转座子Tn916对固氮螺菌Azospirillum brasilense的接合诱变及氨分泌菌株的筛选

以携带质粒pAM120(Ap~r,Tc~r/Tn916)的大肠杆菌(E.coli CG120)为供体菌株,与受体菌巴西固氮螺菌(Azospirillum brasilense)采用滤膜接合法进行接合转移,在选择平板上得到具较高频率的接合子(10~(-5)/每个供体菌,选择四环素抗性)。从846株四环素抗性接合子中进一步用奈氏法筛选得到氨分泌突变株3株。在无氮培养基上,其氨分泌量可达7.5～14.0mmol/L。用乙炔还原法分析氨分泌突变株在不同浓度氮源上的固氮活力,发现20mmol/L NH Cl的存在不抑制其固氮活性,固氮活力与无氮条件下野生株的活力相差不多。无选择压力下细胞分裂50代后的稳定性实验证明,转座子Tn916在氨分泌突变株中的稳定性在50％～80％之间。以固氮螺菌氨分泌突变株为供体菌株,对E.coli HBX1进行反向接合转移实验,证实Tn916确实存在于氨分泌固氮螺菌接合子中。     

转座子Tn916对固氮螺菌Azospirillum brasilense的接合诱变及氨分 …

以携带质粒ｐＡＭ１２０（Ａｐ＾ｒ,Ｔｃ＾ｒ／Ｔｎ９１６）的大肠杆菌（Ｅ．ｃｏｌｉ ＣＧ１２０）为供体菌株,与受体菌巴西固氮螺菌采用膜接合法进行接合转移,在选择平板上得到具较高频率的接合子（１０＾－５／每个供体菌,选择四环素抗性）。从８４６株四环素抗性接合子中进一步用奈氏法筛选得到氨分泌突变株３株。在无氮培养基上,其氨分泌量可达７．５￣１４．０ｍｍｏｌ／Ｌ。用乙炔还原法分析氨分泌突变株在不同浓度氮源     

巴西固氮螺菌的周质蛋白与宿主相关性的研究

本文报道用一种微量的方法研究从小麦、玉米、水稻、谷子等作物根系分离到的30株巴西固氮螺菌的周质蛋白。结果表明:尽管经形态、生理生化等多项指标鉴定确认它们同属于Azospirillum brasilcnse (巴西固氮螺菌),但是来源于不同植物根表的菌株间的周质蛋白扫描图谱有明显的差异,而来自同一种作物根表的菌株间的周质蛋白图谱却很相似。从而表明,巴西固氮螺菌具有宿主特异性。     

糖蜜草(Melinis minutiflora Beauv.)内生固氮菌分离鉴定

糖蜜草（Melinis minutiflora Beauv．）是热带地区的一种优良牧草。采用选择性培养基在厌氧和好氧两种培养条件下,从糖蜜草根、茎中都可分离得到具有较强固氮酶活性的菌株。通过SDS-PAGE全细胞蛋白电泳技术快速聚类分析表明,来源于糖蜜草中的菌株为同一类群。16S rDNA序列分析和总DNA的G＋c％含量进一步确定糖蜜草中所分离的菌株属于固氮螺菌属（Azospirillum）,与产脂固氮螺菌（Azospirillum lipoferum）亲缘关系较近。BIOLOG板测定结果显示,糖蜜草菌株TMCY243对多种碳源具有很强的适应性,与产脂固氮螺菌（A．lipoferum）的模式菌株DSM 1691存在着较大的差异。以上结果表明,糖蜜草内生固氮菌为固氮螺菌属的一个新类群。     

巴西固氮螺菌的周质蛋白与宿主相关性的研究*

本文报道用一种微量的方法研究从小麦、玉米、水稻、谷子等作物根系分离到的30株巴西固氮螺菌的周质蛋白。结果表明:尽管经形态、生理生化等多项指标鉴定确认它们同属于Azospirillum brasilcnse (巴西固氮螺菌),但是来源于不同植物根表的菌株间的周质蛋白扫描图谱有明显的差异,而来自同一种作物根表的菌株间的周质蛋白图谱却很相似。从而表明,巴西固氮螺菌具有宿主特异性。     

