The Rhizobium leguminosarum bv. viciae glnD gene, encoding a uridylyltransferase/uridylyl-removing enzyme, is expressed in the root nodule but is not essential for nitrogen fixation

Abstract

A Rhizobium leguminosarum bv. viciae VF39 gene (glnD) encoding the uridylyltransferase/uridylyl-removing enzyme, which constitutes the sensory component of the nitrogen regulation (ntr) system, was identified, cloned and characterized. The deduced amino acid sequence contains the conserved active site motif of the nucleotidyltransferase superfamily and is highly homologous to the glnD gene products of other bacterial species. Downstream of the VF39 glnD resides an open reading frame with similarity to the Salmonella typhimurium virulence factor gene mviN. Mutation of the glnD gene abolished the ability to use nitrate as a sole nitrogen source but not glutamine. In addition, neither uridylylation of P II nor induction of the ntr-regulated glnII gene (encoding glutamine synthetase II) under ammonium deficiency could be observed in mutant strains. This strongly suggests that glnD mutants harbour a permanently deuridylylated P II protein and as a consequence are unable to activate transcription from NtrC-dependent promoters. The glnD gene itself is expressed constitutively, irrespective of the nitrogen content of the medium. A functional GlnD protein is not essential for nitrogen fixation in R. leguminosarum bv. viciae, but in situ detection of glnD expression in the symbiotic and infection zone of the root nodule and quantitative measurements suggest that at least part of the ntr system functions in symbiosis. The results also indicate that the N-terminal part of GlnD is essential for the cell, as deletions in the 5'-region of the gene appear to be lethal and mutations possibly affecting the expression of the first half of the protein have a significant effect on the vitality of the mutant strain.

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

“…From the data on 15 N accumulation in the shoots at 2 h and 5.5 h after labeling (Fig. 4, tabulated in Table S1), we conclude that 15 N was leaving the mutant nodules, and this export seems to be more rapid than in the wild-type nodules.…”

“…From the data on 15 N accumulation in the shoots at 2 h and 5.5 h after labeling (Fig. 4, tabulated in Table S1), we conclude that 15 N was leaving the mutant nodules, and this export seems to be more rapid than in the wild-type nodules. However, the 15 N levels in nodules, roots, and leaves of plants nodulated by Rm1021⌬glnD-sm2 were very low after 24 h. From the values included in Table S1, we calculate that more than 90% of the total 15 N fixed was lost from the plants (nodules, roots, and leaves) nodulated with Rm1021⌬glnD-sm2 after the 24-h incubation, probably as root exudates.…”

“…Samples were ground to a fine powder, and Ϸ1 mg of material was placed in a tin capsule and combusted in a Costech ECS 4010 elemental analyzer. The resulting N2 gas was injected into a Micromass Isoprime isotope ratio mass spectrometer to determine 15 Western Blot. Crude extracts were prepared from cultures grown for 24 h on MM containing a good (0.5% NH4 ϩ ), intermediate (0.04% glutamine), or poor (0.04% glutamate) nitrogen source.…”

“…We thank the Washington State University Laboratory for Biotechnology and Bioanalysis for sequencing support; Ray Lee for his help with 15 …”

PIISignal Transduction Proteins, Pivotal Players in Microbial Nitrogen Control

“…AF152563), Rhizobium leguminosarum biovar viciae (199), and Sinorhizobium meliloti (accession no. AF227730).…”