Sinorhizobium meliloti nifA is important in fixing nitrogen during symbiosis. A nifA null mutant induces small white invalid nodules in the roots of host plant. The additional phenotypic alterations associated with th...Sinorhizobium meliloti nifA is important in fixing nitrogen during symbiosis. A nifA null mutant induces small white invalid nodules in the roots of host plant. The additional phenotypic alterations associated with the disruption of the nifA gene are reported in this study. Under a free-living state, S. meliloti nifA mutant reduces its ability to swarm on a half-solid plate. Interestingly, the AHL （Acylhomoserine lactones） contents in the nifA mutant are lower than that of the wild type during the lag phase, whereas it is reversed in the logarithmic and stationary phases. Quantitative spectrophotometric assays reveal that the total amount of extracellular proteins of the nifA mutant are lower than that of the wild type. In addition, the mutant abolishes its nodulation competitive ability during symbiosis. These findings indicate that NifA plays a regulatory role in multiple cellular processes in S. meliloti.展开更多
Seventeen Sinorhizobium meliloti strains from seven provinces in China were used to screen highly effective strains for alfalfa cultivar in a greenhouse study and their symbiotic relationship and competitive ability w...Seventeen Sinorhizobium meliloti strains from seven provinces in China were used to screen highly effective strains for alfalfa cultivar in a greenhouse study and their symbiotic relationship and competitive ability were studied in the field. CCBAU30138 was the most effective strain, as evidenced by increase in dry weights. A field experiment showed that the inoculation of alfalfa with CCBAU30138 resulted in increases of 11.9% and 19.6% of dry matter production and crude protein production, respectively, in forage of monocultured plants. The total dry matter yields of alfalfa and tall fescue in binary culture were increased by 16.3% by inoculation of alfalfa with this strain. These results showed that S. rneliloti strain CCBAU30138 was an effective inoculant both in the greenhouse and in the field. The analysis of randomly amplified polymorphic DNA （RAPD） by polymerase chain reaction （PCR） from nodule extracts showed that the strain CCBAU30138 had high competitiveness in the field. It occupied 47.5% of nodules in alfalfa monoculture and 44.4% of nodules in alfalfa-tall fescue binary culture after 20 weeks of growth. In conclusion, a simple system to select highly effective and competitive symbiotic strains specific to alfalfa was established. Using this system, a s.train suitable for the alfalfa cultivar ‘Vector＇ grown in Wuqiao County of Hebei Province was obtained.展开更多
Several studies have demonstrated that the Rhizobium nifA gene is an activator of nitrogen fixation acting in nodule bacteria. To understand the effects of the Sinorhizobium meliloti nifA gene on Alfalfa, the cDNA-AFL...Several studies have demonstrated that the Rhizobium nifA gene is an activator of nitrogen fixation acting in nodule bacteria. To understand the effects of the Sinorhizobium meliloti nifA gene on Alfalfa, the cDNA-AFLP technique was employed to study the changes in gene expression in nifA mutant nodules. Among the approximately 3,000 transcriptderived fragments, 37 had differential expression levels. These expression levels were subsequently confirmed by reverse Northern blot and RT-polymerase chain reaction. Sequence analyses revealed that 21 cDNA fragments corresponded to genes involved in signal communication, protein degradation, nutrient metabolism, cell growth and development.展开更多
Soil pH is a main factor that affects the nodule formation and nitrogen fixation. The largely reduced nitrogen fixation effect caused by acid soil is the ma- jor limitation of alfalfa growth in the south of China. Thu...Soil pH is a main factor that affects the nodule formation and nitrogen fixation. The largely reduced nitrogen fixation effect caused by acid soil is the ma- jor limitation of alfalfa growth in the south of China. Thus selection of anti-acid nodule for inoculating on alfalfa is of great significance in practical production. Using nine candidate rhizobia （ Rhizobium meliloti L. ） as study objects, their colony diameters and the nodule number of each strain inoculated alfalfa variety were investigated at different pH conditions. The YNCY006 strain presented the strongest resistance to acid, while YNCY007 strain presented the weakest. For nedulation, GT13R alfalfa inoculated with YNCY006 or YNCY008 strains was the best symbiosis system in the acidic soil.展开更多
