Role of glucoside transporters in the biology of the heterocyst-forming cyanobacterium anabaena SP.

La aparición de organismos multicelulares supuso una de las mayores transiciones durante el curso de la evolución biológica temprana, habiendo surgido la multicelularidad de manera independiente en los tres dominios de la vida. Las cianobacterias son un amplio grupo monofilético de procariotas fotosintéticos con representantes unicelulares y multicelulares. Muchas cianobacterias filamentosas experimentan procesos de diferenciación celular dando lugar a células o grupos de células con funciones especializadas como son la fijación de nitrógeno, la supervivencia a condiciones adversas y la dispersión. La cianobacteria formadora de heterocistos Anabaena sp. PCC 7120 representa un sistema modelo para estudiar la multicelularidad, la diferenciación cellular y la fijación de nitrógeno. Anabaena crece formando cadenas de células, conocidas como tricomas o filamentos. En ausencia de una fuente de nitrógeno combinado, estos filamentos están formados por dos tipos celulares: células vegetativas que llevan a cabo la fotosíntesis oxigénica y heterocistos que fijan el nitrogeno atmosférico. Las células vegetativas transfieren carbono reducido a los heterocistos, y éstos transfieren nitrógeno a las células vegetativas. La transferencia intercelular de diferentes compuestos es un aspecto muy relevante de la biología de Anabaena que ha sido abordado en esta tesis. Dos vías distintas han sido consideradas para explicar la transferencia de nutrientes y reguladores entre las células del filamento durante la diferenciación de los heterocistos y el crecimiento diazotrófico en estas cianobacterias: una vía indirecta a través del periplasma del filamento y una directa a través de los complejos proteicos llamados “nexos septales” (“septal junctions”). La transferencia molecular intercelular en las cianobacterias filamentosas se ha estudiado con la técnica de análisis Fluorescent Recovery After Photobleaching (FRAP) usando diferentes marcadores fluorescentes: calceína, 5- carboxifluoresceína (5-CF) y esculina, que es un análogo a la sacarosa. En el Capítulo 1, analizamos la dependencia de la temperatura de la transferencia intercelular y concluimos que presenta propiedades de difusión simple. Esta observación es consistente con la presencia de nexos septales responsables de la comunicación intercelular. SepJ, FraC y FraD son proteínas septales identificadas como posibles componentes de los nexos septales, los cuales concluimos que permiten la transferencia intercelular de sustancias por difusión simple. Los nexos septales pueden ser considerados análogos a los conexones de las “gap junctions” de metazoos. Como ha sido mencionado anteriormente, la esculina (análogo fluorescente e la sacarosa) se ha usado para estudiar la transferencia molecular intercelular. En el “Experimental Preamble”, describimos un ensayo que puede ser usado para estudiar la incorporación de esculina a las células de Anabaena. Con este ensayo hemos definido que los transportadores que incorporan esculina en Anabaena son transportadores de α-glucósidos, ya que la incorporación de esculina es inhibida por α- glucósidos como la sacarosa y la maltosa. Los Capítulos 2 y 3 describen la identificación de componentes de transportadores de glucósidos del tipo ABC. Estos componentes son GlsC y GlsD (“nucleotide-binding domain proteins” [NBDs]), GlsP and GlsQ (“transmembrane domain proteins” [TMDs]), y GlsR (“solute-binding protein” [SBP]). Además, HepP, una proteína de la familia MFS, previamente descrita, también contribuye a la incorporación de esculina especialmente en condiciones diazotróficas. Anabaena ha sido considerada un organismo fototrófico estricto durante mucho tiempo. Sin embargo, aunque esta cianobacteria no tiene ningún gen que codifique de forma evidente un transportador de fructosa, trabajos recientes han indicado que puede crecer heterotróficamente con una concentración relativamente alta de fructosa (≥ 50 mM). En el Capítulo 3, describimos el crecimiento fototrófico estimulado por sacarosa, fructosa y glucosa en Anabaena, mostrando la capacidad de crecimiento mixotrófico de este organismo usando estos azúcares. Con objeto de averiguar si los componentes de los transportadores de glucósidos del tipo ABC que hemos identificado están relacionados con el crecimiento estimulado por azúcares, estudiamos el crecimiento mixotrófico