巴西固氮螺菌Yu62 nifA基因克隆、测序及功能分析

用TD－PCR法克隆了巴西固氮螺菌（Azospirillun brasilense)Yu62和nifA基因。序列分析表明它与巴西固氮螺菌Sp7的nifA序列高度同源（96．5％）,其编码的产物NifA蛋白与Sp7菌株NifA的氨基酸序列同源性为97．6％。该基因可以完全互补巴西固氮螺菌Sp7 nifA^-突变株的Nif^-表型。研究了NH4^ 和O2对Yu62 nifA基因的表达及NifA活性的影响。结果表明：nifA基因在Yu62菌株中是部分组成型表达的,氨和氧不能完全阻遏其表达,在5mmol/LNH4Cl4与微氧（0.5%O2)条件下表达最高;NifA蛋白在0.4%-0.5%O2时活性最高,氧分压降低和提高都使NifA活性下降,1mmol/L NH4Cl足以抑制NifA的活性。     

巴西固氮螺菌中吸氢酶基因同源性的分子检测

通过TTC(2 ,3,5 氯化三苯基氮唑 )实验筛选出 12株能产生吸氢酶蛋白 (Hup+ )的巴西固氮螺菌 (Azospir rilumbrasilense)菌株。用Qiagen柱分离提纯含豌豆根瘤菌的hup基因片段的质粒 pHVT10 9和pHVT115 ,并用地高辛标记法标记pHVT115 ,与 12株Hup+ 巴西固氮螺菌的总DNA进行斑点杂交 ,结果显示pHVT115所含的吸氢酶基因 (hup基因 )片段与巴西固氮螺菌的大部分菌株的hup基因同源性不强。这一结果表明hup基因在固氮生物中存在着遗传多样性 ,异源hup基因探针不一定都适宜于探测hup基因。     

固氮螺菌与植物的相互关系研究进展

固氮螺菌与植物的相互关系研究进展阎大来,何路红,李季伦（北京农业大学农业生物技术国家重点实验室,北京１０００９４）固氮螺菌（Ａｚｏｓｐｉｒｉｌｌｕｍ）首先是从巴西热带禾本科牧草俯仰马唐（Ｄｉｇｉｔａｒｉａｄｅｃｕｍｂｅｎｓ）根际分离得到的￣［１,２］...     

巴西固氮螺菌Yu62nifA基因克隆、测序及功能分析

用TD-PCR法克隆了巴西固氮螺菌(Azospirillun brasilense)Yu62的nifA基因.序列分析表明它与巴西固氮螺菌Sp7的nifA序列高度同源(96.5%),其编码的产物NifA蛋白与Sp7菌株NifA的氨基酸序列同源性为97.6%.该基因可以完全互补巴西固氮螺菌Sp7 nifA-突变株的Nif-表型.研究了NH4+和O2对Yu62 nifA基因的表达及NifA活性的影响.结果表明mfA基因在Yu62菌株中是部分组成型表达的,氨和氧不能完全阻遏其表达,在5mmol/LNH4Cl与微氧(0.5%O2)条件下表达最高;NifA蛋白在0.4%～0.5%O2时活性最高,氧分压降低和提高都使NifA活性下降,1mmol/L NH4Cl足以抑制NifA的活性.     

固氮螺菌氢代谢与固氮作用的关系I.固氮螺菌放氢现象与吸氢能力的研究

本文研究了固氮螺菌(Azosptrillum brastlense)的放氢现象和吸氢酶活性以及与固氮作用的关系。测定了57株固氮螺菌的放氢现象及其固氮酶活性,其中不放氢41株,微放氢14株,其放氢量为2·63—31.00n mol C2H4／ml菌液·小时。放氢量较多的2株R38{和R256A都是从水稻根表上分离获得,其放氢量分别为185.75n mol H2／ml 菌液·小时和547．00 moIH2／ml 菌液·小时。测定了53株螺菌的吸氢酶活性,它们均具有吸氢能力,其吸氢量各异,0-63-27·38n mol H2／ml菌液。小时。生长在含有NH4CI培养基上的固氮螺菌既没有固氮能力,也不产氢。在无氮培养基上所产生的氢是固氮过程中放出的氢。实验结果指出,C2H2抑制氢酶的活性。当吸氢的菌株与放氢菌株混合培养时,其固氮酶活性比单株纯培养高,有氢存在时,固氮酶活性比不加氢时高。     

Organic acid utilization,succinate excretion,encystation and oscillating nitrogenase activity inAzospirillum brasilense under microaerobic conditions

