1 Amoeba-resisting bacteria found in multilamellar bodies secreted by Dictyostelium 1 discoideum: social amoebae can also package bacteria 2 3 Valérie E. Paquet 1,2 and Steve J. Charette 1,2,3 * 4 5 1. Institut de Biologie Intégrative et des Systèmes, Pavillon Charles-Eugène-Marchand, 6 Université Laval, Quebec City, QC, Canada 7 2. Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de 8 Québec, Hôpital Laval, Quebec City, QC, Canada 9 3. Département de biochimie, de microbiologie et de bio-informatique, Faculté des 10 sciences et de génie, Université Laval, Quebec City, QC, Canada 11 12 *Corresponding author: 13 Steve J. Charette, 1030 avenue de la medicine, Pavillon Marchand, local 4245, Université 14 Laval, Quebec City, QC, Canada, G1V 0A6, telephone: 1-418-656-2131, ext. 6914, fax: 15 1-418-656-7176, email: [email protected]16 17 Running title (60 characters with space): Packaging of amoeba-resisting bacteria by D. 18 discoideum 19 20 Keywords (6): Multilamellar bodies; Dictyostelium discoideum; packaged bacteria, 21 amoeba-resisting bacteria, Cupriavidus, Rathayibacter 22 23
35
Embed
2 discoideum: social amoebae can also package bacteria ARB... · 1 1 Amoeba-resisting bacteria found in multilamellar bodies secreted by Dictyostelium 2 discoideum: social amoebae
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
1
Amoeba-resisting bacteria found in multilamellar bodies secreted by Dictyostelium 1
discoideum: social amoebae can also package bacteria 2
3
Valérie E. Paquet1,2 and Steve J. Charette1,2,3* 4
5
1. Institut de Biologie Intégrative et des Systèmes, Pavillon Charles-Eugène-Marchand, 6
Université Laval, Quebec City, QC, Canada 7
2. Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de 8
Québec, Hôpital Laval, Quebec City, QC, Canada 9
3. Département de biochimie, de microbiologie et de bio-informatique, Faculté des 10
sciences et de génie, Université Laval, Quebec City, QC, Canada 11
12
*Corresponding author: 13
Steve J. Charette, 1030 avenue de la medicine, Pavillon Marchand, local 4245, Université 14
tetroxide. They were washed three times with sodium cacodylate buffer and were 178
dehydrated for 5 min in 30 % ethanol, 5 min in 50 % ethanol, 5 min in 70 % ethanol, 10 179
min in 95 % ethanol, and 1 h in 100 % ethanol. The samples were then embedded in 180
Epon resin and were incubated overnight at 37 °C followed by 3 days at 60 °C. Very thin 181
slices (60 to 80 nm) were cut and were stained for 8 min with 0.1 % lead citrate and then 182
for 5 min with 3 % uranyl acetate. They were then examined using a transmission 183
electron microscope (JEOL 1230) at 80 kV. 184
185
186
10
RESULTS AND DISCUSSION 187
188
Predation resistance assay 189
D. discoideum is probably the simplest system for assessing bacterial virulence 190
(Hilbi et al., 2007, Froquet et al., 2009). Because medium richness may have an impact 191
on the results of predation resistance assays (Froquet et al., 2007, Filion & Charette, 192
2014), our assays were performed using three different media of varying composition and 193
richness (HL5, SM, SM1/10). 194
195
Phagocytic plaques, which are bacteria-free zones due to amoeba grazing, are 196
produced when amoebae are spotted on lawns of digestible bacteria (Figure 1). 197
Phagocytic plaques were not observed in the presence of ARBs or were observed only for 198
the highest D. discoideum cell concentrations (Figure 1C and D) (Filion & Charette, 199
2014). Ka is used routinely in many phagocytic experiments to feed D. discoideum, 200
which is why we used it as a positive control for amoeba predation (Figure 1B) (Froquet 201
et al., 2009). 202
203
We considered that the isolates were ARBs when 500 or fewer D. discoideum 204
cells were unable to produce phagocytic plaques on the bacterial lawn for at least one of 205
the media tested. For example, it is the case for Cupriavidus sp. and Microbacterium sp. 206
isolates shown in Figure 1C and D. Isolates that allowed the growth of the amoebae with 207
an initial inoculum of 500 D. discoideum cells per drop or less were considered sensitive 208
to amoeba predation and were rejected for subsequent experiments. 209
11
A total of 136 bacterial isolates were screened with the amoeba predation assay to 210
identify those that were potential ARBs. All the experiments were performed twice, and 211
