Search

The TonB-dependent transport of scarcely available substrates across the outer membrane is a conserved feature in Gram-negative bacteria. The plasma membrane-embedded TonB-ExbB-ExbD accomplishes complex functions as an energy transducer by physically interacting with TonB-dependent outer membrane transporters (TBDTs). TonB mediates structural rearrangements in the substrate-loaded TBDTs that are required for substrate translocation into the periplasm. In the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, four TonB-like proteins have been identified. Out of these TonB3 accomplishes the transport of ferric schizokinen, the siderophore which is secreted by Anabaena to scavenge iron. In contrast, TonB1 (SjdR) is exceptionally short and not involved in schizokinen transport. The proposed function of SjdR in peptidoglycan structuring eliminates the protein from the list of TonB proteins in Anabaena . Compared with the well-characterized properties of SjdR and TonB3, the functions of TonB2 and TonB4 are yet unknown. Here, we examined tonB2 and tonB4 mutants for siderophore transport capacities and other specific phenotypic features. Both mutants were not or only slightly affected in schizokinen transport, whereas they showed decreased nitrogenase activity in apparently normal heterocysts. Moreover, the cellular metal concentrations and pigment contents were altered in the mutants, most pronouncedly in the tonB2 mutant. This strain showed an altered susceptibility toward antibiotics and SDS and formed cell aggregates when grown in liquid culture, a phenotype associated with an elevated lipopolysaccharide (LPS) production. Thus, the TonB-like proteins in Anabaena appear to take over distinct functions, and the mutation of TonB2 strongly influences outer membrane integrity. IMPORTANCE The genomes of many organisms encode more than one TonB protein, and their number does not necessarily correlate with that of TonB-dependent outer membrane transporters. Consequently, specific as well as redundant functions of the different TonB proteins have been identified. In addition to a role in uptake of scarcely available nutrients, including iron complexes, TonB proteins are related to virulence, flagellum assembly, pilus localization, or envelope integrity, including antibiotic resistance. The knowledge about the function of TonB proteins in cyanobacteria is limited. Here, we compare the four TonB proteins of Anabaena sp. strain PCC 7120, providing evidence that their functions are in part distinct, since mutants of these proteins exhibit specific features but also show some common impairments.

Cyanobacteria are photosynthetic organisms with a Gram-negative envelope structure. Certain filamentous species such as Anabaena sp. strain PCC 7120 can fix dinitrogen upon depletion of combined nitrogen. Because the nitrogen-fixing enzyme, nitrogenase, is oxygen sensitive, photosynthesis and nitrogen fixation are spatially separated in Anabaena . Nitrogen fixation takes place in specialized cells called heterocysts, which differentiate from vegetative cells. During heterocyst differentiation, a microoxic environment is created by dismantling photosystem II and restructuring the cell wall. Moreover, solute exchange between the different cell types is regulated to limit oxygen influx into the heterocyst. The septal zone containing nanopores for solute exchange is constricted between heterocysts and vegetative cells, and cyanophycin plugs are located at the heterocyst poles. We identified a protein previously annotated as TonB1 that is largely conserved among cyanobacteria. A mutant of the encoding gene formed heterocysts but was impaired in diazotrophic growth. Mutant heterocysts appeared elongated and exhibited abnormal morphological features, including a reduced cyanophycin plug, an enhanced septum size, and a restricted nanopore zone in the septum. In spite of this, the intercellular transfer velocity of the fluorescent marker calcein was increased in the mutant compared to the wild type. Thus, the protein is required for proper formation of septal structures, expanding our emerging understanding of Anabaena peptidoglycan plasticity and intercellular solute exchange, and is therefore renamed SjdR ( s eptal j unction d isk r egulator). Notably, calcium supplementation compensated for the impaired diazotrophic growth and alterations in septal peptidoglycan in the sjdR mutant, emphasizing the importance of calcium for cell wall structure. IMPORTANCE Multicellularity in bacteria confers an improved adaptive capacity to environmental conditions and stresses. This includes an enhanced capability of resource utilization through a distribution of biochemical processes between constituent cells. This specialization results in a mutual dependency of different cell types, as is the case for nitrogen-fixing heterocysts and photosynthetically active vegetative cells in Anabaena . In this cyanobacterium, intercellular solute exchange is facilitated through nanopores in the peptidoglycan between adjacent cells. To ensure functionality of the specialized cells, septal size as well as the position, size, and frequency of nanopores in the septum need to be tightly established. The novel s eptal j unction d isk r egulator SjdR characterized here is conserved in the cyanobacterial phylum. It influences septal size and septal nanopore distribution. Consequently, its absence severely affects the intercellular communication and the strains' growth capacity under nitrogen depletion. Thus, SjdR is involved in septal structure remodeling in cyanobacteria.

N 2 -fixing heterocystous cyanobacteria grow as chains of cells that are connected by proteinaceous septal junctions, which traverse the septal peptidoglycan through nanopores and mediate intercellular molecular transfer. In the model organism Anabaena sp. strain PCC 7120, proteins SepJ, FraC and FraD, which are localized at the cell poles in the intercellular septa, are needed to produce septal junctions. The pentapeptide-repeat, membrane-spanning protein HglK has been described to be involved in the deposition of the heterocyst-specific glycolipid layer, but the hglK mutant also showed intercellular septa broader than in the wild type. Here we found that hglK mutant of Anabaena is impaired in the expression of heterocyst-related genes coxB2A2C2 (cytochrome c oxidase) and nifHDK (nitrogenase), indicating a defect in heterocyst differentiation. HglK was predominantly localized at the intercellular septa and was required to make long filaments, produce a normal number of nanopores and express full intercellular molecular transfer activity. However, the effects of hglK inactivation were not additive to those of the inactivation of sepJ and/or fraC-fraD. We suggest that HglK contributes to the architecture of the intercellular septa with an impact on the function of septal junctions., (© 2020 John Wiley & Sons Ltd.)

Filamentous, heterocyst-forming cyanobacteria are among the simplest multicellular systems in Nature. In the absence of combined nitrogen, the filaments consist of vegetative cells that fix CO 2 through oxygenic photosynthesis and micro-oxic heterocysts specialized for the fixation of N 2 in a proportion of about 10 to 1. The development of a heterocyst-containing filament involves differentiation of vegetative cells into heterocysts in a process that requires a distinct gene expression program. Two transcription factors are strictly required, NtcA and HetR. The CRP-family protein NtcA directly activates the expression of multiple genes during heterocyst differentiation - in some cases assisted by coactivators including HetR - and in mature heterocysts, whereas HetR is needed to build high NtcA levels in differentiating heterocysts and directly activates some particular genes. A few other regulators of gene expression participate at specific differentiation steps, and a specific transcription factor, CnfR, activates nif gene expression under the micro-oxic conditions of the heterocyst., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction, first cleaving the LPXTG motif of one pilin protomer to form an acyl-enzyme intermediate and then joining the terminal Thr to the nucleophilic Lys residue residing within the pilin motif of another pilin protomer. To date, the determinants of class C enzymes that uniquely enable them to construct pili remain unknown. Here, informed by high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and utilizing a structural variant of the enzyme (SrtA 2M ), whose catalytic pocket has been unmasked by activating mutations, we successfully reconstituted in vitro polymerization of the cognate major pilin (SpaA). Mass spectrometry, electron microscopy, and biochemical experiments authenticated that SrtA 2M synthesizes pilus fibers with correct Lys-Thr isopeptide bonds linking individual pilins via a thioacyl intermediate. Structural modeling of the SpaA-SrtA-SpaA polymerization intermediate depicts SrtA 2M sandwiched between the N- and C-terminal domains of SpaA harboring the reactive pilin and LPXTG motifs, respectively. Remarkably, the model uncovered a conserved TP(Y/L)XIN(S/T)H signature sequence following the catalytic Cys, in which the alanine substitutions abrogated cross-linking activity but not cleavage of LPXTG. These insights and our evidence that SrtA 2M can terminate pilus polymerization by joining the terminal pilin SpaB to SpaA and catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile platform for protein engineering and bio-conjugation that has major implications for biotechnology., Competing Interests: The authors declare no conflict of interest.

