Regulation of Nitrogen Fixation in Azoarcus sp Strain BH72

P11 proteins are small signal transduction molecules involved in nitrogen fixation and assimilation. In many Proteobacteria, two different Pn-like proteins occur (Arcondeguy et al. 2001). In contrast, Azoarcus sp. strain BH72 harbors three paralogs of Pn-like proteins (Martin et al. 2000). All three proteins can be covalently modified by uridylylation in response to ammonium starvation (Martin et al. 2000). GlnK and GlnY both occur in wild type cells, whereas GlnY could only be detected in a glnB'-glnK double mutant. As most other Pn-like proteins, GlnK and GlnB occur in their native form under conditions of ammonium excess, or become deuridylylated in when ammonium is added to a N-starved culture. However GlnY is unusual in that the uridylyl residue is not removed under ammonium excess, as demonstrated in the glnK '-glnB ' double mutant (Martin et al. 2000). But what is the differential role of these proteins? Recently it was demonstrated that active nitrogenase of strain BH72 is rapidly inactivated by addition of ammonium (Egener et al. 2001). This ammonium "switch-off' requires a ferredoxin contranscribed with the structural genes of nitrogenase, nifHDK (Egener et al. 2001). In addition, the Pn-like proteins are differentially involved in this process. Mutational analysis has shown that GlnK but not GlnB or GlnY is required for the "switch-off'. The modification of the iron protein of nitrogenase, which occurs concomitantly with the ammonium "switch-off', is in contrast dependent on the presence of both proteins, GlnK and GlnB. Interestingly, a glnB~ mutant did still show ammonium "switch-off', however it did not show modification of the iron protein of nitrogenase any more. This clearly demonstrates that inhibition of nitrogenase activity and its covalent modification are distinct processes.

Moreover, the amtB gene encoding a putative high-affinity ammonium transporter is also involved in the "switch-off' process. In an amtB' mutant, rapid inactivation of nitrogenase activity was not observed any more. Since in this mutant, ammonium was removed from the medium at the same rate as in wild type cells, it is not likely that this effect is due to the lack of ammonium uptake. Western blot analyses of cell fractions of strain BH72 have shown that GlnK occurs not only in the cytoplasm, but also membrane-associated. This is not the case for GlnB. This indicates that GlnK and AmtB might physically interact, as recently hypothesized by others (Thomas et al. 2001). Therefore, AmtB might act as a sensor involved in the signal transduction process of sudden ammonium access. Interestingly, even in an amtB' mutant, GlnK was found to be associated with bacterial membranes, indicating that other membrane proteins in Azoarcus sp. strain BH72 might interact with GlnK, as well.

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