45 isolates were considered as D. discoideum resisting bacteria and, as such, potential 212
candidates for the packaging process (see Table S1). 213
The newly discovered ARBs were not specific to one phylum but belonged to various 214
clades distributed throughout the prokaryotes, which was in agreement with a study by 215
Moliner et al. (Moliner et al., 2010). Table 1 presents the ARBs discovered in the present 216
study. Our results suggested that the adaptation of bacteria to avoid digestion during 217
phagocytosis is widespread in bacteria. Moreover, the term ARB cannot be generalized 218
and be applied to an entire genus or species since bacteria from the same genus or species 219
did not display the same resistance to predation (Table 1). 220
221
Triple co-cultures 222
The 45 newly identified ARBs were co-cultured with digestible bacteria (Ka) and 223
D. discoideum. The goal of this experiment was to assess the growth of amoebae on 224
digestible bacteria (Ka) in the presence of ARBs to determine whether the ARBs were 225
toxic for the amoebae, making it impossible for them to produce packaged bacteria. All 226
the phagocytic plaques with a profile similar to the positive control, that is, with a large 227
bacteria-free zone (black arrow, Figure 2A) due to extensive amoeba growth, were 228
rejected. Similarly, co-cultures where no amoeba growth occurred, as for the negative 229
control, were also rejected. For example, all the Ka:Luteibacter anthropic ratios produced 230
small phagocytic plaques compared to the plaques produced by amoebae grown only on 231
Ka, suggesting that L. anthropic was toxic to the amoebae or markedly limited their 232
12
growth (black arrow, Figure 2B). Conversely, the presence of bacterial colonies in the 233
middle of grazing plaques (black arrow at top, Figure 2C) or substantial growth of the 234
ARB around phagocytic plaques (black arrow at the bottom, Figure 2C) indicated that the 235
ARB was resistant to predation and had no obvious toxicity for D. discoideum. One 236
possibility is that the bacteria passed through the phagocytic pathway and were expelled 237
as packaged bacteria, which then began to grow and form colonies. Three Cupriavidus 238
and 17 other isolates displayed this profile (Table 2). Thus based on the unusual growth 239
pattern of amoebae on their lawns, 20 isolates were considered as ARBs and were 240
retained in order to determine whether they were packageable. 241
242
Bacteria packaging by D. discoideum 243
The next step was to determine whether D. discoideum cells were able to package 244
ARBs. Based on previous packaging assays by Gourabathini et al. with E. coli O157:H7 245
and the ciliate Tetrahymena pyriformis (Gourabathini et al., 2008), packaged bacteria 246
released on a rich medium are able to grow inside the package and break out. Indeed, 247
packaged bacteria are likely a transitory state, allowing the bacteria to survive in harsh 248
conditions (Berk et al., 1998, Marciano-Cabral & Cabral, 2003) until they are released 249
into an environment that is more favorable for bacterial growth. Packaged ARBs were not 250
observed during the triple co-culture experiments using rich medium even after a long 251
period of time probably due to growth of potentially packaged bacteria. On the other 252
hand, starvation media (Smith et al., 2010), which contains only few nutriments to 253
prevent bacterial growth have been also tried, but they induce the multicellular 254
development of amoebae despite the presence of digestible bacteria (data not shown). 255
13
Again, no packaged bacteria were seen because active vegetative D. discoideum cells are 256
required for the packaging process to occur. 257
258
The stimulation of bacteria packaging and secretion was also studied using diluted 259
nutrient agar (SM1/10) to avoid rapid bacterial growth following exocytosis that could 260
break up the packages. We observed amoebae on mixed bacterial lawns of digestible 261
bacteria and ARBs (see ratios and strains in Table 2). Samples collected at the peripheries 262
of the phagocytic plaques were examined by IF with the H36 antibody (Mercanti et al., 263
2006) and by TEM. 264
265
A sample containing potential packaged bacteria had to display combined DAPI and 266
H36 antibody-positive staining for structures smaller than amoebae but bigger than free-267
