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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
37
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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
80
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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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References 471
1. Weinbauer MG, Höfle MG. 1998. Significance of viral lysis and flagellate grazing as 472
factors controlling bacterioplankton production in a eutrophic lake. Applied and 473
Environmental Microbiology 64:431-438. 474
2. Casida Jr L. 1982. Ensifer adhaerens gen. nov., sp. nov.: a bacterial predator of bacteria 475
in soil. International Journal of Systematic and Evolutionary Microbiology 32:339-345. 476
3. Dworkin M. 1999. Fibrils as extracellular appendages of bacteria: Their role in contact‐477
mediated cell‐cell interactions in Myxococcus xanthus. Bioessays 21:590-595. 478
4. Stolp H, Starr M. 1963. Bdellovibrio bacteriovorus gen. et sp. n., a predatory, 479
ectoparasitic, and bacteriolytic microorganism. Antonie Van Leeuwenhoek 29:217-248. 480
5. Burnham JC, Hashimoto T, Conti S. 1968. Electron microscopic observations on the 481
penetration of Bdellovibrio bacteriovorus into gram-negative bacterial hosts. Journal of 482
bacteriology 96:1366-1381. 483
6. Karunker I, Rotem O, Dori-Bachash M, Jurkevitch E, Sorek R. 2013. A global 484
transcriptional switch between the attack and growth forms of Bdellovibrio 485
bacteriovorus. PloS one 8:e61850. 486
7. Davidov Y, Jurkevitch E. 2004. Diversity and evolution of Bdellovibrio-and-like 487
organisms (BALOs), reclassification of Bacteriovorax starrii as Peredibacter starrii gen. 488
nov., comb. nov., and description of the Bacteriovorax–Peredibacter clade as 489
Bacteriovoracaceae fam. nov. International journal of systematic and evolutionary 490
microbiology 54:1439-1452. 491
8. Lambina V, Afinogenova A, Romaĭ SP, Konovalova S, Pushkareva A. 1982. 492
Micavibrio admirandus gen. et sp. nov. Mikrobiologiia 51:114-117. 493
9. Koval SF, Hynes SH, Flannagan RS, Pasternak Z, Davidov Y, Jurkevitch E. 2013. 494
Bdellovibrio exovorus sp. nov., a novel predator of Caulobacter crescentus. International 495
journal of systematic and evolutionary microbiology 63:146-151. 496
10. Pasternak Z, Njagi M, Shani Y, Chanyi R, Rotem O, Lurie-Weinberger M, Koval S, 497
Pietrokovski S, Gophna U, Jurkevitch E. 2014. In and out: an analysis of epibiotic vs 498
periplasmic bacterial predators. The ISME journal 8:625. 499
11. Chanyi RM, Ward C, Pechey A, Koval SF. 2013. To invade or not to invade: two 500
approaches to a prokaryotic predatory life cycle. Canadian journal of microbiology 501
59:273-279. 502
12. Capeness MJ, Lambert C, Lovering AL, Till R, Uchida K, Chaudhuri R, Alderwick 503
LJ, Lee DJ, Swarbreck D, Liddell S. 2013. Activity of Bdellovibrio hit locus proteins, 504
Bd0108 and Bd0109, links Type IVa pilus extrusion/retraction status to prey-independent 505
growth signalling. PloS one 8:e79759. 506
13. Alami M, Lüke I, Deitermann S, Eisner G, Koch H-G, Brunner J, Müller M. 2003. 507
Differential interactions between a twin-arginine signal peptide and its translocase in 508
Escherichia coli. Molecular cell 12:937-946. 509
14. Lerner TR, Lovering AL, Bui NK, Uchida K, Aizawa S-I, Vollmer W, Sockett RE. 510
2012. Specialized peptidoglycan hydrolases sculpt the intra-bacterial niche of predatory 511
Bdellovibrio and increase population fitness. Plos pathogens 8:e1002524. 512
15. Kuru E, Lambert C, Rittichier J, Till R, Ducret A, Derouaux A, Gray J, Biboy J, 513
Vollmer W, VanNieuwenhze M. 2017. Fluorescent D-amino-acids reveal bi-cellular cell 514
on March 28, 2021 by guest
http://jb.asm.org/
Dow
nloaded from
25
wall modifications important for Bdellovibrio bacteriovorus predation. Nature 515
microbiology 2:1648. 516
16. Wen C, Xue M, Zhang J, Huang Y, Zhou S. 2009. The detection of Bdellovibrio-and-517
like organisms in commercial preparations used for aquaculture. Journal of Fisheries of 518
China 33:326-333. 519
17. D'Anteo S, Mannucci A, Meliani M, Verni F, Petroni G, Munz G, Lubello C, Mori 520
G, Vannini C. 2015. Nitrifying biomass characterization and monitoring during 521
bioaugmentation in a membrane bioreactor. Environmental technology 36:3159-3166. 522
18. Duckworth OW, Holmström SJ, Peña J, Sposito G. 2009. Biogeochemistry of iron 523
oxidation in a circumneutral freshwater habitat. Chemical Geology 260:149-158. 524
19. Andersen R, Berges J, Harrison P, Watanabe M. 2005. Recipes for freshwater and 525
seawater media. Algal culturing techniques Elsevier, Amsterdam:429-538. 526
20. Deeg CM, Chow C-ET, Suttle CA. 2018. The kinetoplastid-infecting Bodo saltans virus 527
(BsV), a window into the most abundant giant viruses in the sea. eLife 7:e33014. 528
21. Chin C-S, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum A, 529
Copeland A, Huddleston J, Eichler EE. 2013. Nonhybrid, finished microbial genome 530
assemblies from long-read SMRT sequencing data. Nature methods 10:563-569. 531
22. Parada AE, Needham DM, Fuhrman JA. 2016. Every base matters: assessing small 532
subunit rRNA primers for marine microbiomes with mock communities, time series and 533
global field samples. Environmental microbiology 18:1403-1414. 534
23. Mahmoud KK, McNeely D, Elwood C, Koval SF. 2007. Design and performance of a 535
16S rRNA-targeted oligonucleotide probe for detection of members of the genus 536
Bdellovibrio by fluorescence in situ hybridization. Applied and environmental 537
microbiology 73:7488-7493. 538
24. Delcher AL, Harmon D, Kasif S, White O, Salzberg SL. 1999. Improved microbial 539
gene identification with GLIMMER. Nucleic acids research 27:4636-4641. 540
25. Käll L, Krogh A, Sonnhammer EL. 2004. A combined transmembrane topology and 541
signal peptide prediction method. Journal of molecular biology 338:1027-1036. 542
26. Moriya Y, Itoh M, Okuda S, Yoshizawa AC, Kanehisa M. 2007. KAAS: an automatic 543
genome annotation and pathway reconstruction server. Nucleic acids research 35:W182-544
W185. 545
27. Karp PD, Paley SM, Krummenacker M, Latendresse M, Dale JM, Lee TJ, Kaipa P, 546
Gilham F, Spaulding A, Popescu L. 2009. Pathway Tools version 13.0: integrated 547
software for pathway/genome informatics and systems biology. Briefings in 548
bioinformatics 11:40-79. 549
28. Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high 550
throughput. Nucleic acids research 32:1792-1797. 551
29. Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-552
analysis of large phylogenies. Bioinformatics 30:1312-1313. 553
30. Li L, Stoeckert CJ, Roos DS. 2003. OrthoMCL: identification of ortholog groups for 554
eukaryotic genomes. Genome research 13:2178-2189. 555
556
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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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