Linkage of genes for nitrogenase and nodulation ability on plasmids in Rhizobium leguminosarum and R. phaseoli

Summary The ability to identify genes that specify nitrogenase (nif genes) in Rhizobium depends on the close homology between then and the corresponding nif genes of Klebsiella pneumoniae (Nuti et al. 1979; Ruvkun and Ausubel 1980). Rhizobium plasmids of high molecular weight (>100 Md) were separated on agarose gels, transferred to nitrocellulose filters and tested for their ability to hybridise with radioactively labelled pSA30, containing the nifKDH region of K. pneumoniae. Five large plasmids, each present in different strains of R. leguminosarum or R. phaseoli, were found to hybridise. Each of these plasmids had previously been shown to determine other symbiotic functions such as nodulation ability. The nif genes on three different plasmids appeared to be in conserved DNA regions since they were within an EcoRI restriction fragment of the same size.     

Plasmid-determined alterations of Salmonella typhimurium lipopolysaccharides

Summary Salmonella typhimurium Rc902 infected with derepressed ColIb mutants gave rise to changes in the composition of bacterial lipopolysaccharides (LPS). Bacteria carrying ColIbdrd7, derepressed in transfer, exhibited a marked decrease in the content of all 0-side-chain sugars of LPS. Similar effects were found upon the introduction of R64-11, also derepressed in transfer. In LPS of S. typhimurium containing ColIbdrd2, derepressed in colicin synthesis, a decrease of abequose content associated with an increase of glucose level was observed. Bacteria carrying the wild-type ColIb, the revertant of a drd mutant to the wild type, or the non colicinogenic strain resulting from the elimination of ColIbdrd2, showed no changes in the sugar composition of LPS.     

A mutation that blocks exopolysaccharide synthesis prevents nodulation of peas by Rhizobium leguminosarum but not of beans by R. phaseoli and is corrected by cloned DNA from Rhizobium or the phytopathogen Xanthomonas

Summary A Tn5-induced mutant strain of R. phaseoli which failed to synthesize exopolysaccharide (EPS) was isolated and was shown to induce normal nitrogen-fixing nodules on Phaseolus beans, the host of this Rhizobium species. The corresponding wild-type Rhizobium DNA was cloned in a wide host-range vector and by isolating Tn5 insertions in this cloned DNA, mutations in a gene termed pss (polysaccharide synthesis) were isolated. These were introduced by marker exchange into near-isogenic strains of R. leguminosarum and R. phaseoli which differed only in the identity of their symbiotic plasmids. Whereas the EPS-deficient mutant strain of R. phaseoli induced normal nitrogen-fixing nodules on Phaseolus beans, the same mutation prevented nodulation of peas by a strain of R. leguminosarum which normally nodulates this host. Further, it was found that DNA cloned from the plant pathogen Xanthomonas campestris pathover campestris could correct the defect in EPS synthesis in R. leguminosarum and R. phaseoli and also restored the ability to nodulate peas to the pss::Tn5 mutant strain of R. leguminosarum.     

psi, a plasmid-linked Rhizobium phaseoli gene that inhibits exopolysaccharide production and which is required for symbiotic nitrogen fixation

Summary A strain of R. phaseoli cured of its symbiotic plasmid, pRP2JI, retained the ability to make exopolysaccharide (EPS). However, a region of pRP2JI, when cloned at an increased copy number in wide host-range vectors and transferred to this and other strains of Rhizobium, inhibited EPS synthesis. The gene responsible was termed psi (polysaccharide inhibition) and was located in a region of the symbiotic plasmid close to nodulation and nitrogen fixation genes. psi is important in the symbiosis since a wild-type strain containing psi cloned on a multicopy plasmid failed to form Phaseolus nodules, and mutant strains containing psi::Tn5 mutations failed to fix nitrogen in Phaseolus nodules. It is proposed that the function of psi may be to repress in the bacteriod the expression of genes such as those for EPS synthesis which are normally expressed in free-living culture.     

