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Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential biotechnology models. Recent work has demonstrated that both marine and freshwater cyanobacteria produce extracellular vesicles - small membrane-bound structures released from the outer surface of the microbes. While vesicles likely contribute to diverse biological processes, their specific functional roles in cyanobacterial biology remain largely unknown. To encourage and advance research in this area, a detailed protocol is presented for isolating, concentrating, and purifying cyanobacterial extracellular vesicles. The current work discusses methodologies that have successfully isolated vesicles from large cultures of Prochlorococcus, Synechococcus, and Synechocystis. Methods for quantifying and characterizing vesicle samples from these strains are presented. Approaches for isolating vesicles from aquatic field samples are also described. Finally, typical challenges encountered with cyanobacterial vesicle purification, methodological considerations for different downstream applications, and the trade-offs between approaches are also discussed.

Cyanobacteria are a group of photosynthetic prokaryotes that contribute to primary production on a global scale. These microorganisms release vesicles to the extracellular environment, spherical nanosized structures, derived essentially from the outer membrane. Even though earlier works in model Gram-negative bacteria have hypothesized that outer membrane stability is crucial in vesicle formation, the mechanisms determining vesicle biogenesis in cyanobacteria remain unknown. Here, we report on the identification of six candidate genes encoding outer membrane proteins harboring SLH/OprB-domains in the genome of the model cyanobacterium Synechocystis sp. PCC 6803. Using a genetics-based approach, one gene was found to encode an essential protein (Slr1841), while the remaining five are not essential for growth under standard conditions. Vesicle production was monitored, and it was found that a mutant in the gene encoding the second most abundant SLH/OprB protein in Synechocystis sp. PCC 6803 outer membrane (Slr1908) produces more vesicles than any of the other tested strains. Moreover, the Slr1908-protein was also found to be important for iron uptake. Altogether, our results suggest that proteins containing the SLH/OprB-domains may have dual biological role, related to micronutrient uptake and to outer membrane stability, which, together or alone, seem to be involved in cyanobacterial vesicle biogenesis.

13 Pág., Metal homeostasis is fundamental for optimal performance of cell metabolic pathways. Over the course of evolution, several systems emerged to warrant an intracellular metal equilibrium. When exposed to growth-challenging copper concentrations, Gram-negative bacteria quickly activate copper-detoxification mechanisms, dependent on transmembrane-protein complexes and metallochaperones that mediate metal efflux. Here, we show that vesiculation is also a common bacterial response mechanism to high copper concentrations, and that extracellular vesicles (EVs) play a role in transporting copper. We present evidence that bacteria from different ecological niches release copious amounts of EVs when exposed to copper. Along with the activation of the classical detoxification systems, we demonstrate that copper-stressed cells of the cyanobacterium Synechocystis sp. PCC6803 release EVs loaded with the copper-binding metallochaperone CopM. Under standard growth conditions, CopM-loaded EVs could also be isolated from a Synechocystis strain lacking a functional TolC-protein, which we characterize here as exhibiting a copper-sensitive phenotype. Analyses of Synechocystis tolC-mutant's EVs isolated from cells cultivated under standard conditions indicated the presence of copper therein, in significantly higher levels as compared to those from the wild-type. Altogether, these results suggest that release of EVs in bacteria represent a novel copper-secretion mechanism, shedding light into alternative mechanisms of bacterial metal resistance., This work was financed by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020 Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through Fundação para a Ciência e a Tecnologia/Ministério da Ciência Tecnologia e Ensino Superior in the framework of the project POCI-01–0145-FEDER-029540 (PTDC/BIA-OUT/29540/2017). Fundação para a Ciência e a Tecnologia is also greatly acknowledged for the PhD fellowship SFRH/BD/130478/2017 (SL), the Assistant Researcher contract CEECIND/00259/2017 (CCP), and FCT Investigator grant IF/00256/2015 (PO). Funding from the EPSRC (EP/E036252/1) and BBSRC (BB/M012166/1) (PCW), and Ministerio de Ciencia, Innovación y Universidades (grant PGC2018-098073-A-I00 MCIU/AEI/FEDER) (JG-L) is also acknowledged.

