Members of the major candidate phylum Dependentiae (a.k.a. TM6) are widespread across diverse environments from showerheads to peat bogs; yet, with the exception of two isolates infecting amoebae, they are only known from metagenomic data. The limited knowledge of their biology indicates that they have a long evolutionary history of parasitism. Here, we present Chromulinavorax destructans (Strain SeV1) the first isolate of this phylum to infect a representative from a widespread and ecologically significant group of heterotrophic flagellates, the microzooplankter Spumella elongata (Strain CCAP 955/1). Chromulinavorax destructans has a reduced 1.2 Mb genome that is so specialized for infection that it shows no evidence of complete metabolic pathways, but encodes an extensive transporter system for importing nutrients and energy in the form of ATP from the host. Its replication causes extensive reorganization and expansion of the mitochondrion, effectively surrounding the pathogen, consistent with its dependency on the host for energy. Nearly half (44%) of the inferred proteins contain signal sequences for secretion, including many without recognizable similarity to proteins of known function, as well as 98 copies of proteins with an ankyrin-repeat domain; ankyrin-repeats are known effectors of host modulation, suggesting the presence of an extensive host-manipulation apparatus. These observations help to cement members of this phylum as widespread and diverse parasites infecting a broad range of eukaryotic microbes.

Bdellovibrio and like organisms are abundant environmental parasitoids of prokaryotes that show diverse predation strategies. The vast majority of studied Bdellovibrio bacteria and like organisms deploy intraperiplasmic replication inside the prey cell, while few isolates with smaller genomes consume their prey from the outside in an epibiotic manner. The novel parasitoid “Candidatus Bdellovibrio qaytius” was isolated from a eutrophic freshwater pond in British Columbia, where it was a continual part of the microbial community. “Ca. Bdellovibrio qaytius” was found to preferentially prey on the betaproteobacterium Paraburkholderia fungorum without entering the periplasm. Despite its epibiotic replication strategy, “Ca. Bdellovibrio” encodes a large genomic complement more similar to that of complex periplasmic predators. Functional genomic annotation further revealed several biosynthesis pathways not previously found in epibiotic predators, indicating that “Ca. Bdellovibrio” represents an intermediate phenotype and at the same time narrowing down the genomic complement specific to epibiotic predators. In phylogenetic analysis, “Ca. Bdellovibrio qaytius” occupies a widely distributed, but poorly characterized, basal cluster within the genus Bdellovibrio. This suggests that epibiotic predation might be a common predation type in nature and that epibiotic predation could be the ancestral predation type in the genus. IMPORTANCE Bdellovibrio and like organisms are bacteria that prey on other bacteria and are widespread in the environment. Most of the known Bdellovibrio species enter the space between the inner and outer prey membrane, where they consume their prey cells. However, one Bdellovibrio species has been described that consumes its prey from the outside. Here, we describe “Ca. Bdellovibrio qaytius,” a novel member of the genus Bdellovibrio that also remains outside the prey cell throughout its replication cycle. Unexpectedly, the genome of “Ca. Bdellovibrio” is much more similar to the genomes of intracellular predators than to the species with a similar life cycle. Since “Ca. Bdellovibrio” is also a basal representative of this genus, we hypothesize that extracellular predation could be the ancestral predation strategy.

10Bdellovibrio and like organisms are abundant environmental predators of prokaryotes that 11show a diversity of predation strategies, ranging from intra-periplasmic to epibiotic predation. 12The novel epibiotic predator Bdellovibrio qaytius was isolated from a eutrophic freshwater pond 13 in British Columbia, where it was a continual part of the microbial community. Bdellovibrio 14 qaytius was found to preferentially prey on the beta-proteobacterium Paraburkholderia 15 fungorum. Despite its epibiotic replication strategy, B. qaytius encodes a complex genomic 16 complement more similar to periplasmic predators as well as several biosynthesis pathways not 17 previously found in epibiotic predators. Bdellovibrio qaytius is representative of a widely 18 distributed basal cluster within the genus Bdellovibrio, suggesting that epibiotic predation might 19 be a common predation type in nature and ancestral to the genus. 20 21 Bdellovibrio spp. are delta-proteobacteria predators that use a biphasic lifestyle comprising 32 an attack phase, in which a small, highly motile flagellated cell seeks out prey, and a growth 33 phase, characterized by the predator penetrating the outer membrane of the prey cell and 34 consuming its cytoplasm (5). During the growth phase, the predator forms a characteristic 35 structure in the prey's periplasm known as the bdelloplast, which consists of a rounded, 36 osmotically stabile outer membrane of the prey cell and several replicating Bdellovibrio cells. 37The bdelloplast continues to grow until the resources of the prey cell are exhausted and 38 culminates in the septation and release of several to dozens of new attack-phase cells. This 39 dichotic lifestyle switch is mediated by a highly expressed riboswitch in B. bacterivorous (6). 40The related genera Bacteriovorax and Predibacter are in the family Bacteriovoracea, which is a 41 sister family to the prototypical Bdellovibrionacea within the order Bdellovibrionales (7). 42Curiously, the alpha-proteobacteria genus Micavibrio, which is unrelated to the 43Bdellovibrionales leads a remarkably similar lifestyle to Bdellovibrio species, with high prey 44 specificity. However, these bacteria prey in an epibiotic fashion on the outside of the prey cell 45 instead of penetrating into the periplasm (8). Due to their similar lifestyles, Micavibrio spp. are 46 included into the BALOs. 47