转座子Tn5诱变对三叶草根瘤菌早期侵染和结瘤的影响

对三叶草根瘤菌Rhizobium trifolii野生型菌株ANU843及其转座子Tn5诱导的突变株nod258和nod261的侵染和结瘤能力进行了比较。Ⅲ医结瘤基因nodFE的突变林(nod258)能在地三叶草(Subterranean clover)上正常侵染和结瘤,但瘤数比ANU843所诱导的略有减少。而Ⅱ区结瘤基因nodIJ的突变株(nod261)却表现侵染力减弱,诱导无效瘤和瘤数大大减少。显微镜强察和超微结构研究表明菌株ANU843在接种后24h侵染已经从植物根毛开始,48h侵染线,(infeetioa thread)发展到表皮细胞并开始分支,接种后72h侵染线进一步发展,深入皮层细胞,这时皮层细胞已经大量分裂增生,形成瘤的分生组织。成熟的瘤细胞内充满了类菌体。而nod261突变株侵染植物比ANU843推迟了24h,侵染线的发展受阻碍,接种后72h侵染线仍然停留在根毛细胞中。Nod26l突变株所诱导的瘤细胞内没有或仅有个别类菌体,是无效瘤。这表明结瘤基因nodIJ与侵染线的正常形成和发展密切相关。     

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.     

菜豆根瘤菌sym和mel基因在农杆菌中的转移和表达

豆根瘤菌(Rhizobium phaseoli)共生质粒pSYM3622具有诱导宿主植物(phaseolusvulgaris cv.\"Jamapa\")结瘤固氮的有效基因,以及菜豆根瘤菌特征性的产黑素基因(Melanin production gene)。在诱动因子RP4的存在下,共生质粒能够有效地向三叶草根瘤菌和农杆菌转移。种间和属间杂交子都能诱导菜豆植物形成无效根瘤,并且具备在特定培养基上产生黑素的能力。pSYM3622在三叶草根瘤菌菌株RCR226中具有明显的不亲和性,但是在农杆菌杂交子中,这些结癌和产生黑素的特征在植物根瘤分离菌中能够稳定地保持下去。试验同时研究了pSYM3622向假单胞菌和大肠杆菌的诱动转移。     

转座子Tn5对柠檬酸细菌的诱变

含自杀性质粒 PJB4JI：：Mu：：Tn5的大肠杆菌1830与四株柠檬酸细菌作接合杂交均能获得Kanr转移接合子。其中一株(c-3-1)的Kayr转移接合子中绝大部分对庆大霉素敏感。鉴别了3000个这样的转移接合子菌落获得了2l株营养缺陷型,其中赖氨酸1株,尿嘧啶1株,精氨酸2株,异亮氨酸2株,组氨酸2株,甲硫氨酸株,苯丙氨酸1株,酪氨酸1株,丝氨酸1株,苏氨酸1株,亮氨酸3株,脯氨酸1株,腺嘌呤3株和乳糖利用1株。用琼脂糖电泳检查部分营养缺陷型突变体DNA均未发现自杀性质粒PJB4JI：：Mu：：Tn5,以32p标记的Tn5 DNA为探针与每个营养缺陷型的染色体作杂交均看到了Tn5 DNA同源序列的存在。由此得出结论,这些营养缺陷型产生于转座子Tn5从自杀性质粒PJB4JI到c-3-1染色体的转座。     

转座子Tn916接合转移诱变棒杆菌

采用滤膜接合法,将大肠杆菌中带有Ｔ_ｃ抗性的转座子Ｔｎ９１６接合转移到北京棒杆菌ＧＭ９３中其进行诱变。接合频率在１０^－７－１０^－８之间,在没有选择压力的条件下传代培养,接合子是比较稳定的,仍保持Ｔ_ｃ抗性。Ｔｎ９１６的接合转座作用可以使ＧＭ９３产生营养缺陷型突变,其中氨基酸营养缺陷型占８４％,并得到与Ｌ－Ｉｌｅ产生途径有关的六种氨基酸（Ｔｈｒ,Ｌｙｓ,Ｌｅｕ,Ｍｅｔ,Ｖａｌ,Ｉｌｅ）的缺陷株。     

转座子Tn5诱变慢生大豆根瘤菌

用pSUP1011载体系统,Tn5诱变慢生大豆根瘤菌110、123,其KmR菌落出现频率为5x10-8一5x 10-7。Tn5的SmR。基因能在慢生大豆根瘤菌中表达。用卡那霉素加链霉素来筛选插入变株,可完全排除自然突变的干扰。从4450个KmR变株中检出营养缺陷型7株,其中His-(4)、Glu-(1)和Trp-(2),吸氢功能缺陷型10株,其中2株His-同时是吸氢缺陷(Hup-)和共生固氮缺陷(Nif-),其原养型回变体(回变频率为0.5×10-8)都同时恢复了吸氢与固氮功能,又失去了对卡那霉素的抗性。4株对氧特别敏感的Hup-缺陷型,在培养状态下无吸氢活性,但结瘤固氮时不放氢。     

