Continuous periplasm in a filamentous, heterocyst-forming cyanobacterium

The cyanobacteria bear a Gram-negative type of cell wall that includes a peptidoglycan layer and an outer membrane outside of the cytoplasmic membrane. In filamentous cyanobacteria, the outer membrane appears to be continuous along the filament of cells. In the heterocyst-forming cyanobacteria, two cell types contribute specialized functions for growth: vegetative cells provide reduced carbon to heterocysts, which provide N2-derived fixed nitrogen to vegetative cells. The promoter of the patS gene, which is active specifically in developing proheterocysts and heterocysts of Anabaena sp. PCC 7120, was used to direct the expression of altered versions of the gfp gene. An engineered green fluorescent protein (GFP) that was exported to the periplasm of the proheterocysts through the twin-arginine translocation system was observed also in the periphery of neighbouring vegetative cells. However, if the GFP was anchored to the cytoplasmic membrane, it was observed in the periphery of the producing proheterocysts or heterocysts but not in adjacent vegetative cells. These results show that there is no cytoplasmic membrane continuity between heterocysts and vegetative cells and that the GFP protein can move along the filament in the periplasm, which is functionally continuous and so provides a conduit that can be used for chemical communication between cells.     

The outer membrane of a heterocyst-forming cyanobacterium is a permeability barrier for uptake of metabolites that are exchanged between cells

The multicellular Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can fix N₂ in differentiated cells called heterocysts, which exchange nutritional and regulatory compounds with the neighbour photosynthetic vegetative cells. The outer membrane of this bacterium is continuous along the filament defining a continuous periplasmic space. The Anabaena alr0075 , alr2269 and alr4893 gene products were characterized as Omp85-like proteins, which are generally involved in outer membrane protein biogenesis. Open reading frame alr2269 is the first gene of an operon that also carries genes for lipopolysaccharide lipid A biosynthesis including alr2270 (an lpxC homologue). Strains carrying inactivating alr2269 or alr2270 constructs showed enhanced sensitivity to erythromycin, SDS, lysozyme and proteinase K suggesting that they produce an outer membrane with increased permeability. These strains further exhibited increased uptake of sucrose, glutamate and, to a lesser extent, a few other amino acids. Increased uptake of the same metabolites was obtained by mechanical fragmentation of wild-type Anabaena filaments. These results document that the outer membrane is a permeability barrier for metabolites such as sucrose and glutamate, which are subjected to intercellular exchange in the diazotrophic filament of heterocyst-forming cyanobacteria.     

FraH is required for reorganization of intracellular membranes during heterocyst differentiation in Anabaena sp. strain PCC 7120

In the filamentous, heterocyst-forming cyanobacteria, two different cell types, the CO(2)-fixing vegetative cells and the N(2)-fixing heterocysts, exchange nutrients and regulators for diazotrophic growth. In the model organism Anabaena sp. strain PCC 7120, inactivation of fraH produces filament fragmentation under conditions of combined nitrogen deprivation, releasing numerous isolated heterocysts. Transmission electron microscopy of samples prepared by either high-pressure cryo-fixation or chemical fixation showed that the heterocysts of a ΔfraH mutant lack the intracellular membrane system structured close to the heterocyst poles, known as the honeycomb, that is characteristic of wild-type heterocysts. Using a green fluorescent protein translational fusion to the carboxyl terminus of FraH (FraH-C-GFP), confocal microscopy showed spots of fluorescence located at the periphery of the vegetative cells in filaments grown in the presence of nitrate. After incubation in the absence of combined nitrogen, localization of FraH-C-GFP changed substantially, and the GFP fluorescence was conspicuously located at the cell poles in the heterocysts. Fluorescence microscopy and deconvolution of images showed that GFP fluorescence originated mainly from the region next to the cyanophycin plug present at the heterocyst poles. Intercellular transfer of the fluorescent tracers calcein (622 Da) and 5-carboxyfluorescein (374 Da) was either not impaired or only partially impaired in the ΔfraH mutant, suggesting that FraH is not important for intercellular molecular exchange. Location of FraH close to the honeycomb membrane structure and lack of such structure in the ΔfraH mutant suggest a role of FraH in reorganization of intracellular membranes, which may involve generation of new membranes, during heterocyst differentiation.     