living bacteria (data not shown) due to packaging of bacteria. DAPI would reveal the 268
presence of bacteria in the structures. On its side, H36 antibody has been shown in a 269
previous study to be a specific marker of MLBs by binding to a protein still not 270
characterized (Paquet et al, 2013). The magenta arrows in Figure 3 point to bacteria 271
packages measuring 2 to 3 µm in diameter, and the black arrow indicates a D. discoideum 272
cell. Of the 20 potential candidates tested by IF, three Cupriavidus isolates, two 273
Micrococcus luteus isolates, and one isolate each of Rathayibacter tritici and 274
Microbacterium oxydans presented features suggesting that they were packaged by D. 275
discoideum. 276
277
14
The same co-culture protocol was performed on several samples to formally confirm 278
the presence of expelled packaged bacteria by TEM. For the control condition shown on 279
Figure 4, D. discoideum produced (white arrow, Figure 4B) and secreted empty MLBs 280
(black arrow, Figure 4C) in the presence of digestible bacteria on SM1/10. However, 281
D. discoideum produced fewer MLBs on SM1/10 than on rich HL5 medium (Paquet et al., 282
2013). Despite this, Cupriavidus sp. and R. tritici were found inside secreted MLBs when 283
they were co-cultured with amoeba and digestible bacteria (Figure 4E, F, and I). The 284
TEM observations revealed that some of the tested bacteria could be packaged by D. 285
discoideum. 286
287
Interestingly, R. tritici accumulated inside the amoebae, with up to 50 undigested 288
bacteria visible inside each D. discoideum cell (Figure 4H). It is not clear whether the 289
accumulation was due to rapid bacterial growth inside the amoebae, the inhibition of the 290
exocytic process, or a combination of both. While the mechanism involved is not known, 291
this result suggested that bacteria can also survive in harsh environments by residing 292
inside amoebae. The intracellular survival in protozoa of many bacteria has been 293
described in the past (reviewed in Denoncourt et al., 2014). Many bacteria of the genus 294
Rathayibacter are phytopathogens of terrestrial plants (Hahn et al., 2003, Schaad & 295
Schuenzel, 2010), and it is likely that amoebae and these soil bacteria interact. 296
297
We showed that the packaging of bacteria is possible by D. discoideum amoeba model 298
and that the phenomenon is not restricted to specific genera. Indeed, both Gram-negative 299
and -positive bacteria from various environments, including soil and water, were trapped 300
15
inside the MLBs. Moreover, the outcome of various isolates from a same genera or even 301
a same species regarding packaging is fairly variable. For example, 23 strains of M. 302
luteus were tested using the predation assay and 9 were identified as ARBs, two of which 303
were packaged in MLBs based on the IF results. Thirteen Pseudomonas strains were also 304
tested using the predation assay. While 4 displayed an ARB phenotype, none was 305
packaged in MLBs. These results indicated that bacterial adaptive evolution with respect 306
to protozoa is complex, as has been shown by the farming of different strains of 307
Burkholderia sp. by non-farmer D. discoideum (DiSalvo et al., 2015). Given this, it 308
would be difficult to predict whether a given bacterial isolate can be packaged or can 309
resist predation by a specific protozoan without in vitro testing. It would thus be 310
interesting to determine whether the same ARBs are packaged by different wild-type 311
strains of D. discoideum or other protozoa. 312
313
Lastly, the present study showed that some ARBs are packaged in MLBs and are 314
secreted by D. discoideum in laboratory conditions. Amoeba/bacteria interactions are 315
ubiquitous in natural as well as in man-made environments such as in municipal drinking 316
water storage tank sediments (Lu et al., 2015), the floating and fixed biofilms of spring 317
recreation areas (Hsu et al., 2011), and the surface water of warm water systems and 318
cooling towers (Kuiper et al., 2006). As such, it is likely that bacteria packaging occurs in 319
real conditions, not just in the laboratory. 320
16
CONCLUSION 321
The resistance to predation of 136 bacterial isolates was assessed using a standardized 322
D. discoideum predation assay. Forty-five of these isolates displayed an ARB phenotype 323