en sus mutantes. Mientras que los mutantes glsC y glsD estaban drásticamente afectados en el crecimiento estimulado por sacarosa, los mutantes glsP, glsQ y glsR estaban afectados a menor nivel. Todos los mutantes también estaban afectados de alguna manera en el crecimiento estimulado por fructosa y glucosa, lo cual sugiere que estos azúcares pueden ser asimilados al menos en parte con el concurso de los transportador(es) de glucósidos. El genoma de Anabaena contiene 12 genes que codifican componentes de transportadores de glucósidos tipo ABC: cuatro SBPs, seis TMDs and dos NBDs. Esta información junto con nuestros resultados sugieren la presencia de al menos tres transportadores de glucósidos en Anabaena, uno de los cuales estaría formado por GlsR (SBP), GlsPGlsQ (TMDs) y GlsC-GlsD (NBDs). Otras SBPs podrían contribuir al funcionamiento de este transportador, y las proteínas NBD (GlsC y GlsD) parecen ser compartidas por los tres transportadores como ocurre en otros sistemas ABC. El análisis llevado a cabo mediante Bacterial Adenylate Cyclase Two Hybrid (BACTH) ha mostrado interacciones proteína-proteína consistentes con el modelo propuesto. El efecto de la inactivación de algunos de los genes gls en el crecimiento diazotrófico de Anabaena sugirió la posibilidad de una limitada transferencia de sacarosa a los heterocistos en algunos de estos mutantes. Los mutantes glsC, glsP y hepP estaban afectados en la transferencia intercelular de esculina, por lo que extendimos el estudio a la transferencia de calceína y 5-CF en todos los mutantes gls, así como al mutante hepP. Todos los mutantes ensayados estaban afectados, en particular, en la transferencia intercelular de calceína, lo cual recuerda al fenotipo de la mutación sepJ. Como ya hemos mencionado, SepJ es un posible componente de los nexos septales, y en este trabajo, mediante análisis BACTH, hemos visto que GlsP, GlsQ y HepP interaccionan con SepJ, lo cual sugiere que GlsP, GlsQ and HepP interaccionen con SepJ para un correcto funcionamiento de los nexos septales. Por otro lado, GlsC influye en la localización de SepJ y en el número de nanoporos formados en los discos septales de peptidoglicano. Estos resultados sugieren un papel específico de GlsC influyendo en la maduración de los nexos septales. En conclusión, la cianobacteria formadora de heterocistos Anabaena sp. PCC 7120 expresa transportadores ABC de glucósidos que contribuyen a la asimilación de azúcares haciendo posible un crecimiento mixotrófico. Además, los transportadores de glucósidos estudiados influyen en la comunicación intercelular mediada por los nexos septales.The appearance of multicellular organisms is considered one of the major transitions during the course of early evolution, and multicellularity has been invented independently several times in all three domains of life. The cyanobacteria are a large group of oxygenic photosynthetic prokaryotes that represents a phylum with unicellular and multicellular (filamentous) forms. In many filamentous cyanobacteria, some cells can differentiate into specialized cells or groups of cells supporting different specialized functions, such as nitrogen fixation, survival and dispersion. The heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 represents a model organism to study multicellularity, cellular differentiation and N2 fixation. Anabaena grows as chain of cells, termed trichomes or filaments, which under nitrogen deficiency contain two types of cells: vegetative cells that perform oxygenic photosynthesis and heterocysts that fix nitrogen gas. Vegetative cells transfer reduced carbon to heterocysts, which transfer fixed nitrogen to the vegetative cells. Intercellular molecular exchange is a very relevant aspect of the biology of Anabaena that has been addressed in this thesis. Two different pathways have been considered toxplain the transfer of nutrients and regulators between the cells during heterocyst differentiation and diazotrophic growth in heterocyst-forming cyanobacteria: an indirect pathway via the continuous periplasm of the filament and a direct pathway via septal junction complexes. Intercellular molecular exchange in filamentous cyanobacteria has been studied using Fluorescent Recovery After Photobleaching (FRAP) analysis with different fluorescent markers: calcein, 5-carboxyfluorescein (5-CF) and the sucrose analog esculin. In Chapter 1, we analyzed the temperature-dependence of intercellular