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Identification and Characterization of two nitrogen fixation regulatory regions, nifA and nfrX, in Azotobacter vinelandii and Azotobacter chroococcum
Author(s) -
Santero E.,
Toukdarian A.,
Humphrey R.,
Kennedy C.
Publication year - 1988
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1988.tb00033.x
Subject(s) - azotobacter vinelandii , azotobacter chroococcum , biology , mutant , complementation , azotobacteraceae , microbiology and biotechnology , gene , rhizobium , nitrogenase , genetics , nitrogen fixation , bacteria
Summary 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. vineiandii 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 pLAFRI cosmids pLV163 and pLCI 21. 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.