Alfalfa （Medicago sativa L.） is being grown in harsh environment in Iraq and is mostly subjected to abiotic stresses such as drought, salinity, pH and temperature. Both alfalfa and its nitrogen fixing symbiotic bact...Alfalfa （Medicago sativa L.） is being grown in harsh environment in Iraq and is mostly subjected to abiotic stresses such as drought, salinity, pH and temperature. Both alfalfa and its nitrogen fixing symbiotic bacteria Sinorhizobium meliloti are affected by these environmental stresses. Enhancing nitrogen fixation biologically could be achieved through selection of tolerant strains of S. meliloti to these environmental stresses and inoculating them to the crop, also growing tolerant cultivars. This study examines phenotypic diversity for tolerance to drought, salinity, temperature and pH. Sixty isolates sampled from different areas of Iraq. The results revealed high degree of phenotypic diversity in Sinorhizobium populations. Furthermore, the isolates which showed tolerance to drought stress also showed tolerance to salinity and high degree of temperature, indicating direct relationship between three physiological path ways. Also 58.3% of drought tolerant isolates were alkaline tolerant they tolerated up to pH 9, point to say almost all drought tolerant isolates in this study illustrated strong ＋ positive reaction to catalase enzyme. And 91.6% themes were negative for Gelatinase enzyme test. While only 50% of drought sensitive isolates were negative for drought sensitive isolates could grow at high temperature （42 ℃）.展开更多
Though the majority of bacteria can form structured communities known as biofilms, mutations can cause bacterial strains to vary in their ability to form a biofilm. In this study, the apparent diffusion coefficient of...Though the majority of bacteria can form structured communities known as biofilms, mutations can cause bacterial strains to vary in their ability to form a biofilm. In this study, the apparent diffusion coefficient of polystyrene microspheres 0.29 μm in diameter, which were executing Brownian motion inside bacterial colonies, was used as a quantitative parameter of the ability of a strain to form a biofilm and of the biofilm development. The study was performed using five Sinorhizobium meliloti strains, the biofilm-forming strains Rm8530 expR+, Rm8530 exoY, and Rm9034 expG, and the non-biofilm forming strains Rm1021 and Rm9030-2 expA1. The green fluorescent beads were placed with each strain in a separate channel of a microfluidic device. Thus, as the bacterial colonies grew under identical conditions over a 4-day period, the motion of the fluorescent microspheres was recorded and the diffusion coefficients were measured every 24 hours via particle tracking algorithms. It was found that each strain displayed a unique pattern of change in diffusion coefficient over time. Also, for a given biofilm-forming strain, there was a clear correlation between the value of the diffusion coefficient and the appearance and motility of the bacterial community. Thus, the diffusion coefficient can be used to identify different S. meliloti strains, and for the biofilm-forming strains, it is also a quantitative indicator of the stage of biofilm development.展开更多
Soil bacteria Sinorhizobium meliloti had enormous agricultural value, due to their ability in fixing nitrogen symbiotically with an important forage crop legume--alfalfa. The aims of the present study were （1） to is...Soil bacteria Sinorhizobium meliloti had enormous agricultural value, due to their ability in fixing nitrogen symbiotically with an important forage crop legume--alfalfa. The aims of the present study were （1） to isolate indigenous S. meliloti from different field sites in lraq; （2） to evaluate the isolates tolerance to induced drought using polyethylene glycol-6000; （3） assessing genetic diversity and genetic relationships among isolates of natural population with drought tolerant abilities. Drought tolerance study revealed vast variations between Sinorhizobium isolates, the highest tolerant isolates to drought were 12 from total thirty （40%）, tolerated from -3 up to -4 MPa, while the drought sensitive isolates tolerated up to -1.5 MPa, except isolate Bs 58 which tolerated up to -1 Mpa water potential. The growth declined with the increase of drought stress. Cluster analysis based on Polymerase Chain Reaction-Restriction Fragment Length Polymorphism （PCR-RFLP） of the 16S rDNA showed two divergent groups with 41% similarity, the first group included three drought sensitive isolates （Bs 44, Bs 54 and Bs 58）, the second group comprised the rest nine isolates （moderate and high drought tolerant）, except for Bs 55 which was drought sensitive isolate, all isolates in the two groups showed no differences between them. The PCR-RFLP of 16S rDNA method revealed a genetic variance between the drought sensitive and tolerant isolates.展开更多
The 660 bp region between nodD3 P1 promoter and the following coding region of Rnizopium meliloti has been studied. This region is designated 'downstream sequences' . it consists of two potential open reading ...The 660 bp region between nodD3 P1 promoter and the following coding region of Rnizopium meliloti has been studied. This region is designated 'downstream sequences' . it consists of two potential open reading frames, ORF1 and ORF2. Studies on the role of the downstream sequences on the activity of nooD3 P1 with nod D3(P1)-/acZ fusion show that deletion of the seguences containing ORF2 causes the increase of the activity of the fusion; on the contrary, addition of extra copies of ORF2 markedly decreases the activity of the fusion. These results indicate that the product of ORF2 plays a negative role in the expression of nod D3.展开更多
