Publications by authors named "Enrique Flores"

109 Publications

Heterocyst septa contain large nanopores that are influenced by the Fra proteins in the filamentous cyanobacterium .

J Bacteriol 2021 Apr 12. Epub 2021 Apr 12.

Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and Universidad de Sevilla, Américo Vespucio 49, E-41092 Seville, Spain

Multicellular heterocyst-forming cyanobacteria such as grow as chains of cells forming filaments that, under diazotrophic conditions, contain two cell types: vegetative cells that perform oxygenic photosynthesis and N-fixing heterocysts. Along the filament, the intercellular septa contain a thick peptidoglycan layer that forms septal disks. Proteinaceous septal junctions connect the cells in the filament traversing the septal disks through nanopores. The operon encodes proteins needed to make long filaments in FraC and FraD, located at the intercellular septa, are involved in the formation of septal junctions. Using a superfolder-GFP fusion, here we show that FraE is mainly localized to the poles of the heterocysts, consistent with the requirement of FraE for constriction of the heterocyst poles to form the "heterocyst neck". A insertional mutant was impaired by 22% to 38% in transfer of fluorescent calcein from vegetative cells to heterocysts. Septal disks were inspected in murein sacculi from heterocyst-enriched preparations. Unexpectedly, the diameter of the nanopores in heterocyst septa was about 1.5- to 2-fold larger than in vegetative cell septa. The number of these nanopores was 76% and 6% of the wild-type number in or a mutant, respectively. Our results show that FraE is mainly involved in heterocyst maturation whereas FraC and FraD are needed for the formation of the large nanopores of heterocyst septa as they are for vegetative cell nanopores. Additionally, arrays of small pores conceivably involved in polysaccharide export were observed close to the disks in the heterocyst murein sacculi preparations.Intercellular communication, an essential attribute of multicellularity, is required for diazotrophic growth in heterocyst-forming cyanobacteria such as , in which the cells are connected by proteinaceous septal junctions that are structural analogs of metazoan connexons. The septal junctions allow molecular intercellular diffusion traversing the septal peptidoglycan through nanopores. In the operon encodes septal proteins essential for intercellular communication. FraC and FraD are components of the septal junctions along the filament, whereas here we show that FraE is mainly present at the heterocyst poles. We found that the intercellular septa in murein sacculi from heterocysts contain nanopores that are larger than those in vegetative cells, establishing a previously unknown difference between heterocyst and vegetative cell septa in .
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http://dx.doi.org/10.1128/JB.00081-21DOI Listing
April 2021

Robust, coherent, and synchronized circadian clock-controlled oscillations along filaments.

Elife 2021 Mar 22;10. Epub 2021 Mar 22.

Department of Physics of Complex Systems, Weizmann Institute of Science, Rehovot, Israel.

Circadian clocks display remarkable reliability despite significant stochasticity in biomolecular reactions. We study the dynamics of a circadian clock-controlled gene at the individual cell level in sp. PCC 7120, a multicellular filamentous cyanobacterium. We found significant synchronization and spatial coherence along filaments, clock coupling due to cell-cell communication, and gating of the cell cycle. Furthermore, we observed low-amplitude circadian oscillatory transcription of genes encoding the post-transcriptional core oscillatory circuit and high-amplitude oscillations of coding for the master regulator transducing the core clock output. Transcriptional oscillations of suggest an additional level of regulation. A stochastic one-dimensional toy model of coupled clock cores and their phosphorylation states shows that demographic noise can seed stochastic oscillations outside the region where deterministic limit cycles with circadian periods occur. The model reproduces the observed spatio-temporal coherence along filaments and provides a robust description of coupled circadian clocks in a multicellular organism.
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http://dx.doi.org/10.7554/eLife.64348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8064755PMC
March 2021

Impaired cell-cell communication in the multicellular cyanobacterium affects carbon uptake, photosynthesis, and the cell wall.

iScience 2021 Jan 5;24(1):101977. Epub 2021 Jan 5.

Department of Physics of Complex Systems, Weizmann Institute of Science, Rehovot 76100, Israel.

Cell-cell communication is an essential attribute of multicellular organisms. The effects of perturbed communication were studied in septal protein mutants of the heterocyst-forming filamentous cyanobacterium sp. PCC 7120 model organism. Strains bearing and deletions showed differences in growth, pigment absorption spectra, and spatial patterns of expression of the gene encoding a heterocyst differentiation master regulator. Global changes in gene expression resulting from deletion of those genes were mapped by RNA sequencing analysis of wild-type and mutant strains, both under nitrogen-replete and nitrogen-poor conditions. The effects of and deletions were non-additive, and perturbed cell-cell communication led to significant changes in global gene expression. Most significant effects, related to carbon metabolism, included increased expression of genes encoding carbon uptake systems and components of the photosynthetic apparatus, as well as decreased expression of genes encoding cell wall components related to heterocyst differentiation and to polysaccharide export.
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http://dx.doi.org/10.1016/j.isci.2020.101977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7797909PMC
January 2021

Coexistence of Communicating and Noncommunicating Cells in the Filamentous Cyanobacterium .

mSphere 2021 01 13;6(1). Epub 2021 Jan 13.

Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Seville, Spain

In filamentous heterocyst-forming (N-fixing) cyanobacteria, septal junctions join adjacent cells, mediating intercellular communication, and are thought to traverse the septal peptidoglycan through nanopores. Fluorescence recovery after photobleaching (FRAP) analysis with the fluorescent marker calcein showed that cultures of sp. strain PCC 7120 grown in the presence of combined nitrogen contained a substantial fraction of noncommunicating cells (58% and 80% of the tested vegetative cells in nitrate- and ammonium-grown cultures, respectively), whereas cultures induced for nitrogen fixation contained far fewer noncommunicating cells (16%). A single filament could have communicating and noncommunicating cells. These observations indicate that all (or most of) the septal junctions in a cell can be coordinately regulated and are coherent with the need for intercellular communication, especially under diazotrophic conditions. Consistently, intercellular exchange was observed to increase in response to N deprivation and to decrease rapidly in response to the presence of ammonium in the medium or to nitrate assimilation. Proteins involved in the formation of septal junctions have been identified in and include SepJ, FraC, and FraD. Here, we reevaluated rates of intercellular transfer of calcein and the number of nanopores in mutants lacking these proteins and found a strong positive correlation between the two parameters only in cultures induced for nitrogen fixation. Thus, whereas the presence of a substantial number of noncommunicating cells appears to impair the correlation, data obtained in diazotrophic cultures support the idea that the nanopores are the structures that hold the septal junctions. Multicellularity is found in bacteria as well as in eukaryotes, and the filamentous heterocyst-forming (N-fixing) cyanobacteria represent a simple and ancient paradigm of multicellular organisms. Multicellularity generally involves cell-cell adhesion and communication. The cells in the cyanobacterial filaments are joined by proteinaceous septal junctions that mediate molecular diffusion. The septal junctions traverse the septal peptidoglycan, which bears holes termed nanopores. Our results show that the septal junctions can be coordinately regulated in a cell and emphasize the relationship between septal junctions and nanopores to build intercellular communication structures, which are essential for the multicellular behavior of heterocyst-forming cyanobacteria.
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http://dx.doi.org/10.1128/mSphere.01091-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845620PMC
January 2021

Predicting substrate exchange in marine diatom-heterocystous cyanobacteria symbioses.

Environ Microbiol 2020 06 28;22(6):2027-2052. Epub 2020 Apr 28.

Department of Ecology, Environment and Plant Sciences, Stockholm University, Stockholm, 106 91, Sweden.

In the open ocean, some phytoplankton establish symbiosis with cyanobacteria. Some partnerships involve diatoms as hosts and heterocystous cyanobacteria as symbionts. Heterocysts are specialized cells for nitrogen fixation, and a function of the symbiotic cyanobacteria is to provide the host with nitrogen. However, both partners are photosynthetic and capable of carbon fixation, and the possible metabolites exchanged and mechanisms of transfer are poorly understood. The symbiont cellular location varies from internal to partial to fully external, and this is reflected in the symbiont genome size and content. In order to identify the membrane transporters potentially involved in metabolite exchange, we compare the draft genomes of three differently located symbionts with known transporters mainly from model free-living heterocystous cyanobacteria. The types and numbers of transporters are directly related to the symbiont cellular location: restricted in the endosymbionts and wider in the external symbiont. Three proposed models of metabolite exchange are suggested which take into account the type of transporters in the symbionts and the influence of their cellular location on the available nutrient pools. These models provide a basis for several hypotheses that given the importance of these symbioses in global N and C budgets, warrant future testing.
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http://dx.doi.org/10.1111/1462-2920.15013DOI Listing
June 2020

Arginine catabolism enzyme AgrE/ArgZ likely involves a cyanobacterial specific factor.

Authors:
Enrique Flores

J Biol Chem 2020 03;295(10):2915

Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and Universidad de Sevilla, Avda. Américo Vespucio 49, E-41092 Seville, Spain. Electronic address:

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http://dx.doi.org/10.1074/jbc.L120.012850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062172PMC
March 2020

A novel septal protein of multicellular heterocystous cyanobacteria is associated with the divisome.

Mol Microbiol 2020 06 24;113(6):1140-1154. Epub 2020 Feb 24.

Institute of General Microbiology, Christian-Albrechts-Universität zu Kiel, Kiel, Germany.

