glnA mRNA Research Articles

Glutamine synthetase mRNA releases sRNA from its 3'UTR to regulate carbon/nitrogen metabolic balance in Enterobacteriaceae.

Glutamine synthetase (GS) is the key enzyme of nitrogen assimilation induced under nitrogen limiting conditions. The carbon skeleton of glutamate and glutamine, 2-oxoglutarate, is supplied from the TCA cycle, but how this metabolic flow is controlled in response to nitrogen availability remains unknown. We show that the expression of the E1o component of 2-oxoglutarate dehydrogenase, SucA, is repressed under nitrogen limitation in Salmonella enterica and Escherichia coli. The repression is exerted at the post-transcriptional level by an Hfq-dependent sRNA GlnZ generated from the 3'UTR of the GS-encoding glnA mRNA. Enterobacterial GlnZ variants contain a conserved seed sequence and primarily regulate sucA through base-pairing far upstream of the translation initiation region. During growth on glutamine as the nitrogen source, the glnA 3'UTR deletion mutants expressed SucA at higher levels than the S. enterica and E. coli wild-type strains, respectively. In E. coli, the transcriptional regulator Nac also participates in the repression of sucA. Lastly, this study clarifies that the release of GlnZ from the glnA mRNA by RNase E is essential for the post-transcriptional regulation of sucA. Thus, the mRNA coordinates the two independent functions to balance the supply and demand of the fundamental metabolites.

A 3' UTR-derived small RNA connecting nitrogen and carbon metabolism in enteric bacteria.

Increasing numbers of small, regulatory RNAs (sRNAs) corresponding to 3' untranslated regions (UTR) are being discovered in bacteria. One such sRNA, denoted GlnZ, corresponds to the 3' UTR of the Escherichia coli glnA mRNA encoding glutamine synthetase. Several forms of GlnZ, processed from the glnA mRNA, are detected in cells growing with limiting ammonium. GlnZ levels are regulated transcriptionally by the NtrC transcription factor and post-transcriptionally by RNase III. Consistent with the expression, E. coli cells lacking glnZ show delayed outgrowth from nitrogen starvation compared to wild type cells. Transcriptome-wide RNA-RNA interactome datasets indicated that GlnZ binds to multiple target RNAs. Immunoblots and assays of fusions confirmed GlnZ-mediated repression of glnP and sucA, encoding proteins that contribute to glutamine transport and the citric acid cycle, respectively. Although the overall sequences of GlnZ from E. coli K-12, Enterohemorrhagic E. coli and Salmonella enterica have significant differences due to various sequence insertions, all forms of the sRNA were able to regulate the two targets characterized. Together our data show that GlnZ impacts growth of E. coli under low nitrogen conditions by modulating genes that affect carbon and nitrogen flux.

Cloning of an EF-P homologue from Bacteroides fragilis that increases B. fragilis glutamine synthetase activity in Escherichia coli.

Investigations of possible regulators of Bacteroides fragilis glutamine synthetase (GS) activity were done in Escherichia coli using a compatible dual-plasmid system. The B. fragilis glnA gene, together with upstream and downstream flanking regions, was cloned onto the low copy number plasmid pACYC184 and expressed in the E. coli glnA ntrB ntrC deletion strain, YMC11. GS activity was monitored following co-transformation with a B. fragilis genomic library carried on the compatible plasmid pEcoR251. A gene was cloned that caused a twofold increase in B. fragilis GS activity but did not affect the activity of the E. coli GS enzyme or the B. fragilis sucrase (ScrL). Deletion of the B. fragilis glnA downstream region decreased basal levels of GS activity, but did not affect the ability of the cloned gene to increase the B. fragilis GS activity. Reporter gene analysis, using the B. fragilis glnA promoter region fused to the promoterless Clostridium acetobutylicum endoglucanase gene, showed no increase in reporter gene activity. This demonstrated that the increase in GS activity was not regulated at the transcriptional level, and that the cloned gene product was not affecting the copy number of the plasmid in trans. Sequence data indicated that the cloned gene had good amino acid identity to a range of elongation factor P (EF-P) proteins, the highest being to that of a Synechocystis sp (48%), and the least to Mycobacterium genitalium (27%). Amino acid identity to the E. coli EF-P was intermediate (37%). A possible role for EF-P in enhancing translation of the B. fragilis glnA mRNA is proposed.

