glnD and mviN are genes of an essential operon in Sinorhizobium meliloti.

To evaluate the role of uridylyl-transferase, the Sinorhizobium meliloti glnD gene was isolated by heterologous complementation in Azotobacter vinelandii. The glnD gene is cotranscribed with a gene homologous to Salmonella mviN. glnD1::Omega or mviN1::Omega mutants could not be isolated by a powerful sucrose counterselection procedure unless a complementing cosmid was provided, indicating that glnD and mviN are members of an indispensable operon in S. meliloti.

P(II) signal transduction proteins, pivotal players in microbial nitrogen control.

The P(II) family of signal transduction proteins are among the most widely distributed signal proteins in the bacterial world. First identified in 1969 as a component of the glutamine synthetase regulatory apparatus, P(II) proteins have since been recognized as playing a pivotal role in control of prokaryotic nitrogen metabolism. More recently, members of the family have been found in higher plants, where they also potentially play a role in nitrogen control. The P(II) proteins can function in the regulation of both gene transcription, by modulating the activity of regulatory proteins, and the catalytic activity of enzymes involved in nitrogen metabolism. There is also emerging evidence that they may regulate the activity of proteins required for transport of nitrogen compounds into the cell. In this review we discuss the history of the P(II) proteins, their structures and biochemistry, and their distribution and functions in prokaryotes. We survey data emerging from bacterial genome sequences and consider other likely or potential targets for control by P(II) proteins.

Two residues in the T-loop of GlnK determine NifL-dependent nitrogen control of nif gene expression.

X-ray crystallographic analysis of the Escherichia coli P(II) protein paralogues GlnB and GlnK has shown that they share a superimposable structural core but can differ in conformation of the T-loop, a region of the protein (residues 37-54) that has been shown to be important for interaction with other proteins. In Klebsiella pneumoniae GlnK has been shown to have a clearly defined function in regulating NifL-mediated inhibition of NifA activity in response to the nitrogen status, and GlnB, when expressed from the chromosome, does not substitute for GlnK. Because the T-loops of K. pneumoniae and E. coli GlnB and GlnK differ at just three residues, 43, 52, and 54, we have used a previously constructed heterologous system, in which K. pneumoniae nifLA is expressed in E. coli, to investigate the importance of GlnK residues 43, 52, and 54 for regulation of the NifLA interaction. By site-directed mutagenesis of glnB we have shown that residue 54 is the single most important amino acid in the T-loop in the context of the regulation of NifA activity. Furthermore, a combination of just two changes, in residues 54 and 43, allows GlnB to function as GlnK and completely relieve NifL inhibition of NifA activity.

Studies on the roles of GlnK and GlnB in regulating Klebsiella pneumoniae NifL-dependent nitrogen control.

In Klebsiella pneumoniae, nitrogen fixation (nif) genes are regulated in response to fixed nitrogen and oxygen. The activity of the nif-specific transcriptional activator NifA is modulated by NifL, which mediates both oxygen and nitrogen control. The signal transduction protein GlnK is required to relieve the inhibitory effect of NifL on NifA that occurs when the intracellular N status is high and in a wild-type cell, the action of GlnK cannot be substituted by the structurally related protein PII. We have studied the modulation of NifA activity by NifL in an heterologous system in which the host organism is Escherichia coli. Using a DeltaglnB, DeltaglnK mutant, we have shown that the modulation of NifA activity by NifL is dependent on the concentration of GlnK in the cell and that when overproduced, PII can substitute for GlnK. Furthermore, our data suggest that PII can counteract the positive action of GlnK in relieving NifL-dependent inhibition of NifA activity. This negative effect of PII may be physiologically important in establishing repression of nif gene expression when the intracellular nitrogen status rises.

The Rhizobium meliloti PII protein, which controls bacterial nitrogen metabolism, affects alfalfa nodule development.

Symbiotic nitrogen fixation involves the development of specialized organs called nodules within which plant photosynthates are exchanged for combined nitrogen of bacterial origin. To determine the importance of bacterial nitrogen metabolism in symbiosis, we have characterized a key regulator of this metabolism in Rhizobium meliloti, the uridylylatable P(II) protein encoded by glnB. We have constructed both a glnB null mutant and a point mutant making nonuridylylatable P(II). In free-living conditions, P(II) is required for expression of the ntrC-dependent gene glnII and for adenylylation of glutamine synthetase I. P(II) is also required for efficient infection of alfalfa but not for expression of nitrogenase. However alfalfa plants inoculated with either glnB mutant are nitrogen-starved in the absence of added combined nitrogen. We hypothesize that P(II) controls expression or activity of a bacteroid ammonium transporter required for a functional nitrogen-fixing symbiosis. Therefore, the P(II) protein affects both Rhizobium nitrogen metabolism and alfalfa nodule development.

Symbiotic nitrogen fixation does not require adenylylation of glutamine synthetase I in Rhizobium meliloti.

Symbiotic nitrogen fixation is accompanied by a shift of Rhizobium nitrogen metabolism from ammonium assimilation to ammonium export, which probably involves genetic or metabolic regulation of glutamine synthetase activity. In free-living Rhizobium meliloti glutamine synthetase I (GSI) is regulated post-translationally by reversible adenylylation in response to ammonium addition. Moreover, full expression of the GSI gene glnA requires the transcriptional activator, NtrC. A glnA1 mutant synthesizing a non-adenylylatable GSI produces normal nitrogen-fixing nodules on alfalfa: GSI adenylylation is dispensable for symbiotic nitrogen fixation. This is rationalized by the observation that less GS protein is present in R. meliloti bacteroids than in free-living bacterial cells.