The Cyanobacterium Cylindrospermopsis raciborskii (CYRF-01) Responds to Environmental Stresses with Increased Vesiculation Detected at Single-Cell Resolution.

Victor Zarantonello,Juliana P Gamalier,Natália P Noyma,Mariana M Mello,Marcelo M Marinho,Rossana C N Melo,Thiago P Silva

doi:10.3389/fmicb.2018.00272

Abstract

Secretion of membrane-limited vesicles, collectively termed extracellular vesicles (EVs), is an important biological process of both eukaryotic and prokaryotic cells. This process has been observed in bacteria, but remains to be better characterized at high resolution in cyanobacteria. In the present work, we address the release of EVs by Cylindrospermopsis raciborskii (CYRF-01), a filamentous bloom-forming cyanobacterium, exposed to environmental stressors. First, non-axenic cultures of C. raciborskii (CYRF-01) were exposed to ultraviolet radiation (UVA + UVB) over a 6 h period, which is known to induce structural damage to this species. Second, C. raciborskii was co-cultured in interaction with another cyanobacterium species, Microcystis aeruginosa (MIRF-01), over a 24 h period. After the incubation times, cell density and viability were analyzed, and samples were processed for transmission electron microscopy (TEM). Our ultrastructural analyses revealed that C. raciborskii constitutively releases EVs from the outer membrane during its normal growth and amplifies such ability in response to environmental stressors. Both situations induced significant formation of outer membrane vesicles (OMVs) by C. raciborskii compared to control cells. Quantitative TEM revealed an increase of 48% (UV) and 60% (interaction) in the OMV numbers compared to control groups. Considering all groups, the OMVs ranged in size from 20 to 300 nm in diameter, with most OMVs showing diameters between 20 and 140 nm. Additionally, we detected that OMV formation is accompanied by phosphatidylserine exposure, a molecular event also observed in EV-secreting eukaryotic cells. Altogether, we identified for the first time that C. raciborskii has the competence to secrete OMVs and that under different stress situations the genesis of these vesicles is increased. The amplified ability of cyanobacteria to release OMVs may be associated with adaptive responses to changes in environmental conditions and interspecies cell communication.

Highlights

The bloom-forming cyanobacterium Cylindrospermopsis raciborskii has attracted considerable attention due its widespread distribution and its potential ability to produce diverse toxins, such as hepatotoxins and neurotoxins that can cause detrimental impacts on the environmental health (Molica et al, 2002; Carneiro et al, 2013)
By studying the strain CYRF-01, we showed that this cyanobacterium constitutively releases outer membrane vesicles (OMVs) during its normal growth and amplifies such ability in response to environmental stressors, such as UV radiation and interaction with a potential competitor
Our data are supported by previous works on other cyanobacterial species and different strains which found production of extracellular vesicles (EVs) in response to stressors such as hyperosmotic conditions (Xu et al, 2013), antibiotic supplementation (Oliveira et al, 2016) or reduced temperature (Oliveira et al, 2016)

Summary

Introduction

The bloom-forming cyanobacterium Cylindrospermopsis raciborskii has attracted considerable attention due its widespread distribution and its potential ability to produce diverse toxins, such as hepatotoxins and neurotoxins that can cause detrimental impacts on the environmental health (Molica et al, 2002; Carneiro et al, 2013). The capacity of bacteria to release EVs has been exponentially demonstrated (reviewed in Kulp and Kuehn, 2010; Jan, 2017). These nano-scale vesicles are extruded from the outer membrane of gram-negative bacteria and have been associated with fundamental biological processes such as pathogenesis (Kolling and Matthews, 1999; Rivera et al, 2010); cellular defense (Manning and Kuehn, 2011; Baumgarten et al, 2012), cell communication (Mashburn-Warren et al, 2008) and DNA transfer (Rumbo et al, 2011). Little is still known on the ability of cyanobacteria to secrete outer membrane vesicles (OMVs), their biogenesis and potential functions in aquatic biology

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Conclusion