In Bradyrhizobium japonicum the common nodulation genes, nodABC, are linked to nifA and fixA

Summary Using cloned Rhizobium phaseoli nodulation (nod) genes as hybridization probes homologous restriction fragments were detected in the genome of the slow-growing soybean symbiont, Bradyrhizobium japonicum strain 110. These fragments were isolated from a cosmid library, and were shown to lie 10 kilobasepairs (kb) upstream from the nifA and fixA genes. Specific nod probes from Rhizobium leguminosarum were used to identify nodA-, nodB-, and nodC-like sequences clustered within a 4.5 kb PstI fragment. A mutant was constructed in which the kanamycin resistance gene from Tn5 was inserted into the nodA homologous B. japonicum region. This insertion was precisely located, by DNA sequencing, to near the middle of the nodA gene. B. japonicum mutants carrying this insertion were completely nodulation deficient (Nod^-).     

Competitive nodulation blocking of Afghanistan pea is determined by nodDABC and nodFE alleles in Rhizobium leguminosarum

Summary One well-defined competitive interaction amongst rhizobia is that between compatible and non-compatible strains of Rhizobium leguminosarum with respect to the nodulation of some primitive pea genotypes. The Middle Eastern pea cv Afghanistan is nodulated effectively can R. leguminosarum TOM, but its capacity to nodulate can be blocked if a mixed inoculation is made with R. leguminosarum PF₂. This PF₂ phenotype (Cnb) is encoded by its symbiotic plasmid and cosmid clones thereof. We found that Cnb is also encoded by the well-characterized Sym plasmid pRL1JI of R. leguminosarum strain 248. We have isolated and characterized a 6.9 kb HindIII fragment of pSymPF₂ which confers the Cnb⁺ phentoype on other (Cnb^–) rhizobia. A Tn5 site-directed Cnb^– mutant was constructed by homogenotization and was also found to be Nod^– on the European pea cv Rondo. DNA hybridization and complementation analysis indicated that the 6.9 kb Cnb⁺ fragment contained the nodD, nodABC and nodFE operons. Analysis of the Cnb phenotype of nod::Tn5 alleles of pRL1JI showed that mutations of nodC, nodD or nodE all abolished Cnb activity whereas mutants in nodI and nodJ reduced activity to 50% of the wild-type level.     

Impaired nitrogen fixation and glutamine synthesis in methionine sulfoximine sensitive (MSs) mutants of Rhizobium phaseoli

Summary Random Tn5 mutagenesis of antibiotic-resistant derivatives of Rhizobium phaseoli CFN42 yielded several independent mutants that were sensitive to methionine sulfoximine (MS^s), a specific inhibitor of glutamine synthetase (GS). These MS^s mutants were analyzed for GSI and GSII activities and for their symbiotic properties. Four classes of MS^s mutants have been distinguished. Class I strains are impaired in their synthesis of glutamine and in their symbiotic properties. Class II strains have wild type levels of GSI and GSII activities but have a reduced capacity to fix nitrogen. Class III strains have lost GSII activity, but their symbiotic properties are wild type. In class IV mutants neither glutamine synthesis nor symbiotic properties are affected. Mutants of classes I, III, and IV all have the Tn5 inserted into the chromosome, whereas in class II mutants the Tn5 is located in plasmid p42e, a plasmid different from the previously identified symbiotic plasmid p42d.     

