Abstract
Rheinheimera sp. strain EpRS3, isolated from the rhizosphere of Echinacea purpurea, 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 Euplotes aediculatus (strain EASCc1), which by FISH experiments resulted to harbor in its cytoplasm the obligate bacterial endosymbiont Polynucleobacter necessarius (Betaproteobacteria) and the secondary endosymbiont “Candidatus Nebulobacter yamunensis” (Gammaproteobacteria). 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 Rheinheimera sp. EpRS3 produces and releases in liquid culture one or more bioactive molecules affecting E. aediculatus survival. TEM analysis of control (not treated) ciliates allowed to morphologically characterize both kind of E. aediculatus endosymbionts. In treated ciliates, collected soon after the arising of cell suffering leading to death, TEM observations revealed some ultrastructural damages, indicating that P. necessarius 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 P. necessarius 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 Rheinheimera sp. EpRS3 both inside phagosomes and free in cytoplasm in ciliates after Tq treatment. This finding suggests a putative ability of Rheinheimera sp. EpRS3 to reintroduce itself in the environment avoiding ciliate digestion.
Highlights
Rheinheimera sp. strain EpRS3 is a Gammaproteobacterium belonging to the family Chromatiaceae
Ciliate strain EASCc1 was confirmed in morphological inspections as Euplotes aediculatus as perfectly matching the description of the species (Curds, 1975); molecular analysis based on 18S rRNA gene sequencing confirmed the species assignation by morphological identification
The almost complete 16S rRNA gene sequence of the Gammaproteobacteria endosymbiont was achieved by direct sequencing after PCR on E. aediculatus cells
Summary
Rheinheimera sp. strain EpRS3 is a Gammaproteobacterium belonging to the family Chromatiaceae. Rheinheimera EpRS3 shows resistance to several antibiotic compounds (Mengoni et al, 2014) and can inhibit the growth of various bacteria isolated from both E. purpurea rhizospheric soil and plant tissues (Maida et al, 2016). The toxicity of bacteria belonging to Rheinheimera genus against other organisms has been described in other strains such as GR5, showing antimicrobial activity against Gram-positive and Gram-negative bacteria, yeast, and algae (Chlorophyceae, Trebouxiophyceae) (Chen et al, 2010; Romanenko et al, 2015). The antibacterial toxicity of the marine bacterium Rheinheimera japonica KMM 9513 has been described as well. This strain is able to exert the antimicrobial activities against Bacillus subtilis and/or Enterococcus faecium and Staphylococcus aureus by synthesizing phthalates and diketopiperazines (Kalinovskaya et al, 2017). The anti-quorum sensing activity of the diketopiperazine factor cyclo (Trp-Ser) produced by Rheinheimera aquimaris QSI02 against Chromobacterium violaceum CV026 and Pseudomonas aeruginosa PA01 has been evidenced, focusing the attention on the potentiality of this bacterial genus to interact with other bacterial species in environmental microbial communities (Sun et al, 2016)
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