Arbuscular mycorrhizal fungi: a specialised niche for rhizospheric and endocellular bacteria

Arbuscular mycorrhizal (AM) fungi produce an extensive hyphal network which develops in the soil, producing a specialised niche for bacteria. The aim of this paper is to review briefly the interactions shown by these symbiotic fungi with two bacterial groups: (i) the plant-growth promoting rhizobacteria (PGPRs) which are usually associated with fungal surfaces in the rhizosphere, and (ii) a group of endocellular bacteria, previously identified as being related to Burkholderia on the basis of their ribosomal sequence strains. The endobacteria have been found in the cytoplasm of some isolates of AM fungi belonging to Gigasporaceae and offer a rare example of bacteria living in symbiosis with fungi. This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular cloning and physical mapping of the nitrogenase structural genes from the filamentous, non-heterocystous cyanobacterium Pseudanabaena sp. PCC7409

Abstract Sequences homologous to the structural genes for dinitrogenase ( nifD and nifK ) and nitrogenase reductase ( nifH ) have been cloned from the filamentous, non-heterocystous cyanobacterium Pseudanabaena PCC7409. The nifHDK ⁻ homologous sequences were shown to reside on a 6.5-kb Eco RI restriction fragment by using a restriction fragment encoding the Klebsiella pneumoniae nifHDK genes as a heterologous hybridization probe. This 6.5-kb restriction fragment was cloned from a λ gt.wes Eco RI library of the Paseudanabaena sp. PCC7409 genome. This fragment was subcloned into the plasmid vector pUC9 to generate plasmid pPSU20. A detailed physical map of the insert in plasmid pPSU20 was determined, and relative positions of the nifH, nifD , and nifK homologous sequences on this fragment were determined by hybridization analysis with gene-specific fragments derived from the corresponding Anabaena sp. PCC7120 genes. The results indicate that these genes are contiguous in Pseudanabaena sp. PCC 7409 and are arranged in the order nifH, nifD , and nifK . This arrangement resembles that observed for other non-heterocystous cyanobacteria but differs from that observed for Anabaena, Calothrix , and Nostoc species.     

胃癌细胞中Ras基因的表达、突变分析及新剪接型Kras△E2的发现

目的：传统Ras家族由Kras,Hras和Nras基因组成,这类基因的点突变经常在人类肿瘤中发现,突变热点位于12,13,61位密码子。ERas基因是2003年在鼠胚胎干（ES）细胞中发现的,其cDNA编码的蛋白与Kras,Hras和Nras分别有46％,43％和47％的相似性,故属于新的Ras家族成员,近几年发现ERas基因的表达与胃癌密切相关,而传统Ras基因在胃癌细胞中的表达及突变情况系统报道较少,本文旨在研究传统Ras基因Kras,Hras,Nras及其家族新成员ERas基因在胃癌细胞中的表达和突变情况。方法：选用7株不同来源不同分化程度的胃癌细胞系,利用RT—PCR及real-timePCR检测Ras基因在这些胃癌细胞系中的表达,并通过测序对传统Ras基因突变热点12,13,61位密码子及ERas基因全长进行突变分析。结果：QRas基因在这些胃癌细胞系中均有不同程度的表达,其中Hras和Nms基因在各株细胞中表达水平均一,而Kras和ERas基因则呈差异性表达;②在这些胃癌细胞中传统Ras基因突变热点12,13,61位密码子不存在突变,ERas基因全长亦未检测到突变．③发现Kras基因一新的剪接型,特点为第一、三外显子直接拼接,缺失第二外显子,命名为Kras△E2。结论：与在其他肿瘤中不同,传统Ras基因在胃癌细胞中不存在突变热点,家族新成员ERas基因全长亦无突变,在国际上首次报道新剪接型Kras△E2,从而得出创新性结论：Ras基因家族在胃癌细胞中并不是通过热点突变导致持续活化而致癌,而可能是通过ERas基因表达量的调节或形成新的剪接型KrasAE2而致癌。另外,Kras基因是一被受国际关注的肿瘤基因,新剪接型的发现可能会对Kras基因致癌机制产生新的认识,意义重大。     

盐胁迫下粪产碱菌在水稻根表的定殖和异源固氮基因的表达

异源nif LacZ融合基因在粪产碱菌A15 6 1中的表达活性随盐浓度增加而升高 ,然后逐渐降低 ,nifH LacZ融合基因可以正常表达的盐浓度在 0 .1%～0 .5 %之间 ,盐浓度为 0 .0 5 %时活性最高。A15 6 1在盐浓度为 0 .0 6 %时趋化能力最强 ,随着盐浓度的提高逐渐下降 ,当盐浓度为 3 .0 %时完全丧失趋化能力。一定的盐浓度 (0 .5 % )对固氮粪产碱菌的根表定殖有促进作用 ,该条件下根表定殖的菌体数远大于对照。 3种nif LacZ融合基因在根内的表达部位有显著差异。nifH的表达部位主要分布于根的皮层薄壁组织细胞间隙 ,在条件适宜 (无铵和微量氧 )的部位或某些特殊位置如侧根伸出部位高水平表达。盐胁迫下水稻 耐盐粪产碱菌A15 6 1的联合固氮效率明显高于A15 6 1纯培养物     

