Diversity of Sinorhizobium meliloti from the Central Asian Alfalfa Gene Center

Sinorhizobium meliloti was isolated from nodules and soil from western Tajikistan, a center of diversity of the host plants (Medicago, Melilotus, and Trigonella species). There was evidence of recombination, but significant disequilibrium, between and within the chromosome and megaplasmids. The most frequent alleles matched those in the published genome sequence.     

Diversity of Sinorhizobium meliloti from the Central Asian Alfalfa Gene Center

Sinorhizobium meliloti was isolated from nodules and soil from western Tajikistan, a center of diversity of the host plants (Medicago, Melilotus, and Trigonella species). There was evidence of recombination, but significant disequilibrium, between and within the chromosome and megaplasmids. The most frequent alleles matched those in the published genome sequence.     

Alfalfa and tobacco cells react differently to chitin oligosaccharides and Sinorhizobium meliloti nodulation factors

Alfalfa (Medicago sativa L.) suspension cultures respond to yeast elicitors with a strong alkalinization of the culture medium, a transient synthesis of activated oxygen species, and typical late defence reactions such as phytoalexin accumulation and increased peroxidase activity. The alkalinization reaction as well as the oxidative burst were also observed when tobacco (Nicotiana tabacum L.) cell-suspension cultures were treated with yeast elicitors. Depending on the degree of polymerization, N-acetyl chitin oligomers induced the alkalinization response in both plant cell-suspension cultures, while only tobacco cell cultures developed an oxidative burst. Suspension-cultured tobacco cells responded to Sinorhizobium meliloti nodulation factors with a maximal alkalinization of 0.25 pH units and a remarkable oxidative burst. In contrast, addition of Sinorhizobium meliloti nodulation factors to suspension-cultured alfalfa cells induced a slight acidification of the culture medium, instead of an alkalinization, but no oxidative burst. Received: 23 November 1998 / Accepted: 23 June 1999     

Genetic Diversity and Dynamics of Sinorhizobium meliloti Populations Nodulating Different Alfalfa Cultivars in Italian Soils

We analyzed the genetic diversity of 531 Sinorhizobium meliloti strains isolated from nodules of Medicago sativa cultivars in two different Italian soils during 4 years of plant growth. The isolates were analyzed for DNA polymorphism with the random amplified polymorphic DNA method. The populations showed a high level of genetic polymorphism distributed throughout all the isolates, with 440 different haplotypes. Analysis of molecular variance allowed us to relate the genetic structure of the symbiotic population to various factors, including soil type, alfalfa cultivar, individual plants within a cultivar, and time. Some of these factors significantly affected the genetic structure of the population, and their relative influence changed with time. At the beginning of the experiment, the soil of origin and, even more, the cultivar significantly influenced the distribution of genetic variability of S. meliloti. After 3 years, the rhizobium population was altered; it showed a genetic structure based mainly on differences among plants, while the effects of soil and cultivar were not significant.     

Effects of Engineered Sinorhizobium meliloti on Cytokinin Synthesis and Tolerance of Alfalfa to Extreme Drought Stress

Cytokinin is required for the initiation of leguminous nitrogen fixation nodules elicited by rhizobia and the delay of the leaf senescence induced by drought stress. A few free-living rhizobia have been found to produce cytokinin. However, the effects of engineered rhizobia capable of synthesizing cytokinin on host tolerance to abiotic stresses have not yet been described. In this study, two engineered Sinorhizobium strains overproducing cytokinin were constructed. The tolerance of inoculated alfalfa plants to severe drought stress was assessed. The engineered strains, which expressed the Agrobacterium ipt gene under the control of different promoters, synthesized more zeatins than the control strain under free-living conditions, but their own growth was not affected. After a 4-week inoculation period, the effects of engineered strains on alfalfa growth and nitrogen fixation were similar to those of the control strain under nondrought conditions. After being subjected to severe drought stress, most of the alfalfa plants inoculated with engineered strains survived, and the nitrogenase activity in their root nodules showed no apparent change. A small elevation in zeatin concentration was observed in the leaves of these plants. The expression of antioxidant enzymes increased, and the level of reactive oxygen species decreased correspondingly. Although the ipt gene was transcribed in the bacteroids of engineered strains, the level of cytokinin in alfalfa nodules was identical to that of the control. These findings suggest that engineered Sinorhizobium strains synthesizing more cytokinin could improve the tolerance of alfalfa to severe drought stress without affecting alfalfa nodulation or nitrogen fixation.     