molecular exchange and found that it has properties of simple diffusion. This observation favors a direct pathway for intercellular communication. SepJ, FraC and FraD are septal proteins that have been proposed to be components of septal junction complexes. Septal junctions allow intercellular molecular exchange by simple diffusion and appear to be structures analogous to the connexons of the gap junctions of metazoans. As mentioned above, the fluorescent sucrose analog esculin has been used to study intercellular molecular exchange. In the Experimental Preamble, we describe a specific assay that can be used to study the uptake of esculin into Anabaena. Using this assay, we could define that the transporters that mediate esculin uptake in Anabaena are α-glucoside transporters, since esculin uptake is inhibited by the α- glucosides sucrose and maltose. Chapter 2 and 3 describe the identification of components of ABC-type glucoside transporters. These components are GlsC and GlsD (nucleotide-binding domain proteins; NBDs), GlsP and GlsQ (transmembrane domain proteins; TMDs), and GlsR (a periplasmic solute-binding protein; SBP). Additionally, we found that HepP, a previously described Major Facilitator Superfamily (MFS) protein involved in the formation of the heterocyst-specific polysaccharide layer, also contributes to esculin uptake specifically in diazotrophic conditions. Anabaena has been considered as a strict photoautotroph for a long time. Recent work has indicated however that Anabaena can grow heterotrophically with relatively high concentrations of fructose (≥ 50 mM), although this cyanobacterium does not have any gene predicted to encode a fructose-specific transporter. In Chapter 3, we describe sucrose-, fructose- and glucose-stimulated growth of Anabaena, showing that this cyanobacterium can grow mixotrophically with these sugars. To investigate whether the identified ABC components of glucoside transporters are involved in sugar-stimulated growth, we tested mixotrophic growth in their mutants. Whereas the glsC and glsD mutants were drastically impaired in sucrose-stimulated growth, the glsP, glsQ and glsR mutants were impaired at a lower level. All the mutants were also somewhat impaired in fructose- and glucose-stimulated growth suggesting that these sugars can taken up, at least in part, by the glucoside transporter(s). The Anabaena genome contains 12 genes encoding putative components of ABC glucoside transporters: four SBPs, six TMDs and two NBDs. This information and our results suggest the presence in Anabaena of at least three ABC glucoside transporters, one of which may be constituted by GlsR (SBP), GlsP-GlsQ (TMDs) and GlsC-GlsD (NBDs). Additional SBPs may be used by the membrane complex of this transporter, and the NBD proteins (GlsC and GlsD) appear to be shared by the three glucoside transporters, for which there are precedents in well-known ABC transporters. Bacterial Adenylate Cyclase Two Hybrid (BACTH) analysis has shown protein-protein interactions (GlsC-GlsD; GlsC-GlsQ; GlsD-GlsP) that are consistent with this proposal. The effect of inactivation of some gls genes on the diazotrophic growth of Anabaena suggested the possibility of a limited transfer of sucrose to the heterocysts in some of these mutants. We found that the glsC, glsP and hepP mutants were impaired in the intercellular transfer of esculin, and we then extended our study to the intercellular transfer of calcein and 5–CF in all the gls mutants and the hepP mutant. All the glucoside transporter mutants are especially affected in the intercellular transfer of calcein, resembling the phenotype of sepJ mutants. As mentioned above, SepJ is a putative component of septal junctions, and we found that GlsP, GlsQ and HepP interact with SepJ in BACTH analysis, suggesting that GlsP, GlsQ and HepP are required for the correct function of septal junctions. On the other hand, GlsC influences the localization of SepJ and the number of septal peptidoglycan nanopores formed in Anabaena. These results suggest a specific role of GlsC influencing the formati on of septal junctions. In conclusion, the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 expresses glucoside transporters that have a role in sugar assimilation supporting mixotrophic growth. Additionally, these glucoside transporters influence intercellular communication mediated by the septal junction complexes.Premio Extraordinario de Doctorado U

