PrpJ, a PP2C-type protein phosphatase located on the plasma membrane, is involved in heterocyst maturation in the cyanobacterium Anabaena sp. PCC 7120

Protein phosphatases play important roles in the regulation of cell growth, division and differentiation. The cyanobacterium Anabaena PCC 7120 is able to differentiate heterocysts specialized in nitrogen fixation. To protect the nitrogenase from inactivation by oxygen, heterocyst envelope possesses a layer of polysaccharide and a layer of glycolipids. In the present study, we characterized All1731 (PrpJ), a protein phosphatase from Anabaena PCC 7120. prpJ was constitutively expressed in both vegetative cells and heterocysts. Under diazotrophic conditions, the mutant DeltaprpJ (S20) did not grow, lacked only one of the two heterocyst glycolipids, and fragmented extensively at the junctions between developing cells and vegetative cells. No heterocyst glycolipid layer could be observed in the mutant by electron microscopy. The inactivation of prpJ affected the expression of hglE(A) and nifH, two genes necessary for the formation of the glycolipid layer of heterocysts and the nitrogenase respectively. PrpJ displayed a phosphatase activity characteristic of PP2C-type protein phosphatases, and was localized on the plasma membrane. The function of prpJ establishes a new control point for heterocyst maturation because it regulates the synthesis of only one of the two heterocyst glycolipids while all other genes so far analysed regulate the synthesis of both heterocyst glycolipids.     

Comparative Network Biology Discovers Protein Complexes That Underline Cellular Differentiation in Anabaena sp.

The filamentous cyanobacterium Anabaena sp. PCC 7120 can differentiate into heterocysts to fix atmospheric nitrogen. During cell differentiation, cellular morphology and gene expression undergo a series of significant changes. To uncover the mechanisms responsible for these alterations, we built protein–protein interaction (PPI) networks for these two cell types by cofractionation coupled with mass spectrometry. We predicted 280 and 215 protein complexes, with 6322 and 2791 high-confidence PPIs in vegetative cells and heterocysts, respectively. Most of the proteins in both types of cells presented similar elution profiles, whereas the elution peaks of 438 proteins showed significant changes. We observed that some well-known complexes recruited new members in heterocysts, such as ribosomes, diflavin flavoprotein, and cytochrome c oxidase. Photosynthetic complexes, including photosystem I, photosystem II, and phycobilisome, remained in both vegetative cells and heterocysts for electron transfer and energy generation. Besides that, PPI data also reveal new functions of proteins. For example, the hypothetical protein Alr4359 was found to interact with FraH and Alr4119 in heterocysts and was located on heterocyst poles, thereby influencing the diazotrophic growth of filaments. The overexpression of Alr4359 suspended heterocyst formation and altered the pigment composition and filament length. This work demonstrates the differences in protein assemblies and provides insight into physiological regulation during cell differentiation.     

Anabaena sp. strain PCC 7120 gene devH is required for synthesis of the heterocyst glycolipid layer

In response to deprivation for fixed nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 provides a microoxic intracellular environment for nitrogen fixation through the differentiation of semiregularly spaced vegetative cells into specialized cells called heterocysts. The devH gene is induced during heterocyst development and encodes a product with characteristics of a trans-acting regulatory protein. A devH mutant forms morphologically distinguishable heterocysts but is Fox(-), incapable of nitrogen fixation in the presence of oxygen. We demonstrate that rearrangements of nitrogen fixation genes take place normally in the devH mutant and that it is Fix(+), i.e., has nitrogenase activity under anoxic conditions. The Fox(-) phenotype was shown by ultrastructural studies to be associated with the absence of the glycolipid layer of the heterocyst envelope. The expression of glycolipid biosynthetic genes in the mutant is greatly reduced, and heterocyst glycolipids are undetectable.     

Boron Requirement for Envelope Structure and Function in Anabaena PCC 7119 Heterocysts

The effects of boron deficiency on the ultrastructure and envelopecomposition of heterocysts in the filamentous cyanobacteriumAnabaena PCC 7119 were studied. Microscopic examination of boron-deficientcultures showed changes in heterocyst morphology. When thesecells were isolated and their glycolipid content determined,this specific component of the laminated layer of the heterocystenvelope was found to be lacking. The evidence presented supportsthe view that boron plays an essential role in the structureand function of the heterocyst envelope. Key words: Anabaena, boron, heterocysts, nitrogenase, oxygen-protection     

Identification of the glycosyl transferase required for synthesis of the principal glycolipid characteristic of heterocysts of Anabaena sp. strain PCC 7120

Nitrogenase is oxygen-labile. Cyanobacterial heterocysts can fix N(2) in an oxic milieu because their interior is micro-oxic, for which the glycolipid layer of the heterocyst envelope is required. ORF all5341 of the Anabaena sp. genome predicts a glycosyl transferase. An insertional mutant of all5341 synthesized only a nonglycosylated form of heterocyst envelope glycolipid, and lacked a glycolipid layer. All5341 appears to be the transferase required to glycosylate the glycolipid aglycone.     

