1

From the inside out: An epibiotic Bdellovibrio predator with an 1

expanded genomic complement 2

3

Christoph M. Deeg1, Tan T. Le

1, Matthias M. Zimmer

2$, & Curtis A. Suttle

#1,2,3,4 4

Author affiliation: University of British Columbia, Department of 1: Microbiology and Immunology, 2: Earth, Ocean and Atmospheric 5

Sciences, 3: Botany; 4: Institute for the Oceans and Fisheries 6

# :Corresponding author: suttle@science.ubc.ca; 7

8

Abstract 9

Bdellovibrio and like organisms are abundant environmental parasitoids of prokaryotes 10

that show diverse predation strategies. The vast majority of studied Bdellovibrio and like 11

organisms deploy intra-periplasmic replication inside the prey cell, while few isolates with 12

smaller genomes consume their prey from the outside in an epibiotic manner. The novel 13

parasitoid Bdellovibrio qaytius was isolated from a eutrophic freshwater pond in British 14

Columbia, where it was a continual part of the microbial community. Bdellovibrio qaytius was 15

found to preferentially prey on the beta-proteobacterium Paraburkholderia fungorum without 16

entering the periplasm. Despite its epibiotic replication strategy, B. qaytius encodes a large 17

genomic complement more similar to that of complex periplasmic predators. Functional genomic 18

annotation further revealed several biosynthesis pathways not previously found in epibiotic 19

predators, indicating that B. qaytius represents an intermediate phenotype, at the same time 20

narrowing down the genomic complement specific to epibiotic predators. In phylogenetic 21

analysis, Bdellovibrio qaytius occupies a widely distributed, but poorly characterized basal 22

cluster within the genus Bdellovibrio. This suggests that epibiotic predation might be a common 23

JB Accepted Manuscript Posted Online 3 February 2020J. Bacteriol. doi:10.1128/JB.00565-19Copyright © 2020 American Society for Microbiology. All Rights Reserved.

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predation type in nature and that epibiotic predation could be the ancestral predation type in the 24

genus. 25

26

Importance 27

Bdellovibrio and like organisms are bacteria that prey on other bacteria and are widespread 28

in the environment. Most of the known Bdellovibrio enter the space between the inner and outer 29

prey membrane, where they consume their prey cells. However, one Bdellovibrio species has 30

been described that consume its prey from the outside. Here, we describe Bdellovibrio qaytius, a 31

novel member of the genus Bdellovibrio that also remains outside the prey cell throughout its 32

replication cycle. Unexpectedly, the genome of B. qaytius is much more similar to intracellular 33

predators than to the species with a similar lifecycle. Since B. qaytius is also a basal 34

representative of this genus, we hypothesize that extracellular predation could be the ancestral 35

predation strategy. 36

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Introduction 38

Biotic factors regulating bacterial populations in nature are often assumed to be viral lysis 39

and zooplankton grazing (1); however, an underappreciated cause of mortality is predation by 40

other prokaryotes. Such predators, collectively referred to as Bdellovibrio and like organisms 41

(BALOs), have evolved several times independently and deploy a variety of “hunting strategies”. 42

Many facultative predators with broad host ranges, such as Ensifer adhaerens and Myxococcus 43

xanthus, deploy a “wolfpack strategy” where a prey cell is surrounded by several predators and 44

lysed (2, 3). Other, more specialized obligate predators have a narrower host range and specific 45

predation strategies; for example, Bdellovibrio bacteriovorus strains invade the periplasm of the 46

prey cell from where they extract resources from the cytoplasm of the prey (4, 5). 47

Bdellovibrio spp. are delta-proteobacteria predators that use a biphasic lifestyle comprising 48

an attack phase, in which a small, highly motile flagellated cell seeks out prey, and a growth 49

phase, characterized by the predator penetrating the outer membrane of the prey cell and 50

consuming the resources in the cytoplasm of the prey cell (5). During the growth phase, the 51

predator forms a characteristic structure in the prey’s periplasm. The prey cell is then referred to 52

as a bdelloplast, which consists of a rounded, osmotically stable outer membrane, several 53

replicating Bdellovibrio cells in the periplasm, surrounding the resources of the cytoplasm. The 54

Bdellovibrio cells continue to grow inside the bdelloplast until the resources of the prey cell are 55

exhausted and culminates in the septation and release of several to dozens of new attack-phase 56

cells. This dichotic lifestyle switch is mediated by a highly expressed riboswitch in B. 57

bacterivorous (6). The related genera Bacteriovorax and Predibacter are in the family 58

Bacteriovoracea, which is a sister family to the prototypical Bdellovibrionacea within the order 59

Bdellovibrionales (7). 60

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Curiously, the alpha-proteobacteria genus Micavibrio, which is unrelated to the 61

Bdellovibrionales leads a remarkably similar lifestyle to Bdellovibrio species, with high prey 62

specificity. However, these bacteria prey in an epibiotic fashion on the outside of the prey cell 63

instead of penetrating into the periplasm (8). Due to their similar lifestyles, Micavibrio spp. are 64

included in the BALOs. 65

Recently, an isolate of a newly described species, Bdellovibrio exovorus, in the family 66

Bdellovibrionacea that is closely related to periplasmic bdelloplast-forming Bdellovibrio species, 67

was shown to have an extremely narrow host range, and employ a different epibiotic replication 68

strategy (9). In the attack-phase, cells of Bdellovibrio exovorus resemble those of other 69