颗石藻Pleurochrysis carterae抗捕食特征

颗石藻Pleurochrysis carterae是沿海水域中常见钙化微藻,易形成高密度水华,也是养殖环境致害种之一。抗捕食防御能力可能是其种群增殖优势的一个重要原因。以卤虫作为捕食者,分析了颗石藻P.carterae抗捕食现象,以及在捕食压力下的重要生理生化响应特征,以期为颗石藻P.carterea抗捕食机制研究及其高密度增殖机理提供参考。研究结果显示:(1)当颗石藻P.carterae比例增加时,卤虫对微藻的摄食率显著降低,且存活率显著下降,显示该藻具抗捕食能力。(2)以卤虫饵料微藻球等鞭金藻(Isochrysis galbana)为对照,比较研究发现,相同的捕食压力下,饵料金藻的叶绿素荧光参数(电子传递速率ETR和最大量子产率Fv/Fm)显著降低,但颗石藻P.carterae的ETR和Fv/Fm没有显著变化,显示颗石藻P.carterae对卤虫抗捕食作用。(3)相对于没有捕食压力的对照组,捕食压力下,饵料金藻I.galbana的脂类组成没有显著差异。但是,颗石藻P.carterae的脂类组成则发生了显著变化,主要表现在对细胞叶绿体有重要作用的单半乳糖甘油二酯(MGDG),双半乳糖甘油二酯(DGDG),磷脂酰甘油二酯(PG)含量上升,与促细胞分裂相关的二酰甘油(DAG)和磷脂酰肌醇(PI)也上升。这些脂类代谢物的变化可能在其种群水平上抵抗捕食并实现种群增殖中发挥作用。(4)培养介质中磷的状态对颗石藻P.carterae细胞二甲基巯基丙酸(Dimethyl sulfonio propionate,DMSP)含量有显著影响,且影响颗石藻P.carterae对卤虫的致害效应:缺磷条件下生长的颗石藻P.carterae首先使卤虫受害。当培养液中仅以ATP为磷源时,颗石藻P.carterae的卤虫致害效应则降低。研究证明,颗石藻P.carterae具有抗捕食能力,细胞的脂类代谢物质以及DMSP可能在抗捕食防御中发挥作用。     

Cloned nodulation genes of Rhizobium leguminosarum determine host-range specificity

Summary Three nodulation-deficient (nod) mutants of Rhizobium leguminosarum were isolated following insertion of the transposon Tn5 into pRL1JI, the R. leguminosarum plasmid known to carry the nodulation genes. DNA adjacent to the nod: Tn5 alleles was subcloned and used to probe a cosmid clone bank containing DNA from a Rhizobium strain carrying pRL1JI. Two cosmid clones which showed homology with the probe contained about 10 kb of DNA in common. The R. leguminosarum host-range determinants were found to be present within this 10 kb common region since either of the cosmid clones could enable a cured R. phaseoli strain to nodulate peas instead of Phaseolus beans, its normal host. Electron microscopy of nodules induced by Rhizobium strains cured of their normal symbiotic plasmid but containing either of the two cosmid clones showed bacteroid-forms surrounded by a peri-bacteroid membrane, indicating that normal infection had occurred. Thus it is clear that this 10 kb region of nodDNA carries the genes that determine host range and that relatively few bacterial genes may be involved in nodule and bacteroid development.     

fliL基因显著影响艰难拟梭菌运动功能及产孢能力

鞭毛基底体相关FliL家族蛋白(flagellar basal body-associated FliL family protein,fliL)基因编码FliL蛋白,FliL是一种与鞭毛基体相结合的单跨膜蛋白。为研究艰难拟梭菌fliL基因功能,使用非等长同源臂偶联等位交换(allele-coupled exchange,ACE)方法成功构建了fliL基因缺失(ΔfliL)和回补(::fliL)突变株,研究突变菌株与野生型菌株(CD630)生长曲线、抗生素敏感性、pH耐受性、运动能力及产孢能力等表型的差异。结果显示,菌株ΔfliL生长速率及最大生物量均小于菌株CD630,::fliL回补菌株生长情况回复至野生型。与CD630菌株相比,ΔfliL对阿莫西林、氨苄青霉素、诺氟沙星的敏感性提高,对卡那霉素、四环素敏感性降低,::fliL抗生素敏感性部分回复至野生型水平。与CD630菌株相比,ΔfliL游泳运动能力显著降低,::fliL运动能力超越野生型菌株CD630。相比菌株CD630,菌株ΔfliL在pH值为5时耐受能力显著提高,在pH值为9时,耐受能力显著降低。除此之外,ΔfliL产孢能力较CD630显著降低,::fliL产孢能力部分恢复。以上结果表明,艰难拟梭菌fliL基因与其运动能力、抗生素敏感性、环境耐受能力和产孢能力密切相关,可能进一步影响艰难拟梭菌菌株的致病力。     

