Transcriptional analysis of the glnB-glnA region of Rhizobium leguminosarum biovar viciae.

We report that the glnB and glnA genes of Rhizobium leguminosarum biovar viciae are preceded by promoters located upstream of each gene. We find the presence of a glnB-glnA and a glnA mRNA whose intracellular concentration changes two- to three-fold when R. leguminosarum is grown on different nitrogen sources. Primer extension analysis shows unique transcriptional initiation sites upstream of glnB and glnA. The glnB promoter is rpoN(ntrA)-dependent, while the glnA promoter does not contain a typical consensus sequence for previously described promoters. In Klebsiella pneumoniae the glnB promoter requires active ntrC and ntrA genes and a DNA fragment containing 53 nucleotides upstream of the transcription initiation site shows full promoter activity, thus indicating that no NtrC binding sites are necessary for this activation in the glnB upstream region.

Chiurazzi M ，Iaccarino M 《MOLECULAR MICROBIOLOGY》

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

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).

Patriarca EJ ，Chiurazzi M ，Manco G ，Riccio A ，Lamberti A ，De Paolis A ，Rossi M ，Defez R ，Iaccarino M ... -  《molecular and general genetics》

Characterization of three different nitrogen-regulated promoter regions for the expression of glnB and glnA in Azospirillum brasilense.

The complete nucleotide sequence of the open reading frame (ORF) located upstream of the glnA structural gene for glutamine synthetase (GS) in Azospirillum brasilense Sp7 was determined. This ORF, which codes for a 12 kDa protein, was identified as glnB, the structural gene for the PII protein, a component of the adenylylation cascade involved in the regulation of GS activity in some gram-negative bacteria. Transcription analysis and mRNA mapping of glnB and glnA of A. brasilense was performed with bacteria grown under different physiological conditions. The glnA gene can be transcribed either as a glnB-A mRNA of 2.4 kb or as a glnA mRNA of 1.5 kb. Differential expression of the two mRNAs was found to depend on the nitrogen source. The glnB-A mRNA was the major transcript under nitrogen fixation conditions, while the synthesis of the glnA mRNA was almost completely abolished. The glnA mRNA was predominantly produced in NH4(+)-containing medium. Transcription start site analysis revealed the presence of three different types of nitrogen-regulated promoters. GlnB-A mRNA was transcribed selectively from tandem promoters. One of them is similar to the NtrA-dependent promoter and the other to the Escherichia coli sigma 70 promoter. The synthesis of glnA mRNA was regulated by a promoter, which was repressed (or non-activated) only under conditions of nitrogen fixation, when moleuclar nitrogen was the sole nitrogen source. The transcriptional initiation site in front of glnA is not preceded by a canonical E. coli sigma 70 promoter. A sequence reminiscent of the NtrA-dependent promoter consensus, except for a fundamental mismatch, was found at positions -33 to -21. This sequence overlapped a putative "weak" NtrC-binding site, similar to those identified in enteric bacteria. From these results, it is postulated that glnA mRNA is controlled by a novel type of nitrogen-regulated promoter.

de Zamaroczy M ，Delorme F ，Elmerich C 《molecular and general genetics》

Tandem promoters determine regulation of the Klebsiella pneumoniae glutamine synthetase (glnA) gene.

Transcription of the structural gene for glutamine synthetase (glnA) in Klebsiella pneumoniae is controlled by the nitrogen regulatory genes ntrA, ntrB and ntrC. The nucleotide sequence of the regulatory region upstream of the glnA gene is reported here. High resolution S1 mapping of in vivo transcripts indicates that the regulatory region contains tandem promoters separated by 100 nucleotides. Measurements of beta-galactosidase activities determined in vivo from glnA-lac fusions suggest that the upstream promoter (for RNA2) is negatively regulated by the ntrBC gene products whereas transcription from the downstream promoter (for RNA1) is positively activated by the ntrA gene product in the presence of either the ntrBC or the nifA genes. The nucleotide sequence of the upstream promoter resembles the consensus sequence for E. coli promoters, whereas the downstream promoter shows homology with the nitrogen fixation (nif) promoters of K. pneumoniae.

Dixon R 《-》

Transcription of a Rhizobium leguminosarum biovar phaseoli gene needed for melanin synthesis is activated by nifA of Rhizobium and Klebsiella pneumoniae.

The Rhizobium leguminosarum biovar phaseoli symbiotic plasmid pRP2JI carries a gene, melA, specifying the enzyme tyrosinase, which is responsible for the production of the pigment melanin in these bacteria. Transcription of melA is activated by the nifA gene of Rhizobium and, when the cloned melA gene is transferred to Escherichia coli, melA is expressed if the recipients contain nifA gene of Klebsiella pneumoniae. This nifA-dependent activation was temperature sensitive and required the ntrA gene. The cloned nifA gene of K. pneumoniae, when transferred to a nifA mutant of Rhizobium phaseoli biovar phaseoli, corrected the Mel- but not the Fix- phenotype. nifA of R. leguminosarum biovar phaseoli activated melA at higher levels in cells grown in low concentrations of oxygen. Also, nifA of R. leguminosarum biovar phaseoli activated nifH of K. pneumoniae in Escherichia coli cells grown in low-oxygen concentrations.

Hawkins FK ，Johnston AW 《MOLECULAR MICROBIOLOGY》