Sinorhizobium meliloti Mutants Deficient in Phosphatidylserine Decarboxylase Accumulate Phosphatidylserine and Are Strongly Affected during Symbiosis with Alfalfa

Sinorhizobium meliloti contains phosphatidylglycerol, cardiolipin, phosphatidylcholine, and phosphatidylethanolamine (PE) as major membrane lipids. PE is formed in two steps. In the first step, phosphatidylserine synthase (Pss) condenses serine with CDP-diglyceride to form phosphatidylserine (PS), and in the second step, PS is decarboxylated by phosphatidylserine decarboxylase (Psd) to form PE. In this study we identified the sinorhizobial psd gene coding for Psd. A sinorhizobial mutant deficient in psd is unable to form PE but accumulates the anionic phospholipid PS. Properties of PE-deficient mutants lacking either Pss or Psd were compared with those of the S. meliloti wild type. Whereas both PE-deficient mutants grew in a wild-type-like manner on many complex media, they were unable to grow on minimal medium containing high phosphate concentrations. Surprisingly, the psd-deficient mutant could grow on minimal medium containing low concentrations of inorganic phosphate, while the pss-deficient mutant could not. Addition of choline to the minimal medium rescued growth of the pss-deficient mutant, CS111, to some extent but inhibited growth of the psd-deficient mutant, MAV01. When the two distinct PE-deficient mutants were analyzed for their ability to form a nitrogen-fixing root nodule symbiosis with their alfalfa host plant, they behaved strikingly differently. The Pss-deficient mutant, CS111, initiated nodule formation at about the same time point as the wild type but did form about 30% fewer nodules than the wild type. In contrast, the PS-accumulating mutant, MAV01, initiated nodule formation much later than the wild type and formed 90% fewer nodules than the wild type. The few nodules formed by MAV01 seemed to be almost devoid of bacteria and were unable to fix nitrogen. Leaves of alfalfa plants inoculated with the mutant MAV01 were yellowish, indicating that the plants were starved for nitrogen. Therefore, changes in lipid composition, including the accumulation of bacterial PS, prevent the establishment of a nitrogen-fixing root nodule symbiosis.Rhizobia are soil bacteria able to form a symbiosis with legume plants, which leads to the formation of nitrogen-fixing root nodules. The establishment and functioning of this symbiosis are based on the recognition of signal molecules, which are produced by both the bacterial and plant partners. Known recognition factors of the bacterial endosymbiont include nodulation (Nod) factors, extracellular polysaccharides, lipopolysaccharides, K antigens, and cyclic glucans (24, 53). These factors are required for nodule formation, the infection process, and the colonization of the root nodule. Recently it was demonstrated that adequate levels of phosphatidylcholine (PC) are also required in order to allow the formation of a fully functional symbiosis between Bradyrhizobium japonicum and its soybean host plant (35). Under conditions of phosphate limitation, Sinorhizobium meliloti replaces the majority of its phospholipids with phosphorus-free membrane lipids, such as sulfolipids, ornithine-containing lipids, and diacylglyceryl-N,N,N-trimethylhomoserine lipids (20). Rhizobial mutants lacking the ability to form any one of these phosphorus-free membrane lipids or all three lipids at the same time form effective nitrogen-fixing root nodules (30, 31), demonstrating that not all major bacterial membrane lipids are required for the onset of a successful symbiosis.Escherichia coli is the prokaryote with the best-studied membrane lipid biosynthesis. In E. coli, three major membrane phospholipids, phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL), are