Mutual regulation of ntcA and hetR during heterocyst differentiation requires two similar PP2C-type protein phosphatases, PrpJ1 and PrpJ2, in Anabaena sp. strain PCC 7120

Abstract

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 can form heterocysts for N(2) fixation. Initiation of heterocyst differentiation depends on mutual regulation of ntcA and hetR. Control of hetR expression by NtcA is partially mediated by nrrA, but other factors must be involved in this regulation. Anabaena has two closely related PP2C-type protein phosphatases, PrpJ1 (formerly PrpJ) and PrpJ2; PrpJ1 is involved in heterocyst maturation. In this study, we show that PrpJ2, like PrpJ1, has Mn(2+)-dependent phosphatase activity. We further demonstrate that whereas prpJ2 is dispensable for cell growth under different nitrogen regimens tested, a double mutant with both prpJ1 and prpJ2 disrupted did not initiate heterocyst differentiation. Ectopic expression of hetR in the double mutant could rescue the failure to initiate heterocyst development, but the heterocysts formed, like those of a prpJ1 single mutant, were not mature. The expression of prpJ2 was enhanced during heterocyst development, and the upregulation of the gene was directly under the control of NtcA. Upregulation of both ntcA and hetR was affected in the double mutant. We propose that PrpJ1 and PrpJ2 together are required for mutual regulation of ntcA and hetR and are thus involved in regulation of the initiation of heterocyst differentiation.

FIG. 1.

(A) Different domains of PrpJ1 and PrpJ2. The length of each domain (in number of residues) is indicated in parentheses. TM, putative transmembrane domain. (B) Genomic environment of prpJ2 and strategy for inactivating prpJ2 by insertion of an antibiotic resistance cassette (Neo^r). The arrow for the Neo^r cassette indicates the orientation of the resistance cassette relative to that of prpJ2.

FIG. 2.

Enzymatic activity of PrpJ2. (A) Dephosphorylation of pNPP by PrpJ2 requires Mn²⁺, whereas the other divalent cations tested had much less effect on the activity of PrpJ2. The concentration of the different divalent cations used in these assays was 5 mM, the concentration of pNPP was 5 mM, and the amount of PrpJ2 was 1 μg (15, 29). A.U., arbitrary units. (B) Dephosphorylation of three phosphopeptides by PrpJ2. Filled circles, peptide phosphotyrosine 2 DADE(pY)LIPQQG; triangles, peptide phosphotyrosine 1 END(pY)INASL; open circles, peptide containing phosphothreonine RRA(pT)VA. All of the peptides were purchased from Sigma (15).

FIG. 3.

Heterocyst differentiation observed with a light microscope 24 h after induction. Single mutant MprpJ2 (B) has a phenotype similar to that of the wild type (A), while double mutant MprpJ1prpJ2 (C) forms no heterocysts as determined with a light microscope. All strains were incubated with alcian blue, which stains the polysaccharide layer of heterocysts.

FIG. 4.

Ectopic expression of hetR in double mutant MprpJ1prpJ2 allows initiation of heterocyst differentiation. Plasmid pPH1 bearing hetR was transferred by conjugation into either the wild type (A) or the double mutant (B), and heterocyst differentiation 24 h after removal of nitrogen was observed with a microscope. Note that heterocysts (indicated by arrowheads) in the wild type have polar granules and that there are some double heterocysts, while the heterocysts of the double mutant are not fully mature and lack polar granules. The concentration of copper used to induce the expression of hetR was 0.5 μM.

FIG. 5.

Expression of hetR, ntcA, and nrrA in the wild type (WT) and the double mutant determined by real-time PCR in triplicate. RNAs were extracted at different times after removal of combined nitrogen. After cDNA synthesis, the relative amounts of transcripts of hetR (A), ntcA (B), and nrrA (C) were estimated using real-time PCR. The error bars indicate the fluctuation of data obtained in triplicate. Heterocyst differentiation was induced at time zero after the removal of combined nitrogen.

FIG. 6.

NtcA binds to the upstream region of prpJ2. (A) Upstream region of prpJ2. The putative NtcA-binding site is indicated by bold type and underlining, and the ATG sequence corresponding to the initiation codon is indicated by bold type and italics. (B) EMSA for analysis of the interaction between NtcA and the prpJ2 upstream region. The glnA promoter region was used as a control with (+) and without (−) NtcA. The prpJ2 promoter region (prpJ2) or, as a control, the same region having mutations in the NtcA binding site [prpJ2 (mutant)] was incubated with increasing amounts of NtcA. The lower DNA band corresponds to free DNA, and the upper band corresponds to the NtcA-DNA complex.

FIG. 7.

Expression of prpJ2 during heterocyst differentiation in the wild type (wt) and different mutants (hetR216, nrrA, and ntcA), measured by real-time RT-PCR. The error bars indicate the fluctuation in the data obtained in triplicate. Heterocyst differentiation was induced at time zero after the removal of combined nitrogen.