GlnD Is Essential for NifA Activation, NtrB/NtrC-Regulated Gene Expression, and Posttranslational Regulation of Nitrogenase Activity in the Photosynthetic, Nitrogen-Fixing Bacterium Rhodospirillum rubrum

Abstract

GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme and is thought to be the primary sensor of nitrogen status in the cell. It plays an important role in nitrogen assimilation and metabolism by reversibly regulating the modification of P II proteins, which in turn regulate a variety of other proteins. We report here the characterization of glnD mutants from the photosynthetic, nitrogen-fixing bacterium Rhodospirillum rubrum and the analysis of the roles of GlnD in the regulation of nitrogen fixation. Unlike glnD mutations in Azotobacter vinelandii and some other bacteria, glnD deletion mutations are not lethal in R. rubrum. Such mutants grew well in minimal medium with glutamate as the sole nitrogen source, although they grew slowly with ammonium as the sole nitrogen source (MN medium) and were unable to fix N 2 . The slow growth in MN medium is apparently due to low glutamine synthetase activity, because a ⌬glnD strain with an altered glutamine synthetase that cannot be adenylylated can grow well in MN medium. Various mutation and complementation studies were used to show that the critical uridylyltransferase activity of GlnD is localized to the N-terminal region. Mutants with intermediate levels of uridylyltransferase activity are differentially defective in nif gene expression, the posttranslational regulation of nitrogenase, and NtrB/NtrC function, indicating the complexity of the physiological role of GlnD. These results have implications for the interpretation of results obtained with GlnD in many other organisms.

Effect of AmtB homologues on the post-translational regulation of nitrogenase activity in response to ammonium and energy signals in Rhodospirillum rubrum

“…Whole-cell, cytoplasmic and membrane extracts were loaded such that both protein and P II levels in the cytoplasmic and membrane lanes added up to the levels in the original whole-cell extract. This GlnJ antibody is significantly more responsive to GlnJ than to GlnB, and GlnJ is the most abundant P II homologue in R. rubrum under these conditions (Zhang et al, 2005). We therefore interpret the results below in terms of GlnJ location, but cannot exclude the additional presence of lesser amounts of other P II proteins.…”

“…All of these homologues can properly regulate GlnE to regulate GS activity (Zhang et al, 2001a). However, GlnB is more effective in the regulation of NtrB activity than are the other P II homologues (Zhang et al, 2005). R. rubrum strains lacking both glnB and glnJ grow very poorly on a variety of media, irrespective of the presence of a functional glnK (Zhang et al, 2001a).…”

“…rubrum has three P II homologues, GlnB, GlnK and GlnJ, which play both distinct and overlapping functions by interacting with multiple target proteins in the cell (Zhang et al, 2005(Zhang et al, , 2001a. All of these homologues can properly regulate GlnE to regulate GS activity (Zhang et al, 2001a).…”

Metabolic analysis of Chlorobium chlorochromatii CaD3 reveals clues of the symbiosis in ‘Chlorochromatium aggregatum’

“…As mentioned above, PII regulates GS in bacteria by a post-transcriptional modification via adenylation and deadenylation according to nitrogen availability (Magasanik, 1982;Rudnick et al, 1997;Zhang et al, 2005). In cyanobacteria, PII senses the concentration of 2-oxoglutarate, determining its modified state (phosphorylated) in response to nitrogen availability (Forchhammer et al, 1999;Zhang and Zhao, 2008).…”

Crystal structures of the apo and ATP bound Mycobacterium tuberculosis nitrogen regulatory PII protein

“…6,7 When GlnB is deuridylylated it can interact with a histidine kinase, NtrB, which regulates the phosphorylation state of NtrC, controlling the expression of glnA. Thus GlnB is indispensible to the pathway of nitrogen control in E. coli.…”