Cyanobacteria are generally capable of photoautotrophic growth and are widely distributed on Earth. The model filamentous, heterocyst-forming strain Anabaena sp. PCC 7120 has long been considered as a strict photoautotroph but is now known to be able to assimilate fructose. We have previously described two components of ABC glucoside uptake transporters from Anabaena that are involved in uptake of the sucrose analog esculin: GlsC [a nucleotide-binding domain subunit (NBD)] and GlsP [a transmembrane component (TMD)]. Here, we created Anabaena mutants of genes encoding three further ABC transporter components needed for esculin uptake: GlsD (NBD), GlsQ (TMD) and GlsR (periplasmic substrate-binding protein). Phototrophic growth of Anabaena was significantly stimulated by sucrose, fructose and glucose. Whereas the glsC and glsD mutants were drastically hampered in sucrose-stimulated growth, the different gls mutants were generally impaired in sugar-dependent growth. Our results suggest the participation of Gls and other ABC transporters encoded in the Anabaena genome in sugar-stimulated growth. Additionally, Gls transporter components influence the function of septal junctions in the Anabaena filament. We suggest that mixotrophic growth is important in cyanobacterial physiology and may be relevant for the wide success of these organisms in diverse environments., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Filamentous cyanobacteria have developed a strategy to perform incompatible processes in one filament by differentiating specialized cell types, N 2 -fixing heterocysts and CO 2 -fixing, photosynthetic, vegetative cells. These bacteria can be considered true multicellular organisms with cells exchanging metabolites and signaling molecules via septal junctions, involving the SepJ and FraCD proteins. Previously, it was shown that the cell wall lytic N -acetylmuramyl-L-alanine amidase, AmiC2, is essential for cell-cell communication in Nostoc punctiforme . This enzyme perforates the septal peptidoglycan creating an array of nanopores, which may be the framework for septal junction complexes. In Anabaena sp. PCC 7120, two homologs of AmiC2, encoded by amiC1 and amiC2 , were identified and investigated in two different studies. Here, we compare the function of both AmiC proteins by characterizing different Anabaena amiC mutants, which was not possible in N. punctiforme , because there the amiC1 gene could not be inactivated. This study shows the different impact of each protein on nanopore array formation, the process of cell-cell communication, septal protein localization, and heterocyst differentiation. Inactivation of either amidase resulted in significant reduction in nanopore count and in the rate of fluorescent tracer exchange between neighboring cells measured by FRAP analysis. In an amiC1 amiC2 double mutant, filament morphology was affected and heterocyst differentiation was abolished. Furthermore, the inactivation of amiC1 influenced SepJ localization and prevented the filament-fragmentation phenotype that is characteristic of sepJ or fraC fraD mutants. Our findings suggest that both amidases are to some extent redundant in their function, and describe a functional relationship of AmiC1 and septal proteins SepJ and FraCD.

Filamentous, N2 -fixing, heterocyst-forming cyanobacteria grow as chains of cells that are connected by septal junctions. In the model organism Anabaena sp. strain PCC 7120, the septal protein SepJ is required for filament integrity, normal intercellular molecular exchange, heterocyst differentiation, and diazotrophic growth. An Anabaena strain overexpressing SepJ made wider septa between vegetative cells than the wild type, which correlated with a more spread location of SepJ in the septa as observed with a SepJ-GFP fusion, and contained an increased number of nanopores, the septal peptidoglycan perforations that likely accommodate septal junctions. The septa between heterocysts and vegetative cells, which are narrow in wild-type Anabaena, were notably enlarged in the SepJ-overexpressing mutant. Intercellular molecular exchange tested with fluorescent tracers was increased for the SepJ-overexpressing strain specifically in the case of calcein transfer between vegetative cells and heterocysts. These results support an association between calcein transfer, SepJ-related septal junctions, and septal peptidoglycan nanopores. Under nitrogen deprivation, the SepJ-overexpressing strain produced an increased number of contiguous heterocysts but a decreased percentage of total heterocysts. These effects were lost or altered in patS and hetN mutant backgrounds, supporting a role of SepJ in the intercellular transfer of regulatory signals for heterocyst differentiation., (© 2016 John Wiley & Sons Ltd.)

Many filamentous cyanobacteria respond to the external cue of nitrogen scarcity by the differentiation of heterocysts, cells specialized in the fixation of atmospheric nitrogen in oxic environments. Heterocysts follow a spatial pattern along the filament of two heterocysts separated by ca. 10-15 vegetative cells performing oxygenic photosynthesis. HetR is a transcriptional regulator that directs heterocyst differentiation. In the model strain Anabaena sp. PCC 7120, the HetR protein was observed in various oligomeric forms in vivo, including a tetramer that peaked with maximal hetR expression during differentiation. Tetramers were not detected in a hetR point mutant incapable of differentiation, but were conspicuous in an over-differentiating strain lacking the PatS inhibitor. In differentiated filaments the HetR tetramer was restricted to heterocysts, being undetectable in vegetative cells. HetR co-purified with RNA polymerase from Anabaena mainly as a tetramer. In vitro, purified recombinant HetR was distributed between monomers, dimers, trimers and tetramers, and it was phosphorylated when incubated with (γ-(32)P)ATP. Phosphorylation and PatS hampered the accumulation of HetR tetramers and impaired HetR binding to DNA. In summary, tetrameric HetR appears to represent a functionally relevant form of HetR, whose abundance in the Anabaena filament could be negatively regulated by phosphorylation and by PatS., (© 2015 John Wiley & Sons Ltd.)

