Premium
Mapping and expression of a regulatory nitrogen fixation gene ( fix D) of Rhizobium meliloti
Author(s) -
Weber Gerhard,
Reiländer Helmut,
Pühler Alfred
Publication year - 1985
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1985.tb03999.x
Subject(s) - biology , humanities , philosophy
A 3.5‐kb Hind III fragment from the main nif/fix (nitrogen fixation) gene cluster of Rhizobium meliloti was characterized by studying its expression in Escherichia coli minicells. A coding region for two polypeptides of 68 K and 66 K was mapped using Tn 5 insertions and hybrid fusion polypeptides. DNA sequence analysis of this region revealed the presence of an open reading frame capable of coding for a polypeptide of 59.9 K mol. wt. This coding region was designated fix D. Plasmids, constitutively expressing this fix D gene from vector promoters, activated a nif HD‐ lac Z fusion in E. coli at a low level. Higher levels of activation were obtained following an enhanced expression of the fix D gene in plasmid pRmW541 which was achieved by inducing deletions between the vector promoter and the fix D gene. Sequencing of these deletion mutants showed that, in most cases, fusion polypeptides of the fix D gene product and the aph I (aminoglycoside‐3′‐phosphotransferase) gene product were sufficient for activation. In E. coli the activation is strictly dependent upon a functional gln F ( ntr A) gene.