Rediscovering the genus Lyticum, multiflagellated symbionts of the order Rickettsiales

Abstract

Among the bacterial symbionts harbored by the model organism Paramecium, many still lack a recent investigation that includes a molecular characterization. The genus Lyticum consists of two species of large-sized bacteria displaying numerous flagella, despite their inability to move inside their hosts' cytoplasm. We present a multidisciplinary redescription of both species, using the deposited type strains as well as newly collected material. On the basis of 16S rRNA gene sequences, we assigned Lyticum to the order Rickettsiales, that is intensely studied because of its pathogenic representatives and its position as the extant group most closely related to the mitochondrial ancestor. We provide conclusive proofs that at least some Rickettsiales possess actual flagella, a feature that has been recently predicted from genomic data but never confirmed. We give support to the hypothesis that the mitochondrial ancestor could have been flagellated, and provide the basis for further studies on these ciliate endosymbionts.

Figure 1. Morphology and ultrastructure of Lyticum species.

Negative staining (a) and ultrathin sections (b, c) of L. flagellatum harbored by P. octaurelia strain 299. Negative staining (d) and ultrathin sections (e, f) of L. sinuosum harbored by P. biaurelia strain USBL-36I1. Bars stand for 1 μm. Arrowheads highlight some of the flagella, arrows point at symbiosomal membranes. M, mitochondria.

Figure 2. Genus- and species-specific in situ detection of Lyticum flagellatum and Lyticum sinuosum.

Merge of the signals from probes EUB338 marked with fluorescein (green) and LytiProb_433 marked with Cy3 (red) on P. octaurelia strain 299 (a) and P. biaurelia strain USBL-36I1 (b). The signals coincide, and Lyticum bacteria appear yellowish. Merge of the signals from probes Lflag_268 marked with Cy3 (red) and Lsinu_268 marked with fluorescein (green) on P. octaurelia strain 299 (c) and P. biaurelia strain USBL-36I1 (d). At 20% formamide concentration, the probes used in competition are able to discriminate between the species. Bars stand for 20 μm.

Figure 3. Phylogenetic position of Lyticum species.

Bayesian tree built on the unmodified character matrix (60 sequences, 1331 characters) employing the GTR + I + G model (with the continuous gamma distribution approximated by 4 discrete categories). Numbers associated to each node correspond to ML bootstrap values and posterior probability values (values below 70|0.85 are omitted); numbers inside trapezoids show the number of sequences used to represent that clade. The bar stands for an estimated sequence divergence of 10%. Ca., Candidatus; unc. bac., uncultured bacterium.