Cyanobacteria perform water-splitting photosynthesis and are important primary producers impacting the carbon and nitrogen cycles at global scale. They fix CO2 through ribulose-bisphosphate carboxylase/oxygenase (RuBisCo) and have evolved a distinct CO2 concentrating mechanism (CCM) that builds high CO2 concentrations in the vicinity of RuBisCo favouring its carboxylase activity. Filamentous cyanobacteria such as Anabaena fix CO2 in photosynthetic vegetative cells, which donate photosynthate to heterocysts that rely on a heterotrophic metabolism to fix N2 . CCM elements are induced in response to inorganic carbon limitation, a cue that exposes the photosynthetic apparatus to photodamage by over-reduction. An Anabaena mutant lacking the LysR-type transcription factor All3953 grew poorly and dies under high light. The rbcL operon encoding RuBisCo was induced upon carbon limitation in the wild type but not in the mutant. ChIP-Seq analysis was used to globally identify All3953 targets under carbon limitation. Targets include, besides rbcL, genes encoding CCM elements, photorespiratory pathway- photosystem- and electron transport-related components, and factors, including flavodiiron proteins, with a demonstrated or putative function in photoprotection. Quantitative reverse transcription polymerase chain reaction analysis of selected All3953 targets showed regulation in the wild type but not in the mutant. All3953 (PacR) is a global regulator of carbon assimilation in an oxygenic photoautotroph., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Unlabelled: In the diazotrophic filaments of heterocyst-forming cyanobacteria, two different cell types, the CO2-fixing vegetative cells and the N2-fixing heterocysts, exchange nutrients, including some amino acids. In the model organism Anabaena sp. strain PCC 7120, the SepJ protein, composed of periplasmic and integral membrane (permease) sections, is located at the intercellular septa joining adjacent cells in the filament. The unicellular cyanobacterium Synechococcus elongatus strain PCC 7942 bears a gene, Synpcc7942_1024 (here designated dmeA), encoding a permease homologous to the SepJ permease domain. Synechococcus strains lacking dmeA or lacking dmeA and expressing Anabaena sepJ were constructed. The Synechococcus dmeA mutant showed a significant 22 to 32% decrease in the uptake of aspartate, glutamate, and glutamine, a phenotype that could be partially complemented by Anabaena sepJ. Synechococcus mutants of an ATP-binding-cassette (ABC)-type transporter for polar amino acids showed >98% decreased uptake of glutamate irrespective of the presence of dmeA or Anabaena sepJ in the same strain. Thus, Synechococcus DmeA or Anabaena SepJ is needed to observe full (or close to full) activity of the ABC transporter. An Anabaena sepJ deletion mutant was significantly impaired in glutamate and aspartate uptake, which also in this cyanobacterium requires the activity of an ABC-type transporter for polar amino acids. SepJ appears therefore to generally stimulate the activity of cyanobacterial ABC-type transporters for polar amino acids. Conversely, an Anabaena mutant of three ABC-type transporters for amino acids was impaired in the intercellular transfer of 5-carboxyfluorescein, a SepJ-related property. Our results unravel possible functional interactions in transport elements important for diazotrophic growth., Importance: Membrane transporters are essential for many aspects of cellular life, from uptake and export of substances in unicellular organisms to intercellular molecular exchange in multicellular organisms. Heterocyst-forming cyanobacteria such as Anabaena represent a unique case of multicellularity, in which two cell types exchange nutrients and regulators. The SepJ protein located at the intercellular septa in the filaments of Anabaena contains a permease domain of the drug/metabolite transporter (DMT) superfamily that somehow contributes to intercellular molecular transfer. In this work, we have found that SepJ stimulates the activity of a polar amino acid uptake transporter of the ATP-binding-cassette (ABC) superfamily, which could itself affect an intercellular transfer activity related to SepJ, thus unraveling possible functional interactions between these different transporters., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Nitrate is widely used as a nitrogen source by cyanobacteria, in which the nitrate assimilation structural genes frequently constitute the so-called nirA operon. This operon contains the genes encoding nitrite reductase (nirA), a nitrate/nitrite transporter (frequently an ABC-type transporter; nrtABCD), and nitrate reductase (narB). In the model filamentous cyanobacterium Anabaena sp. strain PCC 7120, which can fix N2 in specialized cells termed heterocysts, the nirA operon is expressed at high levels only in media containing nitrate or nitrite and lacking ammonium, a preferred nitrogen source. Here we examined the genes downstream of the nirA operon in Anabaena and found that a small open reading frame of unknown function, alr0613, can be cotranscribed with the operon. The next gene in the genome, alr0614 (narM), showed an expression pattern similar to that of the nirA operon, implying correlated expression of narM and the operon. A mutant of narM with an insertion mutation failed to produce nitrate reductase activity, consistent with the idea that NarM is required for the maturation of NarB. Both narM and narB mutants were impaired in the nitrate-dependent induction of the nirA operon, suggesting that nitrite is an inducer of the operon in Anabaena. It has previously been shown that the nitrite reductase protein NirA requires NirB, a protein likely involved in protein-protein interactions, to attain maximum activity. Bacterial two-hybrid analysis confirmed possible NirA-NirB and NarB-NarM interactions, suggesting that the development of both nitrite reductase and nitrate reductase activities in cyanobacteria involves physical interaction of the corresponding enzymes with their cognate partners, NirB and NarM, respectively., Importance: Nitrate is an important source of nitrogen for many microorganisms that is utilized through the nitrate assimilation system, which includes nitrate/nitrite membrane transporters and the nitrate and nitrite reductases. Many cyanobacteria assimilate nitrate, but regulation of the nitrate assimilation system varies in different cyanobacterial groups. In the N2-fixing, heterocyst-forming cyanobacteria, the nirA operon, which includes the structural genes for the nitrate assimilation system, is expressed in the presence of nitrate or nitrite if ammonium is not available to the cells. Here we studied the genes required for production of an active nitrate reductase, providing information on the nitrate-dependent induction of the operon, and found evidence for possible protein-protein interactions in the maturation of nitrate reductase and nitrite reductase., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Filamentous, heterocyst-forming cyanobacteria exchange nutrients and regulators between cells for diazotrophic growth. Two alternative modes of exchange have been discussed involving transport either through the periplasm or through septal junctions linking adjacent cells. Septal junctions and channels in the septal peptidoglycan are likely filled with septal junction complexes. While possible proteinaceous factors involved in septal junction formation, SepJ (FraG), FraC, and FraD, have been identified, little is known about peptidoglycan channel formation and septal junction complex anchoring to the peptidoglycan. We describe a factor, SjcF1, involved in regulation of septal junction channel formation in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. SjcF1 interacts with the peptidoglycan layer through two peptidoglycan-binding domains and is localized throughout the cell periphery but at higher levels in the intercellular septa. A strain with an insertion in sjcF1 was not affected in peptidoglycan synthesis but showed an altered morphology of the septal peptidoglycan channels, which were significantly wider in the mutant than in the wild type. The mutant was impaired in intercellular exchange of a fluorescent probe to a similar extent as a sepJ deletion mutant. SjcF1 additionally bears an SH3 domain for protein-protein interactions. SH3 binding domains were identified in SepJ and FraC, and evidence for interaction of SjcF1 with both SepJ and FraC was obtained. SjcF1 represents a novel protein involved in structuring the peptidoglycan layer, which links peptidoglycan channel formation to septal junction complex function in multicellular cyanobacteria. Nonetheless, based on its subcellular distribution, this might not be the only function of SjcF1., Importance: Cell-cell communication is central not only for eukaryotic but also for multicellular prokaryotic systems. Principles of intercellular communication are well established for eukaryotes, but the mechanisms and components involved in bacteria are just emerging. Filamentous heterocyst-forming cyanobacteria behave as multicellular organisms and represent an excellent model to study prokaryotic cell-cell communication. A path for intercellular metabolite exchange appears to involve transfer through molecular structures termed septal junctions. They are reminiscent of metazoan gap junctions that directly link adjacent cells. In cyanobacteria, such structures need to traverse the peptidoglycan layers in the intercellular septa of the filament. Here we describe a factor involved in the formation of channels across the septal peptidoglycan layers, thus contributing to the multicellular behavior of these organisms., (Copyright © 2015 Rudolf et al.)