and were co-cultured with digestible bacteria to stimulate MLB production. Twenty 324
potential candidates were retained based on this screening. The bacteria packaging of 325
seven isolates by D. discoideum was suggested by IF and confirmed for two isolates by 326
TEM. This is the first study to show that D. discoideum can package bacteria. These 327
results open the way to a better understanding of the role of ARBs in microbial ecology 328
and their persistence in many environments. 329
330
331
17
FUNDING 332
This work was supported by grants to S. J. C. from the Fonds de la Recherche du Québec 333
– Nature et Technologies (FRQNT) [2014-PR-173418], the Chaire de pneumologie de la 334
fondation J.-D. Bégin de l’Université Laval, the Fonds Alphonse L’Espérance de la 335
fondation de l’IUCPQ, and the Establishment of young researchers - Juniors 1 program of 336
the Fonds de la Recherche du Québec en Santé (FRQS) [20004]. 337
338
18
Acknowledgements 339
We are grateful to P. Cosson (University of Geneva, Switzerland) for the antibodies and 340
bacterial strains. We warmly thank the teams of J. M. Blatny (FFI, Norway) and M. 341
Filion (University of Moncton, Canada) as well as the USDA, who provided many 342
bacterial strains. We thank A. Denoncourt and A. Vincent (Université Laval, Canada) for 343
their critical reading of the manuscript and Richard Janvier (Plateforme de microscopie, 344
IBIS, Université Laval, Canada) for acquiring the transmission electron 345
microphotographs. 346
347
19
REFERENCES 348
Berk SG, Ting RS, Turner GW & Ashburn RJ (1998) Production of respirable vesicles 349 containing live Legionella pneumophila cells by two Acanthamoeba spp. Appl Environ 350 Microbiol 64: 279-286. 351
Berthiaume C, Gilbert Y, Fournier-Larente J, Pluchon C, Filion G, Jubinville E, Serodes 352 JB, Rodriguez M, Duchaine C & Charette SJ (2014) Identification of dichloroacetic 353 acid degrading Cupriavidus bacteria in a drinking water distribution network model. J 354 Appl Microbiol 116: 208-221. 355
Bonifait L, Charette SJ, Filion G, Gottschalk M & Grenier D (2011) Amoeba host model 356 for evaluation of Streptococcus suis virulence. Appl Environ Microbiol 77: 6271-6273. 357
Brandl MT, Rosenthal BM, Haxo AF et al. (2005) Enhanced survival of Salmonella 358 enterica in vesicles released by a soilborne Tetrahymena species. Appl Environ 359 Microbiol 71:1562-1569. 360
Cateau E, Delafont V, Hechard Y & Rodier MH (2014) Free-living amoebae: what part 361 do they play in healthcare-associated infections? J Hosp Infect 87: 131-140. 362
Cornillon S, Pech E, Benghezal M, Ravanel K, Gaynor E, Letourneur F, Bruckert F & 363 Cosson P (2000) Phg1p is a nine-transmembrane protein superfamily member 364 involved in dictyostelium adhesion and phagocytosis. J Biol Chem 275: 34287-34292. 365
Cosson P & Soldati T (2008) Eat, kill or die: when amoeba meets bacteria. Curr Opin 366 Microbiol 11: 271-276. 367
Cosson P & Lima WC (2014) Intracellular killing of bacteria: is Dictyostelium a model 368 macrophage or an alien? Cell Microbiol 16: 816-823. 369
Dallaire-Dufresne S, Paquet VE & Charette SJ (2011) [Dictyostelium discoideum: a 370 model for the study of bacterial virulence]. Can J Microbiol 57: 699-707. 371
Denoncourt AM, Paquet VE & Charette SJ (2014) Potential role of bacteria packaging by 372 protozoa in the persistence and transmission of pathogenic bacteria. Front Microbiol 373 5: 240. 374
DiSalvo S, Haselkorn TS, Bashir U, Jimenez D, Brock DA, Queller DC & Strassmann JE 375 (2015) Burkholderia bacteria infectiously induce the proto-farming symbiosis of 376 Dictyostelium amoebae and food bacteria. Proc Natl Acad Sci U S A 112: E5029-5037. 377
Dybwad M, Granum PE, Bruheim P & Blatny JM (2012) Characterization of airborne 378 bacteria at an underground subway station. Appl Environ Microbiol 78: 1917-1929. 379
Filion G & Charette SJ (2014) Assessing Pseudomonas aeruginosa virulence using a 380 nonmammalian host: Dictyostelium discoideum. Methods Mol Biol 1149: 671-680. 381
Filion M, Hamelin RC, Bernier L & St-Arnaud M (2004) Molecular profiling of 382 rhizosphere microbial communities associated with healthy and diseased black spruce 383 (Picea mariana) seedlings grown in a nursery. Appl Environ Microbiol 70: 3541-3551. 384
Froquet R, Lelong E, Marchetti A & Cosson P (2009) Dictyostelium discoideum: a model 385 host to measure bacterial virulence. Nat Protoc 4: 25-30. 386
