The "Other" Rickettsiales: an Overview of the Family " Candidatus Midichloriaceae"

Abstract

The family "Candidatus Midichloriaceae" constitutes the most diverse but least studied lineage within the important order of intracellular bacteria Rickettsiales. "Candidatus Midichloriaceae" endosymbionts are found in many hosts, including terrestrial arthropods, aquatic invertebrates, and protists. Representatives of the family are not documented to be pathogenic, but some are associated with diseased fish or corals. Different genera display a range of unusual features, such as full sets of flagellar genes without visible flagella or the ability to invade host mitochondria. Since studies on "Ca. Midichloriaceae" tend to focus on the host, the family is rarely addressed as a unit, and we therefore lack a coherent picture of its diversity. Here, we provide four new midichloriaceae genomes, and we survey molecular and ecological data from the entire family. Features like genome size, ecological context, and host transitions vary considerably even among closely related midichloriaceae, suggesting a high frequency of such shifts, incomplete sampling, or both. Important functional traits involved in energy metabolism, flagella, and secretion systems were independently reduced multiple times with no obvious correspondence to host or habitat, corroborating the idea that many features of these "professional symbionts" are largely independent of host identity. Finally, despite "Ca. Midichloriaceae" being predominantly studied in ticks, our analyses show that the clade is mainly aquatic, with a few terrestrial offshoots. This highlights the importance of considering aquatic hosts, and protists in particular, when reconstructing the evolution of these endosymbionts and by extension all Rickettsiales. IMPORTANCE Among endosymbiotic bacterial lineages, few are as intensely studied as Rickettsiales, which include the causative agents of spotted fever, typhus, and anaplasmosis. However, an important subgroup called "Candidatus Midichloriaceae" receives little attention despite accounting for a third of the diversity of Rickettsiales and harboring a wide range of bacteria with unique features, like the ability to infect mitochondria. Midichloriaceae are found in many hosts, from ticks to corals to unicellular protozoa, and studies on them tend to focus on the host groups. Here, for the first time since the establishment of this clade, we address the genomics, evolution, and ecology of "Ca. Midichloriaceae" as a whole, highlighting trends and patterns, the remaining gaps in our knowledge, and its importance for the understanding of symbiotic processes in intracellular bacteria.

Keywords: Euplotes; Rickettsiales; endosymbionts; “Candidatus Anadelfobacter”; “Candidatus Bandiella”; “Candidatus Cyrtobacter”; “Candidatus Grellia”; “Candidatus Midichloriaceae”.

FIG 1

Hosts and genomic traits of 11 strains of “Ca. Midichloriaceae.” Bacteria are arranged according to their phylogenetic relationships (see Fig. 2). The genomes of “Ca. Cyrtobacter zanobii,” “Ca. Anadelfobacter sociabilis,” and “Ca. Grellia numerosa” (formerly “Ca. Bandiella numerosa”) are new in this study. CDS, coding DNA sequences.

FIG 2

Phylogenomics of “Ca. Midichloriaceae.” Maximum-likelihood phylogenomic trees based on 179 concatenated genes (47,692 characters), obtained under two substitution models. Twelve ingroup plus 10 outgroup (Anaplasmataceae and Rickettsiaceae; not shown) sequences were included; only bootstrap values below 100% are shown for each node, and nodes underlining the difference between the two topologies are circled in red. Bars correspond to an estimated sequence divergence of 0.2. The branch leading to “Ca. Fokinia solitaria” in the LG+C60+I+G4 tree was shortened because of space constraints.

FIG 3

16S rRNA gene phylogeny and ecological traits of “Ca. Midichloriaceae.” Maximum-likelihood single-gene inference obtained after constraining the four main clades recovered by the phylogenomic analysis (labeled on the left). Each sequence in the tree represents a cluster of ≥99% similar sequences, whose numbers are shown in the rightmost column (N.). Host and environment bar plots depict trait frequencies in each cluster. Named and otherwise relevant bacterial taxa are highlighted, and the main hosts are shown as silhouettes with lines colored corresponding to their taxonomy (metazoans, ciliates, amoebozoans, and euglenozoans). Note that for most marine invertebrate hosts, the presence of “Ca. Midichloriaceae” was detected only by high-throughput sequencing, without histological confirmation that the bacterium infects animal tissues (instead of, for example, protist symbionts in the animal). Asterisks mark clusters that include one or more strains with a sequenced genome (Fig. 1 and 2). Bootstrap values below 70% are not shown. The bar corresponds to an estimated sequence divergence of 0.1.

FIG 4

Selected functional traits mapped on the consensus phylogeny of “Ca. Midichloriaceae.” Dashed lines separate the four clades identified in Fig. 2. (A) Energy metabolism-related genes, complexes, and pathways, as annotated by the KEGG Automatic Annotation System (KAAS) and, for oxidative phosphorylation, Prokka. Pathways and complexes were considered complete (green circles) when all essential genes known to be exclusively involved in that pathway/complex were detected, possibly incomplete (yellow circles) when at least half but not all such genes were detected, and likely absent (no circle) otherwise. PPP, pentose phosphate pathway; PDC, pyruvate dehydrogenase complex. (B) Genes involved in T4SS, as annotated by KAAS, Prokka, and TXSScan. Teal circles mark genes detected by at least two analytical tools; gray circles mark genes detected by only one of the three (usually TXSScan, which is optimized for the detection of T4SS genes; see also Fig. S1). Names of genes considered mandatory (73) are in bold. (C) Flagellum and flagellar assembly-related genes, annotated and displayed as in panel B, but with different colors used for different parts of the flagellum, as shown in the schematic drawing above the table. IM, internal membrane; PG, peptidoglycan layer; OM, outer membrane. More details on the functional analysis are shown in Table S2.