Catabolic Function of Compartmentalized Alanine Dehydrogenase in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

In the diazotrophic filaments of heterocyst-forming cyanobacteria, an exchange of metabolites takes place between vegetative cells and heterocysts that results in a net transfer of reduced carbon to the heterocysts and of fixed nitrogen to the vegetative cells. Open reading frame alr2355 of the genome of Anabaena sp. strain PCC 7120 is the ald gene encoding alanine dehydrogenase. A strain carrying a green fluorescent protein (GFP) fusion to the N terminus of Ald (Ald-N-GFP) showed that the ald gene is expressed in differentiating and mature heterocysts. Inactivation of ald resulted in a lack of alanine dehydrogenase activity, a substantially decreased nitrogenase activity, and a 50% reduction in the rate of diazotrophic growth. Whereas production of alanine was not affected in the ald mutant, in vivo labeling with [¹⁴C]alanine (in whole filaments and isolated heterocysts) or [¹⁴C]pyruvate (in whole filaments) showed that alanine catabolism was hampered. Thus, alanine catabolism in the heterocysts is needed for normal diazotrophic growth. Our results extend the significance of a previous work that suggested that alanine is transported from vegetative cells into heterocysts in the diazotrophic Anabaena filament.Cyanobacteria such as those of the genera Anabaena and Nostoc grow as filaments of cells (trichomes) that, when incubated in the absence of a source of combined nitrogen, present two cell types: vegetative cells that perform oxygenic photosynthesis and heterocysts that perform N₂ fixation. Heterocysts carry the oxygen-labile enzyme nitrogenase, and, thus, compartmentalization is the way these organisms separate the incompatible activities of N₂ fixation and O₂-evolving photosynthesis (9). In Anabaena and Nostoc, heterocysts are spaced along the filament so that approximately 1 in 10 to 15 cells is a heterocyst. Heterocysts differentiate from vegetative cells in a process that involves execution of a specific program of gene expression (12, 15, 39). In the N₂-fixing filament, the heterocysts provide the vegetative cells with fixed nitrogen, and the vegetative cells provide the heterocysts with photosynthate (38). Two important aspects of the diazotrophic physiology of heterocyst-forming cyanobacteria that are still under investigation include the actual metabolites that are transferred intercellularly and the mechanism(s) of transfer (10).Because the ammonium produced by nitrogenase is incorporated into glutamate to produce glutamine in the heterocyst and because the heterocyst lacks the main glutamate-synthesizing enzyme, glutamine(amide):2-oxoglutarate amino transferase (GOGAT; also known as glutamate synthase), a physiological exchange of glutamine and glutamate resulting in a net transfer of nitrogen from the heterocysts to the vegetative cells has been suggested (21, 36, 37). On the other hand, a sugar is supposed to be transferred from vegetative cells to heterocysts. Because high invertase activity levels are found in the heterocysts (34) and because overexpression of sucrose-degrading sucrose synthase in Anabaena sp. impairs diazotrophic growth (4), it is possible that sucrose is a transferred carbon source. Indeed, determination of ¹⁴C-labeled metabolites in heterocysts isolated from filaments incubated for short periods of time with [¹⁴C]bicarbonate identified sugars and glutamate as possible compounds transferred from vegetative cells to heterocysts (13). However, this study also identified alanine as a metabolite possibly transported from vegetative cells to heterocysts.The cyanobacteria bear a Gram-negative type of cell envelope, carrying an outer membrane (OM) outside the cytoplasmic membrane (CM) and the peptidoglycan layer (9, 15). In filamentous cyanobacteria, whereas the CM and peptidoglycan layer surround each cell, the OM is continuous along the filament, defining a continuous periplasmic space (10, 19). In Anabaena sp. strain PCC 7120, the OM is a permeability barrier for metabolites such as glutamate and sucrose (27). Two possible pathways for intercellular molecular exchange in heterocyst-forming cyanobacteria have been discussed: the periplasm (10, 19) and cell-to-cell-joining proteinaceous structures (11, 22, 25). Whereas the latter would mediate direct transfer of metabolites between the cytoplasm of adjacent cells, the former would require specific CM permeases to mediate metabolite transfer between the periplasm and the cytoplasm of each cell type (10).In Anabaena sp. strain PCC 7120, two ABC-type amino acid transporters have been identified that are specifically required for diazotrophic growth (29, 30). The N-I transporter (NatABCDE), which shows preference for neutral hydrophobic amino acids, is present exclusively in vegetative cells (30). The N-II transporter (NatFGH-BgtA), which shows preference for acidic and neutral polar amino acids, is present in both vegetative cells and heterocysts (29). A general phenotype of mutants of neutral amino acid transporters in cyanobacteria is release into the culture medium of some hydrophobic amino acids, especially alanine (16, 23, 24), which is accumulated at higher levels in the extracellular medium of cultures incubated in the absence than in the presence of a source of combined nitrogen (30).Thus, alanine is a conspicuous metabolite in the diazotrophic physiology of heterocyst-forming cyanobacteria, and the possibility that it moves in either direction between heterocysts and vegetative cells has been discussed (13, 29, 30). Alanine dehydrogenase, which catalyzes the reversible reductive amination of pyruvate, has been detected in several cyanobacteria (8). In Anabaena spp., alanine dehydrogenase has been found at higher levels or exclusively in diazotrophic cultures (26), and in the diazotrophic filaments of Anabaena cylindrica it is present at higher levels in heterocysts than in vegetative cells (33). Open reading frame (ORF) alr2355 of the Anabaena sp. strain PCC 7120 genome is predicted to encode an alanine dehydrogenase (14). In this work we addressed the expression and inactivation of alr2355, identifying it as the Anabaena ald gene and defining an important catabolic role for alanine dehydrogenase in diazotrophy.     

