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

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 can form heterocysts for N₂ 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 Mn²⁺-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.Many cyanobacteria can fix N₂ when combined nitrogen sources become limiting in the growth medium. The nitrogenase enzymatic complex responsible for nitrogen fixation is very sensitive to oxygen, and oxygen is produced by photosynthesis by cyanobacteria. The strategy used by some filamentous diazotrophic cyanobacteria to resolve this oxygen paradox is to perform photosynthesis and nitrogen fixation in two distinct cell types, differentiated cells called heterocysts that provide a microoxic environment for nitrogenase and vegetative cells which perform oxygenic photosynthesis (22, 36, 39). One such organism is Anabaena sp. strain PCC 7120. In this strain, heterocysts account for 5 to 10% of the cells and appear in a semiregular pattern along each filament. Therefore, the process of heterocyst differentiation provides a prokaryotic model to study developmental pattern formation. Three factors account for the microoxic environment in heterocysts: the heterocyst envelope composed of an inner layer of glycolipid surrounded by an outer layer of polysaccharides that limits oxygen penetration, the lack of oxygen-producing photosystem II, and an increased rate of respiration to consume oxygen (36).The initiation of heterocyst differentiation and the formation of the heterocyst pattern are governed by multiple signals and the concerted actions of several proteins as positive or negative regulators (for a recent review, see 39). The accumulation of 2-oxoglutarate following limitation of combined nitrogen is a trigger that initiates heterocyst development by stimulating the DNA-binding activity of NtcA, a protein involved in the regulation of carbon and nitrogen metabolism, as well as initiation of heterocyst differentiation (7, 12, 13, 18, 20, 32, 35). HetR, a protease with DNA-binding activity, plays a central role in the early steps of heterocyst differentiation (14, 40). Both ntcA and hetR are autoregulated, and the expression of hetR and the expression of ntcA are mutually dependent because upregulation of one of theses genes is dependent on the other gene (3, 4, 23). How HetR regulates the expression of ntcA remains unknown. No NtcA-binding site has been found in the upstream region of hetR, and the regulation of hetR by NtcA could be partially due to the action of the response regulator NrrA (8, 9, 24). However, NrrA cannot be the only link between ntcA and hetR, because when nrrA was inactivated, both heterocyst differentiation and hetR upregulation were only delayed (8). Indeed, ccbP, encoding a calcium-binding protein, is regulated by NtcA, and it has been proposed that the pool of calcium affects the activity of HetR (31).The genome of Anabaena sp. strain PCC 7120 contains a large number of genes encoding two-component signaling systems, protein Ser/Thr and/or Tyr kinases, and phosphatases, including eight genes encoding PP2C-type Ser/Thr phosphatases (16, 26, 34, 38). Some of these genes are involved in heterocyst development, mostly in heterocyst maturation and functioning (8, 11, 17, 19, 21, 25, 30, 37). We have shown previously that PrpJ is a PP2C-type protein phosphatase located on the plasma membrane (15). A prpJ1 mutant (strain S20) failed to grow under diazotrophic conditions and formed heterocysts lacking the major heterocyst-specific glycolipid (HGL), in contrast to other mutants whose mutations affect either the synthesis or the deposition of both the major and minor HGLs (1, 2, 10, 28) or only the minor HGL (30). Therefore, PrpJ represents a new regulatory branch for heterocyst maturation, possibly involving regulation of only a subset of genes involved in glycolipid synthesis. These observations indicate that multiple input pathways participate in the maturation of heterocysts. When proheterocysts were formed, filaments of the prpJ1 mutant, fragmented extensively at the junctions between proheterocysts and vegetative cells, resulting in free nonmature heterocysts and filaments that were 11 cells long on average (15).Open reading frame all2470 encodes one member of the PP2C family of protein phosphatases in Anabaena sp. strain PCC 7120 (35). The deduced amino acid sequence of All2470 is similar to that of PrpJ, and these two proteins have similar architectures, with an N-terminal domain having an unknown function, a central domain similar to the catalytic domains of PP2C-type protein phosphatases, and a C-terminal domain with a putative transmembrane motif (Fig. ​(Fig.1).1). The amino acid sequences of these two proteins share 40% identity overall, and their catalytic domains are 45% identical. Because these two protein phosphatases are very similar, here we use the designations PrpJ1 (formerly PrpJ) for All1731 and PrpJ2 for All2470. In the present study, we show that PrpJ1 and PrpJ2 are involved in the initiation of heterocyst differentiation by acting on the mutual regulation of ntcA and hetR.Open in a separate windowFIG. 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.