four strains of Rhizobium leguminosarum was studied in growth chamber experiments in order to estimate the effects of cultivars, strains and cultivar × strain interaction on the variation in dry weight, N content and dry weight/N ratio. At harvest 63 days after planting, cultivars accounted for 75% of the variation in dry weight, while the Rhizobium strains accounted for 63% of the variation in N-content and 70% of the variation in dry weight/N ratio. Cultivar × strain interactions were statistically significant, but of minor quantitative importance, accounting for 5–15% of the total variation. Rhizobium strains also influenced the partitioning of N between reproductive and vegetative plant parts and between root and shoot biomass.     

Heterologous hybridization of Azospirillum DNA to Rhizobium nod and fix genes

Abstract Probes containing the nod and hsn regions of Rhizobium meliloti and the fixABC genes of Rhizobium japonicum were used to perform hybridization experiments with endonuclease-restricted DNA from Azospirillum brasilense strains and 2 Azospirillum lipoferum strains. Homology to nod, hsn and fixA was found in the 4 Azospirillum strains.     

Lipopolysaccharide core components of Rhizobium etli reacting with a panel of monoclonal antibodies

Monoclonal antibodies that react with Rhizobium leguminosarum lipopolysaccharide core antigens (LPS-2) have been used to investigate LPS-2 structure in Rhizobium etli. The panel of antibodies (JIM 32 - JIM 35, JIM 37, JIM 38) specific for LPS-2 of R. leguminosarum strain 3841 and its core components displays similar reactivities towards isolated LPS-2 from R. etli CE109 (a mutant of wild-type strain R. etli CE3 that displays LPS-2 as its main LPS form on the cell surface). This result suggests the antibodies bind to similar epitopes on both strains and, hence, that R. leguminosarum and R. etli have very similar LPS core and lipid A antigen structures. More detailed analysis of the antibody binding sites with isolated LPS-2 and lipid A from R. etli suggests that some of the antibodies (JIM 32, 33, 34, and MASM-I) bind some part of the core oligosaccharides, while others (JIM 35 and JIM 38) involve lipid A. These antibodies have already proven useful in the biochemical analysis of the LPS antigen forms. For example, the loss of reactivity of certain LPS forms with antibody JIM 37 has led to the discovery of a hitherto unnoticed form of the LPS antigen in a precipitate formed during the phenol/water extraction procedure. This new form reacts with the JIM 37 antibody. Furthermore, the positive reaction of some of the antibodies with only sonicated wild-type R. etli cells suggests that either an effective way of masking the display of core antigens on whole bacterial cells is occurring or that core forms of the LPSs are never displayed on the surface of the bacterial cells. Either possibility, once confirmed, could be important for our picture of the Rhizobium cell surface and could also have some bearing on symbiotic nodule infection and development.Abbreviations LPS lipopolysaccharide