Description of new genera and species of marine cyanobacteria from the Portuguese Atlantic coast.

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.

Isolation and Characterization of Cyanobacterial Extracellular Vesicles.

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.

Extracellular vesicles as an alternative copper-secretion mechanism in bacteria.

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.

Novel protein carrier system based on cyanobacterial nano-sized extracellular vesicles for application in fish.

Aquaculture has been one of the fastest-growing food industry sectors, expanding at the pace of consumers' demands. To promote safe and effective fish growth performance strategies, and to stimulate environmentally friendly solutions to protect fish against disease outbreaks, new approaches are needed to safeguard fish welfare, as well as farmers and consumers interests. Here, we tested the use of cyanobacterial extracellular vesicles (EVs) as a novel nanocarrier system of heterologous proteins for applications in fish. We started by incubating zebrafish larvae with Synechocystis sp. PCC6803 EVs, isolated from selected mutant strains with different cell envelope characteristics. Results show that Synechocystis EVs are biocompatible with fish larvae, regardless of their structural composition, as EVs neither induced fish mortality nor triggered significant inflammatory responses. We establish also that cyanobacteria are amenable to engineering heterologous protein expression and loading into EVs, for which we used the reporter sfGFP. Moreover, upon immersion treatment, we successfully demonstrate that sfGFP-loaded Synechocystis EVs accumulate in the gastrointestinal tract of zebrafish larvae. This work opens the possibility of using cyanobacterial EVs as a novel biotechnological tool in fish, with prospective applications in carrying proteins/enzymes, for example for modulating their nutritional status or stimulating specific adaptive immune responses.

The Role of Outer Membrane Protein(s) Harboring SLH/OprB-Domains in Extracellular Vesicles' Production in Synechocystis sp. PCC 6803.

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.

Extracellular Vesicles: An Overlooked Secretion System in Cyanobacteria.

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.

The secretion signal peptide of the cyanobacterial extracellular protein HesF is located at its C-terminus.

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.