In bacteria, the active transport of material from the interior to the exterior of the cell, or secretion, represents a very important mechanism of adaptation to the surrounding environment. The secretion of various types of biomolecules is mediated by a series of multiprotein complexes that cross the bacterial membrane(s), each complex dedicated to the secretion of specific substrates. In addition, biological material may also be released from the bacterial cell in the form of vesicles. Extracellular vesicles (EVs) are bilayered, nanoscale structures, derived from the bacterial cell envelope, which contain membrane components as well as soluble products. In cyanobacteria, the knowledge regarding EVs is lagging far behind compared to what is known about, for example, other Gram-negative bacteria. Here, we present a summary of the most important findings regarding EVs in Gram-negative bacteria, discussing aspects of their composition, formation processes and biological roles, and highlighting a number of technological applications tested. This lays the groundwork to raise awareness that the release of EVs by cyanobacteria likely represents an important, and yet highly disregarded, survival strategy. Furthermore, we hope to motivate future studies that can further elucidate the role of EVs in cyanobacterial cell biology and physiology. This work was financed by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020 Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal, 2020, and by Portuguese funds through Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project POCI-01-0145-FEDER-029540 (PTDC/BIA-OUT/29540/2017). Fundação para a Ciência e a Tecnologia is also greatly acknowledged for the PhD fellowship SFRH/BD/130478/2017 (SL) and FCT Investigator grant IF/00256/2015 (PO).

SummaryWaterflooding has been commonly used for secondary oil recovery. However, it is well known that the efficiency of oil recovery decreases when the mobility ratio is large, or the reservoir is highly heterogeneous. In these scenarios, the polymer flooding technique arises as an efficient alternative to increase the production curves. The injection of a high viscosity polymer solution reduces the mobility ratio, improving the displacement and sweep efficiency. On the other hand, mechanical retention and adsorption phenomena give rise to formation damage close to the injection wells resulting in injectivity loss. In this context, our main goal is to construct a new computational model based on domain decomposition methods capable of coupling the phenomena in different spatial and time scales during the polymer flooding. From the mathematical point of view, we consider the polymer solution a pseudo-plastic flow with the hydrodynamic model given by a non-linear Darcy’s Law where the injected fluid viscosity depends on the shear rate as suggested by the Carreau’s Law. Furthermore, the polymer movement is quantified making use of a convection-diffusion-reaction transport equation where the non-linear reactive part is due to mechanical retention and adsorption. The studied model takes formation damage into account considering that porosity and permeability depend on the retained polymer concentrations mechanically retained or adsorbed. From the computational point of view, the non-linear mathematical model is discretized making use of the finite element method together with a staggered algorithm and the Newton-Raphson method. The kinetic law for mechanical retention is post-processed by the Runge-Kutta method. It is important to highlight that polymer may accumulate in the neighborhood of the injection well on a fast time scale causing injectivity loss. Contrary to the rest of the reservoir, where large time steps and a coarse spatial mesh can be used, on the neighborhood of the injection wells small time steps and a fine spatial mesh are sometimes required. In this context, we propose the application of domain decomposition techniques to couple the near-well/reservoir domains with accuracy and lower computational cost. To this end, we apply a multi-time step domain decomposition method to couple retention and adsorption near well phenomena with polymer transport in the reservoir. Finally, we propose some numerical simulations to show the efficiency of the domain decomposition as well as to quantify injectivity during polymer flooding.

Cyanobacteria hold great promise as renewable and sustainable microbial cell factories for the production of several chemicals. In the past few years, great efforts have been employed to fine-tune metabolic pathways and fluxes toward product formation. However, focusing exclusively on the biosynthetic pathways of the target molecule, neglects two crucial aspects for the overall production capacity and process efficiency: availability of substrates and product secretion. In fact, the molecular mechanisms mediating substrate import and product export have been largely overlooked in cyanobacteria. In this context, secretion is particularly relevant, as compounds of interest may accumulate in the intracellular space, inhibiting production pathways and/or inducing toxic effects. In this chapter, we wish to raise awareness of the importance of secretion systems in cyanobacteria in the setting of biotechnological applications, highlighting what has been done in closely related bacteria, and identifying cyanobacterial candidates for improving efficient product export.