Tn5-Mob系统诱导耐盐基因转移

利用转座子Tn5-Mob和辅助转移质粒R68.45将放射形土壤杆菌（Agrobacterium radiobacter）生物变型1（biovar 1）菌株PDDCC5785的耐盐基因转移到快生型大豆根瘤菌（Rhizobium fredii）USDA 191中,获得转移接合子,提高了根瘤菌的耐盐能力。随机挑取27个转移接合子进行结瘤试验,其中3株能够结瘤固氨。本研究为根瘤菌的遗传改造提供了一个有用的手段。     

费氏中华根瘤菌共生质粒扩增对结瘤因子组分和共生固氮能力的影响

费氏中华根瘤菌(Sinorhizobium fredii)YC4能在大豆(Glycine max)和野大豆(G．soja)上形成正常固氮的根瘤．人工培养条件下用^14C标记的薄层层析(TLC)法检测根瘤菌产生的结瘤因子(LCOs)的结果表明,与其它4株费氏中华根瘤菌相比,YC4产生的LCOs含有较多的疏水性基团．从YC4菌株中分离到1株共生质粒发生了扩增的自发突变株YSC3,其产生的LCOs中含有较野生型菌株多的1个疏水性组分,28℃培养条件下产生的LCOs量亦较YC4显著增加．结瘤试验结果表明,YSC3菌株只能在大豆和野大豆上形成无效的根瘤．     

Tn5-Mob系统诱导根瘤菌属之间耐盐和共生性状的转移

本实验通过质粒pSUP501l及其辅助质粒RP4将Tn5-Mob随机插入苜蓿根瘤菌(Rhizo-bium meliloti 042B)的基因组中,得到86株接合子SR。随机选取4株SR,通过辅助质粒R68．45的三亲本杂交,将它们的DNA片段引入慢生型大豆根瘤菌(Bradyrhizobium,japo—nicum USDA 110),获得106株接合子BSR。大部分BSR菌株获得了生长快速的特性和耐盐性,一般能耐0．3—0．5m01／L Nacl,其中有些菌株能产生黑色素。将9o株BSR回接大豆和苜蓿植株,发现47株能在大豆和苜蓿植株结瘤,但在苜蓿卜无固氮活性;26株只能在大豆植株结瘤固氮;13株只能在苜蓿植株结瘤而不固氮;4株在大豆和菖蓿植株均不结瘤。其中,获得了4株耐盐性和固氮酶活性强的接合子。     

利用Tn5-1063转座诱变法分离苜蓿中华根瘤菌042BM noeB基因的研究

采用三亲本杂交方法将带有Tn5-1063(含luxAB)的质粒pRL1063a导入苜蓿中华根瘤菌(Sinorhizobium meldoti)042BM,进行转座子插入诱变,在含有氯霉素、卡那霉素的TY平板上筛选接合子。通过结瘤试验,从1000个突变株中,筛选到3个结瘤突变株042BMR5、042BMR11和042BRM29。它们都表现出发光酶活性,表明转座子正向插入到基因组中的某个启动子下游。Southern杂交结果证实,转座子均为单一位点插入。对042BMR5突变株基因组进行反向PCR,扩增位于Tn5-1063两端的侧翼序列。测序结果表明,转座子插入到苜蓿中华根瘤菌的共生质粒pSymA noeB基因内。根据基因组中noeB上游和下游序列扩增出042BM noeB,其与苜蓿中华根瘤菌1021 noeB的同源性为98％,而与NoeB蛋白的氨基酸序列相似性为95％。疏水性分析发现,NoeB是一个跨膜蛋白,在N末端有4个跨膜区,其中包含3个初级螺旋和1个次级螺旋。     

Transposition of Tn1831 to sym plasmids of Rhizobium leguminosarum and Rhizobium trifolii

Abstract All transposon-induced symbiotic mutants of Rhizobium described so far have been obtained using Tn 5 , which codes for kanamycin resistance (Km^R). To enable genetic complementation studies, we tried to find an effective transposon carrying another resistance marker. We report here a method for the apparent random transposition in Rhizobium of Tn 1831 , which codes for resistance against spectinomycin (Sp), streptomycin (Sm) and mercury chloride. When the suicide plasmid pMP12 (RP4::Tn 1831 , Km::Mu) was transferred to Rhizobium , in almost all cases the exconjugants harbour a deleted transfer-deficient R plasmid. From this deleted R plasmid transposition occurred to self-transmissible Sym-plasmids of R. leguminosarum and R. trifolii . Using this method a number of Tn 1831 -induced symbiotic mutants of pRL1JI were isolated.     

