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.

Summary: 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.

Hospital-acquired infections are still a major concern worldwide, being frequently related to bacterial biofilm formation on medical devices, and thus difficult to eradicate with conventional antimicrobial treatments. Therefore, infection-preventive solutions based on natural polymers are being investigated. Recently, a marine cyanobacterium-derived polymeric coating (CyanoCoating) has demonstrated great anti-adhesive potential when immobilized onto gold model substrates. In this work, we took this technology a step closer to an industrial application by covalently immobilizing CyanoCoating onto medical grade polyurethane (PU). This immobilization was developed through the introduction of linkable moieties onto a PU inert surface using different pre-treatments. Besides the application of the polydopamine (pDA) linker layer, other processes frequently found in industrial settings, such as atmospheric plasma (using O₂ or N₂ as reactive gases) and ozone surface activations, were evaluated. From all the pre-treatments tested, the ozone activation was the most promising since the obtained coating not only revealed a homogeneous distribution, but also significantly reduced the adhesion of two relevant etiological bacteria in static conditions (the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli). Moreover, it also impaired E. coli biofilm formation under simulated urinary tract dynamic conditions, reinforcing the potential of CyanoCoating as an antibiotic-free alternative to mitigate medical device-associated infections, particularly in the urinary tract.

Disease outbreaks are a common problem in aquaculture, with serious economic consequences to the sector. Some of the most important bacterial diseases affecting aquaculture are caused by Gram-negative bacteria including Vibrio spp. (vibriosis), Photobacterium damselae (photobacteriosis), Aeromonas spp. (furunculosis; haemorrhagic septicaemia) or Tenacibaculum maritimum (tenacibaculosis). Lipopolysaccharides (LPS) are important components of the outer membrane of Gram-negative bacteria and have been linked to strong immunogenic responses in terrestrial vertebrates, playing a role in disease development. To evaluate LPS effects in fish, we used a hot-phenol procedure to extract LPS from common fish pathogens. A. hydrophila, V. harveyi, T. maritimum and P. damselae purified LPS were tested at different concentrations (50, 100, 250 and 500 µg mL⁻¹) at 3 days post-fertilisation (dpf) Danio rerio larvae, for 5 days. While P. damselae LPS did not cause any mortality under all concentrations tested, A. hydrophila LPS induced 15.5% and V. harveyi LPS induced 58.3% of zebrafish larvae mortality at 500 µg mL⁻¹. LPS from T. maritimum was revealed to be the deadliest, with a zebrafish larvae mortality percentage of 80.6%. Analysis of LPS separated by gel electrophoresis revealed differences in the overall LPS structure between the bacterial species analysed that might be the basis for the different mortalities observed.

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.

Lipids of extracellular vesicles (EVs) are attracting attention due to their crucial biological functions and potential roles in processes such as carcinogenesis. This study compares three commonly used lipid extraction techniques, i.e., liquid-liquid extraction, single-phase extraction, and solid-phase extraction, with a novel direct injection liquid chromatography-mass spectrometry (DI-LC-MS) workflow tailored to EV lipidomics. In the DI-LC-MS approach, EVs are disrupted and released directly in the chromatographic system, enabling the analysis of lipids without a prior extraction step. The applicability of the DI-LC-MS workflow was demonstrated by profiling lipids in mammalian and bacterial EVs. The lipidome coverage and high precision of the DI-LC-MS method (coefficient of variation of peak area lower than 20% for all the identified lipids) enabled identification of differences in lipid profiles of EV samples. The column used in the DI-LC-MS method exhibited a sufficient lifespan and stability for comparative lipidomic studies. Lipidome coverage, lipid species distribution, and precision varied across the studied workflows; our findings highlight the strengths and limitations of these methods. The DI-LC-MS emerges as a sustainable alternative for EV lipidomic studies by eliminating the need for sample preparation and reducing analysis time, solvent use, and chemical noise while requiring less than 1 μL of sample.