Predicting substrate exchange in marine diatom-heterocystous cyanobacteria symbioses

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 exchanges, 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 exchanges 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. This article is protected by copyright. All rights reserved.España, Gobierno BFU2017-88202-

Adaptation to an Intracellular Lifestyle by a Nitrogen-Fixing, Heterocyst-Forming Cyanobacterial Endosymbiont of a Diatom

The symbiosis between the diatom Hemiaulus hauckii and the heterocyst-forming cyanobacterium Richelia intracellularis makes an important contribution to new production in the world’s oceans, but its study is limited by short-term survival in the laboratory. In this symbiosis, R. intracellularis fixes atmospheric dinitrogen in the heterocyst and provides H. hauckii with fixed nitrogen. Here, we conducted an electron microscopy study of H. hauckii and found that the filaments of the R. intracellularis symbiont, typically composed of one terminal heterocyst and three or four vegetative cells, are located in the diatom’s cytoplasm not enclosed by a host membrane. A second prokaryotic cell was also detected in the cytoplasm of H. hauckii, but observations were infrequent. The heterocysts of R. intracellularis differ from those of free-living heterocyst-forming cyanobacteria in that the specific components of the heterocyst envelope seem to be located in the periplasmic space instead of outside the outer membrane. This specialized arrangement of the heterocyst envelope and a possible association of the cyanobacterium with oxygen-respiring mitochondria may be important for protection of the nitrogen-fixing enzyme, nitrogenase, from photosynthetically produced oxygen. The cell envelope of the vegetative cells of R. intracellularis contained numerous membrane vesicles that resemble the outer-inner membrane vesicles of Gram-negative bacteria. These vesicles can export cytoplasmic material from the bacterial cell and, therefore, may represent a vehicle for transfer of fixed nitrogen from R. intracellularis to the diatom’s cytoplasm. The specific morphological features of R. intracellularis described here, together with its known streamlined genome, likely represent specific adaptations of this cyanobacterium to an intracellular lifestyle

Single-Cell Measurements of Fixation and Intercellular Exchange of C and N in the Filaments of the Heterocyst- Forming Cyanobacterium Anabaena sp. Strain PCC 7120

Under diazotrophic conditions, the model filamentous, heterocystforming cyanobacterium Anabaena sp. strain PCC 7120 develops a metabolic strategy based on the physical separation of the processes of oxygenic photosynthesis, in vegetative cells, and N2 fixation, in heterocysts. This strategy requires the exchange of carbon and nitrogen metabolites and their distribution along the filaments, which takes place through molecular diffusion via septal junctions involving FraCD proteins. Here, Anabaena was incubated in a time course (up to 20 h) with [13C]bicarbonate and 15N2 and analyzed by secondary ion mass spectrometry imaging (SIMS) (large-geometry SIMS [LG-SIMS] and NanoSIMS) to quantify C and N assimilation and distribution in the filaments. The 13C/12C and 15N/14N ratios measured in wild-type filaments showed a general increase with time. The enrichment was relatively homogeneous in vegetative cells along individual filaments, while it was reduced in heterocysts. Heterocysts, however, accumulated recently fixed N at their poles, in which the cyanophycin plug [multi-L-arginyl-poly(L-aspartic acid)] is located. In contrast to the rather homogeneous label found along stretches of vegetative cells, 13C/12C and 15N/14N ratios were significantly different between filaments both at the same and different time points, showing high variability in metabolic states. A fraC fraD mutant did not fix N2, and the 13C/12C ratio was homogeneous along the filament, including the heterocyst in contrast to the wild type. Our results show the consumption of reduced C in the heterocysts associated with the fixation and export of fixed N and present an unpredicted heterogeneity of cellular metabolic activity in different filaments of an Anabaena culture under controlled conditions. IMPORTANCE Filamentous, heterocyst-forming cyanobacteria represent a paradigm of multicellularity in the prokaryotic world. Physiological studies at the cellular level in model organisms are crucial to understand metabolic activities and qualify specific aspects related to multicellularity. Here, we used stable isotopes (13C and 15N) coupled to LG-SIMS and NanoSIMS imaging to follow single-cell C and N2 fixation and metabolic dynamics along the filaments in the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. Our results show a close relationship between C and N fixation and distribution in the filaments and indicate that wild-type filaments in a culture can exhibit a substantial variability of metabolic states. This illustrates how some novel properties can be appreciated by studying microbial cultures at the single-cell level.Peer reviewe