Cyanobacteria are unique among the eubacteria as they possess a hybrid Gram phenotype, having an outer membrane but also a comparably thick peptidoglycan sheet. Furthermore, the cyanobacterial divisome includes proteins specific for both the Gram types as well as cyanobacteria-specific proteins. Cells in multicellular cyanobacteria share a continuous periplasm and their cytoplasms are connected by septal junctions that enable communication between cells in the filament. The localization of septal junction proteins depends on interaction with the divisome, however additional yet unknown proteins may be involved in this process. Here, we characterized Alr3364 (termed SepI), a novel septal protein that interacts with the divisome in the multicellular heterocystous cyanobacterium Anabaena sp. strain PCC 7120. SepI localized to the Z-ring and the intercellular septa but did not interact with FtsZ. Instead, SepI interacted with the divisome proteins ZipN, SepF and FtsI and with the septal protein SepJ. The inactivation of sepI led to a defect in cell filament integrity, colony and cell morphology, septum size, nanopore formation and peptidoglycan biogenesis, and inability to differentiate heterocysts. Our results show that SepI plays a role in intercellular communication and furthermore indicate that SepI functions in the coordination of septal junction localization during cell division.
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http://dx.doi.org/10.1111/mmi.14483DOI Listing
June 2020

Pentapeptide-repeat, cytoplasmic-membrane protein HglK influences the septal junctions in the heterocystous cyanobacterium Anabaena.

Mol Microbiol 2020 04 13;113(4):794-806. Epub 2020 Jan 13.

Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Seville, Spain.

N -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.
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http://dx.doi.org/10.1111/mmi.14444DOI Listing
April 2020

Developmental Biology in Cyanobacteria.

Life (Basel) 2019 May 10;9(2). Epub 2019 May 10.

Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas, Universidad de Sevilla, 41004 Seville, Spain.

Filamentous, heterocyst-forming cyanobacteria are phototrophic multicellular organisms in which N-fixing heterocysts and CO-fixing vegetative cells exchange regulators and nutrients [...].
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http://dx.doi.org/10.3390/life9020039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6617022PMC
May 2019

Catabolic pathway of arginine in Anabaena involves a novel bifunctional enzyme that produces proline from arginine.

Mol Microbiol 2019 04 25;111(4):883-897. Epub 2019 Feb 25.

Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and Universidad de Sevilla, Américo Vespucio 49, E-41092, Sevilla, Spain.

Arginine participates widely in metabolic processes. The heterocyst-forming cyanobacterium Anabaena catabolizes arginine to produce proline and glutamate, with concomitant release of ammonium, as major products. Analysis of mutant Anabaena strains showed that this catabolic pathway is the product of two genes, agrE (alr4995) and putA (alr0540). The predicted PutA protein is a conventional, bifunctional proline oxidase that produces glutamate from proline. In contrast, AgrE is a hitherto unrecognized enzyme that contains both an N-terminal α/β propeller domain and a unique C-terminal domain of previously unidentified function. In vitro analysis of the proteins expressed in Escherichia coli or Anabaena showed arginine dihydrolase activity of the N-terminal domain and ornithine cyclodeaminase activity of the C-terminal domain, overall producing proline from arginine. In the diazotrophic filaments of Anabaena, β-aspartyl-arginine dipeptide is transferred from the heterocysts to the vegetative cells, where it is cleaved producing aspartate and arginine. Both agrE and putA were found to be expressed at higher levels in vegetative cells than in heterocysts, implying that arginine is catabolized by the AgrE-PutA pathway mainly in the vegetative cells. Expression in Anabaena of a homolog of the C-terminal domain of AgrE obtained from Methanococcus maripaludis enabled us to identify an archaeal ornithine cyclodeaminase.
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http://dx.doi.org/10.1111/mmi.14203DOI Listing
April 2019

Cyanobacterial Septal Junctions: Properties and Regulation.

Life (Basel) 2018 Dec 20;9(1). Epub 2018 Dec 20.

School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, UK.

Heterocyst-forming cyanobacteria are multicellular organisms that grow as chains of cells (filaments or trichomes) in which the cells exchange regulators and nutrients. In this article, we review the morphological, physiological and genetic data that have led to our current understanding of intercellular communication in these organisms. Intercellular molecular exchange appears to take place by simple diffusion through proteinaceous structures, known as septal junctions, which connect the adjacent cells in the filament and traverse the septal peptidoglycan through perforations known as nanopores. Proteins that are necessary to produce, and that may be components of, the septal junctions-SepJ, FraC and FraD-have been identified in the heterocyst-forming cyanobacterium sp. strain PCC 7120 model. Additionally, several proteins that are necessary to produce a normal number of nanopores and functional septal junctions have been identified, including AmiC-type amidases, peptidoglycan-binding proteins and some membrane transporters. Available reports and reevaluation of intercellular molecular transfer data for some mutants of suggest that the septal junctions can be regulated, likely by a mechanism of gating.
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http://dx.doi.org/10.3390/life9010001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463045PMC
December 2018

Specific mutations in the permease domain of septal protein SepJ differentially affect functions related to multicellularity in the filamentous cyanobacterium .

