bioRxiv preprint doi: https://doi.org/10.1101/506055; this version posted December 26, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 From the inside out: An epibiotic Bdellovibrio predator with an 2 expanded genomic complement 3 4 Christoph M. Deeg1, Tan T. Le1, Matthias M. Zimmer2$, & Curtis A. Suttle*1,2,3,4 5 Author affiliation: University of British Columbia, Department of 1: Microbiology and Immunology, 2: Earth, Oceans and Atmospheric 6 Sciences, 3: Botany; 4: Institute for the Oceans and Fisheries 7 * :Corresponding author: [email protected]; 8 $: M.Z. currently at Helmholtz-Institute for RNA-based Infection Research, Würzburg, Germany 9 10 Abstract 11 Bdellovibrio and like organisms are abundant environmental predators of prokaryotes that 12 show a diversity of predation strategies, ranging from intra-periplasmic to epibiotic predation. 13 The novel epibiotic predator Bdellovibrio qaytius was isolated from a eutrophic freshwater pond 14 in British Columbia, where it was a continual part of the microbial community. Bdellovibrio 15 qaytius was found to preferentially prey on the beta-proteobacterium Paraburkholderia 16 fungorum. Despite its epibiotic replication strategy, B. qaytius encodes a complex genomic 17 complement more similar to periplasmic predators as well as several biosynthesis pathways not 18 previously found in epibiotic predators. Bdellovibrio qaytius is representative of a widely 19 distributed basal cluster within the genus Bdellovibrio, suggesting that epibiotic predation might 20 be a common predation type in nature and ancestral to the genus. 21 22 Introduction 23 Biotic factors regulating bacterial populations in nature are often assumed to be viral lysis 24 and zooplankton grazing (1); however, an underappreciated cause of mortality is predation by 25 other prokaryotes. Such predators, collectively referred to as Bdellovibrio and like organisms 26 (BALOs), have evolved several times independently and deploy a variety of “hunting strategies”. 27 Many facultative predators with broad host ranges, such as Ensifer adhaerens and Myxococcus 28 xanthus, deploy a “wolfpack strategy” where a prey cell is surrounded by several predators and 29 lysed (2, 3). Other, more specialized obligate predators have a narrower host range and specific 30 predation strategies; for example, Bdellovibrio spp. enters the periplasm of the prey cell to 31 consume the prey’s cytoplasm (4, 5). 1 bioRxiv preprint doi: https://doi.org/10.1101/506055; this version posted December 26, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 32 Bdellovibrio spp. are delta-proteobacteria predators that use a biphasic lifestyle comprising 33 an attack phase, in which a small, highly motile flagellated cell seeks out prey, and a growth 34 phase, characterized by the predator penetrating the outer membrane of the prey cell and 35 consuming its cytoplasm (5). During the growth phase, the predator forms a characteristic 36 structure in the prey’s periplasm known as the bdelloplast, which consists of a rounded, 37 osmotically stabile outer membrane of the prey cell and several replicating Bdellovibrio cells. 38 The bdelloplast continues to grow until the resources of the prey cell are exhausted and 39 culminates in the septation and release of several to dozens of new attack-phase cells. This 40 dichotic lifestyle switch is mediated by a highly expressed riboswitch in B. bacterivorous (6). 41 The related genera Bacteriovorax and Predibacter are in the family Bacteriovoracea, which is a 42 sister family to the prototypical Bdellovibrionacea within the order Bdellovibrionales (7). 43 Curiously, the alpha-proteobacteria genus Micavibrio, which is unrelated to the 44 Bdellovibrionales leads a remarkably similar lifestyle to Bdellovibrio species, with high prey 45 specificity. However, these bacteria prey in an epibiotic fashion on the outside of the prey cell 46 instead of penetrating into the periplasm (8). Due to their similar lifestyles, Micavibrio spp. are 47 included into the BALOs. 