The global nitrogen regulator NtcA regulates transcription of the signal transducer PII (GlnB) and influences its phosphorylation level in response to nitrogen and carbon supplies in the Cyanobacterium synechococcus sp. strain PCC 7942

Abstract

The PII protein is encoded by a unique glnB gene in Synechococcus sp. strain PCC 7942. Its expression has been analyzed in the wild type and in NtcA-null mutant cells grown under different conditions of nitrogen and carbon supply. RNA-DNA hybridization experiments revealed the presence of one transcript species 680 nucleotides long, whatever the nutrient conditions tested. A second transcript species, 620 nucleotides long, absent in the NtcA null mutant, was observed in wild-type cells that were nitrogen starved for 2 h under both high and low CO2 and in the presence of nitrate under a high CO2 concentration. Primer extension analysis indicated that the two transcript species are generated from two tandem promoters, a sigma70 Escherichia coli-type promoter and an NtcA-dependent promoter, located 120 and 53 nucleotides, respectively, from the glnB initiation codon. The NtcA-dependent promoter is up-regulated under the conditions mentioned above, while the sigma70 E. coli-type promoter displays constitutive levels of transcripts in the NtcA null mutant and slightly different levels in the wild-type cells, depending on the nitrogen and carbon supplies. In general, a good correlation between the amounts of the two transcript species and that of the PII protein was observed, as revealed by immunodetection with specific antibodies. The phosphorylation level of PII in the wild type is inversely correlated with nitrogen availability and directly correlated with higher CO2 concentration. This regulation is correspondingly less stringent in the NtcA null mutant cells. In contrast, the dephosphorylation of PII is NtcA independent.

FIG. 1

Alignment of NtcA recognition sequences of different genes from Synechococcus sp. strain PCC 7942 (30).

FIG. 2

RNA-DNA hybridization of total RNA from cells of wild-type Synechococcus sp. strain PCC 7942 (WT) and the NtcA-deficient mutant (NtcA⁻) in response to the nature of the nitrogen source and the availability of CO₂. Ammonium-grown cells were transferred for 2 h to BG-11₀ medium containing ammonium (lanes 1), nitrate (lanes 2), or no nitrogen source (lanes 3), under either air or air–3% (vol/vol) CO₂. The same RNA blots were hybridized with a DNA probe internal to the glnB gene encoding the P_II protein and with a DNA probe of the rnpB gene encoding the RNA subunit of RNase P to provide an estimate of the RNA loading.

FIG. 3

Primer extension with the glnB gene. Total RNA (60 μg) from wild-type Synechococcus sp. strain PCC 7942 (WT) and NtcA-deficient mutant (NtcA⁻) cells was annealed with an oligonucleotide specific to the glnB gene and extended with avian myeloblastosis virus reverse transcriptase as indicated in Materials and Methods. The cells were incubated as described in the legend to Fig. 2. Lanes A, C, G, and T contain a dideoxy sequencing ladder of the same DNA region used as a size control of the extension products. The sequences around the 5′ ends are listed on the right. tsp1 and tsp2 are putative transcription start points.

FIG. 4

Gel retardation of DNA fragments from the glnB and glnA promoter regions by cell extracts of an NtcA-overproducing E. coli strain. (A) glnA promoter region, P_glnA (lanes 1 to 3), and glnB promoter region, P_glnB (lanes 4 to 6), incubated with a DNA fragment internal to the glnB gene as the competitor DNA. Lanes 1 and 4, no NtcA-containing extract added; lanes 2 and 5, 5 μg of extract from cells of E. coli DH5α(pTrc99A) added; lanes 3 and 6, 5 μg of NtcA-containing extract from IPTG-induced cells of E. coli DH5α(pCSI26) added. C_glnA and C_glnB are complexes formed after incubation of the DNA fragments carrying P_glnA and P_glnB, respectively, with the NtcA-containing extracts. (B) Same conditions as in panel A, lanes 1 and 4, with various amounts (0 [−] to 8.0 μg) of extract from IPTG-induced cells of E. coli DH5α(pCSI26) added.

FIG. 5

Immunoblot analysis of the P_II protein in cells of wild-type Synechococcus sp. strain PCC 7942 (WT) and of the NtcA-deficient mutant (NtcA⁻) in response to the nature of the nitrogen source and CO₂ availability. The cells were incubated as described in the legend to Fig. 2.

FIG. 6

In vivo phosphorylation of the P_II protein in cells of wild-type Synechococcus sp. strain PCC 7942 (WT) and the NtcA-deficient mutant (NtcA⁻) in response to the nature of the nitrogen source and CO₂ availability. Lanes 1 to 3, the cells were incubated as described in the legend to Fig. 2. Lanes 4, cells starved for nitrogen as in lanes 3 were further incubated for 2 h following addition of ammonium to the culture medium. P_II⁰, P_II¹, P_II², and P_II³ correspond to the four isoforms of the trimeric P_II protein containing zero, one, two, or three phosphorylated monomers.

FIG. 7

Comparison of the physical organization of the promoter regions of the glnB genes from Synechococcus sp. strain PCC 7942 and Synechocystis sp. strain PCC 6803.