Chiellini Carolina, Pasqualetti Chiara, Lanzoni Olivia, Fagorzi Camilla, Bazzocchi Chiara, Fani Renato, Petroni Giulio, Modeo Letizia
Department of Biology, University of Florence, Florence, Italy.
Department of Biology, University of Pisa, Pisa, Italy.
Front Microbiol. 2019 Mar 22;10:510. doi: 10.3389/fmicb.2019.00510. eCollection 2019.
sp. strain EpRS3, isolated from the rhizosphere of , is already known for its ability to produce antibacterial compounds. By use of culture experiments, we verified and demonstrated its harmful effect against the ciliated protist (strain EASCc1), which by FISH experiments resulted to harbor in its cytoplasm the obligate bacterial endosymbiont () and the secondary endosymbiont " Nebulobacter yamunensis" (). In culture experiments, the number of ciliates treated both with liquid broth bacteria-free (Supernatant treatment) and bacteria plus medium (Tq treatment), decreases with respect to control cells, with complete disappearance of ciliates within 6 h after Tq treatment. Results suggest that sp. EpRS3 produces and releases in liquid culture one or more bioactive molecules affecting survival. TEM analysis of control (not treated) ciliates allowed to morphologically characterize both kind of endosymbionts. In treated ciliates, collected soon after the arising of cell suffering leading to death, TEM observations revealed some ultrastructural damages, indicating that endosymbionts went into degradation and vacuolization after both Supernatant and Tq treatments. Additionally, TEM investigation showed that when the ciliate culture was inoculated with Tq treatment, both a notable decrease of number and an increase of damaged and degraded mitochondria occur. FISH experiments performed on treated ciliates confirmed TEM results and, by means of the specific probe herein designed, disclosed the presence of sp. EpRS3 both inside phagosomes and free in cytoplasm in ciliates after Tq treatment. This finding suggests a putative ability of sp. EpRS3 to reintroduce itself in the environment avoiding ciliate digestion.
从[具体植物名称]根际分离出的EpRS3菌株，已因其产生抗菌化合物的能力而闻名。通过培养实验，我们验证并证明了它对纤毛虫（菌株EASCc1）的有害作用，通过荧光原位杂交实验发现该纤毛虫在其细胞质中含有专性细菌内共生体（[具体名称]）和次生内共生体“Nebulobacter yamunensis”（[具体名称]）。在培养实验中，与对照细胞相比，用无细菌液体肉汤处理（上清液处理）和细菌加培养基处理（Tq处理）的纤毛虫数量均减少，在Tq处理后6小时内纤毛虫完全消失。结果表明EpRS3菌株在液体培养中产生并释放一种或多种影响[纤毛虫名称]生存的生物活性分子。对未处理的对照纤毛虫进行透射电镜分析，能够从形态上表征这两种内共生体。在细胞出现病变并导致死亡后不久收集的经处理的纤毛虫中，透射电镜观察发现了一些超微结构损伤，表明在上清液和Tq处理后，内共生体进入降解和空泡化状态。此外，透射电镜研究表明，当纤毛虫培养物接种Tq处理时，[内共生体名称]数量显著减少，受损和降解的线粒体数量增加。对经处理的纤毛虫进行的荧光原位杂交实验证实了透射电镜结果，并通过本文设计的特异性探针，揭示了Tq处理后纤毛虫吞噬体内和细胞质中游离的EpRS3菌株的存在。这一发现表明EpRS3菌株具有一种假定的能力，即能够在避免被纤毛虫消化的情况下重新引入环境中。
