Unmodified GinK Interacts With NifL In Vivo Stimulating NifLmediated Inhibition Of Nitrogenase Expression In Azotobacter Vinelandii

Dept of Plant Pathology, The University of Arizona, Forbes 204, Tucson, AZ 85721 USA

In the y-subgroup of Proteobacteria, represented by A. vinelandii and the enteric bacterium Klebsiella pneumoniae, Mo-dependent nitrogenase, encoded by nif genes, is positively regulated by the a54-dependent activator, NifA, whose activity is controlled by a cognate inhibitor, NifL. In response to inhibitory signals, such as oxygen and fixed nitrogen, a non-activating NifL:NifA complex is formed (Dixon 1998) To elucidate the genetic basis of the fixed nitrogen response in A. vinelandii has been an ongoing project in our lab.

In K. pneumoniae, expression of nifLA is tightly controlled by the ntr system, and hence responsive to fixed nitrogen. However, in A. vinelandii nifLA expression is constitutive, suggesting that control of NifA activity by NifL is the main mechanism controlling the response. In A. vinelandii, mutations that affect the activity of PII-uridylyltransferase (GlnD) prevent NifA activity and these mutations can be rescued by deletion of nifL (Contreras et al. 1991 ; Colnaghi et al. 2001), indicating that GlnD controls an aspect of the NifL-NifA interaction. We sought to determine the role of GlnK, the only PII-like protein in A. vinelandii, in regulating NifA activity. These experiments had been hampered because glnK is essential in A. vinelandii (Meletzus et al. 1998).

In this work, we made use of a glnD null suppressor strain MV72 (gln-71), which carries an ammonium insensitive glutamine synthetase (GS) activity (Colnaghi et al. 2001), to construct a mutation at the site of covalent uridylylation on GlnK (Y51~>F). This allele was only stable in a gln-71 background, indicating that GlnK-UMP is required for GS activity probably by reversing adenylylation of GS by ATase. The glnKY51F mutation was crossed into the chromosome, selecting for a Tetr cassette in the 5' end of the adjacent amtB gene. Transformant glnKY51F strains exhibited severely decreased NifA activity as measured by growth in N-free medium and expression of a nifH-lacZ reporter, when compared to glnKf strains constructed in the same background. To determine if NifL might be a target for GlnK, a yeast two-hybrid system was used to test protein interactions. In this system, GlnK and GlnKY51F interacted with NifL, indicating that NifL controls NifA in response to the uridylylation state of GlnK; under conditions of nitrogen excess, NifL inhibits NifA in response to the binding of unmodified GlnK. This model contrasts to one proposed for regulation of NifA activity in K. pneumoniae where GlnK relieves NifL inhibition of NifA in any uridylylation state (He et al. 1998) and suggests that these organisms have evolved separate mechanisms to control the same regulatory pathway.

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