Studies of Bacterial Species Associated with Ciliated Protozoa Based on Multidisciplinary Approach

Abstract

Ciliates and bacteria occupy the most diverse ecological niches and developed various types of association ranging from mutualism to parasitism, from facultative to obligatory interactions. Many bacterial species belonging to the class of Alpha, Beta, and Gamma-proteobacteria were identified from the different groups of ciliates. Several ciliate samples were collected from diverse habitats and those ciliates were investigated for the isolation of culturable bacterial species. We were able to isolate some bacterial species that belong to the class of Alpha, Beta, and Gamma-proteobacteria from the different ciliates including Paramecium caudatum, Spirostomum cf. yagiui, Stentor tartari, Diaxonella pseudorubra, and Levicoleps biwae jejuensis and a multidisciplinary approach was applied to identify their taxonomic position. Aforesaid results as bacterial sample 1: A Gram-negative, oxidase- and catalase-negative, motile, rod-shaped bacterial strain PCS8T isolated from the ciliate Paramecium caudatum. Based on the distinct phenotypic, phylogenetic, chemotaxonomic, and G+C content results, strain PCS8T represents a currently undescribed species within the genus Pseudaeromonas in the family Aeromonadaceae, for which we suggest the name Pseudaeromonas paramecii sp. nov. and an amendment description of the genus Pseudaeromonas is also provided.; bacterial sample 2: A bacterial strain, designated Sp-1T, was isolated from the heterotrichs ciliate Spirostomum cf. yagiui. On the basis of the polyphasic taxonomic evidence presented in the results section, we concluded that strain Sp-1T is considered to represent a novel species in a new genus within the order Rhizobiales, for which the name Segnisochrobactrum spirostomi gen. nov., sp. nov. is proposed.; bacterial sample 3: A Gram-type negative, rod-shaped bacterium S18 was isolated from the heterotrichs ciliate Stentor tartari the 16S rRNA gene sequence analysis demonstrated that this isolate was included in the genus Pseudomonas within the family Pseudomonadaceae and its closest relative was Pseudomonas koreensis with the greatest degree of similarity (99.86%). The strain was distinguished from its close relatives by phenotypic characteristics. On the basis of the phylogenetic analysis in combination with the physiological biochemical and cellular fatty acids profile and G+C content data, this strain is considered to represent a new species candidate of the genus Pseudomonas.; bacterial sample 4: A Gram-negative, rod-shaped and aerobic bacterial strain D-1 isolated from the ciliate Diaxonella pseudorubra. The morphological, biochemical, physiological properties and chemotaxonomic data of it give us that our new isolate is close to the members of genera Pseudacidovorax and Acidovorax but not matched exactly previously known strains or species. In addition, phylogenetic analyses based on 16S rRNA gene sequences indicate that the closest relative of our bacterial strain is Pseudacidovorax intermedius with 100% sequence similarity. Based on these results, we concluded that this strain represents a currently undescribed taxon belongs to the Pseudacidovorax genus.; and bacterial sample 5: A Gram-stain-negative, motile and rod-shaped bacterium, strain LBJS4, was isolated from the ciliate Levicoleps biwae jejuensis. The strain is distinguishable from closely related members of the genus Aquitalea by its several differential biochemical and physiological characteristics, and distinct fatty acids compositions. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain LBJS4 constituted a tide clade with its closest phylogenetic relative Aquitalea pelogenes with 99.12% of 16S rRNA gene sequence similarity. On the basis of those results, we suggest that this bacterial strain could be assigned as representing a novel taxon within the genus Aquitalea. In addition, fluorescence in situ hybridization (FISH) and Transmission electron microscopy (TEM) showed that symbionts were localized in the host cytoplasm and survived intracellularly. This study also highlights that ciliates may be foremost hosts for different bacterial species. Our present study will enrich the prokaryotic diversity that is associated with the different ciliates through the multidisciplinary strategy.