A mutant GlnD nitrogen sensor protein leads to a nitrogen-fixing but ineffective Sinorhizobium meliloti symbiosis with alfalfa

Abstract

The nitrogen-fixing symbiosis between rhizobia and legume plants is a model of coevolved nutritional complementation. The plants reduce atmospheric CO 2 by photosynthesis and provide carbon compounds to symbiotically associated bacteria; the rhizobia use these compounds to reduce (fix) atmospheric N 2 to ammonia, a form of nitrogen the plants can use. A key feature of symbiotic N 2 fixation is that N2 fixation is uncoupled from bacterial nitrogen stress metabolism so that the rhizobia generate ''excess'' ammonia and release this ammonia to the plant. In the symbiosis between Sinorhizobium meliloti and alfalfa, mutations in GlnD, the major bacterial nitrogen stress response sensor protein, led to a symbiosis in which nitrogen was fixed (Fix ؉ ) but was not effective (Eff ؊ ) in substantially increasing plant growth. Fixed 15 N2 was transported to the shoots, but most fixed 15 N was not present in the plant after 24 h. Analysis of free-living S. meliloti strains with mutations in genes related to nitrogen stress response regulation (glnD, glnB, ntrC, and ntrA) showed that catabolism of various nitrogen-containing compounds depended on the NtrC and GlnD components of the nitrogen stress response cascade. However, only mutants of GlnD with an amino terminal deletion had the unusual Fix ؉ Eff ؊ symbiotic phenotype, and the data suggest that these glnD mutants export fixed nitrogen in a form that the plants cannot use. These results indicate that bacterial nitrogen stress regulation is important to symbiotic productivity and suggest that GlnD may act in a novel way to influence symbiotic behavior.

GlnB/GlnK PII Proteins and Regulation of the Sinorhizobium meliloti Rm1021 Nitrogen Stress Response and Symbiotic Function

“…2. A similar phenotype was observed in Rm1021⌬glnB mutants (45). These data indicate that deletion In enteric bacteria, GlnK is crucial for the ability of cells to survive nitrogen starvation and resume rapid growth when fed ammonia (11).…”

“…Supplementation of the media with 0.5, 1, or 2 g/liter glutamine and/or glutamate did not improve the growth of the Rm1021⌬glnK⌬glnB mutants. This is in contrast to Rm1021⌬ glnD-sm2, which grew much more normally on a favorable nitrogen source, such as glutamine (45). The growth defect of enteric bacteria lacking GlnB and GlnK proteins resulted from inhibition of serine biosynthesis (12).…”

“…A surprisingly high level of total GS activity was seen in Rm1021⌬glnK⌬glnB cells grown in the presence of ammonium (Table 3). Rm1021⌬glnB also had an increased level of GS activity in cells grown under high-nitrogen conditions (Table 3) (45). These data indicate that the presence of GlnB attenuates GS expression/activity in normal S. meliloti Rm1021 metabolism and that GlnK cannot replace it for this function.…”

“…We showed previously that S. meliloti Rm1021 expresses GSI under conditions of both high and low nitrogen stress and that GSI was subject to significant posttranslational modification as a function of the quality of the nitrogen source (45). Using Western blot analyses, we did not detect the presence of a significant amount of GSI protein in extracts prepared from ammonium-or glutamine-grown cultures of Rm1021⌬glnK or Rm1021⌬glnK⌬glnB (data not shown).…”

Biochemical Characterization of a Nitrogen-Type Phosphotransferase System Reveals that Enzyme EI Ntr Integrates Carbon and Nitrogen Signaling in Sinorhizobium meliloti

“…Since these genes encode proteins that regulate glutamine levels in the cell (40), we assayed glutamine for its ability to alter EI Ntr activity. These assays showed that glutamine was a potent inhibitor of EI Ntr , likely coupling the activity of the PTS Ntr to a critical signal of nitrogen levels in both free-living and symbiotic S. meliloti (40 (11). EI Ntr balances this activation against the glutamine availability in the cell, resulting in an intimate link between the availability of carbon and nitrogen.…”