Gene expression profiles of a Si- norhizobium meliloti 1021 nifA mutant and wild type nodule bacteria were compared using whole genome microarrays. The results revealed a large scale al- teration of gene expression (6...Gene expression profiles of a Si- norhizobium meliloti 1021 nifA mutant and wild type nodule bacteria were compared using whole genome microarrays. The results revealed a large scale al- teration of gene expression (601 genes) in the nifA minus background. The loss of NifA altered the ex- pression of many functional groups of genes (mac- romolecular metabolism, TCA cycle and respiration, nodulation and nitrogen fixation) and may lead to quite different life stages of the nodule bacteria. Upregulation of fixK and its associated genes was observed in the nifA mutant nodule bacteria. Addi- tional quantitative real-time PCR experiments re- vealed that the transcript levels of fixLJ were signifi- cantly upshifted in the nifA mutant nodule bacteria. Putative NifA binding sites were predicted by a statis- tical method in the upstream sequences of 13 differ- entially regulated genes from the nifA- transcriptome.展开更多
The interaction between PII and NifA in A. brasilense Sp7 was investigated by using the yeast two-hybrid system. Our experimental results showed that PII directly interacted with the entire NifA protein and its N-term...The interaction between PII and NifA in A. brasilense Sp7 was investigated by using the yeast two-hybrid system. Our experimental results showed that PII directly interacted with the entire NifA protein and its N-terminal domain,but did not interact with the central domain and the C-terminal domain of NifA. No interaction happened if glnB coding for PII was frame-shift mutated. Pz, a homolog of PII, had no interation with NifA.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
Previous investigations have shown that nifA gene is involved in nodulation and symbiotic nitrogen fixation regulation of Rhizobium. We study the role of nifA on nodulation of leguminous plants. We found that Sinorhiz...Previous investigations have shown that nifA gene is involved in nodulation and symbiotic nitrogen fixation regulation of Rhizobium. We study the role of nifA on nodulation of leguminous plants. We found that Sinorhizo-bium fredii harboring multi-copy plasmid carrying the con-stitutively expressed Klebsiella pneumoniae nifA exhibited an increase of nodulation activity and nodulation competitiveness on soybean plants. The Nod-factor secreted by the rhizobia cells containing the multi-copied nifA was assayed, and preliminary results showed that S. fredii containing the multi-copy plasmid carrying nifA produced higher strength of Nod-factor than the rhizobia containing the same plasmid carrying the vector did.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
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
The NifA protein is the central regulator of the nitrogen fixation genes. It activates transcription of nif genes by an alternative holoenzyme form of RNA polymerase containing the σ54 factor. The NifA protein from K...The NifA protein is the central regulator of the nitrogen fixation genes. It activates transcription of nif genes by an alternative holoenzyme form of RNA polymerase containing the σ54 factor. The NifA protein from Klebsiella pneumoniae consists of the N-terminal domain of unknown function, the central catalytic domain with ATPase activity and the C-terminal DNA-binding domain. The Kp NifA protein is sensitive to temperature, while the Enterobacter cloacae NifA protein is less sensitive to temperature than Kp NifA. Our results show that the N-terminal domain of NifA plays the decisive role in the temperature sensitivity of the protein.展开更多
Azospirillum brasilense is a diazotroph associated with many important agricultural crops and shows potential as a biofertilizer. NifA, the transcriptional activator of nitrogen fixation (nif) genes, and GlnB, one of ...Azospirillum brasilense is a diazotroph associated with many important agricultural crops and shows potential as a biofertilizer. NifA, the transcriptional activator of nitrogen fixation (nif) genes, and GlnB, one of PII signal transduction family protein, are key proteins in the regulation of nitrogen fixation in A. brasilense. It was previously reported that the regulation of NifA activity in A. brasilense depends on GlnB. We report here that GlnB was found to interact directly with the N-terminal domain of NifA in vivo under nitrogen-free conditions and the N-terminal mutant of NifA in which the Tyr residues at position 18 and 53 were replaced by Phe (NifA-N-Y18/53F) strengthened the interaction with GlnB. Moreover, we also found that the amino acid residues 66―88 and 165―176 in N-terminus of NifA are responsible for the interaction with GlnB.展开更多
NifA in Azospirillum brasilense plays a key role in regulating the synthesis and activity of nitrogenase in re- sponse to ammonia and oxygen available. In this work we used the yeast two-hybrid system to identify the ...NifA in Azospirillum brasilense plays a key role in regulating the synthesis and activity of nitrogenase in re- sponse to ammonia and oxygen available. In this work we used the yeast two-hybrid system to identify the proteins that interact with NifA. The nifA gene was fused to the yeast two-hybrid vector pGBD-C2, and three A. brasilense Sp7 genomic libraries for use in yeast two-hybrid studies were constructed. Screening of the libraries identified four clones encoding proteins that interact with NifA. The confirmation of the interactions of each gene product of the four clones and NifA were carried out by exchanging the vectors for nifA and the four clones and by mutageneses of the four clones with shift reading frame experiments in yeast two-hybrid studies. DNA sequence analyses showed that two clones en- code proteins containing PAS domains that play an impor- tant role in signal transduction. One clone has high similarity with the fhuE gene of Escherichia coli, whose gene product is involved in iron uptake and transportation, and the other clone encodes an unknown protein.展开更多
基金This work was supported by the Chinese National Key Program for Basic Research （973） （Grant Nos. 001CB108901 ＆ 001CB108902）the National Natural Science Foundation of China （Grant No. 30470940）+1 种基金 Bundesministerium far Bildung und Forschung Germany （Grant No. 031U213D）Deutsche Forschungsgemeinschaft （Grant No. BIZ 7）.
