Shanjiong Shen

The N-terminal domain of NifA determines the temperature sensitivity of NifA in Klebsiella pneumoniae and Enterobacter cloacae

The NifA protein is the central regulator of the nitrogen fixation genes. It activates transcription ofnif genes by an alternative holoenzyme form of RNA polymerase containing the σ54 factor. The NifA protein fromKlebsiella pneumoniae consists of the N-terminal domain of unknown function, the central catalytic domain with ATPase activity and the C-terminal DNA-binding domain. TheKp NifA protein is sensitive to temperature, while theEnterobacter cloacae NifA protein is less sensitive to temperature thanKp NifA. Our results show that the N-terminal domain of NifA plays the decisive role in the temperature sensitivity of the protein.

NifL,an antagonistic regulator of NifA interacting with NifA

Nitrogen fixation (nif) genes of diazotrophic enteric bacteria,Enterobacter cloacae orKlebsiella pneumoniae, are regulated bynif LA operon, in which thenif A product, NifA positively regulatesnif gene transcription, whereas the nifL product NifL represses it under oxygen or in excess of fixed nitrogen. Two-hybrid system was used to detect the possible interaction between NifA and NifL. The preliminary results illustrate that NifL does interact with NifA. The interaction between NifL and NtrC has also been shown.Nitrogen fixation (nif) genes of diazotrophic enteric bacteria,Enterobacter cloacae orKlebsiella pneumoniae, are regulated bynif LA operon, in which thenif A product, NifA positively regulatesnif gene transcription, whereas the nifL product NifL represses it under oxygen or in excess of fixed nitrogen. Two-hybrid system was used to detect the possible interaction between NifA and NifL. The preliminary results illustrate that NifL does interact with NifA. The interaction between NifL and NtrC has also been shown.

Binding of activator SyrM to the site ofnodD3 P1 region ofRhizobium meliloti.

The nodD3 gene ofRhizobium meliloti is transcribed via promoter P1 or P2. Gel retardation assay showed binding of SyrM to the P1 upstream region of nodD3. DNaseI footprint analysis demonstrated that the binding site of SyrM in nodD3 P1 region consists of two inverted repeat sequences arranged in tandem. SyrM seems to bind to DNA in the form of dimer or tetramer and requires the two inverted repeat sequences for binding.

Functional difference between Sinorhizobium meliloti NifA and Enterobacter cloacae NifA

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.

Use of bacterial two-hybrid system to investigate the molecular interaction between the regulators NifA and NifL of Enterobacter cloacae

Expression of the nitrogen fixation (nif) genes is tightly regulated by two proteins NifA and NifL in the γ-subdivision of the proteobacteria. NifA is a transcriptional activator, which can be inactivated by NifL in the presence of oxygen or excess fixed nitrogen. A direct interaction betweenE. cloacae NifL and NifA was detected using the bacterial two-hybrid system. This interaction was accelerated in the presence of fixed nitrogen, while oxygen had no effect. NifL proteins, with their C-terminus being deleted, completely lost the ability to interact with NifA. The data suggest that the C-terminal domain of NifL acts as a sensor of the nitrogen status of the cell and mediates interaction with NifA.

Regulatory role of the sequences downstream fromnodD3 P1 promoter ofRhizobium meliloti

The 660 bp region betweennodD3 P1 promoter and the following coding region ofRhizobium 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 ofnocD3 P1 with nod D3(P1)-IacZ fusion show that deletion of the sequences 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 ofnod D3.

Promoter of soybean early nodulin gene enod2B is induced by rhizobial Nod factors in transgenic rice

Nod factors, which are signaling molecules produced byRhizobia, are the principal determinants of host specificity inRhizobium-legume symbiosis. Nod factors can elicit a number of characteristic developmental responses in the roots of legumes, such as depolarization of the membrane potential in epidermal cells, specific expression of early nodulin genes and changes in the flux of calcium in root hairs, deformation of root hairs, cell division in the root cortex and formation of the nodule primordium. Whether the rice plant can respond to signaling molecules (i.e. Nod factors) is an important question, as it could establish the potential for symbiotic nitrogen fixation in rice. The promoter of the soybean (Glycine max) early nodulin geneGmenod2B fused to the β-glucuronidase (GUS) reporter gene was used as a molecular marker to explore whether Nod factors can be recognized by rice cells as signaling molecules. Transgenic rice plants harboring the chimeric geneGmenod2BP-GUS were obtained via anAgrobacterium tumefaciens-mediated system. NodNGR factors produced by a broad-host-rangeRhizobium strain NGR234(pA28) were used as probes to investigate the activity of theGmenod2B promoter in rice. Our results showed that the early nodulin geneGmenod2B promoter was induced by NodNGR factors in transgenic rice, and that it was specifically expressed in rice plant roots. Moreover, GUS gene expression driven by theGmenod2B promoter in transgenic rice was regulated by nitrogen status. These findings indicated that rice possessed the ability to respond to Nod factor signals, and that this signal transduction system resulted in activation of theGmenod2B promoter. Thus, we predict that the Nod-factor inducible nodulin expression system, which is similar toRhizobium-legume symbiosis, may also exist in rice.

Analysis of the downstream region of nodD3 P1 promoter by deletion and complementation tests in Sinorhizobium meliloti.

In Sinorhizobium meliloti, the nodD3 gene is transcriptionally controlled by two promoters, 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 essential 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.

Cloning and characterization of the glutamate dehydrogenase gene inBacillus licheniformis.

ThegdhA genes of IRC-3 GDH−strain and IRC-8 GDH+ strain were cloned, and they both successfully complemented the nutritional lesion of anE. coli glutamate auxotroph, Q100 GDH−. However, thegdhA gene from the mutant IRC-8 GDH+ strain failed to complement the glutamate deficiency of the wild type strain IRC-3. ThegdhA genes of the wild type and mutant origin were sequenced separately. No nucleotide difference was detected between them. Further investigations indicated that thegdhA genes were actively expressed in both the wild type and the mutant. Additionally, no GDH inhibitor was found in the wild type strain IRC-3. It is thus proposed that the inactivity of GDH in wild type is the result of the deficiency at the post-translational level of thegdhA expression. Examination of the deduced amino acid sequence ofBacillus licheniformis GDH revealed the presence of the motifs characteristic of the family I-type hexameric protein, while the GDH ofBacillus subtilis belongs to family II.

Characterization of sequences downstream from transcriptional start site ofRhizobium meliloti nifHDK promoter.

In freeliving state, the nifHDK promoter P1 ofRhizbium meliloti is induced in response to microaerobiosis 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 freeliving condition by lowering the oxygen tension, and the activity of P2 promoter can also be significantly enhanced inE. 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 freeliving microaerobic condition and inE. coli. The expression of P2 does not depend on the downstream sequences from the promoter element during symbiosis. Primer extension experiments identified the transcriptional 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 regulations of P2 under symbiotic conditions are different from those under freeliving conditions.