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.     

Respiratory terminal oxidases in the facultative chemoheterotrophic and dinitrogen fixing cyanobacterium Anabaena variabilis strain ATCC 29413: characterization of the cox2 locus

Upon nitrogen step-down, some filamentous cyanobacteria differentiate heterocysts, cells specialized for dinitrogen fixation, a highly oxygen sensitive process. Aerobic respiration is one of the mechanisms responsible for a microaerobic environment in heterocysts and respiratory terminal oxidases are the key enzymes of the respiratory chains. We used Anabaena variabilis strain ATCC 29413, because it is one of the few heterocyst-forming facultatively chemoheterotrophic cyanobacteria amenable to genetic manipulation. Using PCR with degenerate primers, we found four gene loci for respiratory terminal oxidases, three of which code for putative cytochrome c oxidases and one whose genes are homologous to cytochrome bd-type quinol oxidases. One cytochrome c oxidase, Cox2, was the only enzyme whose expression, tested by RT-PCR, was evidently up-regulated in diazotrophy, and therefore cloned, sequenced, and characterized. Up-regulation of Cox2 was corroborated by Northern and primer extension analyses. Strains were constructed lacking Cox1 (a previously characterized cytochrome c oxidase), Cox2, or both, which all grew diazotrophically. In vitro cytochrome c oxidase and respiratory activities were determined in all strains, allowing for the first time to estimate the relative contributions to total respiration of the different respiratory electron transport branches under different external conditions. Especially adding fructose to the growth medium led to a dramatic enhancement of in vitro cytochrome c oxidation and in vivo respiratory activity without significantly influencing gene expression.     

Cytochrome c6 is the main respiratory and photosynthetic soluble electron donor in heterocysts of the cyanobacterium Anabaena sp. PCC 7120

Cytochrome c₆ is a soluble electron carrier, present in all known cyanobacteria, that has been replaced by plastocyanin in plants. Despite their high structural differences, both proteins have been reported to be isofunctional in cyanobacteria and green algae, acting as alternative electron carriers from the cytochrome b₆-f complex to photosystem I or terminal oxidases. We have investigated the subcellular localization of both cytochrome c₆ and plastocyanin in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 grown in the presence of combined nitrogen and under diazotrophic conditions. Our studies conclude that cytochrome c₆ is expressed at significant levels in heterocysts, even in the presence of copper, condition in which it is strongly repressed in vegetative cells. However, the copper-dependent regulation of plastocyanin is not altered in heterocysts. In addition, in heterocysts, cytochrome c₆ has shown to be the main soluble electron carrier to cytochrome c oxidase-2 in respiration. A cytochrome c₆ deletion mutant is unable to grow under diazotrophic conditions in the presence of copper, suggesting that cytochrome c₆ plays an essential role in the physiology of heterocysts that cannot be covered by plastocyanin.     

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.     