Microb Cell 2018 Oct 16;5(12):555-565. Epub 2018 Oct 16.

Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and Universidad de Sevilla, Américo Vespucio 49, E-41092 Seville, Spain.

Filamentous, heterocyst-forming cyanobacteria are multicellular organisms in which growth requires the activity of two interdependent cell types that exchange nutrients and regulators. Vegetative cells provide heterocysts with reduced carbon, and heterocysts provide vegetative cells with fixed nitrogen. Additionally, heterocyst differentiation from vegetative cells is regulated by inhibitors of differentiation produced by prospective heterocysts and heterocysts. Proteinaceous structures known as septal junctions join the cells in the filament. The SepJ protein is involved in formation of septal junctions in the model heterocyst-forming cyanobacterium sp. strain PCC 7120. SepJ bears extra-membrane and membrane (permease) domains and is located at the cell poles in the intercellular septa of the filament. Here we created mutants that produce SepJ proteins altered in the permease domain. Some of these mutant SepJ proteins did not provide functions needed for to form long filaments and (in some cases) differentiate heterocysts, identifying amino acids and amino acid stretches that are important for the structure or function of the protein. Some other mutant SepJ proteins fulfilled filamentation and heterocyst differentiation functions but failed to provide normal communication function assessed via the intercellular transfer of the fluorescent marker calcein. These mutant SepJ proteins bore mutations in amino acids located at the cytoplasmic face of the permease, which could affect access of the fluorescent marker to the septal junctions. Overall, the data are consistent with the idea that SepJ carries out multiple roles in the multicellular function of the filament.
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http://dx.doi.org/10.15698/mic2018.12.661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282017PMC
October 2018

Genetic responses to carbon and nitrogen availability in Anabaena.

Environ Microbiol 2019 01 16;21(1):1-17. Epub 2018 Oct 16.

Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and Universidad de Sevilla, Américo Vespucio 49, E-41092, Seville, Spain.

Heterocyst-forming cyanobacteria are filamentous organisms that perform oxygenic photosynthesis and CO fixation in vegetative cells and nitrogen fixation in heterocysts, which are formed under deprivation of combined nitrogen. These organisms can acclimate to use different sources of nitrogen and respond to different levels of CO . Following work mainly done with the best studied heterocyst-forming cyanobacterium, Anabaena, here we summarize the mechanisms of assimilation of ammonium, nitrate, urea and N , the latter involving heterocyst differentiation, and describe aspects of CO assimilation that involves a carbon concentration mechanism. These processes are subjected to regulation establishing a hierarchy in the assimilation of nitrogen sources -with preference for the most reduced nitrogen forms- and a dependence on sufficient carbon. This regulation largely takes place at the level of gene expression and is exerted by a variety of transcription factors, including global and pathway-specific transcriptional regulators. NtcA is a CRP-family protein that adjusts global gene expression in response to the C-to-N balance in the cells, and PacR is a LysR-family transcriptional regulator (LTTR) that extensively acclimates the cells to oxygenic phototrophy. A cyanobacterial-specific transcription factor, HetR, is involved in heterocyst differentiation, and other LTTR factors are specifically involved in nitrate and CO assimilation.
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http://dx.doi.org/10.1111/1462-2920.14370DOI Listing
January 2019

Homospermidine biosynthesis in the cyanobacterium Anabaena requires a deoxyhypusine synthase homologue and is essential for normal diazotrophic growth.

Mol Microbiol 2018 09 4;109(6):763-780. Epub 2018 Oct 4.

Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and Universidad de Sevilla, Avda. Américo Vespucio 49, E-41092, Sevilla, Spain.

Polyamines are primordial, small organic polycations present in almost all cells, but their roles in bacteria are poorly understood. sym-Homospermidine is the dominant polyamine in the filamentous, N -fixing, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Synthesis of homospermidine was dependent on speA (encoding arginine decarboxylase), speB (agmatinase) and speY (deoxyhypusine synthase homologue), which in bacteria is an unprecedented pathway. Inactivation of any of these genes impaired diazotrophic growth. Heterocyst differentiation in the speA mutant was blocked at an early step, after induction of the regulatory gene hetR but before production of heterocyst-specific glycolipids (HGL). In contrast, the speY mutant produced HGL and showed slow diazotrophic growth. Analysis of fusions to green fluorescent protein revealed that SpeA (like SpeB previously described) accumulates at higher levels in vegetative cells than in heterocysts, and that SpeY accumulates in vegetative cells but also at significant levels in heterocysts. The homospermidine biosynthetic pathway is therefore active primarily in vegetative cells but the last step can be completed in heterocysts. Our findings indicate an important role for polyamines in the diazotrophic biology of Anabaena. Furthermore, inactivation of a gene cluster (potADB) encoding a polyamine ABC transporter disrupted diazotrophic growth, corroborating the importance of polyamine homeostasis in Anabaena.
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http://dx.doi.org/10.1111/mmi.14006DOI Listing
September 2018

Transcriptional regulation of development in heterocyst-forming cyanobacteria.