Novel ATP-driven pathway of glycolipid export involving TolC protein

Upon depletion of combined nitrogen, N(2)-fixing heterocysts are formed from vegetative cells in the case of the filamentous cyanobacterium Anabaena sp. strain PCC 7120. A heterocyst-specific layer composed of glycolipids (heterocyst envelope glycolipids (HGLs)) that functions as an O(2) diffusion barrier is deposited over the heterocyst outer membrane and is surrounded by an outermost heterocyst polysaccharide envelope. Mutations in any gene of the devBCA operon or tolC result in the absence of the HGL layer, preventing growth on N(2) used as the sole nitrogen source. However, those mutants do not have impaired HGL synthesis. In this study, we show that DevBCA and TolC form an ATP-driven efflux pump required for the export of HGLs across the Gram-negative cell wall. By performing protein-protein interaction studies (in vivo formaldehyde cross-linking, surface plasmon resonance, and isothermal titration calorimetry), we determined the kinetics and stoichiometric relations for the transport process. For sufficient glycolipid export, the membrane fusion protein DevB had to be in a hexameric form to connect the inner membrane factor DevC and the outer membrane factor TolC. A mutation that impaired the ability of DevB to form a hexameric arrangement abolished the ability of DevC to recognize its substrate. The physiological relevance of a hexameric DevB is shown in complementation studies. We provide insights into a novel pathway of glycolipid export across the Gram-negative cell wall.     

The hglK gene is required for localization of heterocyst-specific glycolipids in the cyanobacterium Anabaena sp. strain PCC 7120.

Mutant strain 543 of the cyanobacterium Anabaena sp. strain PCC 7120 was originally isolated as a Fox- mutant following chemical mutagenesis. Ultrastructural analysis shows that in nitrogen-replete media the vegetative cells of the mutant are more cylindrical and have thicker septa than those of the wild type, while in nitrogen-free media the mutant heterocysts lack the normal glycolipid layer external to the cell wall. Although this layer is absent, strain 543 heterocysts nevertheless contain heterocyst-specific glycolipids, as determined by thin-layer chromatography. The mutation in strain 543 is in a gene we have named hglK, encoding a protein of 727 amino acids. The wild-type HglK protein appears to contain four membrane-spanning regions followed by 36 repeats of a degenerate pentapeptide sequence, AXLXX. The mutation in strain 543 introduces a termination codon immediately upstream of the pentapeptide repeat region. A mutant constructed by insertion of an antibiotic resistance cassette near the beginning of the hglK gene has the same phenotype as strain 543. We propose that hglK encodes a protein necessary for the localization of heterocyst glycolipids and that this function requires the pentapeptide repeats of the HglK protein.     

The role of HetN in maintenance of the heterocyst pattern in Anabaena sp. PCC 7120

The gene hetN encodes a putative oxidoreductase that is known to suppress heterocyst differentiation when present on a multicopy plasmid in Anabaena sp. PCC 7120. To mimic the hetN null phenotype and to examine where HetN acts in the regulatory cascade that controls heterocyst differentiation, we replaced the native chromosomal hetN promoter with the copper-inducible petE promoter. In the presence of copper, heterocyst formation was suppressed in undifferentiated filaments. When hetN expression was turned off by transferring cells to media lacking copper, the filaments initially displayed the wild-type pattern of single heterocysts but, 48 h after the induction of heterocyst formation, a pattern of multiple contiguous heterocysts predominated. Suppression of heterocyst formation by HetN appears to occur both upstream and downstream of the positive regulator HetR: overexpression of hetN in undifferentiated filaments prevents the wild-type pattern of hetR expression as well as the multiheterocyst phenotype normally observed when hetR is expressed from an inducible promoter. Green fluorescent protein fusions show that the expression of hetN in wild-type filaments normally occurs primarily in heterocysts. We propose that HetN is normally involved in the maintenance of heterocyst spacing after the initial heterocyst pattern has been established, but ectopic expression of hetN can also block the initial establishment of the pattern.     

Heterocyst differentiation in the cyanobacterium Mastigocladus laminosus.

The morphological and ultrastructural aspects of heterocyst differentiation in the branching, filamentous cyanobacterium Mastigocladus laminosus were examined with light and electron microscopy. The earliest differentiation stages involved cytoplasmic changes, including (i) rapid degradation of carboxysomes, (ii) degradation of polysaccharide granules, and (iii) accumulation of electron-dense ribosomal or protein material (or both). Intermediate differentiation stages involved synthesis of a homogeneous extra wall layer, development of necks leading to adjacent cells, and elaboration of a complex system of intracytoplasmic membranes. Late differentiation stages included further development of necks and continued elaboration of membranes. Mature heterocysts possessed a uniformly electron-dense cytoplasm that contained large numbers of closely packed membranes, some of which were arranged in lamellar stacks. Mature heterocysts lacked all of the inclusion bodies present in undifferentiated vegetative cells, but contained a number of unusual spherical inclusions of variable electron density. Cells in both narrow and wide filaments were capable of differentiating. No regular heterocyst spacing pattern was observed in the narrow filaments; the number of vegetative cells between consecutive heterocysts of any given filament varied by a factor of 10. Heterocysts developed at a variety of locations in the wide, branching filaments, although the majority of them were situated adjacent to branch points. M. laminosus displayed a marked tendency to produce sets of adjacent heterocysts or proheterocysts (or both) that were not separated from each other by vegetative cells. Groups of four or more adjacent heterocysts or proheterocysts occurred frequently in wide filaments, and in some of these filaments virtually all of the cells appeared to be capable of differentiating into heterocysts.     