Heterocyst-forming cyanobacteria are multicellular organisms that grow as filaments that can be hundreds of cells long. Septal junction complexes, of which SepJ is a possible component, appear to join the cells in the filament. SepJ is a cytoplasmic membrane protein that contains a long predicted periplasmic section and localizes not only to the cell poles in the intercellular septa but also to a position similar to a Z ring when cell division starts suggesting a relation with the divisome. Here, we created a mutant of Anabaena sp. strain PCC 7120 in which the essential divisome gene ftsZ is expressed from a synthetic NtcA-dependent promoter, whose activity depends on the nitrogen source. In the presence of ammonium, low levels of FtsZ were produced, and the subcellular localization of SepJ, which was investigated by immunofluorescence, was impaired. Possible interactions of SepJ with itself and with divisome proteins FtsZ, FtsQ and FtsW were investigated using the bacterial two-hybrid system. We found SepJ self-interaction and a specific interaction with FtsQ, confirmed by co-purification and involving parts of the SepJ and FtsQ periplasmic sections. Therefore, SepJ can form multimers, and in Anabaena, the divisome has a role beyond cell division, localizing a septal protein essential for multicellularity., (© 2015 John Wiley & Sons Ltd.)

Arginine decarboxylase produces agmatine, and arginase and agmatinase are ureohydrolases that catalyze the production of ornithine and putrescine from arginine and agmatine, respectively, releasing urea. In the genome of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, ORF alr2310 putatively encodes an ureohydrolase. Cells of Anabaena supplemented with [(14) C]arginine took up and catabolized this amino acid generating a set of labeled amino acids that included ornithine, proline, and glutamate. In an alr2310 deletion mutant, an agmatine spot appeared and labeled glutamate increased with respect to the wild type, suggesting that Alr2310 is an agmatinase rather than an arginase. As determined in cell-free extracts, agmatinase activity could be detected in the wild type but not in the mutant. Thus, alr2310 is the Anabaena speB gene encoding agmatinase. The ∆alr2310 mutant accumulated large amounts of cyanophycin granule polypeptide, lacked nitrogenase activity, and did not grow diazotrophically. Growth tests in solid media showed that agmatine is inhibitory for Anabaena, especially under diazotrophic conditions, suggesting that growth of the mutant is inhibited by non-metabolized agmatine. Measurements of incorporation of radioactivity from [(14) C]leucine into macromolecules showed, however, a limited inhibition of protein synthesis in the ∆alr2310 mutant. Analysis of an Anabaena strain producing an Alr2310-GFP (green fluorescent protein) fusion showed expression in vegetative cells but much less in heterocysts, implying compartmentalization of the arginine decarboxylation pathway in the diazotrophic filaments of this heterocyst-forming cyanobacterium., (© 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

In the model cyanobacterium Anabaena sp. PCC 7120, cells called heterocysts that are specialized in the fixation of atmospheric nitrogen differentiate from vegetative cells of the filament in the absence of combined nitrogen. Heterocysts follow a specific distribution pattern along the filament, and a number of regulators have been identified that influence the heterocyst pattern. PatS and HetN, expressed in the differentiating cells, inhibit the differentiation of neighboring cells. At least PatS appears to be processed and transferred from cell to cell. HetC is similar to ABC exporters and is required for differentiation. We present an epistasis analysis of these regulatory genes and of genes, hetP and asr2819, successively downstream from hetC, and we have studied the localization of HetC and HetP by use of GFP fusions. Inactivation of patS, but not of hetN, allowed differentiation to proceed in a hetC background, whereas inactivation of hetC in patS or patS hetN backgrounds decreased the frequency of contiguous proheterocysts. A HetC-GFP protein is localized to the heterocysts and especially near their cell poles, and a putative HetC peptidase domain was required for heterocyst differentiation but not for HetC-GFP localization. hetP is also required for heterocyst differentiation. A HetP-GFP protein localized mostly near the heterocyst poles. ORF asr2819, which we denote patC, encodes an 84-residue peptide and is induced upon nitrogen step-down. Inactivation of patC led to a late spreading of the heterocyst pattern. Whereas HetC and HetP appear to have linked functions that allow heterocyst differentiation to progress, PatC may have a role in selecting sites of differentiation, suggesting that these closely positioned genes may be functionally related.

Flavodiiron proteins are known to have crucial and specific roles in photoprotection of photosystems I and II in cyanobacteria. The filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 contains, besides the four flavodiiron proteins Flv1A, Flv2, Flv3A, and Flv4 present in vegetative cells, two heterocyst-specific flavodiiron proteins, Flv1B and Flv3B. Here, we demonstrate that Flv3B is responsible for light-induced O2 uptake in heterocysts, and that the absence of the Flv3B protein severely compromises the growth of filaments in oxic, but not in microoxic, conditions. It is further demonstrated that Flv3B-mediated photosynthetic O2 uptake has a distinct role in heterocysts which cannot be substituted by respiratory O2 uptake in the protection of nitrogenase from oxidative damage and, thus, in an efficient provision of nitrogen to filaments. In line with this conclusion, the Δflv3B strain has reduced amounts of nitrogenase NifHDK subunits and shows multiple symptoms of nitrogen deficiency in the filaments. The apparent imbalance of cytosolic redox state in Δflv3B heterocysts also has a pronounced influence on the amounts of different transcripts and proteins. Therefore, an O2-related mechanism for control of gene expression is suggested to take place in heterocysts.

Heterocyst-forming cyanobacteria are multicellular organisms in which growth requires the activity of two metabolically interdependent cell types, the vegetative cells that perform oxygenic photosynthesis and the dinitrogen-fixing heterocysts. Vegetative cells provide the heterocysts with reduced carbon, and heterocysts provide the vegetative cells with fixed nitrogen. Heterocysts conspicuously accumulate polar granules made of cyanophycin [multi-L-arginyl-poly (L-aspartic acid)], which is synthesized by cyanophycin synthetase and degraded by the concerted action of cyanophycinase (that releases β-aspartyl-arginine) and isoaspartyl dipeptidase (that produces aspartate and arginine). Cyanophycin synthetase and cyanophycinase are present at high levels in the heterocysts. Here we created a deletion mutant of gene all3922 encoding isoaspartyl dipeptidase in the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. The mutant accumulated cyanophycin and β-aspartyl-arginine, and was impaired specifically in diazotrophic growth. Analysis of an Anabaena strain bearing an All3922-GFP (green fluorescent protein) fusion and determination of the enzyme activity in specific cell types showed that isoaspartyl dipeptidase is present at significantly lower levels in heterocysts than in vegetative cells. Consistently, isolated heterocysts released substantial amounts of β-aspartyl-arginine. These observations imply that β-aspartyl-arginine produced from cyanophycin in the heterocysts is transferred intercellularly to be hydrolyzed, producing aspartate and arginine in the vegetative cells. Our results showing compartmentalized metabolism of cyanophycin identify the nitrogen-rich molecule β-aspartyl-arginine as a nitrogen vehicle in the unique multicellular system represented by the heterocyst-forming cyanobacteria.