The nifA gene is an important regulatory gene and its product, NifA protein, regulates the expression of many nif genes involved in the nitrogen fixation process. We introduced multiple copies of the constitutively ex...The nifA gene is an important regulatory gene and its product, NifA protein, regulates the expression of many nif genes involved in the nitrogen fixation process. We introduced multiple copies of the constitutively expressed Sinorhizobium meliloti (Sm) or Enterobacter cloacae (Ec) nifA gene into both the nifA mutant strain SmY and the wild-type strain Sm1021. Root nodules produced by SmY containing a constitutively expressed Sm nifA gene were capable of fixing nitrogen, while nodules produced by SmY containing the Ec nifA gene remained unable to fix nitrogen, as is the case for SmY itself. However, transfer of an additional Sm nifA gene into Sm1021 improved the nitrogen-fixing efficiency of root nodules to a greater extent than that observed upon transfer of the Ec nifA gene into Sm1021. Comparative analysis of amino acid sequences between Sm NifA and Ec NifA showed that the N-terminal domain was the least similar, but this domain is indispensable for complementation of the Fix? phenotype of SmY by Sm NifA. We conclude that more than one domain is involved in determining functional differences between Sm NifA and Ec NifA.展开更多
In Sinorhizobium meliloti, the nodD3 gene is transcriptionally controlled by two promot-ers, P1 and P2. Under P1, there is a 660 bp sequence including a small open reading frame, ORF2, followed by the nodD3 coding reg...In Sinorhizobium meliloti, the nodD3 gene is transcriptionally controlled by two promot-ers, P1 and P2. Under P1, there is a 660 bp sequence including a small open reading frame, ORF2, followed by the nodD3 coding region. Genetic analysis using the different deletions on the 3′ends of P1 downstream sequence showed that the downstream sequence +1—+125nt is es-sential for P1 expression. Complementation, mutations and nodulation tests demonstrated that the ORF2 auto-represses P1 expression, while the P1 downstream sequence +1—+125nt counteracts it.展开更多
Using luc gene as a reporter to study the activation of Rhizobium meliloti nif/fix genes in thedevelopment of symbiosis,the authors observed that nodule development and nitrogen fixation were inhibitedby both multicop...Using luc gene as a reporter to study the activation of Rhizobium meliloti nif/fix genes in thedevelopment of symbiosis,the authors observed that nodule development and nitrogen fixation were inhibitedby both multicopy promoters of nifHDK and fixABCX.The phenotype of R.meliloti containing multicopynif/fix promoters appeared exactly like that of nifA mutant.Using lacZ as a reporter,the authors got the same re-sults.By contrast,the rhizobia containing low-copy promoters of nif/fix genes were normal fornodule development and nitrogen fixation.These results substantiate the evidence that the product of nifAgene not only acts as a transcriptional activator of nif/fix genes,but also plays an important role in thedevelopment of root nodules.展开更多
In free-living state, the nifHDK promoter P1 of Rhizobium meliloti is induced in response to mi-croaerobiosis and expressed to a high level, while the fixABCX promoter P2 is not. The sequences upstream from both P1 an...In free-living state, the nifHDK promoter P1 of Rhizobium meliloti is induced in response to mi-croaerobiosis and expressed to a high level, while the fixABCX promoter P2 is not. The sequences upstream from both P1 and P2 share extended homology (about 85% ), which are about 160 bp in length, but the sequences downstream of the respective transcriptional start site are different. When the downstream sequence (DS) of P2 was replaced by the corresponding fragment from+ 17 to + 61 of P1, the expression of P2 is greatly increased under free-living condi-tion by lowering the oxygen tension, and the activity of P2 promoter can also be significantly enhanced in E. coli by the NifA protein. The difference between the DS regions of P1 and P2 promoter resulted in different expressions of P1 and P2 promoter under free-living microaerobic condition and in E. coli. The expression of P2 does not depend on the downstream sequences from the promoter element during symbiosis. Primer extension experiments identified the tran-scriptional start site of P2. Transcription from P2 was not changed when P2 promoter region was inserted by P1 DS. Under symbiotic conditions, levels of expression of P2 were independent of the P1 DS region. It indicates that the reg-ulations of P2 under symbiotic conditions are different from those under free-living conditions.展开更多
基金This work was supported by the National Grand Fundamental Research 973 Project (No. 2001CB108901)National Science Foundation of China (No. 30400267).
