Induction of the Nitrate Assimilation nirA Operon and Protein-Protein Interactions in the Maturation of Nitrate and Nitrite Reductases in the Cyanobacterium Anabaena sp. Strain PCC 7120

Abstract

Nitrate is widely used as a nitrogen source by cyanobacteria, in which the nitrate assimilation structural genes frequently constitute the so-called nirA operon. This operon contains the genes encoding nitrite reductase (nirA), a nitrate/nitrite transporter (frequently an ABC-type transporter; nrtABCD), and nitrate reductase (narB). In the model filamentous cyanobacterium Anabaena sp. strain PCC 7120, which can fix N2 in specialized cells termed heterocysts, the nirA operon is expressed at high levels only in media containing nitrate or nitrite and lacking ammonium, a preferred nitrogen source. Here we examined the genes downstream of the nirA operon in Anabaena and found that a small open reading frame of unknown function, alr0613, can be cotranscribed with the operon. The next gene in the genome, alr0614 (narM), showed an expression pattern similar to that of the nirA operon, implying correlated expression of narM and the operon. A mutant of narM with an insertion mutation failed to produce nitrate reductase activity, consistent with the idea that NarM is required for the maturation of NarB. Both narM and narB mutants were impaired in the nitrate-dependent induction of the nirA operon, suggesting that nitrite is an inducer of the operon in Anabaena. It has previously been shown that the nitrite reductase protein NirA requires NirB, a protein likely involved in protein-protein interactions, to attain maximum activity. Bacterial two-hybrid analysis confirmed possible NirA-NirB and NarB-NarM interactions, suggesting that the development of both nitrite reductase and nitrate reductase activities in cyanobacteria involves physical interaction of the corresponding enzymes with their cognate partners, NirB and NarM, respectively.

Importance: Nitrate is an important source of nitrogen for many microorganisms that is utilized through the nitrate assimilation system, which includes nitrate/nitrite membrane transporters and the nitrate and nitrite reductases. Many cyanobacteria assimilate nitrate, but regulation of the nitrate assimilation system varies in different cyanobacterial groups. In the N2-fixing, heterocyst-forming cyanobacteria, the nirA operon, which includes the structural genes for the nitrate assimilation system, is expressed in the presence of nitrate or nitrite if ammonium is not available to the cells. Here we studied the genes required for production of an active nitrate reductase, providing information on the nitrate-dependent induction of the operon, and found evidence for possible protein-protein interactions in the maturation of nitrate reductase and nitrite reductase.

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 cassette C.S3 (strains CSE36 and CSE38) was inserted are indicated. The regions deleted from nirA (strain CSE27; see reference 10), alr0613 (strain CSE39), and narB (strain CSE40) are indicated with hatched bars. Abbreviations for some restriction endonuclease sites: E1, EcoRI; P, PvuII; and Sc, ScaI. Lines below the genes denote the probes used for Northern analyses.

FIG 2

Expression of narB, alr0613, and narM (alr0614) in strains PCC 7120 (wild type [WT]), CSE27 (nirA), and CSE36 (nrtB). Hybridization assays were carried out using RNAs isolated from cells grown with ammonium and incubated for 4 h in medium containing nitrate (NO₃⁻), ammonium (NH₄⁺), or no combined nitrogen (−N). Hybridization to rnpB (45) served as a loading and transfer control for each of the filters used and is shown in the image below each panel. The positions of some size markers or identified transcripts (in kilobases) are shown on the left.

FIG 3

RT-PCR analysis of expression of the narB to narM (alr0612 to alr0614) region. (A) The gene region and Anabaena gene/ORF names (46) are depicted. (B) Reverse transcription was carried out with the oligonucleotide primers depicted as closed arrowheads in panel A and with RNA isolated from cells grown with ammonium and incubated for 4 h in medium containing nitrate. (Open arrowheads in panel A represent forward primers used for amplification by PCR.) The positions of the primers used for amplification correspond to the ends of the segments indicated in panel A, which are depicted in black, indicating amplification by RT-PCR, or in gray, indicating no amplification. Lanes + and −, RNA samples treated and RNA samples not treated with RNase A, respectively; lanes p, plasmid pCSE139 used as the template for amplification with the corresponding primers.

FIG 4

Detection of nitrite reductase protein in Anabaena strains. (Top) Western blot analysis with anti-NirA antibody and samples of strains PCC 7120, CSE38 (narM), and CSE40 (narB) grown in ammonium-containing medium (t₀) or grown in ammonium-containing medium and incubated in medium with the indicated source of combined nitrogen: nitrate (NO₃⁻), nitrite (NO₂⁻), no combined nitrogen (−N), or ammonium (NH₄⁺). Lane P, size markers. The arrow points to the nitrite reductase protein band. (Bottom) Portion of the Coomassie blue-stained membrane used in panel A showing the appropriate loading in all lanes.

FIG 5

Expression of the nirA gene in strains PCC 7120 (wild type), CSE38 (narM), and CSE40 (narB). Hybridization assays were carried out using RNA isolated from cells grown with ammonium and incubated for 4 h in medium containing nitrate (NO₃⁻), nitrite (NO₂⁻), ammonium (NH₄⁺), or no combined nitrogen (−N). The signals obtained were normalized with respect to those obtained with a probe of the rnpB gene. Data are in reference to the level of nirA expression for PCC 7120 in nitrate, which is considered 100%, and summarize the results of two to four experiments with independent cultures.

FIG 6

BACTH assays of interactions between Anabaena nitrate assimilation proteins. The interactions of the proteins fused to the adenylate cyclase T18 and T25 fragments cloned in E. coli were measured as the amount of β-galactosidase activity in liquid cultures as described in Materials and Methods and expressed as nanomoles of ONP per milligram of protein per minute. The protein fused to the N or the C terminus of T18 or T25 is indicated in each case (N terminus, protein-T18 or protein-T25; C terminus, T18-protein or T25-protein). The number of independent clones analyzed for each plasmid combination is shown in parentheses. An E. coli strain containing plasmids producing nonfused T18 and T25 was used as a general negative control and showed activity of 10.65 ± 1.45 nmol ONP mg protein⁻¹ min⁻¹ (mean ± SD). Controls with each T18 or T25 fusion protein and nonfused T25 or T18 were also run. All negative controls together rendered a value of 10.14 ± 1.33 nmol ONP mg protein⁻¹ min⁻¹ (mean ± SD). The vertical dotted line marks this background activity. Error bars reflect SDs (only when the number of assays indicated in parentheses was ≥2). *, the difference between the activity obtained with the indicated plasmid pair and the combined activity from all negative controls was significant at a P value of <0.01, as assessed by the Student t test.