Characterization of HetR protein turnover in Anabaena sp. PCC 7120

The hetR gene plays an important role in heterocyst development and pattern formation in heterocystous cyanobacteria. The hetR gene from Anabaena sp. PCC 7120 was overexpressed in Escherichia coli. Antibodies raised against the recombinant HetR protein (rHetR) were used to characterize metabolism of the HetR of Anabaena sp. PCC 7120 in vivo. HetR was present at a low level when Anabaena sp. PCC 7120 was grown in the presence of combined nitrogen. Shifting from nitrogen repletion conditions to nitrogen depletion conditions led to a two fold increase of HetR in total cell extracts, and most of HetR was located in heterocysts. The amount of HetR in total cellular extracts increased rapidly after shifting to nitrogen depletion conditions and reached a maximum level 3 h after the shift. Isoelectrofocusing electrophoresis revealed that the native HetR had a more acidic isoelectric point than did rHetR. After combined nitrogen was added to the nitrogen-depleted cultures, the degradation of HetR depended on culture conditions: before heterocysts were fully developed, HetR was rapidly degraded; after heterocysts were fully developed, HetR was degraded much more slowly. The distribution of HetR in other species of cyanobacteria was also studied. Received: 24 June 1997 / Accepted: 5 December 1997     

Arrangement of nitrogenase structural genes in an aerobic filamentous nonheterocystous cyanobacterium.

Members of the marine filamentous, nonheterocystous cyanobacterial genus Trichodesmium not only are capable of fixing nitrogen aerobically in the light but when grown under a light-dark cycle will fix nitrogen only during the light phase. In this study, we constructed a restriction map of the structural nitrogen fixation genes (nifHDK) in Trichodesmium sp. strain NIBB 1067. We found that the organization of the nif genes in Trichodesmium sp. strain NIBB 1067 is contiguous, as found in other nonheterocystous cyanobacteria and in heterocysts. Furthermore, the nif gene arrangement was identical when the cultures were grown with combined nitrogen or under nitrogen-fixing conditions. Therefore, no gene rearrangements occur, such as those that occur during the development of heterocysts in heterocystous species.     

Cyanobacterial Cell Lineage Analysis of the Spatiotemporal hetR Expression Profile during Heterocyst Pattern Formation in Anabaena sp. PCC 7120

Diazotrophic heterocyst formation in the filamentous cyanobacterium, Anabaena sp. PCC 7120, is one of the simplest pattern formations known to occur in cell differentiation. Most previous studies on heterocyst patterning were based on statistical analysis using cells collected or observed at different times from a liquid culture, which would mask stochastic fluctuations affecting the process of pattern formation dynamics in a single bacterial filament. In order to analyze the spatiotemporal dynamics of heterocyst formation at the single filament level, here we developed a culture system to monitor simultaneously bacterial development, gene expression, and phycobilisome fluorescence. We also developed micro-liquid chamber arrays to analyze multiple Anabaena filaments at the same time. Cell lineage analyses demonstrated that the initial distributions of hetR::gfp and phycobilisome fluorescence signals at nitrogen step-down were not correlated with the resulting distribution of developed heterocysts. Time-lapse observations also revealed a dynamic hetR expression profile at the single-filament level, including transient upregulation accompanying cell division, which did not always lead to heterocyst development. In addition, some cells differentiated into heterocysts without cell division after nitrogen step-down, suggesting that cell division in the mother cells is not an essential requirement for heterocyst differentiation.     

The hetZ gene indirectly regulates heterocyst development at the level of pattern formation in Anabaena sp. strain PCC 7120

Multicellular development requires the careful orchestration of gene expression to correctly create and position specialized cells. In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, nitrogen‐fixing heterocysts are differentiated from vegetative cells in a reproducibly periodic and physiologically relevant pattern. While many genetic factors required for heterocyst development have been identified, the role of HetZ has remained unclear. Here, we present evidence to clarify the requirement of hetZ for heterocyst production and support a model where HetZ functions in the patterning stage of differentiation. We show that a clean, nonpolar deletion of hetZ fails to express the developmental genes hetR, patS, hetP and hetZ correctly and fails to produce heterocysts. Complementation and overexpression of hetZ in a hetP mutant revealed that hetZ was incapable of bypassing hetP, suggesting that it acts upstream of hetP. Complementation and overexpression of hetZ in a hetR mutant, however, demonstrated bypass of hetR, suggesting that it acts downstream of hetR and is capable of bypassing the need for hetR for differentiation irrespective of nitrogen status. Finally, protein–protein interactions were observed between HetZ and HetR, Alr2902 and HetZ itself. Collectively, this work suggests a regulatory role for HetZ in the patterning phase of cellular differentiation in Anabaena.     