被孢霉△~9脂肪酸脱饱和酶基因保守区的cDNA克隆及结构分析

被孢霉被广泛采用用于发酵生产γ-亚麻酸、花生四烯酸和EPA等多不饱和脂肪酸。为了解决发酵产率过低等诸多问题,我们拟采用基因工程技术改造生产菌株。通过对已克隆△~9脂肪酸脱饱和酶基因的分析,合成一组简并引物,PCR扩增了被饱霉△9脂肪酸脱饱和酶基因的保守区。结果表明被抱霉△9脂肪酸脱饱和酶基因保守区由537个核苷酸组成,共编码179个氨基酸。其与迄今为止发表的微生物面△9脂肪酸脱饱和酶基因有很高的同源性。这是被饱霉△~9脂肪酸脱饱和酶基因研究的次次报道。     

ORGANIZATION OF THE nif GENES OF THE NONHETEROCYSTOUS CYANOBACTERIUM TRICHODESMIUM SP. IMS101

An approximately 16-kb fragment of the Trichodesmium sp. IMS101 (a nonheterocystous filamentous cyanobacterium) "conventional" nif gene cluster was cloned and sequenced. The gene organization of the Trichodesmium and Anabaena variabilis vegetative ( nif 2 ) nitrogenase gene clusters spanning the region from nif B to nif W are similar except for the absence of two open reading frames (ORF3 and ORF1) in Trichodesmium . The Trichodesmium nif EN genes encode a fused Nif EN polypeptide that does not appear to be processed into individual Nif E and Nif N polypeptides. Fused nif  EN genes were previously found in the A. variabilis nif 2 genes, but we have found that fused nif EN genes are widespread in the nonheterocystous cyanobacteria. Although the gene organization of the nonheterocystous filamentous Trichodesmium nif gene cluster is very similar to that of the A. variabilis vegetative nif 2 gene cluster, phylogenetic analysis of nif sequences do not support close relatedness of Trichodesmium and A. variabilis vegetative ( nif 2 ) nitrogenase genes.     

Plasmid transfer in natural soil: a case by case study with nitrogen-fixing Enterobacter

Abstract In strains of nitrogen-fixing Enterobacter agglomerans , isolated from the rhizosphere of cereals, the nif genes are located on large plasmids. Plasmid pEA9 (200 kb) is self-transmissible between closely related strains. To collect data on possible uncontrolled gene spread, for planned releases of such bacteria, plasmid pEA9 was labelled with transposons (Tn 1725 and Tn 5 ) and used in mating experiments between homologous Enterobacter strains with soil as substrate. The soil was from a plot into which an actual release was being planned. In the majority of experiments it was not sterilized.
Survival and plasmid transfer is described, as are variations in temperature, time, moisture, pH and soil packing. Further experiments were with or without added energy sources, and with or without plant roots. Under standard conditions (22°C, pH 5.2, 15.5% moisture, loose soil, 2 × 10⁷ inoculated donor and recipient cells each per g soil, 3 days incubation) sterilized soil gave low rates of plasmid transfer (10⁻⁶ per donor) but non-sterilized soil gave none. Adding Luria broth or sucrose to non-sterilized soil elicited strong cell propagation, together with plasmid transfer (optimum after incubation for 1 day: 10⁻⁴ exconjugants per donor). No transfer could be registered in the presence of wheat seedling roots for periods up to 5 weeks.     

Direct detection of nif-gene sequences of Enterobacter agglomerans in soil

Abstract: Specific nif sequences of Enterobacter agglomerans plasmid pEA9 were detected in total DNA recovered from soil 70 days after its inoculation with the bacteria, when these were no longer culturable on agar medium. For this, a modified method of DNA extraction from soil was used. No amplification of DNA sequences by PCR was necessary.     

华葵根瘤菌nifA基因的克隆和功能分析

华葵根瘤菌(Mesorhizobium huakuii即R.astragali)159的nifA基因的序列分析表明,该基因全长1227bp,编码分子量为44734D的Nif A蛋白。与其它NifA蛋白的序列比较发现,华葵根瘤菌NifA蛋白也存在保守的中间结构域和C末端DNA结合结构域,但其氨基端缺失。Tn5定点突变得到的突变体是Nif-表型。构建了nifA基因组成型表达的质粒,此质粒在大肠杆菌中对华葵根瘤菌nifHlacZ有激活作用。     

nifH—gfp表达载体的构建及其在Enterobacter gergoviae 57—7中的表达

采用ＰＣＲ技术,从ＧＦＰｍｕｔ２中扩增得到三位点突变的报告基因ｇｆｐＳ６５Ｔ、Ｖ６８Ｌ、Ｓ７２Ａ片段,并将它和肺炎克氏杆菌（Ｋｌｅｂｓｉｅｌｌａ ｐｎｅｕｍｏｎｉａｅ（Ｓｃｈｒｏｅｅｔｅｒ）Ｔｒｅｖｉｓａｎ）Ｍ５ａ１的固氮酶结构基因ｎｉｆＨ的启动子和其起始密码子相融合,获得ｎｉｆＨ－ｇｆｐ表达载体ｐＭＧＦＰ２;再在ｐＭＧＦＰ２上插入卡那霉素抗性基因,获得可在日勾维肠杆菌（Ｅｎｔｅｒｏｂａｃｔｅｒ