Mobilization of a Rhizobium meliloti megaplasmid carrying nodulation and nitrogen fixation genes into other rhizobia and Agrobacterium

Summary The indigenous megaplasmid pRme41b of Rhizobium meliloti 41 was made susceptible to mobilization with the P-1 type plasmid pJB3JI by inserting the mobilization (mob) region of RP4 into it. First the mob region together with a kanamycin resistance marker was inserted in vitro into a fragment of pRme41b cloned into pBR322. The recombinant plasmids so formed (pAK11 and pAK12) were then mobilized into R. meliloti. Since these recombinant plasmids were unable to replicate in R. meliloti, selection for kanamycin resistant derivatives allowed the isolation of pRme41b::pAK11 or pRme41b::pAK12 cointegrates. It was shown that in the majority of these recombinants, pAK11 or pAK12 was integrated into the homologous fragment of pRme41b. The pRme41b cointegrates were transferred into nod-nif deletion mutants of R. meliloti 41 where it was shown that both Nod⁺ and Fix⁺ phenotypes could be restored. The pRme41b cointegrates were also transferred into two other Rhizobium strains and into Agrobacterium tumefaciens. The Rhizobium strains and A. tumefaciens carrying pRme41b formed nodules of variable size on Medicago sativa roots, indicating that at least the early steps of nodulation of M. sativa are coded by pRme41b and are expressed in these bacteria.     

Three regulatory nodD alleles of diverged flavonoid-specificity are involved in host-dependent nodulation by Rhizobium meliloti

Summary Rhizobium meliloti infective on Medicago, Melilotus and Trigonella plants has three copies of the nodulation regulatory gene nodD. Strains containing mutations in nodD1 exhibited a delayed and/or decreased nodulation on Melilotus albus (Ma), Medicago sativa (Ms), Medicago quasifalcata (Mqu) and Trigonella coerulea (Tc), while on Medicago truncatula (Mt) they nodulated similarly to the wild-type R. meliloti. Delayed nodulation was observed also when nodD2 mutants were inoculated onto Ms, Mt and Tc, but not on Ma and Mqu. A nodD3 mutant exhibited delayed nodulation on Ms and Ma. Using a nodC-lacZ fusion and cloned nodD genes on plasmids, high induction levels were detected in R. meliloti when nodD1 was present with seed exudates from Ms, Ma and Mqu, nodD2 with those from Ms and Mt, and nodD3 with those from Ms, Ma and Mqu. NOne of the nodD copies exhibited high levels of nodC-lacZ induction when present with seed exudate from Tc. Only nodD1 induced nodC-lacZ expression in conjunction with the flavone, luteolin. The plant hosts used in this study exude different flavonoids and correlation between nodulation and nodC-lacZ induction abilities of the host exudates was observed. We concluded that all the three nodD copies of R. meliloti have common nod-promoter activating but diverged flavonoid-recognizing abilities. Thus, the three nodD alleles contribute to the activation of nodulation genes in a host-dependent manner.     

A molecular linkage map of nitrogenase and nodulation genes in Rhizobium trifolii

Summary A molecular map was constructed linking the nitrogenase structural genes (nif) and nodulation genes (nod) in the white clover symbiont, Rhizobium trifolii. In R. trifolii strain ANU843 these two genetic regions are located some 16 kilobases (kb) apart on the 180 kb symbiotic (Sym) plasmid. The molecular linkage of nod and nif genetic regions was established by hybridization analysis using recombinant plasmids containing overlapping cloned sequences. Nodulation genes were located by means of a Tn5-induced nodulation-defective mutant that failed to induce clover root hair curling (Hac^- phenotype). A cloned wild-type DNA fragment was shown to phenotypically correct the Hac^- mutation by complementation. The nifHDK genes were cloned by positive hybridization to another R. trifolii nif-specific probe. Location of the nif genes relative to the nod genes was established by analysis of a Sym plasmid deletion derivative.     