Background: The CRP-family transcription factor NtcA, universally found in cyanobacteria, was initially discovered as a regulator operating N control. It responds to the N regime signaled by the internal 2-oxoglutarate levels, an indicator of the C to N balance of the cells. Canonical NtcA-activated promoters bear an NtcA-consensus binding site (GTAN8TAC) centered at about 41.5 nucleotides upstream from the transcription start point. In strains of the Anabaena/Nostoc genera NtcA is pivotal for the differentiation of heterocysts in response to N stress., Results: In this study, we have used chromatin immunoprecipitation followed by high-throughput sequencing to identify the whole catalog of NtcA-binding sites in cells of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 three hours after the withdrawal of combined N. NtcA has been found to bind to 2,424 DNA regions in the genome of Anabaena, which have been ascribed to 2,153 genes. Interestingly, only a small proportion of those genes are involved in N assimilation and metabolism, and 65% of the binding regions were located intragenically., Conclusions: The distribution of NtcA-binding sites identified here reveals the largest bacterial regulon described to date. Our results show that NtcA has a much wider role in the physiology of the cell than it has been previously thought, acting both as a global transcriptional regulator and possibly also as a factor influencing the superstructure of the chromosome (and plasmids).

The formation of a diazotrophic cyanobacterial filament represents a simple example of biological development. In Anabaena, a non-random pattern of one nitrogen-fixing heterocyst separated by about 10 photosynthetic vegetative cells results from lateral inhibition elicited by the cells differentiating into heterocysts. Key to this process is the patS gene, which has been shown to produce an inhibitor of heterocyst differentiation that involves the C-terminal RGSGR pentapeptide. Complementation of a ΔpatS Anabaena mutant with different versions of PatS, including point mutations or tag fusions, showed that patS is translated into a 17-amino acid polypeptide. Alterations in the N-terminal part of PatS produced inhibition of heterocyst differentiation, thus this part of the peptide appears necessary for proper processing and self-immunity in the producing cells. Alterations in the C-terminal part of PatS led to over-differentiation, thus supporting its role in inhibition of heterocyst differentiation. A polypeptide, produced in proheterocysts, consisting of a methionine followed by the eight, but not the five, terminal amino acids of PatS recreated the full activity of the native peptide. Immunofluorescence detection showed that an RGSGR-containing peptide accumulated in the cells adjacent to the producing proheterocysts, illustrating intercellular transfer of a morphogen in the cyanobacterial filaments., (© 2013 John Wiley & Sons Ltd.)

In cyanobacteria-plant symbioses, the symbiotic nitrogen-fixing cyanobacterium has low photosynthetic activity and is supplemented by sugars provided by the plant partner. Which sugars and cyanobacterial sugar uptake mechanism(s) are involved in the symbiosis, however, is unknown. Mutants of the symbiotically competent, facultatively heterotrophic cyanobacterium Nostoc punctiforme were constructed bearing a neomycin resistance gene cassette replacing genes in a putative sugar transport gene cluster. Results of transport activity assays using (14)C-labeled fructose and glucose and tests of heterotrophic growth with these sugars enabled the identification of an ATP-binding cassette-type transporter for fructose (Frt), a major facilitator permease for glucose (GlcP), and a porin needed for the optimal uptake of both fructose and glucose. Analysis of green fluorescent protein fluorescence in strains of N. punctiforme bearing frt::gfp fusions showed high expression in vegetative cells and akinetes, variable expression in hormogonia, and no expression in heterocysts. The symbiotic efficiency of N. punctiforme sugar transport mutants was investigated by testing their ability to infect a nonvascular plant partner, the hornwort Anthoceros punctatus. Strains that were specifically unable to transport glucose did not infect the plant. These results imply a role for GlcP in establishing symbiosis under the conditions used in this work.

Some filamentous cyanobacteria such as Anabaena sp. strain PCC 7120 produce cells, termed heterocysts, specialized in nitrogen fixation. Heterocysts bear a thick envelope containing an inner layer of glycolipids and an outer layer of polysaccharide that restrict the diffusion of air (including O(2)) into the heterocyst. Anabaena sp. mutants impaired in production of either of those layers show a Fox(-) phenotype (requiring fixed nitrogen for growth under oxic conditions). We have characterized a set of transposon-induced Fox(-) mutants in which transposon Tn5-1063 was inserted into the Anabaena sp. chromosome open reading frame all1711 which encodes a predicted membrane protein that belongs to the major facilitator superfamily (MFS). These mutants showed higher nitrogenase activities under anoxic than under oxic conditions and altered sucrose uptake. Electron microscopy and alcian blue staining showed a lack of the heterocyst envelope polysaccharide (Hep) layer. Northern blot and primer extension analyses showed that, in a manner dependent on the nitrogen-control transcription factor NtcA, all1711 was strongly induced after nitrogen step-down. Confocal microscopy of an Anabaena sp. strain producing an All1711-green fluorescent protein (All1711-GFP) fusion protein showed induction in all cells of the filament but at higher levels in differentiating heterocysts. All1711-GFP was located in the periphery of the cells, consistent with All1711 being a cytoplasmic membrane protein. Expression of all1711 from the P(glnA) promoter in a multicopy plasmid led to production of a presumptive exopolysaccharide by vegetative cells. These results suggest that All1711, which we denote HepP, is involved in transport of glycoside(s), with a specific physiological role in production of Hep.

Heterocyst differentiation is orchestrated by the N control transcriptional regulator NtcA and the differentiation-specific factor HetR. In Anabaena sp. strain PCC 7120, the devBCA operon is expressed from two different promoters activated upon N stepdown. The distal devB promoter (transcription start point [TSP] located at position -704) represents a canonical class II NtcA-activated promoter, including a consensus NtcA-binding site centered 39.5 nucleotides upstream from the TSP. Transcription activation from a second TSP (-454) requires NtcA and is impaired in hetR mutants. In a wild-type background, three different DNA fragments, including both or each individual promoter, directed gfp expression localized mainly to proheterocysts and heterocysts. Expression was undetectable in an ntcA background and, for the fragment including the proximal promoter alone, also in a hetR background. In spite of the absence of consensus NtcA-binding sequences between the two TSPs, NtcA was shown to interact with this DNA region, and NtcA and its effector, 2-oxoglutarate, were necessary and sufficient for in vitro transcription from the -454 TSP. No HetR binding to the DNA or in vitro transcription from the proximal devB TSP promoted by HetR alone were detected. However, a moderate positive effect of HetR on NtcA binding to the DNA between the two devB TSPs was observed. The proximal devB promoter appears to represent a suboptimal NtcA-activated promoter for which HetR may act as a coactivator, with the physiological effect of restricting gene activation to conditions of prevalence of high NtcA and HetR levels, such as those taking place during heterocyst differentiation.

In the model, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120, gene cluster alr2877-alr2880, which encodes an ABC-type transport system, was induced under conditions of carbon limitation and its inactivation impaired the uptake of bicarbonate. Thus, this gene cluster encodes a Cmp bicarbonate transporter. ORF all0862, encoding a LysR-type transcriptional regulator, was expressed under carbon limitation and at higher levels in the absence than in the presence of combined nitrogen, with a positive effect of the N-control transcription factor NtcA. all0862 was expressed from two putative transcription start sites located 164 and 64 bp upstream from the gene respectively. The latter was induced under carbon limitation and was dependent on positive autoregulation by All0862. All0862 was required for the induction of the Cmp bicarbonate transporter, thus representing a CmpR regulator of Anabaena sp. These results show a novel mode of co-regulation by C and N availability through the concerted action of N- and C-responsive transcription factors., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

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.