文摘Sinorhizobium meliloti nifA is important in fixing nitrogen during symbiosis. A nifA null mutant induces small white invalid nodules in the roots of host plant. The additional phenotypic alterations associated with the disruption of the nifA gene are reported in this study. Under a free-living state, S. meliloti nifA mutant reduces its ability to swarm on a half-solid plate. Interestingly, the AHL （Acylhomoserine lactones） contents in the nifA mutant are lower than that of the wild type during the lag phase, whereas it is reversed in the logarithmic and stationary phases. Quantitative spectrophotometric assays reveal that the total amount of extracellular proteins of the nifA mutant are lower than that of the wild type. In addition, the mutant abolishes its nodulation competitive ability during symbiosis. These findings indicate that NifA plays a regulatory role in multiple cellular processes in S. meliloti.
基金Project supported by the National "Eleventh Five Years Plan" Key Project on Science and Technology (No. 2006BAD02A15)the National Natural Science Foundation of China (No. 30671222).
文摘Seventeen Sinorhizobium meliloti strains from seven provinces in China were used to screen highly effective strains for alfalfa cultivar in a greenhouse study and their symbiotic relationship and competitive ability were studied in the field. CCBAU30138 was the most effective strain, as evidenced by increase in dry weights. A field experiment showed that the inoculation of alfalfa with CCBAU30138 resulted in increases of 11.9% and 19.6% of dry matter production and crude protein production, respectively, in forage of monocultured plants. The total dry matter yields of alfalfa and tall fescue in binary culture were increased by 16.3% by inoculation of alfalfa with this strain. These results showed that S. rneliloti strain CCBAU30138 was an effective inoculant both in the greenhouse and in the field. The analysis of randomly amplified polymorphic DNA （RAPD） by polymerase chain reaction （PCR） from nodule extracts showed that the strain CCBAU30138 had high competitiveness in the field. It occupied 47.5% of nodules in alfalfa monoculture and 44.4% of nodules in alfalfa-tall fescue binary culture after 20 weeks of growth. In conclusion, a simple system to select highly effective and competitive symbiotic strains specific to alfalfa was established. Using this system, a s.train suitable for the alfalfa cultivar ‘Vector＇ grown in Wuqiao County of Hebei Province was obtained.
文摘Several studies have demonstrated that the Rhizobium nifA gene is an activator of nitrogen fixation acting in nodule bacteria. To understand the effects of the Sinorhizobium meliloti nifA gene on Alfalfa, the cDNA-AFLP technique was employed to study the changes in gene expression in nifA mutant nodules. Among the approximately 3,000 transcriptderived fragments, 37 had differential expression levels. These expression levels were subsequently confirmed by reverse Northern blot and RT-polymerase chain reaction. Sequence analyses revealed that 21 cDNA fragments corresponded to genes involved in signal communication, protein degradation, nutrient metabolism, cell growth and development.
基金Supported by National Natural Science Foundation of China(31060324)Natural Science Foundation of Yunnan Province(2012FB148,2008CD031)Natural Science Key Fund of Yunnan Provincial Education Department(2012Z021)
文摘Soil pH is a main factor that affects the nodule formation and nitrogen fixation. The largely reduced nitrogen fixation effect caused by acid soil is the ma- jor limitation of alfalfa growth in the south of China. Thus selection of anti-acid nodule for inoculating on alfalfa is of great significance in practical production. Using nine candidate rhizobia （ Rhizobium meliloti L. ） as study objects, their colony diameters and the nodule number of each strain inoculated alfalfa variety were investigated at different pH conditions. The YNCY006 strain presented the strongest resistance to acid, while YNCY007 strain presented the weakest. For nedulation, GT13R alfalfa inoculated with YNCY006 or YNCY008 strains was the best symbiosis system in the acidic soil.