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.     

Tunicamycin suppresses differentiation and stimulates dedifferentiation of heterocysts in the cyanobacteriumAnabaena azollae Stras

The effect of tunicamycin (TM), a specific inhibitor of glycosylation of proteins, on heterocyst differentiation inAnabaena azollae Stras. was investigated. Heterocysts were developed in the presence of TM up to a concentration of 0.2 g ml^–1, whereas at higher concentrations differentiation proceeded only up to the proheterocyst stage. Analysis of lipids by thinlayer chromatography showed that the glycolipid that is specific for the laminated layer of mature heterocysts was synthesized even in the cultures where the differentiation had proceeded only up to the early proheterocyst stage (i.e., at>0.2 g TM ml^–1). Further, deposition of the glycolipid-containing laminated layer in the envelope of the heterocysts differentiated in the presence of TM (i.e., at 0.2 g ml^–1) was incohesive as observed at the ultrastructural level. These findings clearly suggest that the process leading to the transportation of the heterocyst-specific glycolipid and its deposition as a laminated layer are affected by TM treatment. Because of the reported highly selective mode of action of TM, our results implicate a role for protein glycosylation in the process of heterocyst development and maturation.Although the heterocyst is a terminal stage of differentiation, because they normally do not divide/multiply, a considerable percentage of heterocysts that developed in the presence of TM dedifferentiated. The aberrations formed in the laminated layer, probably because of the inhibition of protein glycosylation owing to TM treatment, may have contributed to the instability of heterocyst structure and consequently led to their dedifferentiation.     

The 2A resolution crystal structure of HetL, a pentapeptide repeat protein involved in regulation of heterocyst differentiation in the cyanobacterium Nostoc sp. strain PCC 7120

The hetL gene from the cyanobacterium Nostoc sp. PCC 7120 encodes a 237 amino acid protein (25.6kDa) containing 40 predicted tandem pentapeptide repeats. Nostoc sp. PCC 7120 is a filamentous cyanobacterium that forms heterocysts, specialized cells capable of fixing atmospheric N(2) during nitrogen starvation in its aqueous environment. Under these conditions, heterocysts occur in a regular pattern of approximately one out of every 10-15 vegetative cells. Heterocyst differentiation is highly regulated involving hundreds of genes, one of which encodes PatS, thought to be an intercellular peptide signal made by developing heterocysts to inhibit heterocyst differentiation in neighboring vegetative cells, thus contributing to pattern formation and spacing of heterocysts along the filament. While overexpression of PatS suppresses heterocyst differentiation in Nostoc sp. PCC 7120, overexpression of HetL produces a multiple contiguous heterocyst phenotype with loss of the wild type heterocyst pattern, and strains containing extra copies of hetL allow heterocyst formation even in cells overexpressing PatS. Thus, HetL appears to interfere with heterocyst differentiation inhibition by PatS, however, the mechanism for HetL function remains unknown. As a first step towards exploring the mechanism for its biochemical function, the crystal structure of HetL has been solved at 2.0A resolution using sulfur anomalous scattering.     

LytM factor Alr3353 affects filament morphology and cell–cell communication in the multicellular cyanobacterium Anabaena sp. PCC 7120

Heterocyst‐forming cyanobacteria are organized as multicellular filaments of tightly interacting, functionally specialized cells. N₂‐fixing heterocysts differentiate from vegetative cells under nitrogen limitation in a semi‐regular pattern along the filament. Diazotrophic growth requires metabolite exchange between neighboring cells within the filament. This exchange occurs via cell–cell junction complexes that span the gap between the plasma membranes and thereby cross the septal peptidoglycan through an array of uniform nanopores formed by AmiC‐type cell wall hydrolases. We investigated how the lytic hydrolase AmiC1 (Alr0092) from Anabaena sp. PCC 7120, whose activity needs to be tightly controlled to avoid cell lysis, is regulated by the LytM factor Alr3353. Inactivation of alr3353 resulted in significantly fewer nanopores and as a consequence, a lower rate of fluorescent tracer exchange between cells. The mutant was not able to grow with N₂ as sole nitrogen source, although heterocysts were formed. Alr3353 localized mainly to fully developed intercellular septa of vegetative cells. The purified protein bound to peptidoglycan and enhanced the hydrolytic activity of AmiC1 in vitro. Our data show that the LytM factor Alr3353 regulates nanopore formation and cell–cell communication by directly interacting with AmiC1.