Bdellovibrio bacteriovorus isolates; whereas, in growth-phase the cells do not penetrate into the 70

cytoplasm, but stay attached to the outside of the prey, strongly resembling Micavibrio species. 71

Further, in growth-phase, B. exovorus does not induce a bdelloplast and seems to extract the 72

cytoplasmic contents of the prey across both membranes. Once the resources of the prey are 73

exhausted, growth-phase result in binary fission releasing two progeny attack-phase cells. The 74

comparatively small genome of B. exovorus has been linked to its epibiotic predation strategy 75

and reductionist evolution from an ancestor capable of intra-periplasmic replication (9-11). 76

Bdellovibrio qaytius is the second epibiotic predator within the genus Bdellovibrio. Its genomic 77

complement, phylogenetic placement and environmental distribution broaden our understanding 78

of the ecology, and evolution of this genus. 79

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Results 82

Isolation, host range and distribution 83

A lytic pathogen of bacteria was collected in a mixed microbial assemblage from a 84

temperate eutrophic pond in southwestern British Columbia, Canada. Based on full-genome 85

sequencing and electron microscopy of the infection cycle the pathogen was determined to be a 86

new species of bacterium, here named Candidatus Bdellovibrio qaytius (subsequently referred to 87

as B. qaytius), after “q̓a:yt” (“kill it”) in hən̓q̓əmin̓əm̓ the language of the Musqueam tribe of 88

Coast Salish, the indigenous peoples from whose territory it was isolated. B. qaytius propagated 89

in a mixed microbial assemblage from the sample site (Supplementary Figure 1). Additionally, 90

B. qaytius could also be propagated on a specific isolate from this assemblage that was identified 91

as the beta-proteobacterium Paraburkholderia fungorum where high numbers of putative attack-92

phase B. qaytius cells could be observed under phase-contrast microscopy (Supplementary 93

Figure 1). Inoculation of B. qaytius in cultures of Pseudomonas fluorescens, another isolate from 94

the native mixed assembly, or E. coli resulted in the observation of weak PCR amplification after 95

propagation, presumable due to carry-over (Supplementary Figure 1). Similarly, small clear 96

plaques were only observed on plates of P. fungorum, but not on plates of Pseudomonas 97

fluorescens, or E. coli (Supplementary Figure 2). Bdellovibrio qaytius could not be propagated in 98

an axenic culture and is thus presumed to be an obligate predator. Bdellovibrio qaytius was 99

detected at several time points in DNA extracted from water concentrates from Nitobe Gardens 100

UBC in 2017, four years after the initial isolation, indicating the population persists in the pond 101

(Supplementary Figure 3). 102

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Morphology and replication cycle 103

Bdellovibrio qaytius attack-phase cells are free-swimming highly motile flagellated, 104

slightly bend rods that are about 1 μm by 0.4 μm in size and exhibit a sheathed flagellum (Figure 105

1A). Once attack-phase cells contact a prey cell, they attach irreversibly, preferentially at the 106

prey cell pole, and form a broad predatory synapse and discard the flagellum (Figure 1B, 107

Supplementary Figure 4). No invasion of the prey cell was observed during growth-phase, nor 108

were bdelloplasts, implying that Bdellovibrio qaytius is an epibiotic predator (Figure 1). The 109

growth-phase cell attached to the prey cell empties the cytoplasm of the host cell, leaving behind 110

an empty ghost cell (Figure 1C,E,G). Simultaneously, the growth-phase Bdellovibrio cell grows 111

in size and once the resources of the prey cell are exhausted, the growth-phase cumulates in 112

fission and the production of offspring attack-phase cells that repeat actively searching for new 113

prey cells by rapid locomotion. Throughout the growth-phase, the cell membranes of the prey ,as 114

well as the predator, remain intact and instead of periplasmic invasion, an electron-dense layer is 115

observed on both the prey’s and predator’s membranes suggesting that a high concentration of 116

effector molecules like transmembrane transporters are likely recruited to these sited to facilitate 117

predation (Figure 1F). 118

Bdellovibrio qaytiusBdellovibrio qaytiusBdellovibrio qaytius has a complex genome 119

for an epibiotic predator 120

Genome structure and content 121

The 3,348,710-bp B. qaytius genome is similar in size to periplasmic Bdellovibrio spp., 122

but considerably larger than that of Bdellovibrio exovorus, another epibiotic predator with a 2.66 123

Mb genome and with 38.9% B, qaytius also has the lowest GC content of any species within the 124

genus (Table 1). The GC content is relatively constant and exhibits a dichotomy in GC-skew that 125

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is typical of a circular bacterial genome (Figure 2A). Furthermore, the B. qaytius genome 126

contains one complete rDNA operon, similar to other epibiotic predators, and 31 tRNAS, three 127

non-coding RNAs (ssrS, rnpB and ffs), and three putative riboswitches (Figure 2B, Table 1). A 128

total of 3166 protein-coding genes were identified and are distributed equally between the plus 129

and minus strands (Figure 2A). These proteins represent 22 different functional clusters of 130

orthologous genes (Figure 2 B). 131

Bdellovibrio qaytiusMetabolism 132

The B. qaytius genome encodes a metabolism typical for a predatory bacterium. 133