Transcription of glutamine synthetase genes (glnA and glnN) from the cyanobacterium Synechocystis sp. strain PCC 6803 is differently regulated in response to nitrogen availability.

In the cyanobacterium Synechocystis sp. strain PCC 6803 we have previously reported the presence of two different proteins with glutamine synthetase activity: GSI, encoded by the glnA gene, and GSIII, encoded by the glnN gene. In this work we show that expression of both the glnA and glnN genes is subjected to transcriptional regulation in response to changes in nitrogen availability. Northern blot experiments and transcriptional fusions demonstrated that the glnA gene is highly transcribed in nitrate- or ammonium-grown cells and exhibits two- to fourfold-higher expression in nitrogen-starved cells. In contrast, the glnN gene is highly expressed only under nitrogen deficiency. Half-lives of both mRNAs, calculated after addition of rifampin or ammonium to nitrogen-starved cells, were not significantly different (2.5 or 3.4 min, respectively, for glnA mRNA; 1.9 or 1.4 min, respectively, for glnN mRNA), suggesting that changes in transcript stability are not involved in the regulation of the expression of both genes. Deletions of the glnA and glnN upstream regions were used to delimit the promoter and the regulatory sequences of both genes. Primer extension analysis showed that structure of the glnA gene promoter resembles those of the NtcA-regulated promoters. In addition, mobility shift assays demonstrated that purified, Escherichia coli-expressed Synechocystis NtcA protein binds to the promoter of the glnA gene. Primer extension also revealed the existence of a sequence related to the NtcA binding site upstream from the glnN promoter. However, E. coli-expressed NtcA failed to bind to this site. These findings suggest that an additional modification of NtcA or an additional factor is required for the regulation of glnN gene expression.

Diel variability in transcription of the structural gene for glutamine synthetase ( glnA) in natural populations of the marine diazotrophic cyanobacterium Trichodesmium thiebautii

A 360-bp fragment of the Trichodesmium thiebautii glutamine synthetase gene ( glnA) was amplified from DNA isolated from oceanic populations of this ecologically important, diazotrophic cyanobacterium. The cloned fragment showed high homology to the type 1 glutamine synthetase (GS) enzymes of other cyanobacteria and was used to probe the in situ, temporal variability in transcription of Trichodesmium GS. Three distinct phases in the diel pattern of glnA mRNA abundance were observed. These appear to be correlated with the temporal variability in carbon and nitrogen fixation rates and the effect that these physiological processes have on the size of cellular pools of the primary end-products of N assimilation. Our data show that Trichodesmium thiebautii GS is regulated at the level of transcription and support the hypothesis that GS expression in this organism is subject to similar controls as those established for heterocyst-forming cyanobacteria.

Diel variability in transcription of the structural gene for glutamine synthetase (glnA) in natural populations of the marine diazotrophic cyanobacterium Trichodesmium thiebautii

Abstract A 360-bp fragment of the Trichodesmium thiebautii glutamine synthetase gene (glnA) was amplified from DNA isolated from oceanic populations of this ecologically important, diazotrophic cyanobacterium. The cloned fragment showed high homology to the type 1 glutamine synthetase (GS) enzymes of other cyanobacteria and was used to probe the in situ, temporal variability in transcription of Trichodesmium GS. Three distinct phases in the diel pattern of glnA mRNA abundance were observed. These appear to be correlated with the temporal variability in carbon and nitrogen fixation rates and the effect that these physiological processes have on the size of cellular pools of the primary end-products of N assimilation. Our data show that Trichodesmium thiebautii GS is regulated at the level of transcription and support the hypothesis that GS expression in this organism is subject to similar controls as those established for heterocyst-forming cyanobacteria.

Transcription of the glnB and glnA genes in the photosynthetic bacterium Rhodospirillum rubrum

The PII protein, encoded by glnB, has a central role in the control of nitrogen metabolism in nitrogen-fixing prokaryotes. The glnB gene of Rhodospirillum rubrum was isolated and sequenced. The deduced amino acid sequence had very high sequence identity to other PII proteins. The glnA gene, encoding glutamine synthetase, was located 135 bp downstream of glnB and was partially sequenced. glnB is cotranscribed with glnA from a promoter with high similarity to the sigma 54-dependent promoter consensus sequence. A putative sigma 70 promoter was also identified further upstream of glnB. Northern blotting analyses showed that in addition glnA is either transcribed from an unidentified promoter or, more likely, that the glnBA transcript is processed to give the glnA mRNA. The total level of the two transcripts was much higher in nitrogen-fixing cells than in ammonia-grown cells.