Oscillating nitrogenase activity in long lasting batch cultures ofAzospirillum brasilense ATCC 29145 is independent of the carbon source malate. With fumarate, succinate or pyruvate as sole carbon source nitrogenase activity is also oscillating. Cultivation in a medium with 20-fold the buffer concentration also results in oscillating nitrogenase activity. Nitrogen-fixing cultures ofAzospirillum brasilense ATCC 29145 excrete ammonia into the culture medium varying between 0.02 and 0.04 mM concentrations. This is not sufficient to cause a drop of nitrogenase activity inAzospirillum brasilense after the first maximum. During growth under nitrogen-fixing conditions with malate as carbon source, the cells excrete significant quantities of succinate into the culture medium. Cultures with only 0.05% malate reutilized the excreted succinate as soon as malate disappeared from the medium. Azospirillum brasilense ATCC 29145 is shown to have the capability of encystation. Encysted cells are different from vegetative cells in their resistance to desiccation, by the spherical shape and by immotility. The results indicate that oscillating nitrogenase activity in long lasting cultures reflects the development from vegetative cells to cysts and again to vegetative cells under microaerobic conditions.     

Molecular cloning and sequence analysis of an Azospirilium brasilense indole-3-pyruvate decarboxylase gene

Molecular Genetics and Genomics - Azospirillum brasilense isolated from the rhizosphere of different plants has the ability to excrete indole-3-acetic acid (IAA) into the culture media. Cosmid...     

Effects of heavy metals on plant-associated rhizobacteria: comparison of endophytic and non-endophytic strains of Azospirillum brasilense.

The plant-associated nitrogen-fixing rhizobacterium Azospirillum brasilense attracts world-wide attention owing to its plant growth-promoting activities. Among hundreds of its strains known up to date, wild-type strain Sp245 has been proved to be capable of colonising both the plant-root interior and exterior (i.e. a facultative endophyte), whereas others are non-endophytes colonising the root surface only. Thus, the different ecological niches occupied by these strains in the rhizosphere suggest that their responses to environmental conditions might differ as well. In this study, responses of A. brasilense strains Sp245 and Sp7 to several heavy metals (Co2+, Cu2+, Zn2+), present in the medium at tolerable concentrations (up to 0.2 mmol/l) and taken up by the bacteria, were compared. Fourier transform infrared (FTIR) spectroscopy was used for controlling the compositional features of whole cells. The results obtained show that in strain Sp7 (non-endophyte) the heavy metals induced an enhanced accumulation of polyester compounds (poly-3-hydroxybutyrate; PHB). In contrast, the response of the endophytic strain Sp245 to heavy metal uptake was found to be much less pronounced. These dissimilarities in their behaviour may be caused by different adaptation abilities of these strains to stress conditions owing to their different ecological status. It was also found that adding 0.2 mmol/l Cu2+ or Cd2+ in the culture medium resulted in noticeably reducing the levels of indole-3-acetic acid (IAA, auxin) produced by both the strains of the bacterium. This can directly affect the efficiency of associative plant-bacterial symbioses involving A. brasilense in heavy-metal-contaminated soil.     

Phytostimulatory effect of Azospirillum brasilense wild type and mutant strains altered in IAA production on wheat

Auxin production by Azospirillum is believed to play a major role in the observed plant growth promoting effect. By using different genetically modified strains, the contribution of auxin biosynthesis by A. brasilense in altering root morphology was evaluated in a plate assay. Inoculation with the wild type strains A. brasilense Sp245 and Sp7 resulted in a strong decrease in root length and increase in root hair formation. This effect was abolished when inoculating with an ipdC mutant of A. brasilense. The ipdC gene encodes a key enzyme in the IPyA pathway of IAA synthesis by A. brasilense. On the other hand, the observed auxin effect was further enhanced by adding tryptophan, a precursor of IAA, to the plates and could be mimicked by replacing the Azospirillum cells by a particular concentration of IAA. Furthermore, particular mutants (rpoN, scrp) and transconjugants (extra copy of ipdC) of A. brasilense were tested in the plate assay. Together, these results confirm the important role of IAA produced by Azospirillum in altering root morphology and illustrate the power of combining genetic tools and bioassays to elucidate the mechanism of a beneficial Azospirillum-plant interaction. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plasmid localization and mapping of two Azospirillum brasilense loci that affect exopolysaccharide synthesis

Two Azospirillum brasilense loci that correct Rhizobium meliloti exoB and exoC mutants for exopolysaccharide (EPS) synthesis have been identified previously (K. W. Michiels, J. Vanderleyden, A. P. Van Gool, E. R. Signer, J. Bacteriol., 1988b). A. brasilense exo mutants produce EPS of lower molecular weight than the wild type strain. Here, we show by hybridization that these exo loci are located on a 90-MDa plasmid in A. brasilense Sp7. In four other Azospirillum strains but not in A. lipoferum SpBr17, the loci are likewise located on a plasmid of approximately the same size. Transposon Tn5 insertions in these loci were isolated and mapped on the cloned DNA by restriction analysis. Hybridization of restriction digests of purified 90-MDa plasmid DNA with probes containing the exo loci confirmed their plasmid location. This is the first report on plasmid localization of genes in Azospirillum.     