Glycolysis and the complete TCA cycle, as well as a core set of pentose phosphate pathway 134

genes are present, suggesting B. qaytius is capable of several sugar conversions and is able to 135

provide the precursors for riboflavin biosynthesis (Table 1). Pyruvate metabolism is coded for, 136

but propanoate metabolism is only partially possible. A vitamin B6 biosynthesis pathway is 137

encoded and acetyl-CoA biosynthesis is possible via pantoate. Nicotinamide metabolism and 138

biosynthesis pathways also exist. Oxidative phosphorylation is encoded with the exception of 139

cytochrome C reductase. The presence of core mevalonate pathway enzymes suggests this 140

pathway is functional. 141

Based on inferred CDS, B. qaytius can synthesize pyrimidines and purines de novo, including 142

inosine. A complete DNA polymerase complex facilitates DNA replication. As well, all types of 143

DNA repair pathways are present, including base-excision, nucleotide excision, mismatch repair 144

and homologous recombination, the latter being limited to single-stand-break repair. 145

Bdellovibrio qaytius encodes a complete ribosome except for the non-essential protein 146

L28, and there is a complete set of tRNAs loaded by aminoacyl-tRNA-synthetases for every 147

amino acid. The genome encodes a core RNA degradosome for post-transcriptional regulation 148

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and nucleotide recycling. Amino-acid biosynthesis pathways are limited and only cysteine, 149

methionine, glutamate, lysine, proline, and threonine can be completely synthesized de novo. 150

Glycine and serine can be synthesized via the one-carbon pool pathway using tetrahydrofolate. 151

Aspartate, alanine leucine, isoleucine, valine phenylalanine, and tyrosine can be converted from 152

their direct precursors, which are presumably acquired from the prey. 153

Complete fatty-acid degradation pathways are coded for, but fatty-acid elongation seems limited. 154

Also encoded are complete sec and gsp pathways for protein secretion, as well as a partial tat 155

pathway (subunits tatA, tatC, tatD). Partial lipopolysaccharide biosynthesis pathways and 156

peptidoglycan modification pathways putatively decorate the periplasmic space and cell surface. 157

Regulatory elements 158

Master regulators, such as sigma factor 28 / FliA, which is proposed to enable the switch 159

between attack and growth-phase modes in other Bdellovibrio species, is present in B. qaytius 160

and might work alongside putative riboswitch elements similar to those found in B. 161

bacteriovorus (6). No homolog of the host interaction (“hit”) locus protein bd0108, of B. 162

bacteriovorus, was found in B. qaytius. This protein has been shown to facilitate the switch 163

between host prey-independent and prey-dependent replication in B. bacteriovourus, which is 164

dependent on the presence of a pilus that is used to invade the prey (12). Accordingly, this 165

suggests that B. qaytius might not be able to switch between prey-independent and prey 166

dependent replication. 167

Predatory arsenal 168

The B. qaytius genome shows many adaptations to a predatory lifestyle. A complete 169

biosynthesis pathway for flagellar assembly and regulation provides locomotion in the attack-170

phase. Attack phase cells are likely guided by a canonical, almost, complete chemotaxis pathway 171

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that is only missing cheY. Substrate recognition is putatively mediated by TatC and two copies 172

of von Willebrand factors (13). A type IV pilus appears to be present that could be putatively 173

involved in prey-cell attachment through pilZ, as has been described in B. bacteriovorus (12). 174

However, in the absence of a Hit locus, the pilus attachment is putatively not involved in any 175

change of replication behaviours (12). Prey peptidoglycan modification pathways are deployed 176

by B. bacteriovorus to invade the prey cell, but are only partially present in the epibiotic predator 177

B. exovorus (9, 14, 15). As with previous observations, B. qaytius exhibits an intermediate 178

phenotype, containing more copies of peptidoglycan modifying genes compared to B. exovorus, 179

but lacks homologues to Bd1176 and Bd0886 that appear to be exclusive to periplasmic 180

predators (Table 1). To access resources within the prey, an array of transporters are coded for, 181

many of which show signal peptides that facilitate export, and might insert into the prey-cell 182

membrane. ABC-type transporters likely import phosphate (pst), phosphonate (phn), and 183

lipopolysaccharides (lpt), while there appear to be partial ABC transporter systems for 184

lipoproteins, thiamine, branched-chain amino acids, oligo and dipeptides, microcin, 185

phospholipids, biotin, daunorubiscine, alkylophaosphate, methionine, iron and siderophores, 186

cobalt, sugar and organic solvents. Non-ABC transporter system CDS are present for potassium 187

(kdp), biopolymers (exbD and tol), iron (ofeT), heavy metals (cusA), biotin (bioY), threonine 188

(rhtB), as well as for several multidrug exporters (bcr, cflA, arcB). CDS for low-specificity 189

transporters include MFS and EamE transporters, as well as transporters for ions and cations, 190

macrolide, chromate, as well as sodium-dependent transporters and others of uncharacterized 191

specificity. 192

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Bdellovibrio qaytius exhibits intermediate genomic traits at the intersection of 193

epibiotic and periplasmic Bdellovibrio species 194

The clear epibiotic predation stagey of Bdellovibrio qaytius is at odds with its much more 195

complex genomic complement when compared to other epibiotic predators, such as Bdellovibrio 196

exovorus (Table 1). This intermediate position becomes more apparent when contrasting the 197

abundance and coverage of functionally grouped clusters of orthologous genes (COGs) between 198

the epibiotic B. qaytius and B, exovorus with the periplasmic B. bacteriovorus (Figure 3). 199

Bdellovibrio qaytius shows intermediate coverage, lower than B. bacteriovorus but higher than 200

B. exovorus, of COGs associated with cell wall/membrane/envelope biogenesis, amino-acid 201

transport and metabolism, defense mechanisms, and lipid transport and metabolism. Coverage of 202

