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》

Transcriptional analysis and promoter mapping of the fdxA gene which encodes the 7Fe ferredoxin (FdII) of Rhodobacter capsulatus.

The structural gene (fdxA) coding for ferredoxin II (FdII) of the photosynthetic bacterium Rhodobacter capsulatus has been previously cloned and sequenced. Transcription of the fdxA gene was studied by mRNA analyses and by use of plasmid-borne fdxA::lacZ translational fusions. The transcription start site was mapped 23 bp upstream of the initiation codon, as deduced from analysis of mRNA by mung bean nuclease protection and primer extension experiments. A motif resembling the canonical sequence observed in sigma 70-dependent Escherichia coli promoters is present at the expected distance (-10/-35) from the proposed transcription start site. mRNA analysis by Northern hybridization revealed a fdxA-specific transcript of approximately 0.4 kb, indicating that fdxA is transcribed as a single gene.fdxA expression, as measured by the activity of the fdxA::lacZ fusion, was found to be constant during growth and reached a similar level under all growth conditions tested. These results suggest that FdII is constitutively synthesized in R. capsulatus.

Duport C ，Jouanneau Y ，Vignais PM 《molecular and general genetics》

Expression in Escherichia coli and characterization of a recombinant 7Fe ferredoxin of Rhodobacter capsulatus.

The 7Fe ferredoxin of Rhodobacter capsulatus (FdII) could be expressed in Escherichia coli by cloning the fdxA gene coding for FdII downstream from the lac promoter. The expressed recombinant ferredoxin appeared as a brown protein which was specifically recognized in E. coli cell-free extracts by anti-FdII serum. The purified recombinant ferredoxin was indistinguishable from R. capsulatus FdII on the basis of its molecular, redox and spectroscopic properties. These results indicate that the [3Fe-4S] and [4Fe-4S] clusters were correctly inserted into the recombinant ferredoxin.

Jouanneau Y ，Duport C ，Meyer C ，Gaillard J ... -  《-》

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》

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》