Strain-specific salt tolerance and osmoregulatory mechanisms in Azospirillum brasilense

Salinity stress inhibits the growth and nitrogen fixation ability of the plant growth-promoting rhizobacterium Azospirillum brasilense. Five strains of A. brasilense were isolated from the rhizosphere of Indian cereals and grasses and identified on the basis of their phenotypic features and 16S rRNA gene sequence. The five Indian isolates and two standard strains of A. brasilense, Sp7 and Cd, showed notable differences in growth, acetylene-reducing activity under salt stress, and ability to take up and use glycine betaine for the restoration of growth and acetylene-reducing activity under salt stress. Salt stress also enhanced the production of exopolysaccharides and cell aggregates, the extent of which varied in different strains of A. brasilense at different carbon to nitrogen ratios in the culture medium. It can be concluded that the production of exopolysaccharides and cell aggregates is a more consistent physiological response of A. brasilense to salt stress than is the uptake and osmoprotection by glycine betaine.     

Growth and indole-3-acetic acid biosynthesis of Azospirillum brasilense Sp245 is environmentally controlled

Batch and fed batch cultures of Azospirillum brasilense Sp245 were conducted in a bioreactor. Growth response, IAA biosynthesis and the expression of the ipdC gene were monitored in relation to the environmental conditions (temperature, availability of a carbon source and aeration). A. brasilense can grow and produce IAA in batch cultures between 20 and 38 degrees C in a standard minimal medium (MMAB) containing 2.5 gl(-1)l-malate and 50 microgml(-1) tryptophan. IAA synthesis requires depletion of the carbon source from the growth medium in batch culture, causing growth arrest. No significant amount of IAA can be detected in a fed batch culture. Varying the concentration of tryptophan in batch experiments has an effect on both growth and IAA synthesis. Finally we confirmed that aerobic growth inhibits IAA synthesis. The obtained profile for IAA synthesis coincides with the expression of the indole-3-pyruvate decarboxylase gene (ipdC), encoding a key enzyme in the IAA biosynthesis of A. brasilense.     

Antigenic identity of the capsule lipopolysaccharides,exopolysaccharides, and O-specific polysaccharides in Azospirillum brasilense

The antigenic identity (and close values of electrophoretic mobility) of capsular polysaccharides, exopolysaccharides, and O-specific polysaccharides was revealed in the Azospirillum brasilense strains Sp7 and Sp245 by the immunodiffusion and immunoelectrophoretic methods. Together with the literature data on the identity of the monosaccharides composition of these polymers, this gives evidence of the absence of a specific capsular antigen in the bacteria studied. Thus, extracellular Azospirillum brasilense polysaccharides are likely to represent O-antigenic lipopolysaccharide fragments excreted by the bacteria into the culture medium, and their identification as a capsule or as an exopolysaccharide depends on the strength of the attachment of these polysaccharides to the cell surface.     

Wheat root colonization and nitrogenase activity by Azospirillum isolates from crop plants in Korea

Nitrogen-fixing bacteria were isolated from the rhizosphere of different crops of Korea. A total of 16 isolates were selected and characterized. Thirteen of the isolates produced characteristics similar to those of the reference strains of Azospirillum, and the remaining 3 isolates were found to be Enterobacter spp. The isolates could be categorized into 3 groups based on their ARDRA patterns, and the first 2 groups comprised Azospirillum brasilense and Azospirillum lipoferum. The acetylene reduction activity (ARA) of these isolates was determined for free cultures and in association with wheat roots. There was no correlation between pure culture and plant-associated nitrogenase activity of the different strains. The isolates that showed higher nitrogenase activities in association with wheat roots in each group were selected and sequenced. Isolates of Azospirillum brasilense CW301, Azospirillum brasilense CW903, and Azospirillum lipoferum CW1503 were selected to study colonization in association with wheat roots. We observed higher expression of beta-galactosidase activity in A. brasilense strains than in A. lipoferum strains, which could be attributed to their higher population in association with wheat roots. All strains tested colonized and exhibited the strongest beta-galactosidase activity at the sites of lateral roots emergence.