COGs associated with carbohydrate transport and metabolism, cell-cycle control, cell division, 203

chromosome partitioning, chromatin structure and dynamics, energy production and conversion, 204

inorganic ion transport and metabolism, posttranslational modification, protein turnover, 205

chaperones, RNA processing and modification, signal transduction mechanisms, and 206

transcription resemble the lower coverage observed in B. exovorus (Figure 3). Conversely, COGs 207

for coenzyme transport and metabolism, general function prediction only, nucleotide transport 208

and metabolism, and secondary metabolites biosynthesis, and transport and catabolism are 209

present in the B. qaytius genome at coverages similar to B. bacterivorous and higher than in the 210

B. exovorus genome (Figure 3). Notable is the absence of COGs associated with cytoskeleton in 211

B. exovorus (Figure 3). Differences in abundance of COG classes within each genome are less 212

pronounced, suggesting a more even scaling of COG functions with genome size, and in general 213

resemble the COG coverages (Figure 3). Noteworthy exceptions are present in COGs 214

representing nucleotide transport and metabolism where B. qaytius shows high abundances 215

similar to B. bacteriovorus, despite showing coverage more similar to B. exovorus, suggesting 216

the presence of several homologues within the B. qaytius genome (Figure 3). 217

218

Bdellovibrio qaytius is a basal representative of its genus 219

In phylogenetic analysis of the 16S rDNA locus, B. qaytius occupies a well-supported 220

basal branch within the genus Bdellovibrio; the closest relatives are found in environmental 221

amplicon sequences, as well as in poorly characterized isolates (Figure 4 A). Notably, these 222

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strains from a tight cluster basal to B. exovorus and B. bacteriovorus strains and show rather low 223

boot-strap support within their clade despite being closely related. This cluster appears to be 224

equivalent to the “cluster 2” described by Davidov et al. (7). 225

Bdellovibrio qaytiusBdellovibrio qaytiusShared gene cluster analysis was congruent with 226

the 16S phylogeny with the majority of gene clusters in B. qaytius being shared with other 227

members of the genus Bdellovibrio (Figure 4B). Surprisingly, B. qaytius shares more than 300 228

gene clusters with periplasmic predators, which are not found in the epibiotic predator B. 229

exovorus, despite their similar predation strategy. On the other hand, the epibiotic predators B. 230

qaytius and B. exovorus shared more than 130 genes that are not found in periplasmic predators 231

and might be involved in epibiotic predation. These exclusively epibiotic genes within members 232

of the genus Bdellovibrio include CDS for proteases, peroxiredoxin, glutathione-dependent 233

formaldehyde-activating enzyme, nucleases, hydrolases thioesterase, polysaccharide deacetylase, 234

an amino-acid ABC transporter, as well as many poorly characterized proteins. Gene clusters 235

exclusively shared with B. exovorus, as well as the phylogenetically distant M. aeruginosavorus 236

that also deploys an epibiotic predation strategy, are remarkable as they might highlight a 237

common set of genes are required for epibiotic predation. Six such genes that were identified 238

include anhydro-N-acetylmuramic acid kinase, peptidoglycan translocase, a FAD/NAD binding 239

protein, a cation-transporting P-type ATPase and a pseudouridine synthase. 240

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Discussion 242

Bdellovibrio qaytius is an epibiotic predator of the beta-proteobacterium 243

Paraburkholderia fungorum 244

Electron microscopy and FISH epifluorescence microscopy have shown B. qaytius to be 245

an epibiotic predator, making it the second such spices with a full genomic and phenotypical 246

description. While initially observed in a mixed culture, B. qaytius replication could ultimately 247

only be confirmed using the beta-proteobacterium Paraburkholderia fungorum as prey during a 248

limited host range study using plaque assay and targeted 16S PCR. 249

Despite an epibiotic phenotype, Bdellovibrio qaytius shares many genes with 250

periplasmic predators. 251

Microscopic analysis clearly shows B. qaytius deploying an epibiotic predation strategy. 252

However, abundance and coverage of clusters of orthologous genes grouped by function reveal 253

an intermediate composition of genomic functions that sit between the genomic complements of 254

the epibiotic B. exovorus and the periplasmic B. bacteriovorus (Figure 3). This trend is reflected 255

in B. qaytius’ gene content that shares several features previously identified to be involved in 256

epibiotic predators such as B. exovorus and Micavibrio aeruginosavorus. These include physical 257

features such as the number of rDNA loci, as well as metabolic capabilities based on gene 258

content that suggests limited fatty-acid elongation and the absence of polyhydroxyalkanoate 259

depolymerase and the siderophore aerobactin, all present in periplasmic predators (11). In 260

contrast, B. qaytius also has coding sequences for the biosynthesis of riboflavin and vitamin B6, 261

which had been found in periplasmic but not epibiotic predators reflecting its comparatively 262

large and complex genome (10). The linkage of these genes with periplasmic replication was by 263

association and not for functional reasons; hence, the presence of several of these genes in B. 264

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qaytius may simply reflect its relatively larger genome size. Cluster analysis of orthologous 265

genes in B. qaytius, other epibiotic and periplasmic Bdellovibrio spp., as well as the unrelated 266

BALOs Micavibrio aeruginosavorus (epibiotic) and Halobacteriovorax marinus (periplasmic), 267

reveals just six gene clusters associated with epibiotic predation. Genes involved in prey 268

peptidoglycan modification are thought to facilitate invasion of periplasmic replicating 269

Bdellovibrio species such as B. bacteriovorus. While more copies of these genes were found in 270

