Identification and characterization of two nitrogen fixation regulatory regions, nifA and nfrX, in Azotobacter vinelandii and Azotobacter chroococcum.
Five Tn5-induced Nif- mutants of Azotobacter vinelandii were characterized as regulatory mutants because they were restored to Nif+ by the introduction of constitutively expressed nifA from Klebsiella pneumoniae. The mutants fell into two different classes on the basis of hybridization to a Rhizobium leguminosarum nifA gene probe and by complementation with cosmids isolated from pLAFRI gene banks of A. vinelandii and Azotobacter chroococcum. One mutant, MV3, was located in or near a nifA gene. The others, MV12, MV16, MV18 and MV26, defined a new regulatory gene, which has been called nfrX. The lack of expression of different nif-lacZ fusions confirmed the regulatory phenotype of all five mutant strains. The ability of both nifA and nfrX mutants to grow on nitrogen-free medium with vanadium, but not on medium with molybdenum, suggests that neither gene is required for expression of the alternative V-containing nitrogenase of A. vinelandii. A fragment carrying Tn5 and flanking DNA from MV3 was used as a probe to isolate the nifA region of A. chroococcum. Ligation of two adjacent EcoRI fragments of A. chroococcum yielded an intact nifA gene that activated expression of nifH-lac fusions and also restored MV3 to Nif+. The four nfrX mutants were complemented by pLAFR1 cosmids pLV163 and pLC121. The nfrX gene was subcloned from pLV163 and located within a 3.2 kb fragment. To determine whether nfrX might be found in other nitrogen-fixing organisms, DNA from 13 different species was hybridized to an nfrX probe. The failure to observe hybridization suggests that nfrX may be specific to nif regulation in Azotobacter.
Santero
E
E
AFRC--Institute of Plant Science Research, University of Sussex, Brighton, UK.
Toukdarian
A
A
Humphrey
R
R
Kennedy
C
C
D001395:Azotobacter / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003360:Cosmids; D004274:DNA, Recombinant; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D007711:Klebsiella pneumoniae / Q000235:genetics; D009154:Mutation; D009586:Nitrogen Fixation / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012231:Rhizobium / Q000235:genetics; D013045:Species Specificity
D001426:Bacterial Proteins; D004274:DNA, Recombinant