Cell Envelope Components Influencing Filament Length in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

Heterocyst-forming cyanobacteria grow as chains of cells (known as trichomes or filaments) that can be hundreds of cells long. The filament consists of individual cells surrounded by a cytoplasmic membrane and peptidoglycan layers. The cells, however, share a continuous outer membrane, and septal proteins, such as SepJ, are important for cell-cell contact and filament formation. Here, we addressed a possible role of cell envelope components in filamentation, the process of producing and maintaining filaments, in the model cyanobacterium Anabaena sp. strain PCC 7120. We studied filament length and the response of the filaments to mechanical fragmentation in a number of strains with mutations in genes encoding cell envelope components. Previously published peptidoglycan- and outer membrane-related gene mutants and strains with mutations in two genes (all5045 and alr0718) encoding class B penicillin-binding proteins isolated in this work were used. Our results show that filament length is affected in most cell envelope mutants, but the filaments of alr5045 and alr2270 gene mutants were particularly fragmented. All5045 is a dd-transpeptidase involved in peptidoglycan elongation during cell growth, and Alr2270 is an enzyme involved in the biosynthesis of lipid A, a key component of lipopolysaccharide. These results indicate that both components of the cell envelope, the murein sacculus and the outer membrane, influence filamentation. As deduced from the filament fragmentation phenotypes of their mutants, however, none of these elements is as important for filamentation as the septal protein SepJ.     

Cyanobacterial Heterocysts

Many multicellular cyanobacteria produce specialized nitrogen-fixing heterocysts. During diazotrophic growth of the model organism Anabaena (Nostoc) sp. strain PCC 7120, a regulated developmental pattern of single heterocysts separated by about 10 to 20 photosynthetic vegetative cells is maintained along filaments. Heterocyst structure and metabolic activity function together to accommodate the oxygen-sensitive process of nitrogen fixation. This article focuses on recent research on heterocyst development, including morphogenesis, transport of molecules between cells in a filament, differential gene expression, and pattern formation.Organisms composed of multiple differentiated cell types can possess structures, functions, and behaviors that are more diverse and efficient than those of unicellular organisms. Among multicellular prokaryotes, heterocyst-forming cyanobacteria offer an excellent model for the study of cellular differentiation and multicellular pattern formation. Cyanobacteria are a large group of Gram-negative prokaryotes that perform oxygenic photosynthesis. They have evolved multiple specialized cell types, including nitrogen-fixing heterocysts, spore-like akinetes, and the cells of motile hormogonia filaments. Of these, the development of heterocysts in the filamentous cyanobacterium Anabaena (also Nostoc) sp. strain PCC 7120 (hereafter Anabaena PCC 7120) has been the best studied. Heterocyst development offers a striking example of cellular differentiation and developmental biology in a very simple form: Filaments are composed of only two cell types and these are arrayed in a one-dimensional pattern similar to beads on a string (Figs. 1 and ​and22).Open in a separate windowFigure 1.Heterocyst development in Anabaena PCC 7120. (A) Anabaena PCC 7120 grown in medium containing a source of combined nitrogen grows as filaments of photosynthetic vegetative cells. (B) In the absence of combined nitrogen, heterocysts differentiate at semiregular intervals, forming a developmental pattern of single heterocysts every 10 to 20 vegetative cells along filaments. Heterocysts are often larger than vegetative cells, have a thicker multilayered envelope, and usually contain cyanophycin granules at their poles adjacent to a vegetative cell.Open in a separate windowFigure 2.Heterocyst development in Anabaena PCC 7120. Filaments of the wild type carrying a patS-gfp reporter grown in medium containing nitrate are composed of vegetative cells (A), and have undergone heterocyst development 1 d after transfer to medium without combined nitrogen (B). A patS mutant strain carrying the same patS-gfp reporter grown in media containing nitrate contains a small number of heterocysts (C), and 1 d after transfer to medium without combined nitrogen shows a higher than normal frequency of heterocysts and an abnormal developmental pattern (D). (A, B, C, D) Merged DIC (grayscale), autofluorescence of photosynthetic pigments (red), and patS-gfp reporter fluorescence (green) microscopic images; arrowheads indicate heterocysts; asterisks indicate proheterocysts; size bar, 5 µm. (E, F) Transmission electron micrographs of wild-type vegetative cells (V) and a heterocyst (H) at the end of a filament; T, thylakoid membranes; PS, polysaccharide layer; GL, glycolipid layer; C, polar cyanophycin granule; size bar, 0.2 µm.Many cyanobacterial species are capable of nitrogen fixation. However, oxygenic photosynthesis and nitrogen fixation are incompatible processes because nitrogenase is inactivated by oxygen. Cyanobacteria mainly use two mechanisms to separate these activities: a biological circadian clock to separate them temporally, and multicellularity and cellular differentiation to separate them spatially. For example, the unicellular Cyanothece sp. strain ATCC 51142 stores glycogen during the day and fixes nitrogen at night (Toepel et al. 2008), whereas the filamentous Trichodesmium erythraeum IMS101 fixes nitrogen during the day in groups of specialized cells (Sandh et al. 2009). Heterocyst-forming cyanobacteria differentiate highly specialized cells to provide fixed nitrogen to the vegetative cells in a filament.In the presence of a source of combined nitrogen such as nitrate or ammonium, Anabaena PCC 7120 grows as long filaments containing hundreds of photosynthetic vegetative cells. In the absence of combined nitrogen, it produces heterocysts, which are terminally differentiated nitrogen-fixing cells that form at semiregular intervals between stretches of vegetative cells to produce a multicellular pattern of single heterocysts every ten to twenty vegetative cells along filaments (Figs. 1 and ​and2).2). Some heterocyst-forming cyanobacteria show different regulation or display different developmental patterns but these topics are beyond the scope of this article. Heterocyst development involves integration of multiple external and internal signals, communication between the cells in a filament, and temporal and spatial regulation of genes and cellular processes. The study of heterocyst development in Anabaena PCC 7120 has proven to be an excellent model for the study of cell fate determination, pattern formation, and differential gene expression during prokaryotic multicellular evelopment. Various aspects of heterocyst development, signaling, and regulation have been the subject of several recent reviews (Meeks and Elhai 2002; Forchhammer 2004; Herrero et al. 2004; Zhang et al. 2006; Aldea et al. 2008; Zhao and Wolk 2008).Although beyond the scope of this article, it should be noted that cyanobacteria have recently attracted increased attention because of their important roles in environmental carbon and nitrogen fixation (Montoya et al. 2004), and their potential for providing renewable chemicals and biofuels (Dismukes et al. 2008).     