Froquet R, Cherix N, Burr SE, Frey J, Vilches S, Tomas JM & Cosson P (2007) 387 Alternative host model to evaluate Aeromonas virulence. Appl Environ Microbiol 73: 388 5657-5659. 389
Gourabathini P, Brandl MT, Redding KS, Gunderson JH & Berk SG (2008) Interactions 390 between food-borne pathogens and protozoa isolated from lettuce and spinach. Appl 391 Environ Microbiol 74: 2518-2525. 392
20
Hahn MW, Lunsdorf H, Wu Q, Schauer M, Hofle MG, Boenigk J & Stadler P (2003) 393 Isolation of novel ultramicrobacteria classified as actinobacteria from five freshwater 394 habitats in Europe and Asia. Appl Environ Microbiol 69: 1442-1451. 395
Hilbi H, Weber SS, Ragaz C, Nyfeler Y & Urwyler S (2007) Environmental predators as 396 models for bacterial pathogenesis. Environ Microbiol 9: 563-575. 397
Hsu BM, Huang CC, Chen JS, Chen NH & Huang JT (2011) Comparison of potentially 398 pathogenic free-living amoeba hosts by Legionella spp. in substrate-associated 399 biofilms and floating biofilms from spring environments. Water Res 45: 5171-5183. 400
Kebbi-Beghdadi C & Greub G (2014) Importance of amoebae as a tool to isolate 401 amoeba-resisting microorganisms and for their ecology and evolution: the Chlamydia 402 paradigm. Environ Microbiol Rep 6: 309-324. 403
Kuiper MW, Valster RM, Wullings BA, Boonstra H, Smidt H & van der Kooij D (2006) 404 Quantitative detection of the free-living amoeba Hartmannella vermiformis in surface 405 water by using real-time PCR. Appl Environ Microbiol 72: 5750-5756. 406
Loret JF, Jousset M, Robert S, Saucedo G, Ribas F, Thomas V & Greub G (2008) 407 Amoebae-resisting bacteria in drinking water: risk assessment and management. Water 408 Sci Technol 58: 571-577. 409
Lu J, Struewing I, Yelton S & Ashbolt N (2015) Molecular Survey of Occurrence and 410 Quantity of Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa and 411 Amoeba Hosts in Municipal Drinking Water Storage Tank Sediments. J Appl 412 Microbiol. 119:278-88 413
Marciano-Cabral F & Cabral G (2003) Acanthamoeba spp. as agents of disease in 414 humans. Clin Microbiol Rev 16: 273-307. 415
Mercanti V, Charette SJ, Bennett N, Ryckewaert JJ, Letourneur F & Cosson P (2006) 416 Selective membrane exclusion in phagocytic and macropinocytic cups. J Cell Sci 119: 417 4079-4087. 418
Moliner C, Fournier PE & Raoult D (2010) Genome analysis of microorganisms living in 419 amoebae reveals a melting pot of evolution. FEMS Microbiol Rev 34: 281-294. 420
Pagnier I, Merchat M & La Scola B (2009) Potentially pathogenic amoeba-associated 421 microorganisms in cooling towers and their control. Future Microbiol 4: 615-629. 422
Paquet VE, Lessire R, Domergue F, Fouillen L, Filion G, Sedighi A & Charette SJ (2013) 423 Lipid composition of multilamellar bodies secreted by Dictyostelium discoideum reveals 424 their amoebal origin. Eukaryot Cell 12: 1326-1334. 425 Raghu Nadhanan R, Thomas CJ (2014) Colpoda secrete viable Listeria monocytogenes 426
within faecal pellets. Environ Microbiol 16:396-404. 427 Rodriguez-Zaragoza S (1994) Ecology of free-living amoebae. Crit Rev Microbiol 20: 428
225-241. 429 Schaad N & Schuenzel E (2010) Sensitive molecular diagnostic assays to mitigate the 430
risks of asymptomatic bacterial diseases of plants. Crit Rev Immunol 30: 271-275. 431 Siddiqui R & Khan NA (2012) Biology and pathogenesis of Acanthamoeba. Parasit 432
Vectors 5: 6. 433 Smith EW, Lima WC, Charette SJ & Cosson P (2010) Effect of starvation on the 434
endocytic pathway in Dictyostelium cells. Eukaryot Cell 9: 387-392. 435 Thomas JM & Ashbolt NJ (2011) Do free-living amoebae in treated drinking water 436
systems present an emerging health risk? Environ Sci Technol 45: 860-869. 437
21
Walker PL, Prociv P, Gardiner WG & Moorhouse DE (1986) Isolation of free-living 438 amoebae from air samples and an air-conditioner filter in Brisbane. Med J Aust 145: 439 175. 440
441
22
Table 1. Taxonomic grouping of new ARBs identified by the predation assay 442 Gram Class a Order Familia Genera Species No. of
Table 2. ARBs identified after co-culture assays as potential candidates for bacteria 444 packaging. 445 Strains Ratio KA:ARB Observations and comments Cupriavidus basilensis 1:1 Based on the morphology and
color of the colonies at the center and periphery of the phagocytic plaques A few fruiting bodies, with colored spores at the top.