B. qaytius than in B. exovorus, the present genes are presumed to be functional equivalents of the 271

one found in B. exovorus based on sequence homology (Table 1). Key genes found in B. 272

bacteriovorus such as Bd1176 and Bd0886 were missing, with no pseudogenes detected, 273

suggesting that these genes are in fact essential to periplasmic invasion in Bdellovibrio 274

bacteriovorus. This also suggests that the prey peptidoglycan is salvaged by N-acetylmuramic-275

acid kinase as well as a peptidoglycan translocase in that is specific to epibiotic predators. Since 276

this limited complement of genes was found between distantly related taxa, there might not be a 277

clear functional separation between epibiotic and periplasmic predators. This is consistent with 278

epibiotic predation evolving independently within the genera Bdellovibrio and Micavibrio. 279

Therefore, conclusions regarding function based on gene-cluster analysis across larger 280

taxonomic separations should be interpreted with caution, especially since functionally 281

equivalent proteins can group into different gene clusters, and thus may escape detection in such 282

analysis. Accordingly, gene-cluster comparison among species in the same genus that deploy 283

different predation strategies is more informative and revealed a specialized complement of 284

proteases nucleases, hydrolases and detoxifying enzymes in epibiotic Bdellovibrio species 285

(Figure B). Notably, the addition of B. qaytius as a second epibiotic Bdellovibrio species greatly 286

decreased the number of genes associated with epibiotic predation, as its large genome has 287

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greater overlap with periplasmic Bdellovibio species. Further, this suggests that different 288

mechanisms can be deployed in both, epibiotic and periplasmic predation. 289

Epibiotic predation could be a common strategy of environmental Bdellovibrio 290

species and could be the ancestral phenotype in the genus 291

The closest known relatives to B. qaytius are poorly characterized isolates and 292

environmental 16S rRNA sequences from diverse environments. These environments include 293

“commercial aquaculture preparations”, soils, waste-water activated sludge, and iron-oxidizing 294

freshwater environments (7, 16-18). This broad diversity of habitats suggests that Bdellovibrio 295

species that are closely related to B. qaytius are widely distributed in freshwaters, and could be a 296

major contributor to global BALO diversity. Because of the phylogenetic placement and the 297

broad distribution of related isolates, epibiotic predation might be common among BALOs, 298

despite being underrepresented in isolates. The recurrent detection of populations of B. qaytius in 299

the pond from which it was isolated confirms that is part if the natural community and therefore 300

supports the idea that epibiotic predation might be widespread in nature. Additionally, both 301

characterized epibiotic Bdellovibrio species, B. qaytius and B. exovorus, occupy well supported 302

basal branches within the genus Bdellovibrio, compared to the periplasmic predators, B. 303

bacteriovorus strains HD 100 and Tiberius (Figure 4A). This suggests that periplasmic 304

replication could be a derived trait with epibiotic predation representing the ancestral predation 305

type in the genus. 306

Detailed knowledge of environmental BALO diversity is still missing. The discovery and 307

analysis of Bdellovibrio qaytius provide new insights into this fascinating group. Its intermediate 308

genomic complement blurs the lines between what is required for epibiotic and periplasmic 309

replication. Moreover, as a representative of a basal and widespread member within the genus 310

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Bdellovibrio, it suggests that epibiotic predation is common in the environment, and might be the 311

ancestral form of predation in this genus. 312

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Materials and Methods 314

Isolation and Culturing 315

An isolate of a lytic bacterium, here named Candidatus Bdellovibrio qaytius sp. nov., 316

was obtained from a water sample collected near the sediment surface of a eutrophic pond in 317

Nitobe Memorial Garden at the University of British Columbia, Canada (49°15'58"N, 318

123°15'34"W). As part of a bioassay for pathogens infecting heterotrophic protists a subsample 319

of the water was inoculated into modified DY-V artificial freshwater medium (2.03x10-7

M 320

MgSO4, 4.02x10-8

M KCl, 5.01x10-8

M NH4Cl, 2.35x10

-7 M NaNO3, 1.00x10

-8 M Na2-ß-glycero-321

phosphate, 1.29x10-8

M H3BO3, 4.93x10-8

M Na2SiO3, 5.10x10-7

M CaCl2, 3.70x10-9

M FeCl3, 322

1.53x10-8

M Na2EDTA, 3.84x10-10

M MnCl2, 1.39x10-10

M ZnSO4, 3.36x10-11

M CoCl2, 323

2.77x10-11

M Na2MoO4, 9.2x10-12

M Na3VO4, 3.10x10-11

M H2SeO3, 9.56x10-7

M MOPS, 324

2.96x10-7

M thiamine, 2.05x10-9

M biotin, and 3.69x10-10

M cyanocobalamin at pH 6.8) with 325

yeast extract and a wheat grain (19). 326

Genome sequencing 327

For PacBio sequencing, exponentially growing mixed cultures containing B. qaytius was 328

centrifuged in a Sorvall SLC-6000 for 20 min and 5000 rpm at 4°C to remove eukaryotic cells 329

(20). Particles in the supernatant concentrated approximately 100-fold by tangential flow 330

ultrafiltration with at 30kDa cut-off (Vivaflow 200, PES) cartridge. To concentrate the cells 331

further they were centrifuged at 28,000 rpm, 15°C for 8 h in a Beckman ultracentrifuge using a 332

Ti90 fixed-angle rotor (Beckman-Coulter, Brea, California, USA), and then sedimented onto a 333