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．     

The sym plasmid pRP2JI and at least two other loci of Rhizobium leguminosarum biovar phaseoli can confer resistance to infection by the virulent bacteriophage RL38

Abstract The virulent Rhizobium bacteriophage RL38 did not form plaques on R.leguminosarum by phaseoli but did so at high efficiency on a derivative of that strain lacking its symbiotic plasmid pRP2JI. Other strains with large deletions in pRP2JI which removed many nod and nif genes retained resistance to RL38, showing that the gene which confers phage resistance lies elsewhere on the plasmid. Although the wild-type strain of R. leguminosarum bv. phaseoli failed to plate RL38, it was possible to transduce chromosomal markers into this strain, indicating that the 'block' was not at an early stage in the infection process. Two different recombinant plasmids obtained from a clone bank of genomic DNA of R. leguminosarum bv. phaseoli , which appeared to have no DNA in common, both conferred resistance to RL38. Surprisingly, the DNA cloned in each of these plasmids did not originate from pRP2JI. Therefore, several different loci both on the Sym plasmid and elsewhere on the bacterial genome can be involved in conferring resistance to this bacteriophage.     

Symbiotic effectiveness of strains of Rhizobium leguminosarum biovar phaseoli isolated from soils of Rwanda

We have isolated 48 strains of Rhizobium leguminosarum biovar phaseoli from nodules of Phaseolus vulgaris L. cultivated on 32 different soils at 22 various locations in Rwanda, Central Africa. The symbiotic effectiveness of the strains was appraised in the greenhouse by measuring shoots dry matter and total plant nitrogen content after six weeks of growth. Of the strains tested 19%, 58% and 23% were rated very effective, effective and ineffective, respectively. A very significant correlation (r=0.96, P<0.01) was observed between shoots dry matter and total N content. By using the total nitrogen balance method, it was estimated that in the presence of a very effective strain, up to 86% of the N present in the shoots comes from N₂ fixation. No significant correlations were observed between the symbiotic effectiveness of the strains and the pH of the soils from which they originated, the tolerance of the strains to acidity or their ability to produce organic acids. The nine very effective strains selected were highly competitive against two ineffective strains with the two P. vulgaris cultivars Rubona-5 and Kiryumukwe.Contribution no 367, Station de recherches, Agriculture Canada.Contribution no 367, Station de recherches, Agriculture Canada.     

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．     

Spread and evolution of natural plasmids harboring transposon Tn5

Abstract: The presence of transposon Tn 5 was studied in 730 Enterobacteriaceae strains from clinical and sewage origin. From these strains, twenty-five conjugative plasmids harboring transposon Tn 5 were isolated. These plasmids were compared with pJR67 and pRYC119, the only previously studied plasmids harboring Tn 5 . A phylogenetic tree of the evolution of all different plasmids was proposed. Irrespective of their bacterial host and geographical place of isolation, some of the plasmids were shown to be identical. All of them can be included in only eight different prototypical plasmid species. Twenty-two plasmids (88%) carried an IncI1 incompatibility determinant as judged form DNA hybridization experiments. The presence of some other common resistance genes suggested that these plasmids are descendants of a common ancestor. These IncI1 plasmids could be grouped in six prototypical species. The results presented here suggest that Tn 5 spread in nature may be dependent on the conjugative ability of the IncI plasmids harboring the transposon, rather than on the efficiency of Tn 5 transposition between different replicons.     

Root colonization of maize and lettuce by bioluminescent Rhizobium leguminosarum biovar phaseoli.

Two strains of Rhizobium leguminosarum bv. phaseoli and three other plant growth-promoting rhizobacteria (PGPR) were examined for the potential of maize and lettuce root colonization. All of these strains were selected in vitro for their phosphate-solubilizing abilities. Maize and lettuce seeds were treated with derivatives of all strains marked with lux genes for bioluminescence and resistance to kanamycin and rifampin prior to planting in nonsterile Promix and natural soil. The introduced bacterial strains were quantified on roots by dilution plating on antibiotic media together with observation of bioluminescence. Rhizobia were superior colonizers compared with other tested bacteria; rhizobial root populations averaged log 4.1 CFU/g (fresh weight) on maize roots 4 weeks after seeding and log 3.7 CFU/g (fresh weight) on lettuce roots 5 weeks after seeding. The average populations of the recovered PGPR strains were log 3.5 and log 3.0 CFU/g (fresh weight) on maize and lettuce roots, respectively. One of the three PGPR was not recovered later than the first week after seeding in Promix. Bioluminescence also permitted visualization of in situ root colonization in rhizoboxes and demonstrated the efficiency of rhizobial strains to colonize and survive on maize and lettuce roots.     