文摘Gene expression profiles of a Si- norhizobium meliloti 1021 nifA mutant and wild type nodule bacteria were compared using whole genome microarrays. The results revealed a large scale al- teration of gene expression (601 genes) in the nifA minus background. The loss of NifA altered the ex- pression of many functional groups of genes (mac- romolecular metabolism, TCA cycle and respiration, nodulation and nitrogen fixation) and may lead to quite different life stages of the nodule bacteria. Upregulation of fixK and its associated genes was observed in the nifA mutant nodule bacteria. Addi- tional quantitative real-time PCR experiments re- vealed that the transcript levels of fixLJ were signifi- cantly upshifted in the nifA mutant nodule bacteria. Putative NifA binding sites were predicted by a statis- tical method in the upstream sequences of 13 differ- entially regulated genes from the nifA- transcriptome.
基金This work was supported by the National 73Project (Grant No. 001CB108904) and the National Natural Science Foundation of China (Grant No.30070407).
文摘The interaction between PII and NifA in A. brasilense Sp7 was investigated by using the yeast two-hybrid system. Our experimental results showed that PII directly interacted with the entire NifA protein and its N-terminal domain,but did not interact with the central domain and the C-terminal domain of NifA. No interaction happened if glnB coding for PII was frame-shift mutated. Pz, a homolog of PII, had no interation with NifA.
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This work was supported by the State "863" High-Tech Programs and the fund of the Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences.
文摘Previous investigations have shown that nifA gene is involved in nodulation and symbiotic nitrogen fixation regulation of Rhizobium. We study the role of nifA on nodulation of leguminous plants. We found that Sinorhizo-bium fredii harboring multi-copy plasmid carrying the con-stitutively expressed Klebsiella pneumoniae nifA exhibited an increase of nodulation activity and nodulation competitiveness on soybean plants. The Nod-factor secreted by the rhizobia cells containing the multi-copied nifA was assayed, and preliminary results showed that S. fredii containing the multi-copy plasmid carrying nifA produced higher strength of Nod-factor than the rhizobia containing the same plasmid carrying the vector did.
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.
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.
文摘The NifA protein is the central regulator of the nitrogen fixation genes. It activates transcription of nif genes by an alternative holoenzyme form of RNA polymerase containing the σ54 factor. The NifA protein from Klebsiella pneumoniae consists of the N-terminal domain of unknown function, the central catalytic domain with ATPase activity and the C-terminal DNA-binding domain. The Kp NifA protein is sensitive to temperature, while the Enterobacter cloacae NifA protein is less sensitive to temperature than Kp NifA. Our results show that the N-terminal domain of NifA plays the decisive role in the temperature sensitivity of the protein.
基金the National Basic Research Program of China (Grant No. 2001CB108904)
文摘Azospirillum brasilense is a diazotroph associated with many important agricultural crops and shows potential as a biofertilizer. NifA, the transcriptional activator of nitrogen fixation (nif) genes, and GlnB, one of PII signal transduction family protein, are key proteins in the regulation of nitrogen fixation in A. brasilense. It was previously reported that the regulation of NifA activity in A. brasilense depends on GlnB. We report here that GlnB was found to interact directly with the N-terminal domain of NifA in vivo under nitrogen-free conditions and the N-terminal mutant of NifA in which the Tyr residues at position 18 and 53 were replaced by Phe (NifA-N-Y18/53F) strengthened the interaction with GlnB. Moreover, we also found that the amino acid residues 66―88 and 165―176 in N-terminus of NifA are responsible for the interaction with GlnB.
基金supported by the National Natural Science Foundation of China(Grant No.30170020)by the Doctoral Fellowship of the Education Ministry of China(Grant No.20010019002).
文摘NifA in Azospirillum brasilense plays a key role in regulating the synthesis and activity of nitrogenase in re- sponse to ammonia and oxygen available. In this work we used the yeast two-hybrid system to identify the proteins that interact with NifA. The nifA gene was fused to the yeast two-hybrid vector pGBD-C2, and three A. brasilense Sp7 genomic libraries for use in yeast two-hybrid studies were constructed. Screening of the libraries identified four clones encoding proteins that interact with NifA. The confirmation of the interactions of each gene product of the four clones and NifA were carried out by exchanging the vectors for nifA and the four clones and by mutageneses of the four clones with shift reading frame experiments in yeast two-hybrid studies. DNA sequence analyses showed that two clones en- code proteins containing PAS domains that play an impor- tant role in signal transduction. One clone has high similarity with the fhuE gene of Escherichia coli, whose gene product is involved in iron uptake and transportation, and the other clone encodes an unknown protein.
