Activation of the Rhizobium leguminosarum glnII gene by NtrC is dependent on upstream DNA sequences

Abstract

The cloning and sequence determination is reported of the DNA region of Rhizobium leguminosarum coding for glutamine synthetase II (GSII). An open reading frame (ORF) encoding 326 amino acids was defined as the glnII gene on the basis of its similarity to other glnII genes and the ability of a DNA fragment carrying this ORF to complement the glutamine auxotrophy of a Klebsiella pneumoniae glnA mutant. We find that the glnII gene in R. leguminosarum is transcribed as a monocistronic unit from a single promoter, which shows structural features characteristic of rpoN (ntrA)-dependent promoters. In K. pneumoniae, such promoters require the ntrC and rpoN (ntrA) gene products for transcription. The intracellular level of glnII mRNA changes when R. leguminosarum is grown on different nitrogen sources, as expected for regulation by the nitrogen regulatory system. Promoter deletion analysis has shown that an extensive upstream DNA sequence (316 bp) is essential for in vivo activation of the glnII promoter in different biovars of R. leguminosarum. This DNA region requires a wild-type ntrC gene for activity and includes two conserved putative NtrC-binding site sequences. The results conclusively show that transcription from the R. leguminosarum glnII promoter is fully dependent on positive control by NtrC protein and on an upstream activator sequence (UAS).

Key Role of Bacterial NH 4 + Metabolism in Rhizobium-Plant Symbiosis

“…GSII, encoded by the glnII gene, is a heat-sensitive (relative to GSI) octameric enzyme (103). The expression of the glnII gene is activated under N deficiency (for a review, see reference 54) and requires an upstream activating sequence (140). When R. leguminosarum is switched to an NH 4 ϩ -containing medium, glnII transcription is 10-fold repressed, but GSII activity and the GSII protein become undetectable (103,140).…”

“…The expression of the glnII gene is activated under N deficiency (for a review, see reference 54) and requires an upstream activating sequence (140). When R. leguminosarum is switched to an NH 4 ϩ -containing medium, glnII transcription is 10-fold repressed, but GSII activity and the GSII protein become undetectable (103,140). That this is due to a posttranscriptional control mechanism of glnII expression has recently been demonstrated (172,183).…”

“…It is possible that lack of growth at low nitrate concentrations is a consequence of a reduced bacterial capacity to maintain NH 4 ϩ inside the cells and/or to assimilate it. In fact, the genes involved in NH 4 ϩ uptake and assimilation (like amtB and glnII) are highly expressed in an NtrC-dependent mode in bacteria grown on nitrate (140,143,179). Moreover, it has been reported that the majority of the N limitationinduced genes are not regulated by the ntr system (119).…”

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

“…Since glnII (GSII) is switched off in the root nodule, GSI is probably the only glutamine synthetase present in bacteroids (Shatters et al, 1989 ;Patriarca et al, 1992 ;Amar et al, 1994 ;Espin et al, 1994). Therefore, one could hypothesize that GlnD could play a role in the regulation pathway leading to GSI adenylylation, i.e.…”

In vitro characterization of the ORF1‐ntrBC promoter of Rhizobium etli

“…In Rhizobium etli NtrC is essential for expression of glnlI, R. etli wild-type strain CE3 and strain CFN2012, an ntrC: :Tn5 the structural gene for a glutamine synthetase [4], and the derivative [11], were grown at 30°C in TYR rich medium [12] or in RMM minimal medium [13] with 1 g 1-1 NH4C1 or KNO3 as sole ntrBC genes are part of an ORFI-ntrBC operon, where nitrogen source. When NH4C1 was used, 100 mM 3-{N-morpholino} ORF1 is an open reading frame of unknown function [5].…”