40% Optiprep 50 mM Tris-Cl, pH 8.0, 2mM MgCl2 cushion for 30 min at 28,000 rpm, and 15°C 334

in a SW40Ti swing-out rotor. The Optiprep gradient was created by underlaying a 10% Optiprep 335

solution in 50 mM Tris-Cl, pH 8.0, 2 mM MgCl2 with a 30% solution followed by a 50% 336

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solution and equilibration overnight at 4°C. One ml of concentrate from the 40% cushion was 337

added atop the gradient and the concentrate was fractionated by centrifugation in an SW40 rotor 338

for 4 h at 25000 rpm and 18°C. The fraction corresponding to the predator was extracted from 339

the gradient with a syringe and washed twice with 50 mM Tris-Cl, pH 8.0, 2 mM MgCl2 340

followed by centrifugation in an SW40 rotor for 20 min at 7200 rpm and 18°C and were finally 341

collected by centrifugation in an SW40 rotor for 30 min at 7800 rpm and 18°C. Purity of the 342

concentrate was verified by fluorescence vs SSC of SYBR-Green stained samples (Invitrogen 343

Carlsbad, California, USA) on a FACScalibur flow cytometer (Becton-Dickinson, Franklin 344

Lakes, New Jersey, USA). High molecular weight genomic DNA was extracted using phenol-345

chloroform-chloroform extraction. Length and purity were confirmed by gel electrophoresis and 346

by using a Bioanalyzer 2100 with the HS DNA kit (Agilent Technology). PacBio RSII 20kb 347

sequencing was performed by the sequencing center of the University of Delaware. Reads were 348

assembled using PacBio HGAP3 software with 20 kb seed reads resulting in a single contig of 349

3,376,027 bp, 97.08 x coverage, 99.92% called bases and a consensus concordance of 99.9954 % 350

(21). 351

Propagation and host range studies 352

Plaque assays were performed by mixing 0.5 ml putative host cultures in logarithmic 353

growth stage and 10μl of Bdellovibrio qaytius stock culture with 4.5 ml molten 0.5% DY-V agar 354

and incubation for 48h. Propagation of Bdellovibrio qaytius in liquid culture was monitored by 355

PCR with custom primers set specific to B. qaytius 16S rDNA (Forward-5’-356

AGTCGAACGGGTAGCAATAC-3’, Reverse-5’-CTGACTTAGAAGCCCACCTAC-3’) as 357

well as a BALO-specific primer set by Davidov et al. (7). To obtain a pure isolate of prey cells 358

present in the mixed microbial assemblage, culture samples were streaked onto a DY-V agar 359

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plate and incubated at room temperature. Distinct colonies were picked and propagated in liquid 360

DY-V medium. Propagation of Bdellovibrio qaytius using these cultures as hosts was confirmed 361

by PCR. The identity of the prey cell cultures was confirmed by universal 16S rDNA Sanger 362

sequencing (515F-5’-GTGYCAGCMGCCGCGGTAA-3’, 926R-5’-363

CCGYCAATTYMTTTRAGTTT-3’) (22). To clean up the predator culture, E. coli (Thermo 364

Fisher) cells were grown in LB medium and pelleted at 3,900 x g (4500 rpm) for 10 min, washed 365

with 10 ml of HEPES/CaCl2 buffer (25 mM HEPES, 2 mM CaCl2), centrifuged in a fixed angle 366

rotor centrifuge at 3,900 x g for 5 min, and re-suspended in 19 ml of HEPES/CaCl2 buffer. This 367

cell suspension was then inoculated with 1 ml of 0.8-μm PVDF membrane filtered lysate of the 368

Bdellovibrio containing culture and B. qaytius propagation was monitored by PCR. Bdellovibrio 369

remained viable at 4°C storage for up to two years and glycerol stocks of the native community 370

containing Bdellovibrio as well as an inoculated E.coli TOP10 culture was stored at -80°C for 371

archival purposes. 372

Environmental sampling 373

The presence of B. qaytius in the Nitobe-Garden pond was determined in 20-L water 374

samples that were taken bimonthly during spring and summer 2017 filtered through GF-A filters 375

(Millipore, Bedford, MA, USA; nominal pore size 1.1 μm) laid over a 0.8-μm pore-size PES 376

membrane (Sterlitech, Kent, WA, USA). The remaining particulate material was concentrated 377

into 250 ml using a 30-kDa MW cut-off tangential flow filtration cartridge (Millipore, Bedford, 378

MA, USA). DNA from these concentrates was extracted using phenol-chlorophorm extraction 379

and subjected to PCR using Bdellovibrio qaytius specific 16S rDNA primers to confirm its 380

presence. 381

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Microscopy 382

Negative staining transmission electron microscopy 383

Cultures of Escherichia coli TOP10 were inoculated with B. qaytius at two hour time 384

intervals and infected cultures, as well as an uninfected control were diluted tenfold and fixed in 385

4% glutaraldehyde. Next, the samples were applied to the carbon side of formvar carbon-coated 386

400-mesh copper grids (TedPella, CA, USA) and incubated at 4°C in the dark overnight under 387

high humidity. The liquid was then removed and the grids stained with 1% uranyl acetate for 388