present. Certain functions have been defined for specific membrane phospholipids in E. coli. Anionic phospholipids (PG and CL) were shown to be involved in the initiation of DNA replication (60) and in the translocation of outer membrane precursor proteins (27). The zwitterionic PE is essential for a proper functioning of the electron transfer chain (34), for the assembly and functionality of lactose permease (4, 5), and for motility and chemotaxis (47). Certain specific functions have also been shown for other membrane lipids. Recently PC has been shown to be required for pathogenesis of Legionella pneumophila, Brucella abortus, and Agrobacterium tumefaciens on their hosts (7, 8, 9, 59). The cationic membrane lipid lysyl-phosphatidylglycerol is involved in conferring resistance to cationic antimicrobial peptides of the host''s innate immune system to Staphylococcus aureus (40), and the presence of LPG in Rhizobium tropici also increases resistance to the cationic peptide polymyxin B (52).In the initial step of the pathway leading to PE formation, phosphatidylserine (PS) synthase (Pss) is responsible for the formation of PS from CDP-diacylglycerol and serine (EC 2.7.8.8) (Fig. ​(Fig.1).1). In the subsequent step, PS is decarboxylated by PS decarboxylase (Psd) (EC 4.1.1.65) to yield PE (17, 58). In S. meliloti, PE is a substrate for the enzyme phospholipid N-methyltransferase (PmtA) (15), leading to the formation of PC. A gene coding for the Pss enzyme (pssA) has been found and cloned from prokaryotes (11, 19, 38, 51), lower eukaryotes, such as Saccharomyces cerevisiae (28, 37), and plants (12). In a previous work we described the construction and characterization of an S. meliloti mutant deficient in Pss (51).Open in a separate windowFIG. 1.Biosynthesis of phospholipids in Sinorhizobium meliloti. SAM, S-adenosylmethionine; SAHC, S-adenosylhomocysteine; PgsA, phosphatidylglycerolphosphate synthase; Pgp, phosphatidylglycerolphosphate phosphatase; Cls, cardiolipin synthase; Pss, phosphatidylserine synthase; Psd, phosphatidylserine decarboxylase; PmtA, phospholipid N-methyltransferase; Pcs, phosphatidylcholine synthase.Psds have been described and characterized for a wide range of organisms, including bacteria, such as E. coli (22, 23, 29) and Bacillus subtilis (32), lower eukaryotes, such as S. cerevisiae (6, 54-56) and Plasmodium falciparum (1), plants, such as Arabidopsis thaliana (36, 41), and mammals (CHO [Chinese hamster ovary] cells) (26). All Psd sequences identified so far seem to be phylogenically related (see Fig. S1A in the supplemental material). Interestingly, S. meliloti lacks a good homologue to any of the above-mentioned Psds.Here we describe the identification and characterization of the sinorhizobial psd gene coding for Psd. The mutant MAV01, in which the sinorhizobial psd gene is deleted, accumulated PS to about 20% of total lipids when grown in complex growth medium. We compared the mutant MAV01 to a sinorhizobial mutant deficient in Pss (CS111) (51) under free-living conditions and during symbiosis. The Pss-deficient mutant, CS111, forms about 30% fewer nodules than the wild type on its alfalfa host plant, whereas the PS-accumulating mutant, MAV01, forms 90% fewer nodules than the wild type. Nodule formation in the mutant MAV01 sets in about 20 days later than that in the wild type. The few nodules formed by the psd-deficient mutant seem to be almost devoid of bacteria and are not able to fix nitrogen. Leaves of alfalfa plants inoculated with the mutant MAV01 are yellowish, indicating that the plants are starved for nitrogen. The accumulation of PS, therefore, although allowing wild-type-like growth in different growth media, strongly interferes with nodule development.     

