Molecular Identification of Rickettsial Endosymbionts in the Non-Phagotrophic Volvocalean Green Algae

Abstract

BackgroundThe order Rickettsiales comprises Gram-negative obligate intracellular bacteria (also called rickettsias) that are mainly associated with arthropod hosts. This group is medically important because it contains human-pathogenic species that cause dangerous diseases. Until now, there has been no report of non-phagotrophic photosynthetic eukaryotes, such as green plants, harboring rickettsias.Methodology/Principal FindingsWe examined the bacterial endosymbionts of two freshwater volvocalean green algae: unicellular Carteria cerasiformis and colonial Pleodorina japonica. Epifluorescence microscopy using 4′-6-deamidino-2-phenylindole staining revealed the presence of endosymbionts in all C. cerasiformis NIES-425 cells, and demonstrated a positive correlation between host cell size and the number of endosymbionts. Strains both containing and lacking endosymbionts of C. cerasiformis (NIES-425 and NIES-424) showed a >10-fold increase in cell number and typical sigmoid growth curves over 192 h. A phylogenetic analysis of 16 S ribosomal (r)RNA gene sequences from the endosymbionts of C. cerasiformis and P. japonica demonstrated that they formed a robust clade (hydra group) with endosymbionts of various non-arthropod hosts within the family Rickettsiaceae. There were significantly fewer differences in the 16 S rRNA sequences of the rickettsiacean endosymbionts between C. cerasiformis and P. japonica than in the chloroplast 16 S rRNA or 18 S rRNA of the host volvocalean cells. Fluorescence in situ hybridization demonstrated the existence of the rickettsiacean endosymbionts in the cytoplasm of two volvocalean species.Conclusions/SignificanceThe rickettsiacean endosymbionts are likely not harmful to their volvocalean hosts and may have been recently transmitted from other non-arthropod organisms. Because rickettsias are the closest relatives of mitochondria, incipient stages of mitochondrial endosymbiosis may be deduced using both strains with and without C. cerasiformis endosymbionts.

“Candidatus Finniella” (Rickettsiales, Alphaproteobacteria), Novel Endosymbionts of Viridiraptorid Amoeboflagellates (Cercozoa, Rhizaria)

“…A Rickettsia species was found in the cytoplasm of the naked, filose amoeba Nuclearia pattersonii (Nucleariidae, Opisthokonta) (73), and representatives of the "Ca. Megaira" clade (Rickettsiaceae) colonize volvocalean green algae such as Carteria, Pleodorina, and Volvox (Chlorophyta, Viridiplantae) (74)(75)(76). There is also one report on "Ca.…”

Permanent residents or temporary lodgers: characterizing intracellular bacterial communities in the siphonous green algaBryopsis

“…We presume that Bryopsis also hosts Rhizobiaceae species with nitrogen fixing capacities, as we were able to amplify Ensifer-like nitrogenase reductase genes (EMBL accession numbers HE649370-HE649371) from Bryopsis samples 4718 and MZ4 [51]. Obligate intracellular Rickettsia species, on the other hand, have not previously been described from macroalgae but have been characterized through 16S rRNA gene analysis within freshwater green microalgae [52], marine ciliates [53] and coral tissue [54].…”

A rickettsial endosymbiont inhabiting the cytoplasm of Volvox carteri (Volvocales, Chlorophyceae)

“…Recently, Kawafune et al (2012) reported the molecular identification of intracellular bacteria in the green algae P. japónica and C. cerasiformis. In the first report of rickettsial endosymbionts within photosynthetic eukaryotic cells, they demonstrated that rickettsial endosymbionts reside in the cytoplasm of P. japónica and C. cerasiformis (Kawafune et al 2012). The rickettsiae [i.e.…”

“…bacteria belonging to the order Rickettsiales (phylum Proteobacteria, class Alphaproteobacteria)} are Gram-negative obligate intracellular bacteria and zoonotic pathogens of tick-borne diseases (Mansueto et al 2012). Rickettsial endosymbionts found in the two green algal hosts are positioned within the bacterial family Rickettsiaceae and form a small clade with rickettsial endosymbionts hosted by freshwater hydra and freshwater and brackish ciliates (Kawafune et al 2012), which is called the "hydra group" (defined by Weinert et al 2009). However, the endosymbionts in V. carteri have not been identified using molecular methods.…”

“…Total DNA from strain UTEX 2180 was extracted as described by Kawafune et al (2012). To sequence bacterial 16S rRNA genes, PCR was performed using total DNA with the universal prirners 9F and 1492R (Weisburg et al 1991) as described previously (Nozaki et cd.…”