Biochim Biophys Acta Gene Regul Mech 2019 07 30;1862(7):673-684. Epub 2018 Apr 30.

Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC, Universidad de Sevilla, Seville, Spain.

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 through oxygenic photosynthesis and micro-oxic heterocysts specialized for the fixation of N 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.
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http://dx.doi.org/10.1016/j.bbagrm.2018.04.006DOI Listing
July 2019

Multiple ABC glucoside transporters mediate sugar-stimulated growth in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

Environ Microbiol Rep 2018 02 14;10(1):40-48. Epub 2017 Dec 14.

Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and Universidad de Sevilla, Américo Vespucio 49, 41092, Seville, Spain.

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.
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http://dx.doi.org/10.1111/1758-2229.12603DOI Listing
February 2018

Septal protein SepJ from the heterocyst-forming cyanobacterium forms multimers and interacts with peptidoglycan.

FEBS Open Bio 2017 10 30;7(10):1515-1526. Epub 2017 Aug 30.

Instituto de Bioquímica Vegetal y Fotosíntesis CSIC Universidad de Sevilla Spain.

Heterocyst-forming cyanobacteria grow as filaments that can be hundreds of cells long. Proteinaceous septal junctions provide cell-cell binding and communication functions in the filament. In sp. strain PCC 7120, the SepJ protein is important for the formation of septal junctions. SepJ consists of integral membrane and extramembrane sections - the latter including linker and coiled-coil domains. SepJ (predicted MW, 81.3 kDa) solubilized from membranes was found in complexes of about 296-334 kDa, suggesting that SepJ forms multimeric complexes. We constructed an strain producing a double-tagged SepJ protein (SepJ-GFP-His) and isolated the tagged protein by a two-step affinity chromatography procedure. Analysis of the purified protein preparation provided no indication of the presence of specific SepJ partners, but suggested that SepJ is processed to remove an N-terminal fragment. Additionally, pull-down experiments showed that His-tagged versions of SepJ and of the SepJ coiled-coil domain interact with peptidoglycan (PG). Our results indicate that SepJ forms multimers, that it interacts with PG, and that the coiled-coil domain is involved in this interaction. These observations support the idea that SepJ is a component of the septal junctions that join the cells in the filament.
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http://dx.doi.org/10.1002/2211-5463.12280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5623728PMC
October 2017

Role of Two Cell Wall Amidases in Septal Junction and Nanopore Formation in the Multicellular Cyanobacterium sp. PCC 7120.

Front Cell Infect Microbiol 2017 5;7:386. Epub 2017 Sep 5.

Interfaculty Institute of Microbiology and Infection Medicine Tübingen, Organismic Interactions, University of TübingenTübingen, Germany.

Filamentous cyanobacteria have developed a strategy to perform incompatible processes in one filament by differentiating specialized cell types, N-fixing heterocysts and CO-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 -acetylmuramyl-L-alanine amidase, AmiC2, is essential for cell-cell communication in . This enzyme perforates the septal peptidoglycan creating an array of nanopores, which may be the framework for septal junction complexes. In sp. PCC 7120, two homologs of AmiC2, encoded by and , were identified and investigated in two different studies. Here, we compare the function of both AmiC proteins by characterizing different mutants, which was not possible in , because there the 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 double mutant, filament morphology was affected and heterocyst differentiation was abolished. Furthermore, the inactivation of influenced SepJ localization and prevented the filament-fragmentation phenotype that is characteristic of or 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.
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http://dx.doi.org/10.3389/fcimb.2017.00386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5591844PMC
July 2018

The multicellular nature of filamentous heterocyst-forming cyanobacteria.

FEMS Microbiol Rev 2016 11;40(6):831-854

Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and Universidad de Sevilla, Seville, Spain.