Cyanobacteria are photosynthetic prokaryotes, capable of sustaining their growth by converting sunlight into chemical energy by fixing CO2 into organic matter. The cyanobacterium Anabaena sp. PCC 7120 is also capable of fixing atmospheric nitrogen, a metabolic process that occurs in specialized cells, the heterocysts. During the process of heterocyst differentiation, drastic morphological changes occur to prepare the future differentiated cell to accommodate the nitrogen fixation metabolism, which is a highly O2-sensitive process. Recently, we identified an unknown extracellular protein (termed HesF) in Anabaena sp. PCC 7120 and found it to be required for the proper deposition of the polysaccharide layers in the heterocyst cell wall. HesF is a non-classical type I secretion system (T1SS)-dependent secreted substrate, and its secretion signal remained elusive. Here, we report that the secretion signal of HesF is located in its C-terminus. We present evidence that a heterologous reporter protein fused with HesF's secretion signal could be successfully expressed in heterocysts and secreted to the extracellular medium, following hesF's native regulation. This represents the first time that the secretion signal of a cyanobacterial T1SS-dependent substrate is identified, and demonstrates the feasibility of using cyanobacteria for selected protein expression and secretion.

Aiming at increasing the knowledge on marine cyanobacteria from temperate regions, we previously isolated and characterized 60 strains from the Portuguese foreshore and evaluate their potential to produce secondary metabolites. About 15% of the obtained 16S rRNA gene sequences showed less than 97% similarity to sequences in the databases revealing novel biodiversity. Herein, seven of these strains were extensively characterized and their classification was re-evaluated. The present study led to the proposal of five new taxa, three genera (Geminobacterium, Lusitaniella, and Calenema) and two species (Hyella patelloides and Jaaginema litorale). Geminobacterium atlanticum LEGE 07459 is a chroococcalean that shares morphological characteristics with other unicellular cyanobacterial genera but has a distinct phylogenetic position and particular ultrastructural features. The description of the Pleurocapsales Hyella patelloides LEGE 07179 includes novel molecular data for members of this genus. The filamentous isolates of Lusitaniella coriacea - LEGE 07167, 07157 and 06111 - constitute a very distinct lineage, and seem to be ubiquitous on the Portuguese coast. Jaaginema litorale LEGE 07176 has distinct characteristics compared to their marine counterparts, and our analysis indicates that this genus is polyphyletic. The Synechococcales Calenema singularis possess wider trichomes than Leptolyngbya, and its phylogenetic position reinforces the establishment of this new genus. © 2017 Elsevier This work was funded by National Funds through FCT - Fundação para a Ciência e a Tecnologia, grants SFRH/BD/70284/2010 (to AB), SFRH/BD/80153/2011 (to VR), SFRH/BD/84914/2012 (to RM), and GACR 15-11912S (to JK) and by the project NORTE-01-0145-FEDER-000012, Structured Programme on Bioengineering Therapies for Infectious Diseases and Tissue Regeneration, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work was also supported by the Strategic Funding UID/Multi/04423/2013 through national funds provided by FCT - Fundação para a Ciência e a Tecnologia and the Structured Program of R&D&I INNOVMAR - Innovation and Sustainability in the Management and Exploitation of Marine Resources (reference NORTE-01-0145-FEDER-000035, Research Line NOVELMAR), funded by the Northern Regional Operational Program (NORTE2020) through the European Regional Development Fund (ERDF). We are grateful to Daniela Silva from Centro de Materiais da Universidade do Porto (CEMUP) - Porto - Portugal for technical assistance with SEM, to Paulo Oliveira from IBMC for the help with the GC measurements, and Sílvia Coimbra and Mário Costa from FCUP for providing access to the Leica DMLB microscope.