Two mutations that block heterocyst differentiation have different effects on akinete differentiation in Nostoc ellipsosporum

Evident differentiation of vegetative cells into hetero-cysts in Anabaena sp. strain PCC 7120 is prevented by Insertions in genes hetR and hetP. Nostoc ellipsosporum possesses single copies of genes that hybridize with hetR and hetP. In mutant NE2 of N. ellipsosporum, in which hetR is interrupted by an insert, and in a double recombinant of wild-type N. ellipsosporum with a plasmid that bears an interrupted copy of hetR, neither heterocysts nor akinetes are formed. When an intact copy of hetR from Anabaena sp. strain PCC 7120 was added to NE2 the ability to form both heterocysts and akinetes was restored, in contrast to the hetR mutant, a hetP mutant of N. ellipsosporum could form akinetes, but heterocyst formation was blocked. Use of luxAB, encoding luciferase, as a reporter, and use of luxC, luxD and luxE to generate aldehyde (a substrate for the luciferase reaction), permitted visualization of the expression of hetR at the level of single cells; hetR was expressed in akinetes.     

ppGpp Metabolism Is Involved in Heterocyst Development in the Cyanobacterium Anabaena sp. Strain PCC 7120

When deprived of a combined-nitrogen source in the growth medium, the filamentous cyanobacterium Anabaena sp. PCC 7120 (Anabaena) can form heterocysts capable of nitrogen fixation. The process of heterocyst differentiation takes about 20 to 24 h, during which extensive metabolic and morphological changes take place. Guanosine tetraphosphate (ppGpp) is the signal of the stringent response that ensures cell survival by adjusting major cellular activities in response to nutrient starvation in bacteria, and ppGpp accumulates at the early stage of heterocyst differentiation (J. Akinyanju, R. J. Smith, FEBS Lett. 107:173–176, 1979; J Akinyanju, R. J. Smith, New Phytol. 105:117–122, 1987). Here we show that all1549 (here designated rel_ana) in Anabaena, homologous to relA/spoT, is upregulated in response to nitrogen deprivation and predominantly localized in vegetative cells. The disruption of rel_ana strongly affects the synthesis of ppGpp, and the resulting mutant, all1549Ωsp/sm, fails to form heterocysts and to grow in the absence of a combined-nitrogen source. This phenotype can be complemented by a wild-type copy of rel_ana. Although the upregulation of hetR is affected in the mutant, ectopic overexpression of hetR cannot rescue the phenotype. However, we found that the mutant rapidly loses its viability, within a time window of 3 to 6 h, following the deprivation of combined nitrogen. We propose that ppGpp plays a major role in rebalancing the metabolic activities of the cells in the absence of the nitrogen source supply and that this regulation is necessary for filament survival and consequently for the success of heterocyst differentiation.     

Crystal structure of Alr1298, a pentapeptide repeat protein from the cyanobacterium Nostoc sp. PCC 7120, determined at 2.1 Å resolution

Nostoc sp. PCC 7120 are filamentous cyanobacteria capable of both oxygenic photosynthesis and nitrogen fixation, with the latter taking place in specialized cells known as heterocysts that terminally differentiate from vegetative cells under conditions of nitrogen starvation. Cyanobacteria have existed on earth for more than 2 billion years and are thought to be responsible for oxygenation of the earth's atmosphere. Filamentous cyanobacteria such as Nostoc sp. PCC 7120 may also represent the oldest multicellular organisms on earth that undergo cell differentiation. Pentapeptide repeat proteins (PRPs), which occur most abundantly in cyanobacteria, adopt a right-handed quadrilateral β-helical structure, also referred to as a repeat five residue (Rfr) fold, with four-consecutive pentapeptide repeats constituting a single coil in the β-helical structure. PRPs are predicted to exist in all compartments within cyanobacteria including the thylakoid and cell-wall membranes as well as the cytoplasm and thylakoid periplasmic space. Despite their intriguing structure and importance to understanding ancient cyanobacteria, the biochemical function of PRPs in cyanobacteria remains largely unknown. Here we report the crystal structure of Alr1298, a PRP from Nostoc sp. PCC 7120 predicted to reside in the cytoplasm. The structure displays the typical right-handed quadrilateral β-helical structure and includes a four-α-helix cluster capping the N-terminus and a single α-helix capping the C-terminus. A gene cluster analysis indicated that Alr1298 may belong to an operon linked to cell proliferation and/or thylakoid biogenesis. Elevated alr1298 gene expression following nitrogen starvation indicates that Alr1298 may play a role in response to nitrogen starvation and/or heterocyst differentiation.     