FurC (PerR) contributes to the regulation of peptidoglycan remodeling and intercellular molecular transfer in the cyanobacterium Anabaena sp. strain PCC 7120

Microbial extracellular proteins and metabolites provide valuable information concerning how microbes adapt to changing environments. In cyanobacteria, dynamic acclimation strategies involve a variety of regulatory mechanisms, being ferric uptake regulator proteins as key players in this process. In the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120, FurC (PerR) is a global regulator that modulates the peroxide response and several genes involved in photosynthesis and nitrogen metabolism. To investigate the possible role of FurC in shaping the extracellular environment of Anabaena, the analysis of the extracellular metabolites and proteins of a furC-overexpressing variant was compared to that of the wild-type strain. There were 96 differentially abundant proteins, 78 of which were found for the first time in the extracellular fraction of Anabaena. While these proteins belong to different functional categories, most of them are predicted to be secreted or have a peripheral location. Several stress-related proteins, including PrxA, flavodoxin, and the Dps homolog All1173, accumulated in the exoproteome of furC-overexpressing cells, while decreased levels of FurA and a subset of membrane proteins, including several export proteins and amiC gene products, responsible for nanopore formation, were detected. Direct repression by FurC of some of those genes, including amiC1 and amiC2, could account for odd septal nanopore formation and impaired intercellular molecular transfer observed in the furC-overexpressing variant. Assessment of the exometabolome from both strains revealed the release of two peptidoglycan fragments in furC-overexpressing cells, namely 1,6-anhydro-N-acetyl-β-D-muramic acid (anhydroMurNAc) and its associated disaccharide (β-D-GlcNAc-(1-4)-anhydroMurNAc), suggesting alterations in peptidoglycan breakdown and recycling

SepT, a novel protein specific to multicellular cyanobacteria, influences peptidoglycan growth and septal nanopore formation in Anabaena sp. PCC 7120

ABSTRACT Anabaena sp. PCC 7120 grows by forming filaments of communicating cells and is considered a paradigm of bacterial multicellularity. Molecular exchanges between contiguous cells in the filament take place through multiprotein channels that traverse the septal peptidoglycan through nanopores connecting their cytoplasms. Besides, the septal-junction complexes contribute to strengthen the filament. In search for proteins with coiled-coil domains that could provide for cytoskeletal functions in Anabaena, we identified SepT (All2460). SepT is characteristic of the phylogenetic clade of filamentous cyanobacteria with the ability to undergo cell differentiation. SepT-GFP fusions indicate that the protein is located at the cell periphery and, conspicuously, in the intercellular septa. During cell division, the protein is found at midcell resembling the position of the divisome. The bacterial adenylate cyclase two-hybrid analysis shows SepT interactions with itself and putative elongasome (MreB, RodA), divisome (FtsW, SepF, ZipN), and septal-junction (SepJ)-related proteins. Thus, SepT appears to rely on the divisome for localization at mature intercellular septa to form part of intercellular protein complexes. Two independently obtained mutants lacking SepT showed alterations in cell size and impaired septal and peripheral peptidoglycan incorporation during cell growth and division. Notably, both mutants showed conspicuous alterations in the array of nanopores present in the intercellular peptidoglycan disks, including aberrant nanopore morphology, number, and distribution. SepT appears, therefore, to be involved in the control of peptidoglycan growth and the formation of cell-cell communication structures that are at the basis of the multicellular character of this group of cyanobacteria. IMPORTANCE Multicellular organization is a requirement for the development of complex organisms, and filamentous cyanobacteria such as Anabaena represent a paradigmatic case of bacterial multicellularity. The Anabaena filament can include hundreds of communicated cells that exchange nutrients and regulators and, depending on environmental conditions, can include different cell types specialized in distinct biological functions. Hence, the specific features of the Anabaena filament and how they are propagated during cell division represent outstanding biological issues. Here, we studied SepT, a novel coiled-coil-rich protein of Anabaena that is located in the intercellular septa and influences the formation of the septal specialized structures that allow communication between neighboring cells along the filament, a fundamental trait for the performance of Anabaena as a multicellular organism