Cyanobacteria carry out oxygenic photosynthesis, play a key role in the cycling of carbon and nitrogen in the biosphere, and have had a large impact on the evolution of life and the Earth itself. Many cyanobacterial strains exhibit a multicellular lifestyle, growing as filaments that can be hundreds of cells long and endowed with intercellular communication. Furthermore, under depletion of combined nitrogen, filament growth requires the activity of two interdependent cell types: vegetative cells that fix CO2 and heterocysts that fix N2. Intercellular molecular transfer is essential for signaling involved in the regulation of heterocyst differentiation and for reciprocal nutrition of heterocysts and vegetative cells. Here we review various aspects of multicellularity in cyanobacterial filaments and their differentiation, including filament architecture with emphasis on the structures used for intercellular communication; we survey theoretical models that have been put forward to understand heterocyst patterning and discuss the factors that need to be considered for these models to reflect the biological entity; and finally, since cell division in filamentous cyanobacteria has the peculiarity of producing linked instead of independent cells, we review distinct aspects of cell division in these organisms.
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http://dx.doi.org/10.1093/femsre/fuw029DOI Listing
November 2016

Specific Glucoside Transporters Influence Septal Structure and Function in the Filamentous, Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120.

J Bacteriol 2017 04 14;199(7). Epub 2017 Mar 14.

Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and Universidad de Sevilla, Seville, Spain

When deprived of combined nitrogen, some filamentous cyanobacteria contain two cell types: vegetative cells that fix CO through oxygenic photosynthesis and heterocysts that are specialized in N fixation. In the diazotrophic filament, the vegetative cells provide the heterocysts with reduced carbon (mainly in the form of sucrose) and heterocysts provide the vegetative cells with combined nitrogen. Septal junctions traverse peptidoglycan through structures known as nanopores and appear to mediate intercellular molecular transfer that can be traced with fluorescent markers, including the sucrose analog esculin (a coumarin glucoside) that is incorporated into the cells. Uptake of esculin by the model heterocyst-forming cyanobacterium sp. strain PCC 7120 was inhibited by the α-glucosides sucrose and maltose. Analysis of mutants identified components of three glucoside transporters that move esculin into the cells: GlsC (Alr4781) and GlsP (All0261) are an ATP-binding subunit and a permease subunit of two different ABC transporters, respectively, and HepP (All1711) is a major facilitator superfamily (MFS) protein that was shown previously to be involved in formation of the heterocyst envelope. Transfer of fluorescent markers (especially calcein) between vegetative cells of was impaired by mutation of glucoside transporter genes. GlsP and HepP interact in bacterial two-hybrid assays with the septal junction-related protein SepJ, and GlsC was found to be necessary for the formation of a normal number of septal peptidoglycan nanopores and for normal subcellular localization of SepJ. Therefore, beyond their possible role in nutrient uptake in , glucoside transporters influence the structure and function of septal junctions. Heterocyst-forming cyanobacteria have the ability to perform oxygenic photosynthesis and to assimilate atmospheric CO and N These organisms grow as filaments that fix these gases specifically in vegetative cells and heterocysts, respectively. For the filaments to grow, these types of cells exchange nutrients, including sucrose, which serves as a source of reducing power and of carbon skeletons for the heterocysts. Movement of sucrose between cells in the filament takes place through septal junctions and has been traced with a fluorescent sucrose analog, esculin, that can be taken up by the cells. Here, we identified α-glucoside transporters of that mediate uptake of esculin and, notably, influence septal structure and the function of septal junctions.
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http://dx.doi.org/10.1128/JB.00876-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5350280PMC
April 2017

Molecular Diffusion through Cyanobacterial Septal Junctions.

mBio 2017 01 3;8(1). Epub 2017 Jan 3.

Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Seville, Spain

Heterocyst-forming cyanobacteria grow as filaments in which intercellular molecular exchange takes place. During the differentiation of N-fixing heterocysts, regulators are transferred between cells. In the diazotrophic filament, vegetative cells that fix CO through oxygenic photosynthesis provide the heterocysts with reduced carbon and heterocysts provide the vegetative cells with fixed nitrogen. Intercellular molecular transfer has been traced with fluorescent markers, including calcein, 5-carboxyfluorescein, and the sucrose analogue esculin, which are observed to move down their concentration gradient. In this work, we used fluorescence recovery after photobleaching (FRAP) assays in the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 to measure the temperature dependence of intercellular transfer of fluorescent markers. We find that the transfer rate constants are directly proportional to the absolute temperature. This indicates that the "septal junctions" (formerly known as "microplasmodesmata") linking the cells in the filament allow molecular exchange by simple diffusion, without any activated intermediate state. This constitutes a novel mechanism for molecular transfer across the bacterial cytoplasmic membrane, in addition to previously characterized mechanisms for active transport and facilitated diffusion. Cyanobacterial septal junctions are functionally analogous to the gap junctions of metazoans.

