Characterization of the glnK-amtB operon of Azotobacter vinelandii

Abstract

To determine whether in Azotobacter vinelandii the PII protein influences the regulation of nif gene expression in response to fluxes in the ammonium supply, the gene encoding PII was isolated and characterized. Its deduced translation product was highly similar to PII proteins from other organisms, with the greatest degree of relatedness being exhibited to the Escherichia coli glnK gene product. A gene designated amtB was found downstream of and was contranscribed with glnK as in E. coli. The AmtB protein is similar to functionally characterized ammonium transport proteins from a few other eukaryotes and one other prokaryote. glnK and amtB comprise an operon. Attempts to isolate a stable glnK mutant strain were unsuccessful, suggesting that glnK, like glnA, is an essential gene in A. vinelandii. amtB mutants were isolated, and although growth on limiting amounts of ammonium was similar in the mutant and wild-type strains, the mutants were unable to transport [14C]methylammonium.

FIG. 1

The 2.3-kb glnK-amtB region of A. vinelandii. The locations of KIXX and lacZ interposon constructs and their directions of insertion are shown by the hatched rectangles and arrows below the line, respectively. The resulting plasmids are named along with (where appropriate) the stable mutant strain of A. vinelandii in which the glnK::KIXX or glnK::lacZ region replaced the wild-type glnK gene. The fragments used as probes to identify RNA fragments on the Northern blots (see Fig. 2) are shown as thin lines. Abbreviations for restriction sites: B, BamHI; E, EcoRI; H, HincII; Sn, StuI; SnaBI; P, PstI.

FIG. 2

Northern blot analysis of the glnK-amtB operon. mRNA from ammonium-grown (lanes a, c, and e) or N₂-grown (lanes b, d, and f) cultures was prepared by using the Qiagen (Chatsworth, Calif.) RNeasey Kit (catalog no. 74904). Samples containing approximately equal amounts of total RNA were separated on 0.8% formaldehyde–agarose gels by electrophoresis; this was followed by capillary blotting of the RNA onto nylon membranes. Blots were hybridized to three ³²P-labeled probes from the glnK-amtB region: a 360-bp HincII fragment (glnK only), a 1.2-kb StuI fragment (glnK plus amtB), and a 0.9-kb StuI fragment (amtB only). L, DNA fragment standards.

FIG. 3

[¹⁴C]Methylammonium uptake by wild-type (▪) and amtB mutant (▴) strains. Overnight cultures in Burke’s N-free sucrose medium were diluted 50-fold and grown to an optical density at 600 nm of 0.5 to 0.7. The details of the uptake experiments were described by Jayakumar and Barnes (21).