30 s. 389

Ultra-thin sectioning transmission electron microscopy 390

For higher resolution images, cells of E. coli infected with Bdellovibrio qaytius were 391

harvested at at 4h intervals, as well as from uninfected control cultures. Cells from 10 ml of 392

culture were pelleted at 5000 xg in a Beckmann tabletop centrifuge using a fixed angle rotor. The 393

pellet was resuspended in 0.2 M Na-cacodylate buffer, 0.2 M sucrose, 5% EM-grade 394

glutaraldehyde, pH 7.4 and incubated for 2 h on ice. After washing in 0.2 M Na-cacodylate 395

buffer, cells were post-fixed with 1% Osmium tetroxide. Samples were dehydrated through 396

water/ethanol gradients and ethanol was substituted by acetone. Samples were embedded in an 397

equal part mixture of Spurr’s and Gembed embedding and the resin was polymerized at 60°C 398

overnight. Fifty-nm thin sections were prepared using a Diatome ultra 45° knife (Diatome, 399

Switzerland) on an ultra-microtome. The sections were collected on a 400x copper grid and 400

stained for 10 min in 2% aqueous uranyl acetate and 5 min in Reynold’s lead citrate. Image data 401

were recorded on a Hitachi H7600 transmission electron microscope at 80 kV. Image J 402

(RRID:SCR_003070) was used to compile all TEM images. Adjustments to contrast and 403

brightness levels were applied equally to all parts of the image. 404

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Fluorescence In Situ Hybridization Epifluorescence Microscopy 405

To confirm epibiotic predation, cultures for fluorescence in-situ hybridization (FISH) 406

were prepared as outlined below. Two 10-ml volumes of E. coli TOP10 were centrifuged at 3900 407

xg in a Beckman tabletop fixed angle centrifuge (4500 rpm) for 10 min, washed with 5 ml of 408

HEPES/CaCl2 buffer, centrifuged at 3900 g for 5 min, and re-suspended in 9 ml of 409

HEPES/CaCl2 buffer. One ml of B. qaytius containing culture was added to the resuspended E. 410

coli while another served as a control. Both cultures were incubated at room temperature for 24 h 411

and were centrifuged again at 3900 x g (4500 rpm) for 10 min, washed with 10 ml of PBS, 412

centrifuged at 3,900 x g for 5 min, and re-suspended in 5 ml of PBS. Two ml of the cultures were 413

fixed in a 1:3 dilution of 10% buffered formalin (pH 7.0; 10 ml of 37% formaldehyde, 0.65 g 414

Na₂HPO₄, 0.4 g NaH2PO4. 90 ml of Milli-QTM H2O) at 4°C for 3 h. Cells were then centrifuged 415

again at 3,900 x g, washed twice in 10 ml of PBS, re-suspended in 10 ml of a mixture of PBS 416

and 96% EtOH (1:1), and vortexed. In order to localize the predator an Alexa-488 tagged probe 417

specific to Bdellovibrio 16S rDNA was designed (5’-418

/5Alexa488N/TGCTGCCTCCCGTAGGAGT-3’) based on Mahmoud et al. which also served 419

as a template for the incubation protocol (23). Ten μl of sample was spotted onto a 70% EtOH-420

cleaned slide, dried at room temperature, and then taken through a dehydration series of 50%, 421

80%, and 95% EtOH. 25 μl of the hybridization master mix (20 mM Tris-HCl [pH 7.4], 0.1 % 422

SDS, 5 mM EDTA, 0.8 M NaCl, 37% formalin, 1 ng/μl of final probe concentration) was added 423

onto the sample. A cover glass was placed onto each sample and the slides incubated for 2 h at 424

46°C. With the cover slip removed, the slides were subsequently submerged into a bath of wash 425

buffer and incubated at 48°C for 30 min. Slides were rinsed with sterile deionized H2O and dried 426

at room temperature. A drop of ProLongTM Diamond Antifade Mountant with DAPI (4,6-427

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diamidine-2-phenylindole) was spotted onto a new cover glass and placed on the sample. Finally, 428

the slides were incubated at room temperature in the dark for 24 h prior to observation on an 429

Olympus FV 1000 system. 430

Annotation 431

The genome was circular and 3,348,710 bp in length. Genome annotation was performed 432

using the automated NCBI Prokaryotic Genome Annotation Pipeline (PGAAP). In parallel, open 433

reading frames were predicted using GLIMMER (RRID:SCR_011931) with default settings 434

(24). Translated proteins were analyzed using BLASTp, CDD RPS-BLAST, and pfam HMMER. 435

These results were used to refine the PGAAP annotation. Signal peptides and trans-membrane 436

domains were predicted using Phobius (25). The annotated complete genome is available under 437

the accession number CP025734. Metabolic pathways were predicted using the Kyoto 438

Encyclopedia of Genes and Genomes (KEGG RRID:SCR_012773) automatic annotation server 439

KAAS and Pathway Tools (RRID:SCR_013786) (26, 27). 440

Phylogenetic analysis 441

Full length 16S rDNA sequences of completely sequenced isolates of Bdellovibrio spp., 442

as well as full-length uncultured top BLAST hits were downloaded from NCBI. Alignments of 443

rDNA sequences were performed in Geneious R9 (RRID:SCR_010519) using MUSCLE with 444

default parameters (RRID:SCR_011812)(28). Maximum likelihood trees were constructed with 445

RAxML ML search with 1000 rapid bootstraps using GTR+GAMMA (29). 446

Phylogenetic analysis of the genome content by orthologous gene clusters was performed by 447

OrthoMCL (RRID:SCR_007839) (30) using whole-genome sequences downloaded from NCBI. 448

OrthoMCL was run with standard parameters (Blast E-value cutoff = 10−5 and mcl inflation 449

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factor = 1.5) on all protein-coding genes of length ≥ 100 aa. This resulted in the definition of 450