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., (© 2011 Blackwell Publishing Ltd.)

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., (© 2010 Blackwell Publishing Ltd.)

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.

In the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, expression of the nitrate assimilation nirA operon takes place in the absence of ammonium and the presence of nitrate or nitrite. Several positive-action proteins that are required for expression of the nirA operon have been identified. Whereas NtcA and NtcB exert their action by direct binding to the nirA operon promoter, CnaT acts by an as yet unknown mechanism. In the genome of this cyanobacterium, open reading frame (ORF) all0605 (the nirB gene) is found between the nirA (encoding nitrite reductase) and ntcB genes. A nirB mutant was able to grow at the expense of nitrate as a nitrogen source and showed abnormally high levels of nirA operon mRNA both in the presence and in the absence of nitrate. This mutant showed increased nitrate reductase activity but decreased nitrite reductase activity, an imbalance that resulted in excretion of nitrite, which accumulated in the extracellular medium, when the nirB mutant was grown in the presence of nitrate. A nirA in-frame deletion mutant also showed a phenotype of increased expression of the nirA operon in the absence of ammonium, independent of the presence of nitrate in the medium. Both NirB and NirA are therefore needed to keep low levels of expression of the nirA operon in the absence of an inducer. Because NirB is also needed to attain high levels of nitrite reductase activity, NirA appears to be a negative element in the nitrate regulation of expression of the nirA operon in Anabaena sp. strain PCC 7120.

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can fix N(2) in differentiated cells called heterocysts, which exchange nutritional and regulatory compounds with the neighbouring photosynthetic vegetative cells. The cells in the filament appear to be joined by some protein structures, of which SepJ (FraG) that is located at the cell poles in the intercellular septa and is needed for filament integrity seems to be a component. Other known proteins required for filament integrity include FraC and FraH. Whereas fraC (alr2392) was constitutively expressed as an operon together with two downstream genes, alr2393 (fraD) and alr2394 (fraE), fraH (alr1603) was induced under nitrogen deprivation. Single mutants of these genes showed filament fragmentation under nitrogen deprivation and did not grow diazotrophically, although they formed heterocysts. The fraC and fraD mutants showed an impaired localization of SepJ at the intercellular septa and were hampered in the intercellular transfer of the fluorescent probe calcein. As shown with GFP fusions, FraC and FraD are also located at the intercellular septa. Therefore, at least three different proteins, SepJ, FraC and FraD, influence the architecture and function of the intercellular septa in the Anabaena filaments.

Transcription of the hetC gene, whose product is required for heterocyst differentiation, takes place from a long promoter region that includes the previously described HetR-independent, NtcA-activated promoter producing transcripts with a 5' end corresponding to position -571 with respect to the translational start site of hetC. Northern blot analysis indicated that the accumulation of hetC transcripts depends on HetR, and a second transcriptional start site located at position -293 that leads to NtcA-dependent, HetR-dependent inducible transcription of hetC was identified. Upon nitrogen stepdown, expression of a P(hetC)::gfp fusion was transiently induced in specific cells that were differentiating into heterocysts, both when the whole promoter region (containing transcription start points -571 and -293) or a short version (containing only the transcription start point -293) was used. Expression of hetC from the -293 position was delayed in a strain bearing a deleted promoter region lacking sequences upstream from position -570. Such a strain was still able to differentiate functional heterocysts and to grow diazotrophically, although diazotrophic growth was impaired under certain conditions. Similarly, a second, NtcA-dependent, HetR-dependent transcriptional start site was identified at position -454 in the promoter region upstream from the devBCA operon encoding an ABC transport system involved in heterocyst maturation, in which an NtcA-dependent promoter producing transcripts starting at position -704 had been previously noted. Thus, the hetC and devBCA promoter regions exhibit similar tandem promoter arrangements.

In the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, the glnB gene is expressed at considerable levels both in the presence and in the absence of combined nitrogen, although induction, influenced by NtcA, takes place upon combined-nitrogen deprivation likely localized to vegetative cells. In spite of extensive efforts, a derivative of PCC 7120 lacking a functional glnB gene could be obtained only with constructs that lead to overexpression of a downstream open reading frames (ORF), particularly all2318. Strain CSP10 [glnB all2318(Con)] exhibited growth rates similar to those of the wild type when it was using nitrate or ammonium, but its diazotrophic growth was impaired. However, it differentiated heterocysts with a time course and distribution pattern similar to those of the wild type, although with no cyanophycin-containing polar granules, and exhibited impaired nitrogenase activity under oxic conditions, but not under microoxic conditions. In the mutant, NtcA-dependent induction of the hetC and nifH genes was unaltered, but induction of the urtA gene and urea transport activity were increased. Active uptake of nitrite was also increased and insensitive to the ammonium-promoted inhibition observed for the wild type. Thus, regulation of the nitrite transport activity requires the glnB gene product. In the presence of a wild-type glnB gene, neither inactivation nor overexpression of all2318 produced an apparent phenotype. Thus, in an otherwise wild-type background, the glnB gene appears to be essential for growth of strain PCC 7120. For growth with combined nitrogen but not for diazotrophic growth, the requirement for glnB can be overridden by increasing the expression of all2318 (and/or ORFs downstream of it).

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that fixes N(2) in specialized cells called heterocysts, which differentiate from vegetative cells in a process that requires the nitrogen control transcription factor NtcA. 2-Oxoglutarate-stimulated binding of purified NtcA to wild-type and modified versions of the ntcA gene promoter from Anabaena sp. was analyzed by mobility shift and DNase I footprinting assays, and the role of NtcA-binding sites in the expression of the ntcA gene during heterocyst differentiation was studied in vivo by using an ntcA-gfp translational fusion and primer extension analysis. Mutation of neither of the two identified NtcA-binding sites eliminated localized expression of ntcA in proheterocysts, but mutation of both sites led to very low, nonlocalized expression.

The genome of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 bears a gene cluster including three amt genes that, based on homology of their protein products, we designate amt4, amt1, and amtB. Expression of the three genes took place upon ammonium withdrawal in combined nitrogen-free medium and was NtcA dependent. The genes were transcribed independently, but an amt4-amt1 dicistronic transcript was also produced, and expression was highest for the amt1 gene. A mutant with the whole amt region removed could grow under laboratory conditions using ammonium, nitrate, or dinitrogen as the nitrogen source.

In Anabaena sp. strain PCC 7120, differentiation of heterocysts takes place in response to the external cue of combined nitrogen deprivation, allowing the organism to fix atmospheric nitrogen in oxic environments. NtcA, a global transcriptional regulator of cyanobacteria, is required for activation of the expression of multiple genes involved in heterocyst differentiation, including key regulators that are specific to the process. We have set up a fully defined in vitro system, which includes the purified Anabaena RNA polymerase, and have studied the effects of NtcA and its signaling effector 2-oxoglutarate on RNA polymerase binding, open complex formation, and transcript production from promoters of the hetC, nrrA, and devB genes that are activated by NtcA at different stages of heterocyst differentiation. Both RNA polymerase and NtcA could specifically bind to the target DNA in the absence of any effector. 2-Oxoglutarate had a moderate positive effect on NtcA binding, and NtcA had a limited positive effect on RNA polymerase recruitment at the promoters. However, a stringent requirement of both NtcA and 2-oxoglutarate was observed for the detection of open complexes and transcript production at the three investigated promoters. These results support a key role for 2-oxoglutarate in transcription activation in the developing heterocyst.