48 Recently, an isolate of a newly described species, Bdellovibrio exovorus, in the family 49 Bdellovibrionacea that is closely related to periplasmic bdelloplast-forming Bdellovibrio species, 50 was shown to have an extremely narrow host range, and employ a different epibiotic replication 51 strategy (9). In the attack-phase, cells of Bdellovibrio exovorus resemble those of other 52 Bdellovibrio isolates; whereas, in growth-phase the cells do not penetrate into the cytoplasm, but 53 stay attached to the outside of the prey, strongly resembling Micavibrio species. Further, in 54 growth-phase, B. exovorus does not induce a bdelloplast and seems to extract the cytoplasmic 55 contents of the prey across both membranes. Once the resources of the prey are exhausted, 56 growth-phase result in binary fission releasing two progeny attack-phase cells. The 57 comparatively small genome of B. exovorus has been linked to its epibiotic predation strategy 58 and reductionist evolution from an ancestor capable of intra-periplasmic replication (9-11). 59 Bdellovibrio qaytius is the second epibiotic predator within the genus Bdellovibrio. Its genomic 60 complement, phylogenetic placement and environmental distribution broaden our understanding 61 of the ecology and evolution of this genus. 62 2 bioRxiv preprint doi: https://doi.org/10.1101/506055; this version posted December 26, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 63 Materials and Methods 64 Isolation and Culturing 65 An isolate of a lytic bacterium, here named Bdellovibrio qaytius sp. nov., was obtained 66 from a water sample collected near the sediment surface of a eutrophic pond in Nitobe Memorial 67 Garden at the University of British Columbia, Canada (49°15'58"N, 123°15'34"W).As part of a 68 bioassay for pathogens infecting heterotrophic protists a subsample of the water was inoculated 69 into modified DY-V artificial freshwater medium with yeast extract and a wheat grain (12). 70 Genome sequencing 71 For PacBio sequencing, exponentially growing mixed cultures containing B. qaytius as 72 well as Bodo saltans NG1 and its virus BsV were centrifuged in a Sorvall SLC-6000 for 20 min 73 and 5000 rpm at 4°C to remove eukaryotic cells (13). Particles in the supernatant concentrated 74 approximately 100-fold by tangential flow ultrafiltration with at 30kDa cut-off (Vivaflow 200, 75 PES) cartridge. To concentrate the cells further they were centrifuged at 28,000 rpm, 15°C for 8 76 h in a Beckman ultracentrifuge using a Ti90 fixed-angle rotor (Beckman-Coulter, Brea, 77 California, USA), and then sedimented onto a 40% Optiprep 50 mM Tris-Cl, pH 8.0, 2mM 78 MgCl2 cushion for 30 min at 28,000 rpm, and 15°C in a SW40Ti swing-out rotor. The Optiprep 79 gradient was created by underlaying a 10% Optiprep solution in 50 mM Tris-Cl, pH 8.0, 2 mM 80 MgCl2 with a 30% solution followed by a 50% solution and equilibration overnight at 4°C. One 81 ml of concentrate from the 40% cushion was added atop the gradient and the concentrate was 82 fractionated by centrifugation in an SW40 rotor for 4 h at 25000 rpm and 18°C. The fraction 83 corresponding to the pathogen was extracted from the gradient with a syringe and washed twice 84 with 50 mM Tris-Cl, pH 8.0, 2 mM MgCl2 followed by centrifugation in an SW40 rotor for 20 85 min at 7200 rpm and 18°C and were finally collected by centrifugation in an SW40 rotor for 30 86 min at 7800 rpm and 18°C. Purity of the concentrate was verified by fluorescence vs SSC of 87 SYBR-Green stained samples (Invitrogen Carlsbad, California, USA) on a FACScalibur flow 88 cytometer (Becton-Dickinson, Franklin Lakes, New Jersey, USA). High molecular weight 89 genomic DNA was extracted using phenol-chloroform-chloroform extraction. Length and purity 90 were confirmed by gel electrophoresis and by using a Bioanalyzer 2100 with the HS DNA kit 91 (Agilent Technology). PacBio RSII 20kb sequencing was performed by the sequencing center of 92 the University of Delaware. Reads were assembled using PacBio HGAP3 software with 20 kb 3 bioRxiv preprint doi: https://doi.org/10.1101/506055; this version posted December 26, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 93 seed reads resulting in a single contig of 3,376,027 bp, 97.08 x coverage, 99.92% called bases 94 and a consensus concordance of 99.9954 % (14). 95 Propagation and host range studies 96 Plaque assays were performed by mixing 0.5 ml putative host cultures in logarithmic 97 growth stage and 10μl of Bdellovibrio qaytius stock culture with 4.5 ml molten 0.5% DY-V agar 98 and incubation for 48h. Propagation of Bdellovibrio qaytius in liquid culture was monitored by 99 PCR with custom primers set specific to B. qaytius 16S rDNA (Forward-5’- 100 AGTCGAACGGGTAGCAATAC-3’, Reverse-5’-CTGACTTAGAAGCCCACCTAC-3’) as 101 well as a BALO-specific primer set by Davidov et al. (7). To obtain a pure isolate of prey cells 102 present in the mixed microbial assemblage, culture samples were streaked onto a DY-V agar 103 plate and incubated at room temperature.