Negative regulation of expression of the nitrate assimilation nirA operon in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120

Abstract

In the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, expression of the nitrate assimilation nirA operon takes place in the absence of ammonium and the presence of nitrate or nitrite. Several positive-action proteins that are required for expression of the nirA operon have been identified. Whereas NtcA and NtcB exert their action by direct binding to the nirA operon promoter, CnaT acts by an as yet unknown mechanism. In the genome of this cyanobacterium, open reading frame (ORF) all0605 (the nirB gene) is found between the nirA (encoding nitrite reductase) and ntcB genes. A nirB mutant was able to grow at the expense of nitrate as a nitrogen source and showed abnormally high levels of nirA operon mRNA both in the presence and in the absence of nitrate. This mutant showed increased nitrate reductase activity but decreased nitrite reductase activity, an imbalance that resulted in excretion of nitrite, which accumulated in the extracellular medium, when the nirB mutant was grown in the presence of nitrate. A nirA in-frame deletion mutant also showed a phenotype of increased expression of the nirA operon in the absence of ammonium, independent of the presence of nitrate in the medium. Both NirB and NirA are therefore needed to keep low levels of expression of the nirA operon in the absence of an inducer. Because NirB is also needed to attain high levels of nitrite reductase activity, NirA appears to be a negative element in the nitrate regulation of expression of the nirA operon in Anabaena sp. strain PCC 7120.

FIG. 1.

Genomic region of Anabaena sp. strain PCC 7120 bearing the nitrate assimilation gene cluster. Genes and ORFs are indicated by thick arrows, which also show the direction of transcription. Black arrows correspond to the ORFs investigated in this work. The locations of the restriction sites into which gene cassettes (C.S3 for strains CSE17 and CSE23 and C.K1 for strains EF116B and CSE27B) were inserted are indicated. The region deleted from nirA in strain CSE27 is indicated with a hatched bar. Abbreviations for some restriction endonuclease sites: B, BglI; C, ClaI; E5, EcoRV; H, HindIII; P, PvuII; S, SpeI; Sc, ScaI; and X, XbaI. Horizontal lines below the genes denote probes used for Northern analyses.

FIG. 2.

Northern analysis of the expression of all0604 in strains PCC 7120, CSE17, and CSE172. Hybridization assays were carried out using RNAs isolated from cells grown with ammonium (NH₄⁺) or grown with ammonium and incubated for 4 or 24 h in medium containing nitrate (NO₃⁻) or no combined nitrogen (−N). Hybridization to rnpB (40) served as a loading and transfer control (lower panel). The positions and sizes (in kb) of the detected transcripts are shown on the left.

FIG. 3.

Changes in levels of nitrate and nitrite reductase activities and nitrite accumulation in the culture medium after transfer of ammonium-grown cells to nitrate-containing medium. (A) Strain PCC 7120 (WT); (B) strain CSE17. Diamonds, nitrate reductase activity; squares, nitrite reductase activity; triangles, nitrite concentration in the medium.

FIG. 4.

Changes in levels of nitrate reductase activity after transfer of ammonium-grown cells to a medium containing no combined nitrogen. Circles, strain PCC 7120; squares, strain CSE17; triangles, strain CSE172.

FIG. 5.

Northern analysis of expression of the nirA operon and of some regulatory genes in strains PCC 7120, CSE17, and CSE172. Hybridization assays were carried out using RNAs isolated from cells grown with ammonium (NH₄⁺) or grown with ammonium and incubated for 4 or 24 h in medium containing nitrate (NO₃⁻) or no combined nitrogen (−N). The hybridization probes used (see Materials and Methods for details) corresponded to narB (A), cnaT (B), nirA (C; fragment a in Fig. 1), ntcA (D), and ntcB (E). Hybridization to rnpB (40) served as a loading and transfer control for each of the two filters used. The positions of some size markers (A and C) and identified transcripts (B, D, and E) are shown (in kb) on the left.

FIG. 6.

Primer extension analysis of expression of the nirA gene in strains PCC 7120 and CSE17. Primer extension assays were carried out using oligonucleotide nir-1 as a primer and RNAs isolated from cells grown with ammonium (NH₄⁺) or grown with ammonium and incubated for 4 h in medium containing nitrate (NO₃⁻) or no combined nitrogen (−N). The arrowhead points to the extension product identifying the Anabaena nirA operon tsp. The sequencing ladders presented were generated with the same primer used in the primer extension reactions, with plasmid pCSE26 as template.

FIG. 7.

Northern analysis of expression of nirA in strains PCC 7120, CSE17, and CSE27. Hybridization assays were carried out using RNAs isolated from cells grown with ammonium (NH₄⁺) or grown with ammonium and incubated for 4 h in medium containing nitrate (NO₃⁻) or no combined nitrogen (−N). Fragment b in Fig. 1 was used as a nirA probe (see Materials and Methods for details). Hybridization to rnpB (40) served as a loading and transfer control (lower panel). The positions and sizes (in kb) of some size standards are shown on the left. The small change in the pattern of hybridization bands for strain CSE27 compared to that for strains CSE17 and PCC 7120 resulted from the deletion of 666 bp from nirA in CSE27.

FIG. 8.

Genomic structure of nirA complementing constructs in strains CSE271 and CSE272 and Northern analysis of the expression of narB and nirA in strains PCC 7120, CSE27, CSE271, and CSE272. The complementing constructs included ORF all0606, the nirA operon promoter region, and the nirA gene incorporated into the nucA-nuiA region of the alpha megaplasmid (A) or into the chromosomal nirA region (B). Primers (A and B): 1, orf398-4; 2, nir-7120-6; 3, nir-7120-27; 4, nrtA-7120-3; 5, nrtA-7120-2; 6, nui-7120-4; 7, nir-7120-28; and 8, ntcs3 (18). Hybridization assays were carried out using RNAs isolated from cells of the indicated strains grown with ammonium (NH₄⁺) or grown with ammonium and incubated for 4 h in medium containing nitrate (NO₃⁻) or no combined nitrogen (−N). The hybridization probes used (see Materials and Methods) corresponded to narB (C) and nirA (D; fragment c in Fig. 1). Hybridization to rnpB (40) served as a loading and transfer control. The positions and sizes (in kb) of some size standards are shown on the left.

FIG. 9.

Nitrate uptake and Northern analysis of expression of nirA in strains PCC 7120, EF116B, CSE27, and CSE27B. (A) Ammonium-grown cells of strains PCC7120 (closed diamonds), EF116B (open diamonds), CSE27 (closed triangles), and CSE27B (open triangles) were washed, resuspended in BG11 medium (17.6 mM NaNO₃), and incubated for 4 h as indicated in Materials and Methods for derepression of the nitrate assimilation system. Cells were then harvested and used for nitrate uptake assays (see Materials and Methods). At the indicated times, nitrate was measured by HPLC, using aliquots withdrawn from the assay mixture. (B) Hybridization assays were carried out using RNAs isolated from cells of the indicated strains grown with ammonium (NH₄⁺) or grown with ammonium and incubated for 4 h in medium containing nitrate (NO₃⁻) or no combined nitrogen (−N). Fragment b in Fig. 1 was used as the nirA probe (see Materials and Methods). Hybridization to rnpB (40) served as a loading and transfer control (lower panel). The positions and sizes (in kb) of some size standards are shown on the left.