Growth promotion of maize and lettuce by phosphate-solubilizing Rhizobium leguminosarum biovar. phaseoli

Rhizobium leguminosarum bv. phaseoli strains P31 and R1, Serratia sp. strain 22b, Pseudomonas sp. strain 24 and Rhizopus sp. strain 68 were examined for their plant growth-promoting potential on lettuce and forage maize. All these phosphate solubilizing microorganisms (PSM) were isolated from Québec soils. The plants were grown in field conditions in three sites having high to low amounts of available P. In site 1 (very fertile soil), strains R1 and 22b tended to increase the dry matter yield of lettuce shoots (p≤0.10). Lettuce inoculated with rhizobia R1 had a 6% higher P concentration (p≤0.10) than the uninoculated control. In site 2 (poorly fertile soil), the dry matter of lettuce shoots was significantly increased (p≤0.05) by inoculation with strain P31 and 24 plus 35 kg ha^-1 P-superphosphate, or with strain 68 plus 70 kg ha^-1 P-superphosphate. In site 3 (moderately fertile soil), the dry matter of maize shoots was significantly increased (p≤0.05) by inoculation with strain 24 plus 17.5 kg ha^-1 P-superphosphate, or with strain P31 plus 35 kg ha^-1 P-superphosphate. Inoculation with PSM did not affect lettuce P uptake in the less fertile soil in site 2. In site 3 with the moderately fertile soil, maize plants inoculated with strain R1 had 8% higher P concentration than the uninoculated control (p≤0.01), and 6% with strains P31 and 68 (p≤0.05). The results clearly demonstrate that rhizobia specifically selected for P solubilization function as plant growth promoting rhizobacteria with the nonlegumes lettuce and maize. The P solubilization effect seems to be the most important mechanism of plant growth promotion in moderately fertile and very fertile soils when P uptake was increased with rhizobia and other PSM.     

Dissection of Nodule Development by Supplementation of Rhizobium leguminosarum biovar phaseoli Purine Auxotrophs with 4-Aminoimidazole-5-Carboxamide Riboside

Purine auxotrophs of Rhizobium leguminosarum biovar phaseoli CFN42 elicit uninfected pseudonodules on bean (Phaseolus vulgaris L.). Addition of 4-aminoimidazole-5-carboxamide (AICA) riboside to the root medium during incubation of the plant with these mutants leads to enhanced nodule development, although nitrogenase activity is not detected. Nodules elicited in this manner had infection threads and anatomical features characteristic of normal nodules, such as peripheral vasculature rather than the central vasculature of the pseudonodules that were elicited without AICA riboside supplementation. Although 10⁵ to 10⁶ bacteria could be recovered from these nodules after full development, bacteria were not observed in the interior nodule cells. Instead, large cells with extensive internal membranes were present. Approximately 5% of the normal amount of leghemoglobin and 10% of the normal amount of uricase were detected in these nodules. To promote the development of true nodules rather than pseudonodules, AICA riboside was required no later than the second day through no more than the sixth day following inoculation. After this period, removal of AICA riboside from the root medium did not prevent the formation of true nodules. This observation suggests that there is a critical stage of infection, reached before nodule emergence, at which development becomes committed to forming a true nodule rather than a pseudonodule.     

Mobilization ofEscherichia coli R1 silver-resistance plasmid pJT1 by Tn5-Mob intoEscherichia coli C600

Summary Escherichia coli Rl is an Ag⁺-resistant strain that, as we have shown recently, harbours at least two large plasmids, pJT1 (83 kb) and pJT2 (77 kb). Tn5-Mob was introduced into theE. coli Rl host replicon via conjugation on membrane filters. The transfer functions of plasmid RP4-4 were provided in this process and Tn5-Mob clones mated withE. coli C600 yielded Ag⁺-resistant transconjugants. This mobilization procedure allowed transfer and expression of pJT1 Ag⁺ resistance inE. coli C600. Prior to use of Tn5-Mob mobilization, it was not possible to transfer Ag⁺-resistant determinant(s) intoE. coli by conjugation or transformation including high-voltage electroporation.E. coli C600 containing PJTI and PJT2 displayed decreased accumulation of Ag⁺ similar toE. coli R1.E. coli C600 could not tolerate 0.1 and 0.5 mM Ag⁺, rapidly accumulated Ag⁺ and became non-viable. Tn5-Mob mobilization may be useful in the study of metal resistance in bacteria, especially in strains not studied for resistance mechanisms.