Importance: Although bacteria are frequently considered just as unicellular organisms, there are bacteria that behave as true multicellular organisms. The heterocyst-forming cyanobacteria grow as filaments in which cells communicate. Intercellular molecular exchange is thought to be mediated by septal junctions. Here, we show that intercellular transfer of fluorescent markers in the cyanobacterial filament has the physical properties of simple diffusion. Thus, cyanobacterial septal junctions are functionally analogous to metazoan gap junctions, although their molecular components appear unrelated. Like metazoan gap junctions, the septal junctions of cyanobacteria allow the rapid intercellular exchange of small molecules, without stringent selectivity. Our finding expands the repertoire of mechanisms for molecular transfer across the plasma membrane in prokaryotes.
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http://dx.doi.org/10.1128/mBio.01756-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210496PMC
January 2017

Overexpression of SepJ alters septal morphology and heterocyst pattern regulated by diffusible signals in Anabaena.

Mol Microbiol 2016 09 18;101(6):968-81. Epub 2016 Jul 18.

Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Américo Vespucio 49, Seville, E-41092, Spain.

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.
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http://dx.doi.org/10.1111/mmi.13436DOI Listing
September 2016

Septal Junctions in Filamentous Heterocyst-Forming Cyanobacteria.

Trends Microbiol 2016 Feb 31;24(2):79-82. Epub 2015 Dec 31.

Department of Microbiology/Organismic Interactions, University of Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany.

In the filaments of heterocyst-forming cyanobacteria, septal junctions that traverse the septal peptidoglycan join adjacent cells, allowing intercellular communication. Perforations in the septal peptidoglycan have been observed, and proteins involved in the formation of such perforations and putative protein components of the septal junctions have been identified, but their relationships are debated.
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http://dx.doi.org/10.1016/j.tim.2015.11.011DOI Listing
February 2016

The heterocyst differentiation transcriptional regulator HetR of the filamentous cyanobacterium Anabaena forms tetramers and can be regulated by phosphorylation.

Mol Microbiol 2016 Feb 9;99(4):808-19. Epub 2015 Dec 9.

Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas, Universidad de Sevilla, Américo Vespucio 49, E-41092, Seville, Spain.

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.
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http://dx.doi.org/10.1111/mmi.13268DOI Listing
February 2016

The Peptidoglycan-Binding Protein SjcF1 Influences Septal Junction Function and Channel Formation in the Filamentous Cyanobacterium Anabaena.

mBio 2015 Jun 30;6(4):e00376. Epub 2015 Jun 30.

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.
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http://dx.doi.org/10.1128/mBio.00376-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4488944PMC
June 2015

Functional Dependence between Septal Protein SepJ from Anabaena sp. Strain PCC 7120 and an Amino Acid ABC-Type Uptake Transporter.

J Bacteriol 2015 Aug 15;197(16):2721-30. Epub 2015 Jun 15.

Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Seville, Spain

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.
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http://dx.doi.org/10.1128/JB.00289-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4507348PMC
August 2015

Induction of the Nitrate Assimilation nirA Operon and Protein-Protein Interactions in the Maturation of Nitrate and Nitrite Reductases in the Cyanobacterium Anabaena sp. Strain PCC 7120.

J Bacteriol 2015 Jul 11;197(14):2442-52. Epub 2015 May 11.

Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Seville, Spain

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.
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http://dx.doi.org/10.1128/JB.00198-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524197PMC
July 2015

Amino Acid Transporters and Release of Hydrophobic Amino Acids in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120.

Life (Basel) 2015 Apr 23;5(2):1282-300. Epub 2015 Apr 23.

Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Américo Vespucio 49, E-41092 Seville, Spain.

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can use inorganic compounds such as nitrate or ammonium as nitrogen sources. In the absence of combined nitrogen, it can fix N2 in differentiated cells called heterocysts. Anabaena also shows substantial activities of amino acid uptake, and three ABC-type transporters for amino acids have been previously characterized. Seven new loci encoding predicted amino acid transporters were identified in the Anabaena genomic sequence and inactivated. Two of them were involved in amino acid uptake. Locus alr2535-alr2541 encodes the elements of a hydrophobic amino acid ABC-type transporter that is mainly involved in the uptake of glycine. ORF all0342 encodes a putative transporter from the dicarboxylate/amino acid:cation symporter (DAACS) family whose inactivation resulted in an increased uptake of a broad range of amino acids. An assay to study amino acid release from Anabaena filaments to the external medium was set up. Net release of the alanine analogue α-aminoisobutyric acid (AIB) was observed when transport system N-I (a hydrophobic amino acid ABC-type transporter) was engaged in the uptake of a specific substrate. The rate of AIB release was directly proportional to the intracellular AIB concentration, suggesting leakage from the cells by diffusion.
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http://dx.doi.org/10.3390/life5021282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4500139PMC
April 2015

Spatial fluctuations in expression of the heterocyst differentiation regulatory gene hetR in Anabaena filaments.