苜蓿根瘤菌自生与共生状态下核糖体蛋白的比较

苜蓿根瘤菌在与宿主植物建立共生关系的过程中,以自生状态进入宿主植物细胞,经过分化发育转变为共生状态的类菌体(Bacteroid)。由于生存环境发生了变化,类菌体在形态、结构和功能方面都产生了很大的改变,其中最为明显的改变是类菌体获得了共生固氮的能力。此时,类菌体中许多与共生相关的基因被激活,蛋白的表达量显著增加。为了探明这种改变是否与合成蛋白质的细胞器-核糖体有关,比较分析了苜蓿根瘤菌在自生和共生状态下核糖体蛋白的表达谱。蛋白质双向电泳结果显示二者之间没有明显的差别,说明类菌体的分化发育过程中核糖体蛋白的形成没有改变。     

Formate-dependent autotrophic growth in Sinorhizobium meliloti

It was found that S. meliloti strain SmA818, which is cured of pSymA, could not grow on defined medium containing only formate and bicarbonate as carbon sources. Growth experiments showed that Rm1021 was capable of formate/bicarbonate-dependent growth, suggesting that it was capable of autotrophic-type growth. The annotated genome of S. meliloti Rm1021 contains three formate dehydrogenase genes. A systematic disruption of each of the three formate dehydrogenase genes, as well as the genes encoding determinants of the Calvin-Benson-Bassham, cycle was carried out to determine which of these determinants played a role in growth on this defined medium. The results showed that S. meliloti is capable of formate-dependent autotrophic growth. Formate-dependent autotrophic growth is dependent on the presence of the chromosomally located fdsABCDG operon, as well as the cbb operon carried by pSymB. Growth was also dependent on the presence of either of the two triose-phosphate isomerase genes (tpiA or tpiB) that are found in the genome. In addition, it was found that fdoGHI carried by pSymA encodes a formate dehydrogenase that allows Rm1021 to carry out formate-dependent respiration. Taken together, the data allow us to present a model of how S. meliloti can grow on defined medium containing only formate and bicarbonate as carbon sources.     

苜蓿中华根瘤菌氮代谢调控相关非编码RNA的研究

为了探究非编码RNA（non-coding RNA,ncRNA）在氮代谢调控过程的作用,以苜蓿中华根瘤菌（Sinorhizobium meliloti）为出发菌株,鉴定并获得了与ntrC基因表达相关的ncRNA,利用lncPRO软件分析苜蓿中华根瘤菌中ncRNA与调控蛋白NtrC相互作用的可能性,最终确定了评分较高的4个基因,即SM2011_c06191、SM2011_c06248、SM2011_c07102和SM2011_c07132,并对其中得分最高的SM2011_c06248基因进行研究。利用Northern blot验证ncRNA的表达,发现富氮处理后其表达水平呈先升高后降低的趋势。实验构建了SM2011_c06248基因敲除菌株,通过qRT-PCR发现SM2011_c06248在自然生长条件下表达无明显规律,富氮处理后,野生型菌株ncRNA表达水平呈先降低后升高再降低的趋势,与自然生长相比,ncRNA在20 min后表达水平明显上调;野生型菌株ntrC、nar基因表达水平呈先降低后升高再降低的趋势;与野生型相比,SM2011_c06248基因敲除菌株SM∷248中ntrC、nar基因表达水平明显下调。实验中构建SM2011_c06248、NtrC双突变菌株SM∷248-NtrC和SM2011_c06248过表达菌株SM:dld-248,qRT-PCR分析表明,与野生型相比,富氮处理后,SM∷248-NtrC菌株nar基因表达量无明显变化,SM:dld-248菌株ntrC、nar基因表达水平显著上调。ncRNA SM2011_c06248可响应环境中氮元素信号变化,对ntrC、nar基因的表达有正调控作用,且ntrC对nar有上位基因效应。     

The biological activity of the Sinorhizobium meliloti glucan

The study of the effect of the periplasmic glucan isolated from the root-nodule bacterium S. meliloti CXM1-188 on the symbiosis of another strain (441) of the same root-nodule bacterium with alfalfa plants showed that this effect depends on the treatment procedure. The pretreatment of alfalfa seedlings with the glucan followed by their bacterization with S. meliloti 441 insignificantly influenced the nodulation parameters of symbiosis (the number of root nodules and their nitrogen-fixing activity) but induced a statistically significant increase in the efficiency of symbiosis (expressed as the masses of the alfalfa overground parts and roots). At the same time, the pretreatment of S. meliloti 441 cells with the glucan brought about a considerable decrease in the nodulation parameters of symbiosis (the number of the root nodules and their nitrogen-fixing activity decreased by 2.5-11 and 7 times, respectively). These data suggest that the stimulating effect of rhizobia on host plants may be due not only to symbiotrophic nitrogen fixation but also to other factors. Depending on the experimental conditions, the treatment of alfalfa plants with the glucan and their bacterization with rhizobial cells enhanced the activity of peroxidase in the alfalfa roots and leaves by 10-39 and 12-27%, respectively.     