文摘Alfalfa （Medicago sativa L.） is being grown in harsh environment in Iraq and is mostly subjected to abiotic stresses such as drought, salinity, pH and temperature. Both alfalfa and its nitrogen fixing symbiotic bacteria Sinorhizobium meliloti are affected by these environmental stresses. Enhancing nitrogen fixation biologically could be achieved through selection of tolerant strains of S. meliloti to these environmental stresses and inoculating them to the crop, also growing tolerant cultivars. This study examines phenotypic diversity for tolerance to drought, salinity, temperature and pH. Sixty isolates sampled from different areas of Iraq. The results revealed high degree of phenotypic diversity in Sinorhizobium populations. Furthermore, the isolates which showed tolerance to drought stress also showed tolerance to salinity and high degree of temperature, indicating direct relationship between three physiological path ways. Also 58.3% of drought tolerant isolates were alkaline tolerant they tolerated up to pH 9, point to say almost all drought tolerant isolates in this study illustrated strong ＋ positive reaction to catalase enzyme. And 91.6% themes were negative for Gelatinase enzyme test. While only 50% of drought sensitive isolates were negative for drought sensitive isolates could grow at high temperature （42 ℃）.
文摘Though the majority of bacteria can form structured communities known as biofilms, mutations can cause bacterial strains to vary in their ability to form a biofilm. In this study, the apparent diffusion coefficient of polystyrene microspheres 0.29 μm in diameter, which were executing Brownian motion inside bacterial colonies, was used as a quantitative parameter of the ability of a strain to form a biofilm and of the biofilm development. The study was performed using five Sinorhizobium meliloti strains, the biofilm-forming strains Rm8530 expR+, Rm8530 exoY, and Rm9034 expG, and the non-biofilm forming strains Rm1021 and Rm9030-2 expA1. The green fluorescent beads were placed with each strain in a separate channel of a microfluidic device. Thus, as the bacterial colonies grew under identical conditions over a 4-day period, the motion of the fluorescent microspheres was recorded and the diffusion coefficients were measured every 24 hours via particle tracking algorithms. It was found that each strain displayed a unique pattern of change in diffusion coefficient over time. Also, for a given biofilm-forming strain, there was a clear correlation between the value of the diffusion coefficient and the appearance and motility of the bacterial community. Thus, the diffusion coefficient can be used to identify different S. meliloti strains, and for the biofilm-forming strains, it is also a quantitative indicator of the stage of biofilm development.
文摘Soil bacteria Sinorhizobium meliloti had enormous agricultural value, due to their ability in fixing nitrogen symbiotically with an important forage crop legume--alfalfa. The aims of the present study were （1） to isolate indigenous S. meliloti from different field sites in lraq; （2） to evaluate the isolates tolerance to induced drought using polyethylene glycol-6000; （3） assessing genetic diversity and genetic relationships among isolates of natural population with drought tolerant abilities. Drought tolerance study revealed vast variations between Sinorhizobium isolates, the highest tolerant isolates to drought were 12 from total thirty （40%）, tolerated from -3 up to -4 MPa, while the drought sensitive isolates tolerated up to -1.5 MPa, except isolate Bs 58 which tolerated up to -1 Mpa water potential. The growth declined with the increase of drought stress. Cluster analysis based on Polymerase Chain Reaction-Restriction Fragment Length Polymorphism （PCR-RFLP） of the 16S rDNA showed two divergent groups with 41% similarity, the first group included three drought sensitive isolates （Bs 44, Bs 54 and Bs 58）, the second group comprised the rest nine isolates （moderate and high drought tolerant）, except for Bs 55 which was drought sensitive isolate, all isolates in the two groups showed no differences between them. The PCR-RFLP of 16S rDNA method revealed a genetic variance between the drought sensitive and tolerant isolates.
