Synthesis of the ferredoxin-like protein FdxN from Rhizobium meliloti bacteroids as a fusion protein in Escherichia coli.

To analyze the overexpression of the Rhizobium meliloti fdxN gene in Escherichia coli, different translational and transcriptional fusions were constructed. The translational signals of R. meliloti fdxN were recognized in E. coli as demonstrated by the use of in-frame lac fusions. Translational fusions consisting of the lacZ or the lpp gene fused in frame to the 3' end of the entire fdxN gene were expressed at high levels in E. coli. In contrast, the wild-type R. meliloti FdxN protein without a C-terminal fusion could only be detected using the very sensitive T7 promoter-polymerase system and not in immunoblots with antibodies against an FdxN-LacZ hybrid protein. Evidently, translational fusions to the 3' end of fdxN had a stabilizing effect on the expression of the fdxN gene. A constitutively expressed transcriptional fdxN fusion, which did not mediate detectable amounts of FdxN protein either in E. coli or in free-living R. meliloti cells, complemented the Fix- phenotype of an R. meliloti fdxN::[Tc] mutant strain to wild-type levels. Therefore, either low amounts of the wild-type FdxN protein are sufficient for symbiotic nitrogen fixation or there are stabilizing factors, which are present only in R. meliloti bacteroids but not in free-living R. meliloti cells. Fusion proteins consisting of FdxN and LacZ or a partial Lpp protein restored the Fix- phenotype of an R. meliloti fdxN mutant to 3 and 11%, respectively, indicating that a C-terminal fusion did not completely abolish the function of FdxN.

Riedel KU ，Masepohl B ，Klipp W ，Pühler A ... -  《CANADIAN JOURNAL OF MICROBIOLOGY》

Functional analysis of the cysteine motifs in the ferredoxin-like protein FdxN of Rhizobium meliloti involved in symbiotic nitrogen fixation.

The Rhizobium meliloti fdxN gene, which is part of the nifA-nifB-fdxN operon, is absolutely required for symbiotic nitrogen fixation. The deduced sequence of the FdxN protein is characterized by two cysteine motifs typical of bacterial-type ferredoxins. The Fix-phenotype of an R. meliloti fdxN::[Tc] mutant could be rescued by the R. leguminosarum fdxN gene, whereas no complementation was observed with nif-associated genes encoding ferredoxins from Bradyrhizobium japonicum, Azotobacter vinelandii, A. chroococcum and Rhodobacter capsulatus. In addition to these heterologous genes, several R. meliloti fdxN mutant genes constructed by site-directed mutagenesis were analyzed. Not only a cysteine residue within the second cysteine motif (position 42), which is known to coordinate the Fe-S cluster in homologous proteins, but also a cysteine located down-stream of this motif (position 61), was found to be essential for the activity of the R. meliloti FdxN protein. Changing the amino acid residue proline in position 56 into methionine resulted in a FdxN mutant protein with decreased activity, whereas changes in positions 35 (Asp35Glu) and 45 (Gly45Glu) had no significant effect on the function of the FdxN mutant proteins. In contrast to bacterial-type ferredoxins, which contain two identical cysteine motifs of the form C-X2-C-X2-C-X3-C, nif-associated ferredoxins, including R. meliloti FdxN, are characterized by two different cysteine motifs. Six "additional" amino acids separate the second (Cys42) and the third cysteine (Cys51) in the C-terminal motif (C-X2-C-X8-C-X3-C).(ABSTRACT TRUNCATED AT 250 WORDS)

Masepohl B ，Kutsche M ，Riedel KU ，Schmehl M ，Klipp W ，Pühler A ... -  《molecular and general genetics》

A Rhizobium meliloti ferredoxin (FdxN) purified from Escherichia coli donates electrons to Rhodobacter capsulatus nitrogenase.

The fdxN gene from Rhizobium meliloti encoding a bacterial-type ferredoxin (FdxN) was expressed in Escherichia coli under the control of the lac promoter. The fdxN gene product was purified under anaerobic conditions by ion-exchange chromatography and gel-filtration steps using an antiserum raised against an FdxN-LacZ fusion protein as a detection system. The purified ferredoxin was shown to be identical to the predicted R. meliloti FdxN protein in its amino acid composition and N-terminal amino acid sequence. Chemical determination of the iron content revealed 8.6 +/- 0.6 mol Fe/mol FdxN. The ultraviolet/visible absorption spectrum of the FdxN protein in the oxidized form exhibited maxima at 284 nm and 378 nm, with an A378/A284 ratio of 0.7. EPR spectroscopy revealed a rhombic signal when FdxN was partially reduced, and a broad signal indicative of spin-spin interaction when fully reduced, suggesting the presence of two Fe-S cluster/ferredoxin polypeptide. Our data suggest that FdxN contains two [4Fe-4S] clusters. Purified FdxN was able to mediate electron transport between illuminated chloroplasts and Rhodobacter capsulatus nitrogenase in vitro.

Riedel KU ，Jouanneau Y ，Masepohl B ，Pühler A ，Klipp W ... -  《european journal of biochemistry》

The Rhizobium meliloti fdxN gene encoding a ferredoxin-like protein is necessary for nitrogen fixation and is cotranscribed with nifA and nifB.

Sequencing of the Rhizobium meliloti DNA region downstream of nifA revealed the existence of nifB, fdxN and ORF3. The molecular weight of the fdxN protein (Mr 6830) and the distribution of cysteine residues in its deduced amino acid sequence is typical for low molecular weight bacterial ferredoxins. Interposon insertion and plasmid integration mutagenesis demonstrated that FdxN is essential for nitrogen fixation in R. meliloti, whereas the predicted translation product of ORF3 (Mr 8708) is not necessary for this process. In contrast, ferredoxin-like proteins, which are encoded by nifB-associated genes, are not required for nitrogen fixation in all other organisms analysed so far. Plasmid integration mutagenesis additionally revealed that nifA, nifB and fdxN form one transcriptional unit. This result was confirmed by complementation analysis of polar interposon insertion mutants of nifA, nifB and fdxN and by complementation of a non-polar nifA deletion mutant. A DNA sequence resembling a typical nif consensus promoter, which is preceded by two putative NifA-binding sites, is located in front of nifB. This nifB promoter can be activated in Escherichia coli by the nifA gene product of Klebsiella pneumoniae to the same level as that of the R. meliloti nifH promoter. In contrast, R. meliloti NifA stimulates the nifH promoter more efficiently than the nifB promoter. This low-level activation of the nifB promoter may be the reason why transcription of nifB and fdxN is initiated primarily at a promoter in front of nifA.

Klipp W ，Reiländer H ，Schlüter A ，Krey R ，Pühler A ... -  《molecular and general genetics》

Transcriptional analysis of two Rhodobacter capsulatus ferredoxins by translational fusion to Escherichia coli lacZ.

Plasmids which contained the translational fusion of Escherichia coli lacZ to Rhodobacter capsulatus ferredoxin genes, fdxN and fdxA, were constructed. Effects of growth conditions on the expression of each ferredoxin were analyzed by measuring the beta-galactosidase activity in R. capsulatus which harbored a corresponding plasmid. Transcription of fdxN::lacZ, the ferredoxin I fusion gene, was regulated at least 100-fold by either NH4+ or O2 but not by illumination, confirming that fdxN belongs to the nif-gene family. Transcription of fdxA::lacZ, the ferredoxin II fusion gene, however, was constant under all the conditions surveyed, suggesting that the protein has some constitutive function(s).

Suetsugu Y ，Saeki K ，Matsubara H 《FEBS LETTERS》