The basis of ammonium release in nifL mutants of Azotobacter vinelandii

Abstract

In Azotobacter vinelandii, nitrogen fixation is regulated at the transcriptional level by an unusual two-component system encoded by nifLA. Certain mutations in nifL result in the bacterium releasing large quantities of ammonium into the medium, and earlier work suggested that this occurs by a mechanism that does not involve NifA, the activator of nif gene transcription. We have investigated a number of possible alternative mechanisms and find no evidence for their involvement in ammonium release. Enhancement of NifA-mediated transcription, on the other hand, by either elimination of nifL or overexpression of nifA, resulted in ammonium release, correlating with enhanced levels of nifH mRNA, raised levels of nitrogenase and acetylene-reducing activity, and increased concentrations of intracellular ammonium. Up to 35 mM ammonium can accumulate in the medium. Where measured, intracellular levels exceeded extracellular levels, indicating that rather than being actively transported, ammonium is lost from the cell passively, possibly by reversal of an NH(4)(+) uptake system. The data also indicate that in the wild type the bulk of NifA is inactivated by NifL during steady-state growth on dinitrogen.

FIG. 1

Map of the nifLA region of A. vinelandii showing restriction sites used for manipulations and the positions of KIXX inserts. The arrows marked with strain numbers indicate the directions of transcription of the KIXX promoters in the respective strains. Strain designations in parentheses designate constructs that could not be obtained in a genetically homogeneous form. The BclI site is not unique.

FIG. 2

Optical densities (OD) (A), pHs of the medium (B), and ammonium concentrations in the medium (C) for various A. vinelandii strains as a function of time following derepression.

FIG. 3

Relative nitrogenase activities (A) and ammonium concentrations in the medium (B) for the wild type (wt) and nifL::KIXX mutants.

FIG. 4

Strategy for obtaining regulated overexpression of nifA from the chromosome in A. vinelandii.

FIG. 5

Activities of a nifA-lacZ reporter in various nif regulatory mutants. Expression of nifA in MV496.1 was induced with 200 μM IPTG at the start of derepression.

FIG. 6

Relative levels of nifH mRNAs in various A. vinelandii strains. Expression of nifA in MV496 was induced with 200 μM IPTG at the start of derepression. wt, wild type.

FIG. 7

Sodium dodecyl sulfate-polyacrylamide gel showing overexpression of nitrogenase polypeptides in various A. vinelandii strains 30 h after derepression. Duplicated lanes represent separate cultures of the same strain. The lane on the extreme right contains purified Fe protein. Expression of nifA in MV496 was induced with 200 μM IPTG at the start of derepression.

FIG. 8

Intracellular (Int) and extracellular (Ex) ammonium concentrations at various times following derepression. Expression of nifA in MV496 was induced with 200 μM IPTG at the start of derepression.