菜豆根瘤菌对土壤钾的活化作用

以土壤为钾源,通过液体培养试验研究了8株菜豆根瘤菌对土壤钾的活化作用。结果表明,菜豆根瘤菌能释放大量的氢离子,使液体培养基的pH值大幅度降低,氢离子的浓度至少提高22倍以上。根瘤菌分泌有机酸的种类与数量因菌株不同而异,这些有机酸包括甲酸、乙酸、草酸、丁二酸、柠檬酸、苹果酸和乳酸等,其中全部菌株均能分泌草酸和苹果酸,大部分菌株能分泌乙酸。在接种根瘤菌的液体培养基中,可溶性钾含量显著高于不接种的液体培养基,土壤矿物结构钾则显著降低。由于土壤是培养基钾的唯一来源,故根瘤菌可促进土壤无效钾的溶解。相关分析表明,土壤矿物结构钾与有机酸分泌总量呈极显著负相关(r=-0.878**,n=9),与培养液pH值呈极显著正相关(r=0.863**,n=9),说明根瘤菌分泌的有机酸和氢离子可能溶解土壤无效钾。考虑到根瘤菌草酸分泌量大,络合钙、镁、铁、铝的能力强,且与有机酸分泌总量呈极显著正相关(r=0.870**,n=9),推测草酸分泌在活化土壤无效钾的过程中起重要作用。此外,根瘤菌分泌的有机酸电离产生的氢离子仅占培养液氢离子的4.15%—27.56%,推测根瘤菌直接分泌的氢离子可能是造成培养液pH值降低的主要原因之一。     

Transfer of nodulation ability in Rhizobium using R68.45 derived plasmids

Summary Nodulation ability was transferred from Rhizobium meliloti L5.30 to the non-nodulating mutant Rhizobium trifolii 24K using plasmid R68.45. Transconjugants selected for the carbenicillin resistance (cb ^r) marker became simultaneously capable of nodulating clover and showed changes in phage sensitivity. Besides the indigenous plasmid of 90 MD (pUCS201), the nodulating transconjugants harbored the newly introduced plasmid pUCS202 (ca. 40 MD). After treatment of the transconjugants with curing agents the simultaneous loss of antibiotic resistance and ability to form nodules were associated with the disappearance of pUCS202. nod and cb ^r genes were cotransferred into R. trifolii strains by conjugation and transformation. There is genetic evidence that the nod gene(s) was integrated into R68.45.     

The role of Rhizobium conserved and host specific nodulation genes in the infection of the non-legume Parasponia andersonii

Summary Rhizobium and Bradyrhizobium bacteria gain intercellular entry into roots of the non-legume Parasponia andersonii by stimulating localized sites of cell division which disrupt the epidermis. Infection threads are then initiated from intercellular colonies within the cortex. Infection via the information of infection threads within curled root hairs, which commonly occurs in legumes, was not observed in Parasponia. The conserved nodulation genes nodABC, necded for the curling of legume root hairs, were not essential for the initiation of infection, however, these genes were required for Parasponia prenodule development. In contrast, the nodD gene of Rhizobium strain NGR234 was essential for the initiation of infection. In addition, successful infection required not only nodD but a region of the NGR234 symbiotic plasmid which is not needed for the nodulation of legumes. Agrobacterium tumefaciens carrying this Parasponia specific region, as well as legume nod genes, was able to form nodules on Parasponia which reached an advanced stage of development.     

The nodulation and nitrogen fixation of isolated roots of Phaseolus vulgaris L.

Summary The exposure of isolated bean roots to white light immediately before inoculation with nodule bacteria stimulated nodulation during a subsequent 7-day dark period. Strong white light applied to inoculated roots for 3 days or longer suppressed nodulation except when the light was applied immediately after inoculation and the duration of the light period did not exceed 3 days. Alternating periods of 12 hours light and 12 hours darkness were almost as inhibitory to nodulation as a continuous exposure to light. A combination of blue and far-red light inhibited nodule formation more than a combination of red and far-red light. Light treatments that inhibited nodulation also suppressed the production of lateral roots, but to a lesser extent. The effect of light on nodulation and lateral root formation was not influenced by the presence or absence of an attached hypocotyl segment in the explant.