Petroni, Giulio

ABSTRACT “ Candidatus Midichloria mitochondrii” is an intramitochondrial bacterium of the order Rickettsiales associated with the sheep tick Ixodes ricinus . Bacteria phylogenetically related to “ Ca . Midichloria mitochondrii” (midichloria and like organisms [MALOs]) have been shown to be associated with a wide range of hosts, from amoebae to a variety of animals, including humans. Despite numerous studies focused on specific members of the MALO group, no comprehensive phylogenetic and statistical analyses have so far been performed on the group as a whole. Here, we present a multidisciplinary investigation based on 16S rRNA gene sequences using both phylogenetic and statistical methods, thereby analyzing MALOs in the overall framework of the Rickettsiales . This study revealed that (i) MALOs form a monophyletic group; (ii) the MALO group is structured into distinct subgroups, verifying current genera as significant evolutionary units and identifying several subclades that could represent novel genera; (iii) the MALO group ranks at the level of described Rickettsiales families, leading to the proposal of the novel family “ Candidatus Midichloriaceae.” In addition, based on the phylogenetic trees generated, we present an evolutionary scenario to interpret the distribution and life history transitions of these microorganisms associated with highly divergent eukaryotic hosts: we suggest that aquatic/environmental protista have acted as evolutionary reservoirs for members of this novel family, from which one or more lineages with the capacity of infecting metazoa have evolved.

ABSTRACTThe orderRickettsiales(Alphaproteobacteria) is a well-known group containing obligate endocellular prokaryotes. The order encompasses three families (Rickettsiaceae,Anaplasmataceae, andHolosporaceae) and a fourth, family-level cluster, which includes only one candidate species, “CandidatusMidichloria mitochondrii,” as well as several unnamed bacterial symbionts. The broad host range exhibited by the members of the “CandidatusMidichloria” clade suggests their eventual relevance for a better understanding of the evolution of symbiosis and host specificity ofRickettsiales. In this paper, two new bacteria belonging to the “CandidatusMidichloria” clade, hosted by two different strains of the ciliate protistEuplotes harpa, are described on the basis of ultrastructural observations, comparative 16S rRNA gene sequence analysis, and an estimation of the percentage of infection. Ultrastructure of these bacteria shows some unusual features: one has an electron-dense cytoplasm, and the other one lacks a symbiosomal membrane. The latter was up to now considered an exclusive feature of bacteria belonging to the familyRickettsiaceae. 16S rRNA gene phylogenetic analysis unambiguously places the new bacteria in the “CandidatusMidichloria” clade, although their phylogenetic relationships with other members of the clade are not clearly resolved. This is the first report of a ciliate-borne bacterium belonging to the “CandidatusMidichloria” clade. On the basis of the data obtained, the two bacteria are proposed as two new candidate genera and species, “CandidatusAnadelfobacter veles” and “CandidatusCyrtobacter comes.”

ABSTRACT. Rickettsia‐like organisms (RLO) are obligate, often highly fastidious, intracellular bacterial parasites associated with a variety of vertebrate and invertebrate hosts. Despite their importance as causative agents of severe mortality outbreaks in farmed aquatic species, little is known about their life cycle and their host range. The present work reports the characterization of “Candidatus Cryptoprodotis polytropus,” a novel Rickettsia‐like bacterium associated with the common ciliate species Pseudomicrothorax dubius by means of the “Full‐Cycle rRNA Approach” and ultrastructural observations. The morphological description by in vivo and scanning electron microscopy and the 18S rRNA gene sequence of the host species is provided as well. Phylogenetic analysis based on the 16S rRNA gene supports the inclusion of “Candidatus Cryptoprodotis polytropus” within the family Rickettsiaceae (cl. Alphaproteobacteria) together with the genera Rickettsia and Orientia. Observations on natural ciliate populations account for the occasional nature of this likely parasitic association. The presence of a previously unknown RLO in ciliates sheds a new light on the possible role of protists as transient hosts, vectors or natural reservoir for some economically important pathogens.