PLoS Genet 2015 Apr 1;11(4):e1005031. Epub 2015 Apr 1.

Department of Physics of Complex Systems, Weizmann Institute of Science, Rehovot, Israel.

Under nitrogen deprivation, filaments of the cyanobacterium Anabaena undergo a process of development, resulting in a one-dimensional pattern of nitrogen-fixing heterocysts separated by about ten photosynthetic vegetative cells. Many aspects of gene expression before nitrogen deprivation and during the developmental process remain to be elucidated. Furthermore, the coupling of gene expression fluctuations between cells along a multicellular filament is unknown. We studied the statistics of fluctuations of gene expression of HetR, a transcription factor essential for heterocyst differentiation, both under steady-state growth in nitrogen-rich conditions and at different times following nitrogen deprivation, using a chromosomally-encoded translational hetR-gfp fusion. Statistical analysis of fluorescence at the individual cell level in wild-type and mutant filaments demonstrates that expression fluctuations of hetR in nearby cells are coupled, with a characteristic spatial range of circa two to three cells, setting the scale for cellular interactions along a filament. Correlations between cells predominantly arise from intercellular molecular transfer and less from cell division. Fluctuations after nitrogen step-down can build up on those under nitrogen-replete conditions. We found that under nitrogen-rich conditions, basal, steady-state expression of the HetR inhibitor PatS, cell-cell communication influenced by the septal protein SepJ and positive HetR auto-regulation are essential determinants of fluctuations in hetR expression and its distribution along filaments. A comparison between the expression of hetR-gfp under nitrogen-rich and nitrogen-poor conditions highlights the differences between the two HetR inhibitors PatS and HetN, as well as the differences in specificity between the septal proteins SepJ and FraC/FraD. Activation, inhibition and cell-cell communication lie at the heart of developmental processes. Our results show that proteins involved in these basic ingredients combine together in the presence of inevitable stochasticity in gene expression, to control the coupled fluctuations of gene expression that give rise to a one-dimensional developmental pattern in this organism.
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http://dx.doi.org/10.1371/journal.pgen.1005031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4382288PMC
April 2015

Intercellular diffusion of a fluorescent sucrose analog via the septal junctions in a filamentous cyanobacterium.

mBio 2015 Mar 17;6(2):e02109. Epub 2015 Mar 17.

School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom

Unlabelled: Many filamentous cyanobacteria produce specialized nitrogen-fixing cells called heterocysts, which are located at semiregular intervals along the filament with about 10 to 20 photosynthetic vegetative cells in between. Nitrogen fixation in these complex multicellular bacteria depends on metabolite exchange between the two cell types, with the heterocysts supplying combined-nitrogen compounds but dependent on the vegetative cells for photosynthetically produced carbon compounds. Here, we used a fluorescent tracer to probe intercellular metabolite exchange in the filamentous heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. We show that esculin, a fluorescent sucrose analog, is incorporated by a sucrose import system into the cytoplasm of Anabaena cells. The cytoplasmic esculin is rapidly and reversibly exchanged across vegetative-vegetative and vegetative-heterocyst cell junctions. Our measurements reveal the kinetics of esculin exchange and also show that intercellular metabolic communication is lost in a significant fraction of older heterocysts. SepJ, FraC, and FraD are proteins located at the intercellular septa and are suggested to form structures analogous to gap junctions. We show that a ΔsepJ ΔfraC ΔfraD triple mutant shows an altered septum structure with thinner septa but a denser peptidoglycan layer. Intercellular diffusion of esculin and fluorescein derivatives is impaired in this mutant, which also shows a greatly reduced frequency of nanopores in the intercellular septal cross walls. These findings suggest that FraC, FraD, and SepJ are important for the formation of junctional structures that constitute the major pathway for feeding heterocysts with sucrose.

Importance: Anabaena and its relatives are filamentous cyanobacteria that exhibit a sophisticated form of prokaryotic multicellularity, with the formation of differentiated cell types, including normal photosynthetic cells and specialized nitrogen-fixing cells called heterocysts. The question of how heterocysts communicate and exchange metabolites with other cells in the filament is key to understanding this form of bacterial multicellularity. Here we provide the first information on the intercellular exchange of a physiologically important molecule, sucrose. We show that a fluorescent sucrose analog can be imported into the Anabaena cytoplasm by a sucrose import system. Once in the cytoplasm, it is rapidly and reversibly exchanged among all of the cells in the filament by diffusion across the septal junctions. Photosynthetically produced sucrose likely follows the same route from cytoplasm to cytoplasm. We identify some of the septal proteins involved in sucrose exchange, and our results indicate that these proteins form structures functionally analogous to metazoan gap junctions.
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http://dx.doi.org/10.1128/mBio.02109-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4453526PMC
March 2015