文摘The 660 bp region between nodD3 P1 promoter and the following coding region of Rnizopium meliloti has been studied. This region is designated 'downstream sequences' . it consists of two potential open reading frames, ORF1 and ORF2. Studies on the role of the downstream sequences on the activity of nooD3 P1 with nod D3(P1)-/acZ fusion show that deletion of the seguences containing ORF2 causes the increase of the activity of the fusion; on the contrary, addition of extra copies of ORF2 markedly decreases the activity of the fusion. These results indicate that the product of ORF2 plays a negative role in the expression of nod D3.
文摘The nifA gene is an important regulatory gene and its product, NifA protein, regulates the expression of many nif genes involved in the nitrogen fixation process. We introduced multiple copies of the constitutively expressed Sinorhizobium meliloti (Sm) or Enterobacter cloacae (Ec) nifA gene into both the nifA mutant strain SmY and the wild-type strain Sm1021. Root nodules produced by SmY containing a constitutively expressed Sm nifA gene were capable of fixing nitrogen, while nodules produced by SmY containing the Ec nifA gene remained unable to fix nitrogen, as is the case for SmY itself. However, transfer of an additional Sm nifA gene into Sm1021 improved the nitrogen-fixing efficiency of root nodules to a greater extent than that observed upon transfer of the Ec nifA gene into Sm1021. Comparative analysis of amino acid sequences between Sm NifA and Ec NifA showed that the N-terminal domain was the least similar, but this domain is indispensable for complementation of the Fix? phenotype of SmY by Sm NifA. We conclude that more than one domain is involved in determining functional differences between Sm NifA and Ec NifA.
基金This work was supported by the National High Technology 863 Programs of China the 973 Project of the Ministry of Science and Technology (Grant No. 001CB108901) and the fund of the Shanghai Institutes of Biological Sciences.
文摘In Sinorhizobium meliloti, the nodD3 gene is transcriptionally controlled by two promot-ers, P1 and P2. Under P1, there is a 660 bp sequence including a small open reading frame, ORF2, followed by the nodD3 coding region. Genetic analysis using the different deletions on the 3′ends of P1 downstream sequence showed that the downstream sequence +1—+125nt is es-sential for P1 expression. Complementation, mutations and nodulation tests demonstrated that the ORF2 auto-represses P1 expression, while the P1 downstream sequence +1—+125nt counteracts it.
基金the National Natural Science Foundation of China.
文摘Using luc gene as a reporter to study the activation of Rhizobium meliloti nif/fix genes in thedevelopment of symbiosis,the authors observed that nodule development and nitrogen fixation were inhibitedby both multicopy promoters of nifHDK and fixABCX.The phenotype of R.meliloti containing multicopynif/fix promoters appeared exactly like that of nifA mutant.Using lacZ as a reporter,the authors got the same re-sults.By contrast,the rhizobia containing low-copy promoters of nif/fix genes were normal fornodule development and nitrogen fixation.These results substantiate the evidence that the product of nifAgene not only acts as a transcriptional activator of nif/fix genes,but also plays an important role in thedevelopment of root nodules.
基金Project supported by the National Natural Science Foundation of China.
文摘In free-living state, the nifHDK promoter P1 of Rhizobium meliloti is induced in response to mi-croaerobiosis and expressed to a high level, while the fixABCX promoter P2 is not. The sequences upstream from both P1 and P2 share extended homology (about 85% ), which are about 160 bp in length, but the sequences downstream of the respective transcriptional start site are different. When the downstream sequence (DS) of P2 was replaced by the corresponding fragment from+ 17 to + 61 of P1, the expression of P2 is greatly increased under free-living condi-tion by lowering the oxygen tension, and the activity of P2 promoter can also be significantly enhanced in E. coli by the NifA protein. The difference between the DS regions of P1 and P2 promoter resulted in different expressions of P1 and P2 promoter under free-living microaerobic condition and in E. coli. The expression of P2 does not depend on the downstream sequences from the promoter element during symbiosis. Primer extension experiments identified the tran-scriptional start site of P2. Transcription from P2 was not changed when P2 promoter region was inserted by P1 DS. Under symbiotic conditions, levels of expression of P2 were independent of the P1 DS region. It indicates that the reg-ulations of P2 under symbiotic conditions are different from those under free-living conditions.