The Amt proteins constitute a ubiquitous family of transmembrane ammonia channels that permit the net uptake of ammonium by cells. In many organisms, there is more than one amt gene, and these genes are subjected to nitrogen control. The mature Amt protein is a homo- or heterooligomer of three Amt subunits. We previously characterized an amt1 gene in the unicellular cyanobacterium Synechococcus elongatus strain PCC 7942. In this work, we describe the presence in this organism of a second amt gene, amtB, which encodes a protein more similar to the bacterial AmtB proteins than to any other characterized cyanobacterial Amt protein. The expression of amtB took place in response to nitrogen step-down, required the NtcA transcription factor, and occurred parallel to the expression of amt1. However, the transcript levels of amtB measured after 2 h of nitrogen deprivation were about 100-fold lower than those of amt1. An S. elongatus amtB insertional mutant exhibited an activity for uptake of [14C]methylammonium that was about 55% of that observed in the wild type, but inactivation of amtB had no noticeable effect on the uptake of ammonium when it was supplied at a concentration of 100 microM or more. Because an S. elongatus amt1 mutant is essentially devoid of [14C]methylammonium uptake activity, the mature Amt transporter is functional in the absence of AmtB subunits but not in the absence of Amt1 subunits. However, the S. elongatus amtB mutant could not concentrate [14C]methylammonium within the cells to the same extent as the wild type. Therefore, AmtB is necessary for full methylammonium uptake activity in S. elongatus.

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 forms heterocysts in a semiregular pattern when it is grown on N2 as the sole nitrogen source. The transition from vegetative cells to heterocysts requires marked metabolic and morphological changes. We show that a trimeric pore-forming outer membrane beta-barrel protein belonging to the TolC family, Alr2887, is up-regulated in developing heterocysts and is essential for diazotrophic growth. Mutants defective in Alr2887 did not form the specific glycolipid layer of the heterocyst cell wall, which is necessary to protect nitrogenase from external oxygen. Comparison of the glycolipid contents of wild-type and mutant cells indicated that the protein is not involved in the synthesis of glycolipids but might instead serve as an exporter for the glycolipid moieties or enzymes involved in glycolipid attachment. We propose that Alr2887, together with an ABC transporter like DevBCA, is part of a protein export system essential for assembly of the heterocyst glycolipid layer. We designate the alr2887 gene hgdD (heterocyst glycolipid deposition protein).

Heterocyst development was analyzed in mutants of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 bearing inactivated cox2 and/or cox3 genes, encoding heterocyst-specific terminal respiratory oxidases. At the morphological level, the cox2 cox3 double mutant (strain CSAV141) was impaired in membrane reorganization involving the so-called honeycomb system that in the wild-type strain is largely or exclusively devoted to respiration, accumulated glycogen granules at conspicuously higher levels than the wild type (in both vegetative cells and heterocysts), and showed a delay in carboxysome degradation upon combined nitrogen deprivation. Consistently, chemical analysis confirmed higher accumulation of glycogen in strain CSAV141 than in the wild type. No impairment was observed in the formation of the glycolipid or polysaccharide layers of the heterocyst envelope, consistent with the chemical detection of heterocyst-specific glycolipids, or in the expression of the heterocyst-specific genes nifHDK and fdxH. However, nitrogenase activity under oxic conditions was impaired in strain CSAV135 (cox3) and undetectable in strain CSAV141 (cox2 cox3). These results show that these dedicated oxidases are required for normal development and performance of the heterocysts and indicate a central role of Cox2 and, especially, of Cox3 in the respiratory activity of the heterocysts, decisively contributing to protection of the N(2) fixation machinery against oxygen. However, in contrast to the case for other diazotrophic bacteria, expression of nif genes in Anabaena seems not to be affected by oxygen.

Heterocysts, formed when filamentous cyanobacteria, such as Anabaena sp. strain PCC 7120, are grown in the absence of combined nitrogen, are cells that are specialized in fixing atmospheric nitrogen (N(2)) under oxic conditions and that transfer fixed nitrogen to the vegetative cells of the filament. Anabaena sp. mutants whose sepJ gene (open reading frame alr2338 of the Anabaena sp. genome) was affected showed filament fragmentation and arrested heterocyst differentiation at an early stage. In a sepJ insertional mutant, a layer similar to a heterocyst polysaccharide layer was formed, but the heterocyst-specific glycolipids were not synthesized. The sepJ mutant did not exhibit nitrogenase activity even when assayed under anoxic conditions. In contrast to proheterocysts produced in the wild type, those produced in the sepJ mutant still divided. SepJ is a multidomain protein whose N-terminal region is predicted to be periplasmic and whose C-terminal domain resembles an export permease. Using a green fluorescent protein translationally fused to the carboxyl terminus of SepJ, we observed that in mature heterocysts and vegetative cells, the protein is localized at the intercellular septa, and when cell division starts, it is localized in a ring whose position is similar to that of a Z ring. SepJ is a novel composite protein needed for filament integrity, proper heterocyst development, and diazotrophic growth.

The ntcA gene encodes an N-control transcriptional regulator in cyanobacteria. In the N(2)-fixing, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, ntcA is an autoregulatory gene that is transcribed from a complex promoter region that includes a constitutive promoter (P(2)) and promoters that are induced upon N step-down (P(1) and P(3)). Expression of ntcA was investigated with the use of an ntcA-gfp translational fusion, which was introduced both in the natural ntcA locus and in a heterologous genomic place. Induction of ntcA-gfp took place after N step-down in all the cells of the filament, but at especially high levels in developing heterocysts. Localized induction could be driven independently by P(3) and P(1).

All4312, encoded by open reading frame all4312 in the genome of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, exhibits a CheY-like receiver domain and an output domain similar to that of OmpR, characteristic of two-component response regulators. Expression of all4312 was directly regulated by NtcA, the global transcriptional regulator of nitrogen assimilation in cyanobacteria. Features characteristic of NtcA-activated promoters were also found upstream from genes encoding All4312 homologues in several other cyanobacterial genomes. Expression of all4312 was however unaffected in a mutant of hetR, which encodes a regulator triggering heterocyst development. The function of All4312 may be related to the cellular response to nitrogen deprivation.

Ferric uptake regulation (Fur) proteins are prokaryotic transcriptional regulators that integrate iron metabolism with several environmental stress responses. The regulatory network that governs Fur proteins is rather complex. Control at several stages from gene transcription to post-translational binding of different ligands has been reported in Fur from Escherichia coli. In the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 FurA is the product of open reading frame all1691 that is located between sigC and alr1690, the latter encoding a putative cell wall-binding protein. Anabaena FurA is an autoregulated protein whose expression increases slightly under iron deprivation. Northern blot analysis of furA expression showed an unexpected transcription pattern that consisted of two transcripts. The short transcript corresponded to furA mRNA, whereas the longer transcript contained the alr1690 mRNA and a large region that overlapped the complete furA gene and was complementary to the furA mRNA. Increased expression of FurA in a mutant unable to produce the longer message showed that this transcript acted as an antisense RNA (alpha-furA RNA) interfering with furA transcript translation thus contributing to determine cellular levels of FurA protein.

