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
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
NifA in Azospirillum brasilense plays a key role in regulating the synthesis of nitrogenase in response to ammonia and oxygen available. Recently, our laboratory has identified four clones, whose gene prodcuts interac...NifA in Azospirillum brasilense plays a key role in regulating the synthesis of nitrogenase in response to ammonia and oxygen available. Recently, our laboratory has identified four clones, whose gene prodcuts interact with NifA, from A. brasilense Sp7 genomic libraries by using the yeast two-hybrid sys- tem with NifA as bait. We are interested in clone S35, one of the four clones, because it contains a PAS-domain coding region. The entire open reading frame (ORF) for the PAS domain-containing protein was isolated and designated as org35 here. org35 gene is 2211-bp long and encodes a protein of 736 aa with a predicted molecular weight of about 78.4 kD. The predicted amino acid sequence of org35 has similarity to some two-component sensor kinase/response regulator hybrids of bacteria. Struc- tural analyses showed that Org35 comprises at least three discrete conserved domains: the N-terminal PAS, the central histidine protein kinase (HPK) and the C-terminal response regulator (RR). The PAS domain of the deduced Org35 protein was found to interact directly with NifA, but the central HPK and the C-terminal RR domains of Org35 were not. These results indicated that interaction between NifA and Org35 was mediated by PAS domain.展开更多
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.
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.
基金This work was supported by the National Natural Science Foundation of China （Grant No. 30470028）.
文摘NifA in Azospirillum brasilense plays a key role in regulating the synthesis of nitrogenase in response to ammonia and oxygen available. Recently, our laboratory has identified four clones, whose gene prodcuts interact with NifA, from A. brasilense Sp7 genomic libraries by using the yeast two-hybrid sys- tem with NifA as bait. We are interested in clone S35, one of the four clones, because it contains a PAS-domain coding region. The entire open reading frame (ORF) for the PAS domain-containing protein was isolated and designated as org35 here. org35 gene is 2211-bp long and encodes a protein of 736 aa with a predicted molecular weight of about 78.4 kD. The predicted amino acid sequence of org35 has similarity to some two-component sensor kinase/response regulator hybrids of bacteria. Struc- tural analyses showed that Org35 comprises at least three discrete conserved domains: the N-terminal PAS, the central histidine protein kinase (HPK) and the C-terminal response regulator (RR). The PAS domain of the deduced Org35 protein was found to interact directly with NifA, but the central HPK and the C-terminal RR domains of Org35 were not. These results indicated that interaction between NifA and Org35 was mediated by PAS domain.
