A novel nitrate/nitrite permease in the marine Cyanobacterium synechococcus sp. strain PCC 7002

Abstract

The nrtP and narB genes, encoding nitrate/nitrite permease and nitrate reductase, respectively, were isolated from the marine cyanobacterium Synechococcus sp. strain PCC 7002 and characterized. NrtP is a member of the major facilitator superfamily and is unrelated to the ATP-binding cassette-type nitrate transporters that previously have been described for freshwater strains of cyanobacteria. However, NrtP is similar to the NRT2-type nitrate transporters found in diverse organisms. An nrtP mutant strain consumes nitrate at a 4.5-fold-lower rate than the wild type, and this mutant grew exponentially on a medium containing 12 mM nitrate at a rate approximately 2-fold lower than that of the wild type. The nrtP mutant cells could not consume nitrite as rapidly as the wild type at pH 10, suggesting that NrtP also functions in nitrite uptake. A narB mutant was unable to grow on a medium containing nitrate as a nitrogen source, although this mutant could grow on media containing urea or nitrite with rates similar to those of the wild type. Exogenously added nitrite enhanced the in vivo activity of nitrite reductase in the narB mutant; this suggests that nitrite acts as a positive effector of nitrite reductase. Transcripts of the nrtP and narB genes were detected in cells grown on nitrate but were not detected in cells grown on urea or ammonia. Transcription of the nrtP and narB genes is probably controlled by the NtcA transcription factor for global nitrogen control. The discovery of a nitrate/nitrite permease in Synechococcus sp. strain PCC 7002 suggests that significant differences in nutrient transporters may occur in marine and freshwater cyanobacteria.

FIG. 1

(A) Physical map of the 7.2-kb genomic region of the nrtP and narB genes in Synechococcus sp. strain PCC 7002. Arrows indicate the direction of transcription for the following open reading frames: sll1258, which is similar to hypothetical gene in Synechocystis sp. strain PCC 6803; petM, encoding a subunit of b₆f complex; psbW2, encoding a subunit of photosystem II; merA, encoding mercuric reductase; and tmk, encoding thymidylate kinase. (B) The construct for the insertional inactivation of the nrtP gene in Synechococcus sp. strain PCC 7002. The kanamycin resistance gene cartridge (aphII gene) of 1.4 kb was inserted at the SpeI site in the nrtP gene with the same transcription orientation. The EcoRI-to-BglII DNA fragment was cloned between the EcoRI and BamHI sites of pUC19. Arrowheads indicate the positions of the primers for the PCR analysis. (C) The construct for insertional inactivation of the narB gene in Synechococcus sp. strain PCC 7002. The kanamycin resistance gene cartridge (aphII gene) of 1.4 kb was inserted into the XbaI site in the narB gene with the same transcription orientation. The HindIII-to-BamHI DNA fragment was cloned into the HindIII and BamHI sites of pUC19. Arrowheads indicate the positions of the primers for the PCR analysis. (D) Evaluation of gene replacement in the chromosomal DNAs of the nrtP mutant and the narB mutant by PCR analysis. Genomic DNAs from the wild type (lanes 1 and 3), the nrtP mutant (lane 2), and the narB mutant (lane 4) were used as the template for PCR with primers specific for the nrtP gene (lane 1 and 2) and for the narB gene (lanes 3 and 4). PCR products were separated by agarose gel electrophoresis and detected by ethidium bromide staining.

FIG. 2

Sequence alignment of the NrtP protein of Synechococcus sp. strain PCC 7002 to other Nrt2-type nitrate transporter sequences. Amino acids identical and similar to those in the NrtP protein of Synechococcus sp. strain PCC 7002 are marked by shading. Amino acids that are identical in more than five of the nine proteins are shown in boldface. Twelve putative hydrophobic transmembrane regions are indicated by the numbered lines. Osci, partial sequence of the open reading frame in the 5′ upstream region of the narB gene in Oscillatoria chalybea (accession number of the protein sequence, S57965); NrtP, Synechococcus sp. strain PCC 7002 (this study); Chlamy, Chlamydomonas reinhardtii (S40142); Rice, Oryza sativa (BAA33382); Arabi, Arabidopsis thaliana (CAB09794); Ynt1, Pichia angusta (CAA93631); CrnA, Aspergillus nidulans (P22152); NarK, Bacillus subtilis (P46907); NasA, B. subtilis (P42432).

FIG. 3

Growth curves for the wild-type strain, the nrtP mutant strain, and the narB mutant strain on urea and after transfer to nitrate growth conditions. Cells grown on medium AU₁₀Ni containing urea as a nitrogen source were collected by centrifugation and washed with nitrogen-free medium. The cells were then inoculated into fresh medium A⁺ containing nitrate as a nitrogen source (open circles) and into medium AU₁₀Ni containing urea as a nitrogen source (closed circles) and were cultured at 38°C under 250 μE m⁻² s⁻¹ with aeration with 1% CO₂-enriched air. After growth in medium A⁺ for 24 h, the cells of the nrtP and narB mutants were diluted into fresh medium A⁺ and cultured under identical growth conditions (squares). The data shown are derived from a single experiment; however, this experiment was repeated three times, and essentially identical results were obtained in each case.

FIG. 4

Nitrite consumption by whole cells of the wild-type strain and the nrtP mutant strain of Synechococcus sp. strain PCC 7002. The disappearance of nitrite from the assay medium was measured for wild-type cells at pH 7, wild-type cells at pH 10, nrtP mutant cells at pH 7, and nrtP mutant cells at pH 10. The samples were preilluminated for 15 min under 250 μE m⁻² s⁻¹ at 38°C in the presence of the GOGAT inhibitor DON. At time zero, 100 μM NaNO₂ was added to initiate the reaction. The assay medium contained 25 mM HEPES-NaOH (pH 7.0) or 25 mM CAPS-NaOH (pH 10.0), 1 mM KCl, 10 mM NaHCO₃, 0.5 mM DON, 100 μM NaNO₂, and cells equivalent to 10 μg of chlorophyll ml⁻¹. The data shown are the average values from two independent experiments.

FIG. 5

Nitrogen source-dependent expression of the nrtP and narB genes in Synechococcus sp. strain PCC 7002. Total RNA (10 μg per lane) was isolated from cells grown on nitrate (lane 1), urea (lane 2), or ammonium (lane 3) as a sole nitrogen source and transferred to nylon membrane filters. The membranes were hybridized with probes specific for the nrtP or narB genes. After washing to remove the excess probe DNA, the membranes were exposed to X-ray films for 1 day for the nrtP gene and for 5 days for the narB gene. The 23S (2.8-kb) and 16S (1.5-kb) rRNA bands and the in vivo cleavage products of the 23S rRNA (2.3 and 0.5 kb) were detected as nonspecific hybridization background (arrows). The data shown are from a single experiment which was repeated twice, and identical results were obtained in each experiment.

FIG. 6

The 5′ endpoint of the nrtP mRNA and the putative NtcA-dependent promoter sequence of the nrtP gene in Synechococcus sp. strain PCC 7002. The 5′ endpoint of the mRNA was determined by the sequence of RT-PCR products as described in Materials and Methods. The numbering for the sequence corresponds to the nucleotide numbering of the sequence under GenBank accession no. AF089813. The consensus sequence for an NtcA-dependent promoter is that described by Flores et al. (8).