Figure 1. Predation resistance assay. A. Serial dilutions of D. discoideum cells 450
(500,000 to 5 cells/5 µL) were spotted counter clockwise on bacterial lawns on HL5 agar 451
plates. The plates were incubated for 7 days. The negative control (HL5 medium only) 452
was spotted in the middle of the lawn. B. Klebsiella aerogenes is sensitive to predation by 453
amoebae. It was used as a positive control for amoeba predation. Cupriavidus sp. (C) and 454
Microbacterium sp. (D) were resistant to predation and were considered as potential 455
ARBs.456
25
Figure 2. Triple co-cultures. Example of potential ARB isolates co-cultured with 457
digestible bacteria (Ka) and 30 D. discoideum cells on SM agar. A. A lawn of Ka was 458
used as positive control for phagocytic plaque formation (clear zones in the bacterial 459
lawn; black arrow). B. A lawn of co-cultured Ka and Luteibacter anthropic [ratio 1:9]. 460
After the same incubation time, the amoebae were unable to farm the bacterial lawn, and 461
the plaques (black arrow) were much smaller than those of the negative control. This 462
bacterial species was not retained for subsequent analyses. C. A lawn of co-cultured Ka 463
and Cupriavidus sp. [ratio 1:9]. Pigmented colonies corresponding to the Cupriavidus sp. 464
can be seen in the middle of the phagocytic plaques (upper black arrow). Pigmented 465
colonies can also seen around the plaques (lower black arrow). This isolate was 466
considered as an ARB. 467
468
26
Figure 3. Immunofluorescence of bacteria packaged by D. discoideum. Material from 469
the peripheries of phagocytic plaques on lawns of co-cultured bacteria (see ratio in Table 470
2) on SM1/10 agar spotted with D. discoideum were processed for IF and were observed 471
under an epifluorescence microscope. For each ARB tested, the differential interference 472
contrast (DIC) is shown on the left while DAPI (blue), which targets the DNA of bacteria 473
and amoebae, and the H36 antibody (red), which targets MLBs and the amoeba 474
membrane, staining are presented on the right. D. discoideum (black arrow in A) 475
produced and secreted a few packaged Cupriavidus sp. (magenta arrow) into the 476
extracellular milieu. The bacteria shown on the images (A and B) were coated and 477
recognized by the H36 antibody. In C and D, only a fraction of the M. luteus and R. tritici 478
cells were in H36-positive structures. 479
480
27
Figure 4. Transmission electron microscopy of bacteria packaged and secreted by D. 481
discoideum. The peripheries of phagocytic plaques from co-cultured bacteria (see ratio in 482
Table 2) on SM1/10 agar spotted with D. discoideum were processed and were observed 483
by TEM. A, D, and G. Bacteria grown alone on rich medium. B and C. D. discoideum 484
produces (white arrow) and secretes (black arrow) MLBs with digestible bacteria on 485
SM1/10. No Ka were seen inside the MLBs. E, F, and I. Cupriavidus sp. and R. tritici 486
were packaged by D. discoideum and were exocytosed into the extracellular milieu. H. 487
More than 50 undigested R. tritici can be seen inside a D. discoideum cell. 488
Table S1. Compilation of the results of the predation resistance assays. One hundred thirty-six soil and water isolates were plated on three types of medium: HL5 = rich medium, SM = nutrient medium, and SM1/10 = nutrient-poor medium. Serial dilutions of D. discoideum cells (500,000 to 5 cells/5 µL) were spotted and spread on bacterial lawns. The plates were incubated for 7 days at 21°C. The phagocytic plaques were counted to determine the resistance of each isolate to predation by D. discoideum. The potential ARBs (underlined in yellow) are located in the magenta spectrum. The predation-sensitive strains are located in the cyan spectrum. A brown box indicates that the bacterial isolate did not grow on that medium after two or more tries.
Legend: > 500,000 > 50,000 > 5,000 > 500 > 50 > 5
No resistance ARBs No growth
Isolates HL5 SM SM1/10 Aeromonas hydrophila M15918-11