The Biological Activity of the Sinorhizobium meliloti Glucan

The study of the effect of periplasmic glucan isolated from the root-nodule bacterium Sinorhizobium meliloti CXM1-188 on the symbiosis of another strain (441) of the same root-nodule bacterium with alfalfa plants showed that this effect depends on the treatment procedure. The pretreatment of alfalfa seedlings with glucan followed by their bacterization with S. meliloti 441 insignificantly influenced the nodulation parameters of symbiosis (the number of root nodules and their nitrogen-fixing activity) but induced a statistically significant increase in the efficiency of symbiosis (expressed as the masses of the alfalfa overground parts and roots). At the same time, the pretreatment of S. meliloti 441 cells with glucan brought about a considerable decrease in the nodulation parameters of symbiosis (the number of root nodules and their nitrogen-fixing activity decreased by 2.5–11 and 7 times, respectively). These data suggest that the stimulating effect of rhizobia on host plants may be due not only to symbiotrophic nitrogen fixation but also to other factors. Depending on the experimental conditions, the treatment of alfalfa plants with glucan and their bacterization with rhizobial cells enhanced the activity of peroxidase in the alfalfa roots and leaves by 10–39 and 12–27%, respectively.     

Increase in Alfalfa Nodulation, Nitrogen Fixation, and Plant Growth by Specific DNA Amplification in Sinorhizobium meliloti

To improve symbiotic nitrogen fixation on alfalfa plants, Sinorhizobium meliloti strains containing different average copy numbers of a symbiotic DNA region were constructed by specific DNA amplification (SDA). A DNA fragment containing a regulatory gene (nodD1), the common nodulation genes (nodABC), and an operon essential for nitrogen fixation (nifN) from the nod regulon region of the symbiotic plasmid pSyma of S. meliloti was cloned into a plasmid unable to replicate in this organism. The plasmid then was integrated into the homologous DNA region of S. meliloti strains 41 and 1021, which resulted in a duplication of the symbiotic region. Sinorhizobium derivatives carrying further amplification were selected by growing the bacteria in increased concentrations of an antibiotic marker present in the integrated vector. Derivatives of strain 41 containing averages of 3 and 6 copies and a derivative of strain 1021 containing an average of 2.5 copies of the symbiotic region were obtained. In addition, the same region was introduced into both strains as a multicopy plasmid, yielding derivatives with an average of seven copies per cell. Nodulation, nitrogenase activity, plant nitrogen content, and plant growth were analyzed in alfalfa plants inoculated with the different strains. The copy number of the symbiotic region was critical in determining the plant phenotype. In the case of the strains with a moderate increase in copy number, symbiotic properties were improved significantly. The inoculation of alfalfa with these strains resulted in an enhancement of plant growth.     

Alfalfa forage digestibility, quality and yield under future climate change scenarios vary with Sinorhizobium meliloti strain

Elevated CO(2) may decrease alfalfa forage quality and in vitro digestibility through a drop in crude protein and an enhancement of fibre content. The aim of the present study was to analyse the effect of elevated CO(2), elevated temperature and Sinorhizobium meliloti strains (102F78, 102F34 and 1032 GMI) on alfalfa yield, forage quality and in vitro dry matter digestibility. This objective is in line with the selection of S. meliloti strains in order to maintain high forage yield and quality under future climate conditions. Plants inoculated with the 102F34 strain showed more DM production than those inoculated with 1032GMI; however, these strains did not show significant differences with 102F78 plants. Neutral or acid detergent fibres were not enhanced in plants inoculated with the 102F34 strain under elevated CO(2) or temperature and hence, in vitro dry matter digestibility was unaffected. Crude protein content, an indicator of forage quality, was negatively related to shoot yield. Plants inoculated with 102F78 showed a similar shoot yield to those inoculated with 102F34, but had higher crude protein content at elevated CO(2) and temperature. Under these climate change conditions, 102F78 inoculated plants produced higher quality forage. However, the higher digestibility of plants inoculated with the 102F34 strain under any CO(2) or temperature conditions makes them more suitable for growing under climate change conditions. In general, elevated CO(2) in combination with high temperature (Climate Change scenario) reduced IVDMD and CP content and enhanced fibre content, which means that animal production will be negatively affected.