Nucleotide sequence and genetic analysis of the region essential for functional expression of the gene for ferredoxin I, fdxN, in Rhodobacter capsulatus: sharing of one upstream activator sequence in opposite directions by two operons related to nitrogen

Nucleotide sequencing of the region upstream of two ferredoxin genes, fdxC and fdxN, of Rhodobacter capsulatus revealed the existence of one open reading frame (ORF), ORFU1, in the same orientation as these genes and two other ORFs, ORFU2 and ORFU3, in the opposite orientation. Two potential -24/-12 promoters were found in front of ORFU1 and ORFU2, respectively, and there was a putative upstream activator sequence (UAS) or NifA-binding site between them. The ORFs corresponded to no known nif genes. However, analysis of their putative products showed that the product of ORFU1 (M(r) 47,912) and that of ORFU3 (M(r) 19,090) had a flavodoxin-like domain and a 2[4Fe-4S] ferredoxin-like domain, respectively, and that the product of ORFU2 (M(r) 20,424) was a hydrophobic protein with six potential membrane-spanning portions. Results of interposon mutagenesis and complementation experiments indicated that ORFU2 but not ORFU1 is essential for nitrogen fixation and that additional gene(s) essential for nitrogen fixation must be present in the unsequenced region adjacent to ORFU3. Translational fusion analysis involving lacZYA and fdxN or ORFU3 provided evidence that the putative UAS is responsible for regulation of both ORFU1-fdxC-fdxN and ORFU2-ORFU3 operons in opposite orientations, and that the control of the latter is stricter than that of the former.

Saeki K ，Tokuda K ，Fujiwara T ，Matsubara H ... -  《PLANT AND CELL PHYSIOLOGY》

Genetic analysis of functional differences among distinct ferredoxins in Rhodobacter capsulatus.

Rhodobacter capsulatus has been known to possess two ferredoxins (I and II) with distinct physicochemical and structural properties: ferredoxin I is a 2[4Fe-4S] type and the other is a [3Fe-4S] [4Fe-4S] type. To analyze their possible functional differences, their genes (fdxN and fdxA) were cloned, sequenced, and subjected to interposon mutagenesis experiments. The former gene was adjacent to a gene encoding a chloroplast-type [2Fe-2S] ferredoxin (fdxC). Mutants with inactivated fdxN and/or fdxC were obtained, and they showed virtually no growth under nitrogen-fixing conditions. Complementation experiments confirmed that both fdxN and fdxC were required for nitrogen fixation. On the other hand, we have not been able to disrupt fdxA under the screening conditions surveyed, including conditions that do not require nitrogenase activity for growth, suggesting that ferredoxin II could have an unknown essential role(s). These indicate functional differences among multiple ferredoxins in one bacterium other than in cyanobacterial heterocysts and indispensability of certain ferredoxins in nitrogen fixation other than Rhizobium meliloti FdxN.

Saeki K ，Suetsugu Y ，Tokuda K ，Miyatake Y ，Young DA ，Marrs BL ，Matsubara H ... -  《JOURNAL OF BIOLOGICAL CHEMISTRY》

Identification of a new class of nitrogen fixation genes in Rhodobacter capsulatus: a putative membrane complex involved in electron transport to nitrogenase.

DNA sequence analysis of a 12236 bp fragment, which is located upstream of nifE in Rhodobacter capsulatus nif region A, revealed the presence of ten open reading frames. With the exception of fdxC and fdxN, which encode a plant-type and a bacterial-type ferredoxin, the deduced products of these coding regions exhibited no significant homology to known proteins. Analysis of defined insertion and deletion mutants demonstrated that six of these genes were required for nitrogen fixation. Therefore, we propose to call these genes rnfA, rnfB, rnfC, rnfD, rnfE and rnfF (for Rhodobacter nitrogen fixation). Secondary structure predictions suggested that the rnf genes encode four potential membrane proteins and two putative iron-sulphur proteins, which contain cysteine motifs (C-X2-C-X2-C-X3-C-P) typical for [4Fe--4S] proteins. Comparison of the in vivo and in vitro nitrogenase activities of fdxN and rnf mutants suggested that the products encoded by these genes are involved in electron transport to nitrogenase. In addition, these mutants were shown to contain significantly reduced amounts of nitrogenase. The hypothesis that this new class of nitrogen fixation genes encodes components of an electron transfer system to nitrogenase was corroborated by analysing the effect of metronidazole. Both the fdxN and rnf mutants had higher growth yields in the presence of metronidazole than the wild type, suggesting that these mutants contained lower amounts of reduced ferredoxins.

Schmehl M ，Jahn A ，Meyer zu Vilsendorf A ，Hennecke S ，Masepohl B ，Schuppler M ，Marxer M ，Oelze J ，Klipp W ... -  《molecular and general genetics》

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》

A new [2Fe-2S] ferredoxin from Rhodobacter capsulatus. Coexpression with a 2[4Fe-4S] ferredoxin in Escherichia coli.

A 285-base pair open reading frame was found immediately upstream of the fdxN gene (encoding ferredoxin I) of Rhodobacter capsulatus and coded for a 95-amino acid protein with a predicted molecular weight of 10,156. The deduced amino acid sequence contained 5 cysteines, 4 of which exhibited spacing characteristic of [2Fe-2S] plant and cyanobacterial ferredoxins. The amino acid sequence was found to share approximately 25% amino acid similarity with plant-type ferredoxins. The gene was named fdxC. Expression of the fdxC and fdxN genes together in Escherichia coli was accomplished by subcloning the genes in the vector pUC18 downstream of the lac promoter. Cells containing this plasmid produced a red and a brown protein corresponding to the fdxC and fdxN gene products, respectively. EPR and UV-visible absorption spectroscopy confirmed that the FdxC protein contained a [2Fe-2S] cluster and the FdxN protein contained two [4Fe-4S] clusters and that the centers were correctly assembled and inserted in the ferredoxins expressed in E. coli. Transcription (Northern blot) analysis showed that the genes were transcribed only under nitrogen-limiting (nif-derepressing) growth conditions.

Grabau C ，Schatt E ，Jouanneau Y ，Vignais PM ... -  《JOURNAL OF BIOLOGICAL CHEMISTRY》