Electron transport controls transcription of the glutamine synthetase gene (glnA) from the cyanobacterium Synechocystis sp. PCC 6803

The glnA gene, encoding type I glutamine synthetase (GS) in Synechocystis sp. PCC 6803, showed a high sequence similarity with other cyanobacterial glnA genes. A dramatic decrease in the amount of glnA mRNA, a single transcript of about 1.6 kb, was observed after transfer to darkness, or after incubation with the electron transport inhibitors DCMU or DBMIB. The levels of glnA transcript were fully recovered after 5 min of reillumination. The glnA mRNA was found to be equally stable both in the light and the dark (half-life about 2.5 min). Unlike the glnA messenger, the amount of GS protein was not reduced in the dark. Synthesis of the glnA transcript in the dark required the presence of glucose. In addition, glnA transcription in a Synechocystis psbE-psbF mutant lacking photosystem II required the presence of glucose even when grown in the light. These observations indicate that glnA transcription is under the control of the redox state of the cell. Finally, nitrogen starvation provoked a delay in the decrease of glnA transcript in darkness, suggesting a connection between nitrogen and redox controls of glnA transcript levels.

Expression of glnA in the cyanobacterium Synechococcus sp. strain PCC 7942 is initiated from a single nif-like promoter under various nitrogen conditions

The glnA mRNA, encoding glutamine synthetase, is differentially accumulated in the cyanobacterium Synechococcus sp. strain PCC 7942 in media containing different nitrogen sources. With the different nitrogen compounds, transcription of glnA initiated at a single site located -146 nucleotides upstream of the translation start site of the gene. A similarity of the nif-like promoter of the glnA gene of Anabaena sp. strain PCC 7120 and a binding-site sequence for the Synechococcus sp. strain PCC 7942 transcription regulator, NtcA, were found upstream of the transcription initiation site.

Differential expression of a Clostridium acetobutylicum antisense RNA: implications for regulation of glutamine synthetase.

The Clostridium acetobutylicum glutamine synthetase (GS) DNA region is characterized by a downstream promoter, P3, oriented toward the glnA gene, which controls the transcription of an RNA complementary to the start of the glnA mRNA. Expression of the predicted 43-base antisense RNA was demonstrated in C. acetobutylicum and Escherichia coli cells containing the cloned glnA DNA. Antisense RNA transcription from P3 was not regulated by nitrogen in E. coli cells, but the expression of antisense RNA was associated with decreased levels of GS activity. In C. acetobutylicum, GS activity and the transcription of glnA mRNA and antisense RNA were regulated by nitrogen. GS activity and glnA mRNA were repressed in cells grown in nitrogen-rich medium. Repression ratios for GS activity varied from 1.6 to 9.0, depending on the sampling time. The relative number of glnA transcripts was approximately 25% lower in cells grown for 72 h in nitrogen-rich medium than in cells grown in nitrogen-limiting medium. This finding contrasted with the expression of antisense RNA, which was repressed in nitrogen-limiting medium but induced in nitrogen-rich medium. The relative number of antisense RNA transcripts was increased approximately sixfold in cells grown in nitrogen-rich medium. There was a 1.6-fold excess of antisense RNA over glnA mRNA under conditions that repressed GS activity. Under conditions that induced GS activity, glnA mRNA transcripts exceeded antisense RNA transcripts by fivefold.

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.

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.

Glutamine synthetase gene of Bacillus subtilis

The glutamine synthetase gene ( glnA) of Bacillus subtilis was purified from a library of B. subtillis DNA cloned in phage λ. By mapping the locations of previously identified mutations in the glnA locus it was possible to correlate the genetic and physical maps. Mutations known to affect expression of the glnA gene and other genes were mapped within the coding region for glutamine synthetase, as determined by measuring the sizes of truncated, immunologically cross-reacting polypeptides coded for by various sub-cloned regions of the glnA gene. When the entire B. subtilis glnA gene was present on a plasmid it was capable of directing synthesis in Escherichia coli of B. subtilis glutamine synthetase as judged by enzymatic activity, antigenicity, and ability to allow growth of a glutamine auxotroph. By use of the cloned B. subtilis glnA gene as a hybridization probe, it was shown that the known variability of glutamine synthetase specific activity during growth in various nitrogen sources is fully accounted for by changes in glnA mRNA levels.