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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.