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can fix N2 in differentiated cells called heterocysts. The products of Anabaena open reading frames (ORFs) all1046, all1047, all1284, alr1834 and all2912 were identified as putative elements of a neutral amino acid permease. Anabaena mutants of these ORFs were strongly affected (1-12% of the wild-type activity) in the transport of Pro, Phe, Leu and Gly and also impaired (17-30% of the wild-type activity) in the transport of Ala and Ser. These results identified those ORFs as the nat genes encoding the N-I neutral amino acid permease. According to amino acid sequence homologies, natA (all1046) and natE (all2912) encode ATPases, natC (all1047) and natD (all1284) encode transmembrane proteins, and natB (alr1834) encodes a periplasmic substrate-binding protein of an ABC-type uptake transporter. The natA, natC, natD and natE mutants showed defects in Gln and His uptake that were not observed in the natB mutant suggesting that NatB is not a binding protein for Gln or His. The nat mutants released hydrophobic amino acids to the medium, and amino acid release took place at higher levels in cultures incubated in the absence of combined N than in the presence of nitrate. Alanine was the amino acid released at highest levels, and its release was impaired in a mutant unable to develop heterocysts. The nat mutants were also impaired in diazotrophic growth, with natA, natC, natD and natE mutants showing more severe defects than the natB mutant. Expression of natA and natC, which constitute an operon, natCA, as well as of natB was studied and found to take place in vegetative cells but not in the heterocysts. These results indicate that the N-I permease is necessary for normal growth of Anabaena sp. strain PCC 7120 on N2, and that this permease has a role in the diazotrophic filament specifically in the vegetative cells.

Heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120 depends on both the global nitrogen control transcription factor NtcA and the cell differentiation regulatory protein HetR, with expression of ntcA and hetR being dependent on each other. In this study we constructed strains that constitutively express the ntcA gene leading to high levels of NtcA protein irrespective of the nitrogen source, and we analyzed the effects of such NtcA levels on heterocyst differentiation. In the NtcA-overproducing strain, heterocyst differentiation, induction of NtcA-dependent heterocyst development genes or operons such as devBCA or the cox2 operon, and NtcA-dependent excision of the 11-kb nifD-intervening element only took place under nitrogen deficiency. Although functional heterocysts were produced in response to nitrogen step-down, the NtcA overproducing strain could not grow diazotrophically. Overexpression of ntcA in a hetR background promoted expression of devBCA in response to ammonium withdrawal and excision of the 11-kb element even in the presence of combined nitrogen. Our results show that some NtcA-dependent heterocyst-related genes can be expressed independently of HetR.

Some filamentous cyanobacteria can undergo a variety of cellular differentiation processes that permit their better adaptation to certain environmental conditions. These processes include the differentiation of hormogonia, short filaments aimed at the dispersal of the organism in the environment, of akinetes, cells resistant to various stress conditions, and of heterocysts, cells specialized in the fixation of atmospheric nitrogen in oxic environments. NtcA is a transcriptional regulator that operates global nitrogen control in cyanobacteria by activating (and in some cases repressing) many genes involved in nitrogen assimilation. NtcA is required for the triggering of heterocyst differentiation and for subsequent steps of its development and function. This requirement is based on the role of NtcA as an activator of the expression of hetR and other multiple genes at specific steps of the differentiation process. The products of these genes effect development as well as the distinct metabolism of the mature heterocyst. The different features found in the NtcA-dependent promoters, together with the cellular level of active NtcA protein, should have a role in the determination of the hierarchy of gene activation during the process of heterocyst differentiation.

The cyanobacterial protein NtcA is a global transcriptional regulator of genes involved in nitrogen assimilation that are subjected to ammonium-promoted repression and is itself controlled by positive autoregulation. Strain CSI70 derived from Synechococcus sp. strain PCC 7942 was constructed to overexpress an additional ntcA gene copy from a constitutive promoter. This strain exhibited high levels of the NtcA protein both in the presence and in the absence of ammonium. However, expression of the NtcA-dependent nir operon and glnA gene (tested by RNA/DNA hybridization and enzyme activity) was still subjected to nitrogen regulation. These results indicate in vivo regulation of the activity of NtcA at activation of transcription of nitrogen-regulated genes as a function of the nitrogen status of the cell.

Two gene clusters each encoding the cyanophycin-metabolism enzymes cyanophycin synthetase and cyanophycinase are found in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. In cluster cph1, the genes cphB1 and cphA1 were expressed in media containing ammonium, nitrate, or N(2) as nitrogen sources, but expression was higher in the absence of combined nitrogen taking place both in vegetative cells and heterocysts. Both genes were cotranscribed from three putative promoters located upstream of cphB1, and, additionally, the cphA1 gene was expressed monocistronically from at least two promoters located in the intergenic cphB1-cphA1 region. Both constitutive promoters and promoters dependent on the global nitrogen control transcriptional regulator NtcA were identified. In cluster cph2, the cphB2 and cphA2 genes, which are found in opposite orientations, were expressed as monocistronic messages in media containing ammonium, nitrate, or N(2), but expression was higher in the absence of ammonium. Expression of the cph2 genes was lower than that of cph1 genes. Analysis of cph gene insertional mutants indicated that cluster cph1 genes contributed more than cluster cph2 genes to cyanophycin accumulation in the whole filament as well as in heterocysts. Diazotrophic growth was more severely impaired in cyanophycinase than in cyanophycin synthetase mutants, indicating that cyanophycin, although normally synthesized in the heterocysts, is not required for heterocyst function and that the inability to degrade this polymer is detrimental for the diazotrophic growth of the cyanobacterium.

Expression of the nitrate assimilation nir operon in the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 requires the action of both the global nitrogen control transcription factor NtcA and the pathway-specific transcriptional regulator NtcB. In the genome of this cyanobacterium, the ntcB gene is found in a cluster of genes located in the complementary strand, upstream from the nir operon. Just downstream of ntcB, there is an open reading frame, all0601 (previously designated orf356 and now designated the cnaT gene), that putatively encodes a protein similar to proteins with glycosyl transferase activity and that is also present clustered together with ntcB homologues or nitrate assimilation structural genes in other cyanobacterial genomes. An insertional mutant of cnaT was generated and found to be unable to assimilate nitrate, although it could use ammonium or dinitrogen as a source of nitrogen for growth. In the mutant, under derepression conditions, nir operon mRNA (as determined by RNA-DNA hybridization and primer extension analysis) and enzymes of the nitrate reduction system (i.e., nitrate reductase and nitrite reductase) were expressed at low or undetectable levels. Inactivation of cnaT did not impair expression of ntcB, and expression of cnaT itself was constitutive and regulated by neither NtcA nor NtcB. Regulation of expression of the nir operon in Anabaena sp. strain PCC 7120 by CnaT and the previously described regulatory elements, NtcA and NtcB, is discussed.