4242 distinct gene clusters. 451

Data availability 452

The annotated complete genome of Bdellovibrio qaytius is available at GenBank under 453

the accession number CP025734. Mixed cultures containing Bdellovibrio qaytius and its prey 454

Paraburkholderia fungorum are available upon request from the authors. 455

456

457

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Acknowledgements: 458

The work was supported by grants to C. S. from the Natural Sciences and Engineering 459

Research Council of Canada (NSERC; 05896), Canada Foundation for Innovation (25412), 460

British Columbia Knowledge Development Fund, and the Canadian Institute for Advanced 461

Research (IMB). C. D. was supported in part by a fellowship from the German Academic 462

Exchange Service (DAAD). T. L. was supported in part by an award by the Natural Sciences and 463

Engineering Research Council of Canada. The authors would like to thank Jill Campbell, the 464

coordinator for the Musqueam Language and Culture Department 465

(https://fnel.arts.ubc.ca/community/musqueam-nation/musqueam-language-and-culture/), for her 466

guidance in identifying a suitable species name based on the hən̓q̓əmin̓əm̓ language. Finally, the 467

authors would like to thank the UBC Bioimaging Facility for feedback and assistance with 468

microscopy. 469

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Figures 558

559

Figure 1: Bdellovibrio qaytius predation strategy and replication. A: Negative staining electron 560

micrograph of an attack-phase cell showing the characteristic sheathed flagellum (dark outline). 561

B: Negative staining electron micrograph of an early growth-phase cell (arrow) attaching to a 562

prey cell (arrowhead) with a broad predatory synapse. C: Negative staining electron micrograph 563

of a late growth-phase Bdellovibrio qaytius (arrow) next to a ghost cell of a prey cell 564

(arrowhead). The flagellum is detached in preparation for binary fission. D: FISH 565

epifluorescence micrograph of B. qaytius (red with specific FISH probe, arrow) attached to host 566

cell (green DAPI stained, arrowhead). E: Thin-section electron micrograph of growth-phase B. 567

qaytius (black arrow) attached to a prey cell (black arrowhead). The prey cell has an emptied 568

cytoplasm and shows an invagination of the membrane (white arrowhead).F: Thin section 569

micrograph close-up of the predatory synapse shown in E. The membrane of the predator (arrow) 570

and the prey cell (arrowhead) remain intact, but show electron-dense signatures. G: growth-phase 571

B. qaytius (black arrow) showing polar attachment to a prey cell (black arrowhead) that also 572

shows an invaginating cell membrane (white arrowhead). Scale bar in D: 2.5 μm, other scale bars 573

+ 500 nm. 574

575

Figure 2: Bdellovibrio qaytius genome. A: Genomic map of B. qaytius. From outside to inwards: 576

Plus-strand CDS (light blue), Minus-strand CDS (dark blue), tRNAs (black), rRNAs (red), and 577

non-coding RNAs (pink), GC-content (purple/mustard), and GC-skew (red/green). B: 578

Abundance of 866 identified functional clusters of orthologous genes in the B. qaytius genome 579

580

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581

582

Table 1: Comparison of Bdellovibrio qaytius, Bdellovibrio exovorus JSS, and Bdellovibrio 583

bacteriovorus HD100 584

585

586

587

588

Figure 3: Comparison of Clusters of Orthologous Genes (COG) abundance and coverage of 589

Bdellovibrio qaytius, Bdellovibrio exovorus JSS, and Bdellovibrio bacteriovorus HD100 590

591

592

593

594

Figure 4: Bdellovibrio qaytius phylogenetic placement. A: 16S maximum likelihood 595

phylogenetic tree showing the completely sequenced Bdellovibrio species as well as the top 596

BLAST hits to Bdellovibrio qaytius from uncharacterized isolates, as well as from metagenomic 597

data. B: Shared gene cluster analysis of complete Bdellovibrio genomes comparing epibiotic (B. 598

exovorus, B. qaytius) and periplasmic species (B. bacteriovorus strains HD 100 and Tiberius). 599

600

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B. qaytius B. exovorus JSS B. bacteriovorus HD 100

Size μm 1 x 0.4 0.5-1.4x0.5 . – . x . – .Shape rod to comma-shaped comma-shaped comma-shaped

Flagellum width (nm) 29 29 28

Intracellular growth no no yes

Host dependence obligate obligate faculatative

Bdelloplast formation no no yes

Prey range narrow? Claudobacter wide

Size 3.35 Mb 2.66 Mb 3.78 Mb

GC content 38.90% 41.90% 50.60%

# of protein coding genes 3166 2604 3551

# of tRNAs 31 33 36

# of rRNAs 3 3 6

Bd0108 HIT locus - - +

FilA/ Sigma 28 + + +

(D-)D-alanyl-D-alanine_carboxypeptidase3 (AZZ35680.1, AZZ35389.1,

AZZ36481.1)1 (AGH96257.1) 2 (Bd0816, Bd3459 )

polysaccharide_deacetylase_family_protein 1 (AZZ36320.1) 1 (AGH96081.1) 2 (Bd3279, Bd0468)

hypothetical protein 0 0 2 (Bd1176, Bd0886)

TCA + + +

Glycolysis + + (+)

PPP + + +

Riboflavin + - +

Polyhydroxyalkanoate depolymerase - - +

Aaerobactin - - +Misc

Appearance

Re

pli

cati

on

Ge

no

me

Regulators

Prey

peptidoglycan

modification

Pa

thw

ay

s

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