Role of NtcB in activation of nitrate assimilation genes in the cyanobacterium Synechocystis sp. strain PCC 6803

Abstract

In Synechocystis sp. strain PCC 6803, the genes encoding the proteins involved in nitrate assimilation are organized into two transcription units, nrtABCD-narB and nirA, the expression of which was repressed by ammonium and induced by inhibition of ammonium assimilation, suggesting involvement of NtcA in the transcriptional regulation. Under inducing conditions, expression of the two transcription units was enhanced by nitrite, suggesting regulation by NtcB, the nitrite-responsive transcriptional enhancer we previously identified in Synechococcus sp. strain PCC 7942. The slr0395 gene, which encodes a protein 47% identical to Synechococcus NtcB, was identified as the Synechocystis ntcB gene, on the basis of the inability of an slr0395 mutant to rapidly accumulate the transcripts of the nitrate assimilation genes upon induction and to respond to nitrite. While Synechococcus NtcB strictly requires nitrite for its action, Synechocystis NtcB enhanced transcription significantly even in the absence of nitrite. Whereas the Synechococcus ntcB mutant expresses the nitrate assimilation genes to a significant level in an NtcA-dependent manner, the Synechocystis ntcB mutant showed only low-level expression of the nitrate assimilation genes, indicating that NtcA by itself cannot efficiently promote expression of these genes in Synechocystis. Activities of the nitrate assimilation enzymes in the Synechocystis ntcB mutant were consequently low, being 40 to 50% of the wild-type level, and the cells grew on nitrate at a rate approximately threefold lower than that of the wild-type strain. These results showed that the contribution of NtcB to the expression of nitrate assimilation capability varies considerably among different strains of cyanobacteria.

FIG. 1

(A) Map of the nrt and nir regions of the genome of Synechocystis sp. strain PCC 6803. The genes encoding NRT, NR, and NiR are indicated by checkered bars. The putative molybdenum cofactor biosynthesis genes are indicated by hatched bars. The bar above the sll1454 gene shows the region replaced by an antibiotic resistance gene cassette to construct the SNR1 mutant. (B) Structure of the slr0395 genomic region of the wild-type strain (WT) and the SNIC1 mutant of Synechocystis sp. strain PCC 6803. The open bar represents the kanamycin resistance gene cassette, with the hatched bar showing the location and orientation of the kanamycin resistance gene (npt). Abbreviations for restriction endonuclease sites: S, StyI; N, NheI. The gene organization in Synechocystis was obtained from CyanoBase (http://www.kazusa.or.jp/cyano/cyano.html). (C) Electrophoretic profiles showing the PCR products amplified from chromosomal DNAs of the wild-type strain and the slr0395 mutant SNIC 1, using a forward primer specific to the slr0394-slr0395 intergenic region and a reverse primer specific to the 3′ region of slr0362. Lane M shows the molecular size markers (1-kbp ladder; BRL).

FIG. 2

Northern blot analysis of RNA from Synechocystis sp. strain PCC 6803 showing the effects of MSX and nitrite on transcription of nirA (A), nrtC (B), and ntcA (C) in the wild-type (WT) strain (lanes 1 to 3) and the SNIC1 mutant (lanes 4 to 6). Cells were grown with ammonium, the culture was separated into three portions, and total RNA was extracted from the cells before (lanes 1 and 4) and 60 min after the following treatments: addition of MSX (lanes 2 and 5) and addition of MSX plus nitrite (lanes 3 and 6). The numbers in parentheses indicate relative abundances of mRNAs as determined by quantitation of radioactivity using a Bio-Image analyzer (Fuji Photo Film). Asterisks between panels B and C indicate the positions of the rRNA bands as determined by staining of the blots with methylene blue.

FIG. 3

Changes in the abundance of the nirA (A) and nrtC (B) transcripts after addition of MSX to the ammonium-grown cultures of the wild-type strain (○ and ●) and the mutant (▵ and ▴), with (● and ▴) and without (○ and ▵) simultaneous addition of nitrite. The amounts of the nirA and nrtC transcripts were quantitated by dot hybridization analysis with 1.25 μg of RNA per dot and are shown relative to the maximum level in the wild-type cells treated with MSX and nitrite.

FIG. 4

Growth curves of the wild-type strain (● and ○) and the SNIC1 mutant (▴ and ▵) of Synechocystis sp. strain PCC 6803 in media containing nitrate (● and ▴) and ammonium (○ and ▵) as the sole source of nitrogen.

FIG. 5

Expression of the nitrate assimilation activities in the wild-type strain and the ntcB-deficient mutant. (A) Northern hybridization analysis of total RNA, comparing the abundance of mRNA from the nitrate assimilation genes in the two strains growing with nitrate. Cells grown with ammonium were transferred to nitrate-containing medium, and total RNA was isolated after 18 h of growth in nitrate-containing medium. (B) NR and NiR activities of wild-type and SNIC1 cells grown under different nitrogen conditions. Cells grown with ammonium (open bars), ammonium-grown cells subsequently grown for 18 h in nitrate containing medium (gray bars), and ammonium-grown cells subjected to 4 h of nitrogen starvation (hatched bars) were used for the assays. (C) Nitrate uptake activity of intact cells of the wild-type strain and the SNIC1 mutant after 18-h incubation in nitrate-containing medium (gray bars) or 4-h incubation in nitrogen-free medium (hatched bars) of ammonium-grown cells. The results shown in panels B and C are the averages and standard deviations (error bars) from three separate experiments with independent cultures.

FIG. 6

(A) Primer extension analysis of the expression of the nirA (a) and nrtA (b) genes in the wild-type strain of Synechocystis sp. strain PCC 6803, showing the effects of MSX and nitrite. Cells were grown with ammonium, the culture was separated into three portions, and total RNAs extracted from the cells before (lanes 1) and 60 min after the following treatments were used for the assays: addition of MSX (lanes 2) and addition of MSX plus nitrite (lanes 3). The arrows indicate the extension products and the deduced transcription start sites. (B) Alignment of the promoters of the cyanobacterial nitrate assimilation genes. Only those promoters known to be regulated by NtcB and/or nitrite are included. The regions of the putative NtcB-binding site with a LysR motif (T-N₁₁-A), the NtcA-binding site, and the −10 sequence are boxed. The transcription start position is underlined. The nucleotides forming an inverted repeat with the LysR motif are shaded. Asterisks indicate the nucleotides conserved in the five promoter sequences. Gaps have been introduced into the sequences to maintain optimal alignment. The numbers to the right of the sequences indicate the positions of the rightward-most bases with respect to the translation start site. Strains: 7942, Synechococcus sp. strain PCC 7942; 6803, Synechocystis sp. strain PCC 6803; 7120, Anabaena sp. strain PCC 7120; P.b., Plectonema boryanum.