Subcellular Localization and Clues for the Function of the HetN Factor Influencing Heterocyst Distribution in Anabaena sp. Strain PCC 7120

In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, heterocysts are formed in the absence of combined nitrogen, following a specific distribution pattern along the filament. The PatS and HetN factors contribute to the heterocyst pattern by inhibiting the formation of consecutive heterocysts. Thus, inactivation of any of these factors produces the multiple contiguous heterocyst (Mch) phenotype. Upon N stepdown, a HetN protein with its C terminus fused to a superfolder version of green fluorescent protein (sf-GFP) or to GFP-mut2 was observed, localized first throughout the whole area of differentiating cells and later specifically on the peripheries and in the polar regions of mature heterocysts, coinciding with the location of the thylakoids. Polar localization required an N-terminal stretch comprising residues 2 to 27 that may represent an unconventional signal peptide. Anabaena strains expressing a version of HetN lacking this fragment from a mutant gene placed at the native hetN locus exhibited a mild Mch phenotype. In agreement with previous results, deletion of an internal ERGSGR sequence, which is identical to the C-terminal sequence of PatS, also led to the Mch phenotype. The subcellular localization in heterocysts of fluorescence resulting from the fusion of GFP to the C terminus of HetN suggests that a full HetN protein is present in these cells. Furthermore, the full HetN protein is more conserved among cyanobacteria than the internal ERGSGR sequence. These observations suggest that HetN anchored to thylakoid membranes in heterocysts may serve a function besides that of generating a regulatory (ERGSGR) peptide.     

FraC/FraD-dependent intercellular molecular exchange in the filaments of a heterocyst-forming cyanobacterium, Anabaena sp

The filamentous, heterocyst‐forming cyanobacteria are multicellular organisms in which two different cell types, the CO₂‐fixing vegetative cells and the N₂‐fixing heterocysts, exchange nutrients and regulators. In Anabaena sp. strain PCC 7120, inactivation of sepJ or genes in the fraC operon (fraC, fraD and fraE) produce filament fragmentation. SepJ, FraC and FraD are cytoplasmic membrane proteins located in the filament's intercellular septa that are needed for intercellular exchange of the fluorescent tracer calcein (622 Da). Transmission electron microscopy showed an alteration in the heterocyst cytoplasmic membrane at the vegetative cell‐heterocyst septa in ΔfraC and ΔfraD mutants. Immunogold labelling of FraD confirmed its localization in the intercellular septa and clearly showed the presence of part of the protein between the cytoplasmic membranes of the adjacent cells. This localization seemed to be affected in the ΔfraC mutant but was not impaired in a ΔsepJ mutant. Intercellular transfer of a smaller fluorescent tracer, 5‐carboxyfluorescein (374 Da), was largely impaired in ΔfraC, ΔfraD and double ΔfraC‐ΔfraD mutants, but much less in the ΔsepJ mutant. These results show the existence in the Anabaena filaments of a FraC/FraD‐dependent intercellular molecular exchange that does not require SepJ.