Molecular cloning and complementation analysis of nifV gene from Frankia EuIK1 strain.
The nifV gene from the Frankia EuIK1 strain, a symbiont of Elaeagnus umbellata, was cloned and a complementation test using the Klebsiella pneumoniae nifV mutant was performed to verify its function. The nifV ORF consists of 1245 bp, which encodes 414 amino acids. However, the putative promoter and Shine-Dalgarno sequences were not found in the 5' region of the ORF. The Frankia EuIK1 nifV ORF showed about a 70% nucleotide identity and 80% amino acid similarity with that of Frankia sp. FaC1. In the upstream region of the nifV, a putative ORF that showed a 51% nucleotide identity with the afcD gene from Burkholderia cepacia BC11 was found. The other partial ORF that showed a 59% identity with the pkaD gene from Streptomyces coelicolor A(3) was found in the downstream region. In this respect, Frankia EuIK1 nifV has an unusual location on the genome, considering the nif gene organization. A phylogenetic analysis revealed that the NifV from Frankia EuIK1 was close to those from two Alnus-infective Frankia species, and they were grouped with those of the alpha-class proteobacteria, supporting the vertical descent of nifV. The transcription and function of Frankia EuIK1 nifV were verified by a RT-PCR analysis and complementation test with the K. pneumoniae mutant, respectively. These results suggested that Frankia EuIK1 nifV is a functional gene.
Oh
Chang Jae
CJ
School of Biological Sciences, Seoul National University, Seoul 151-742, Korea.
Kim
Ho Bang
HB
An
Chung Sun
CS
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology; D001482:Base Composition; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D040161:Frankia / Q000235:genetics*; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009586:Nitrogen Fixation / Q000235:genetics*; D016366:Open Reading Frames; D007652:Oxo-Acid-Lyases / Q000235:genetics* / Q000502:physiology; D010802:Phylogeny; D011993:Recombinant Fusion Proteins / Q000502:physiology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D003062:Codon; D011993:Recombinant Fusion Proteins; C021365:homocitrate synthase; D007652:Oxo-Acid-Lyases