Signal transduction protein P(II) is required for NtcA-regulated gene expression during nitrogen deprivation in the cyanobacterium Synechococcus elongatus strain PCC 7942

Abstract

The transcription factor of the cyclic AMP receptor protein/FNR family, NtcA, and the P(II) signaling protein play central roles in global nitrogen control in cyanobacteria. A dependence on P(II) for NtcA-regulated transcription, however, has not been observed. In the present investigation, we examined alterations in gene expression following nitrogen deprivation in Synechococcus elongatus strain PCC 7942 and specifically the roles of NtcA and P(II). Global changes in de novo protein synthesis following combined-nitrogen deprivation were visualized by in vivo [(35)S]methionine labeling and two-dimensional polyacrylamide gel electrophoresis analysis. Nearly all proteins whose synthesis responded specifically to combined-nitrogen deprivation in wild-type cells of S. elongatus failed to respond in P(II)- and NtcA-deficient mutants. One of the proteins whose synthesis was down-regulated in a P(II)- and NtcA-dependent manner was RbcS, the small subunit of RubisCO. Quantification of its mRNA revealed that the abundance of the rbcLS transcript following combined-nitrogen deprivation rapidly declined in wild-type cells but not in P(II) and NtcA mutant cells. To investigate further the relationship between P(II) and NtcA, fusions of the promotorless luxAB reporter genes to the NtcA-regulated glnB gene were constructed and these constructs were used to transform wild-type cells and P(II)(-) and NtcA(-) mutants. Determination of bioluminescence under different growth conditions showed that NtcA represses gene expression in the presence of ammonium in a P(II)-independent manner. By contrast, NtcA-dependent activation of glnB expression following combined-nitrogen deprivation was impaired in the absence of P(II). Together, these results suggest that under conditions of combined-nitrogen deprivation, the regulation of NtcA-dependent gene expression requires the P(II) signal transduction protein.

FIG. 1.

Protein synthesis patterns of S. elongatus PCC 7942 wild-type cells revealed by in vivo [³⁵S]Met labeling and 2-D PAGE analysis. Cells were grown in ammonium-replete medium until they reached the mid-exponential phase of growth (OD₇₅₀ = 0.5), then aliquots were shifted to the following media: BG11 with ammonium (as a control for nutrient-replete cells) (A), combined-nitrogen-deprived medium (B), and sulfur-deprived medium (C). After 5 h of preincubation, labeling was performed as described in Materials and Methods. Spots which could be classified into the categories Gsi, Gsr, Nsi, Nsr, and Ssi (for details, see the text) are indicated. In addition, the spots corresponding to GroEL, EF-Tu, thioredoxin M (TrxM), and plastocyanin (PC), which had been identified previously (33), are also shown. The pH of the isoelectric focusing gradient (pI) in the first dimension is indicated on the top of each gel, and the positions (in kilodaltons) of molecular size standards separated in the second dimension are indicated at the left.

FIG. 1.

Protein synthesis patterns of S. elongatus PCC 7942 wild-type cells revealed by in vivo [³⁵S]Met labeling and 2-D PAGE analysis. Cells were grown in ammonium-replete medium until they reached the mid-exponential phase of growth (OD₇₅₀ = 0.5), then aliquots were shifted to the following media: BG11 with ammonium (as a control for nutrient-replete cells) (A), combined-nitrogen-deprived medium (B), and sulfur-deprived medium (C). After 5 h of preincubation, labeling was performed as described in Materials and Methods. Spots which could be classified into the categories Gsi, Gsr, Nsi, Nsr, and Ssi (for details, see the text) are indicated. In addition, the spots corresponding to GroEL, EF-Tu, thioredoxin M (TrxM), and plastocyanin (PC), which had been identified previously (33), are also shown. The pH of the isoelectric focusing gradient (pI) in the first dimension is indicated on the top of each gel, and the positions (in kilodaltons) of molecular size standards separated in the second dimension are indicated at the left.

FIG. 2.

Comparison of spots which are differentially regulated by nitrogen starvation (−N) or sulfur starvation (−S) in S. elongatus wild-type cells (wt) and in mutants deficient in P_II (MP2) and NtcA (MNtcA). Labeling and 2-D PAGE analysis were carried out with MP2 and MNtcA cells as shown in Fig. 1 for wild-type cells. Sectors containing relevant spots were aligned to facilitate the comparison. C, control.

FIG. 3.

Quantitative representation of a Northern blot analysis of rbcLS transcript abundance. RNA was extracted from wild-type, MP2, and MNtcA cells of S. elongatus prior to nitrogen step-down and from cells 2 and 8 h after transfer to combined-nitrogen-deprived medium. Twelve micrograms of RNA was loaded per lane, and the blot was hybridized with a radioactively labeled rbcL DNA probe. Visualization and quantification of the signals were carried out by phosphorimaging. Solid line, MNtcA cells; dashed line, MP2 cells; dotted line, wild-type cells.

FIG. 4.

Expression of glnB::luxAB fusions which were recombined into S. elongatus wild-type (WT), P_II-deficient (MP2), and NtcA-deficient (MNtcA) cells. The bars represent the DNA inserts that contained the 5′ end of the glnB gene and upstream sequences and were cloned in front of the luxAB reporter genes of plasmid pAM1580 (2). For details of the constructs, see Materials and Methods. The first T from the TTG initiation codon of the glnB gene was defined as position +1. The locations of relevant regions in the glnB upstream region, promoter elements, and transcriptional start sites as determined in reference are indicated above the bars. The numbers indicate the positions of the first 5′ nucleotides of the respective sequence elements relative to the TTG initiation codon. The reporter strains were grown for several generations in either nitrate- or ammonium-supplemented BG11 medium, and bioluminescence was recorded as described in Materials and Methods. Relative light units (RLU) from three to four independent determinations, together with the standard deviations, are shown. Bioluminescence values from the promoterless control strain WT-AM1580 are shown at the bottom.

FIG. 5.

Time course of glnB::luxAB induction following nitrogen step-down in reporter strains of S. elongatus in wild-type (WT) (A and B), MP2 (C and D), and MNtcA (E) backgrounds. After the initiation of nitrogen deprivation at time point 0, bioluminescence from the reporter strains was recorded over a time period of 100 h. Experiments with shifts from nitrate-supplemented to nitrogen-deprived (−N) conditions (A and C) and from ammonium-supplemented to nitrogen-deprived media (B, D, and E) were performed. Dotted lines, FAM1 reporter strains; solid lines, FAM2 reporter strains. Independent experiments yielded similar results; for each condition, results from a representative time course experiment are shown.

FIG. 5.

Time course of glnB::luxAB induction following nitrogen step-down in reporter strains of S. elongatus in wild-type (WT) (A and B), MP2 (C and D), and MNtcA (E) backgrounds. After the initiation of nitrogen deprivation at time point 0, bioluminescence from the reporter strains was recorded over a time period of 100 h. Experiments with shifts from nitrate-supplemented to nitrogen-deprived (−N) conditions (A and C) and from ammonium-supplemented to nitrogen-deprived media (B, D, and E) were performed. Dotted lines, FAM1 reporter strains; solid lines, FAM2 reporter strains. Independent experiments yielded similar results; for each condition, results from a representative time course experiment are shown.