Aerobic nitrogen fixation is confined to a subset of cells in the non-heterocystous cyanobacterium Symploca PCC 8002

Symploca PCC 8002 Kützing is a filamentous cyanobacterium that lacks the specialized cells, known as heterocysts, that protect nitrogenase from O₂ in most aerobic N₂-fixing cyanobacteria. Nevertheless, Symploca is able to carry out N₂ fixation in the light under aerobic conditions. When cultures were grown under light/dark cycles, nitrogenase activity commenced and increased in the light phase and declined towards zero in the dark. Immunolocalization of dinitrogenase reductase in sectioned Symploca trichomes showed that the enzyme was present only in 9% of the cells. These cells lacked any obvious mechanical protection against atmospheric O₂ and their ultrastructural characteristics were similar to those of cells that did not contain any dinitrogenase reductase. The nitrogenase-containing cells possessed carboxysomes that were rich in ribulose-1,5-bisphosphate carboxylase/oxygenase and phycoerythrin, a light harvesting pigment of PS II. This indicates that these cells had a capacity for both N₂ fixation and photosynthesis. The significance of the localization pattern for dinitrogenase reductase is discussed in the context of N₂ fixation in Symploca PCC 8002.     

Characterization of Trichodesmium spp. by Genetic Techniques

The genetic diversity of Trichodesmium spp. from natural populations (off Bermuda in the Sargasso Sea and off North Australia in the Arafura and Coral Seas) and of culture isolates from two regions (Sargasso Sea and Indian Ocean) was investigated. Three independent techniques were used, including a DNA fingerprinting method based on a highly iterated palindrome (HIP1), denaturing gradient gel electrophoresis of a hetR fragment, and sequencing of the internal transcribed spacer (ITS) of the 16S-23S rDNA region. Low genetic diversity was observed in natural populations of Trichodesmium spp. from the two hemispheres. Culture isolates of Trichodesmium thiebautii, Trichodesmium hildebrandtii, Trichodesmium tenue, and Katagnymene spiralis displayed remarkable similarity when these techniques were used, suggesting that K. spiralis is very closely related to the genus Trichodesmium. The largest genetic variation was found between Trichodesmium erythraeum and all other species of Trichodesmium, including a species of Katagnymene. Our data obtained with all three techniques suggest that there are two major clades of Trichodesmium spp. The HIP1 fingerprinting and ITS sequence analyses allowed the closely related species to be distinguished. This is the first report of the presence of HIP1 in marine cyanobacteria.     

Functional dissection of the three-domain SepJ protein joining the cells in cyanobacterial trichomes

Heterocyst-forming cyanobacteria grow as filaments of cells (trichomes) in which, under nitrogen limitation, two interdependent cell types, the vegetative cells performing oxygenic photosynthesis and the nitrogen-fixing heterocysts, exchange metabolites and regulatory compounds. SepJ is a protein conspicuously located at the cell poles in the intercellular septa of the filaments that has three well-defined domains: an N-terminal coiled-coil domain, a central linker and a C-terminal permease domain. Mutants of Anabaena sp. strain PCC 7120 carrying SepJ proteins with specific deletions showed that, whereas the linker domain is dispensable, the coiled-coil domain is required for polar localization of SepJ, filament integrity, normal intercellular transfer of small fluorescent tracers and diazotrophy. An Anabaena strain carrying the SepJ protein from the filamentous, non-heterocyst-forming cyanobacterium Trichodesmium erythraeum, which lacks the linker domain, made long filaments in the presence of combined nitrogen but fragmented extensively under nitrogen deprivation and did not grow diazotrophically. In contrast, a chimera made of the Trichodesmium coiled-coil domain and the Anabaena permease allowed heterocyst differentiation and diazotrophic growth. Thus, SepJ provides filamentous cyanobacteria with a cell-cell anchoring function, but the permease domain has evolved in heterocyst formers to provide intercellular molecular exchange functions required for diazotrophy.     

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.