2 downloaded from 3 //aem.asm.org/content/aem/early/2011/09/23/aem.05956-11.full.pdf · 62 arb...

1

Recovery of as-yet uncultured soil Acidobacteria on dilute solid media. 1

2

Isabelle F. George 1$*, Manuela Hartmann2, Mark R. Liles3, and Spiros N. Agathos1 3

4

1 Laboratoire de Génie Biologique, Earth and Life Institute (ELI), Université Catholique de 5

Louvain, Place Croix du Sud 2/19, 1348 Louvain-la-Neuve, Belgium 6

2 Ocean Biogeochemistry and Ecosystems Research Group, National Oceanography Centre, 7

European Way, Waterfront Campus, Southampton, United Kingdom 8

3 Department of Biological Sciences, Auburn University, Rouse Life Sciences Building, 120 9

W. Samford Avenue, Auburn, AL 36849, USA 10

11

$ Present address : Université Libre de Bruxelles, Laboratoire d’Ecologie des Systèmes 12

Aquatiques, Campus de la Plaine CP 221, Boulevard du Triomphe, 1050 Bruxelles, 13

Belgium ; Laboratoire de Biologie Marine, Campus du Solbosch, CP 160/15, 50 Av. F. D. 14

Roosevelt, 1050 Bruxelles, Belgium. 15

16

* Corresponding author. Mailing address: Université Libre de Bruxelles, Laboratoire 17

d’Ecologie des Systèmes Aquatiques, Campus de la Plaine CP 221, Boulevard du Triomphe, 18

1050 Bruxelles, Belgium. Phone : +32 2 6505715, Fax +32 2 6505993, Email: 19

[email protected] 20

21

Running title: Novel Acidobacteria recovered on dilute solid media 22

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.05956-11 AEM Accepts, published online ahead of print on 23 September 2011

on March 15, 2020 by guest

http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

2

ABSTRACT 23

A growing number of Acidobacteria strains have been isolated from environments worldwide, 24

with most isolates derived from acidic samples and affiliated with subdivision 1. We 25

recovered eighteen Acidobacteria strains from an alkaline soil, among which eleven belonged 26

to the previously uncultured subdivision 6. Various media formulations were tested for their 27

effects on Acidobacteria growth. 28

29


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

3

It has been known for more than a century that the great majority of environmental 30

microorganisms are not amenable to cultivation on/in standard laboratory media (6). Among 31

the taxa highly recalcitrant to cultivation, the bacterial division Acidobacteria has been the 32

focus of attention, because it is the second most abundant phylum in soils based on 16S rRNA 33

(gene) surveys (10). Yet very little is known of the ecology and the role of its members in 34

global biogeochemical cycles. The first Acidobacterium strain was isolated in 1991 (15). Since 35

then a growing number of isolates have been cultured from soils (or sediments) (2, 4, 5, 11, 13, 36

16-18, 21, 22, 31, 32) and there are currently 212 sequences of Acidobacteria strains listed in 37

the Ribosomal Database Project (RDP, Release 10, v.21, http://rdp.cme.msu.edu/). Modified 38

media formulations were proposed to improve their recovery, such as solid rather than liquid 39

media (29), polymeric growth substrates like xylan instead of easily degradable carbon sources 40

(4, 13, 27), gellan gum instead of agar (4, 11), or culturing with air enriched in CO2 (5, 31). 41

Notably, most sampled environments were acidic and the great majority of those isolates 42

(71%) belong to subdivision 1, a group favoured by (slightly) acidic conditions (5, 26). 43

Subdivision 6, which is the most phylogenetically diverse and numerous Acidobacteria 44

subgroup (it represents 29% of all Acidobacteria sequences contained in the RDP), completely 45

lacks any cultured representative. We present here the successful recovery of Acidobacteria 46

isolates from a slightly alkaline soil that were mostly affiliated with subdivision 6. 47

48

Samples were obtained from a surface soil (“soil UCL”) at the university campus of Louvain-49

la-Neuve, Belgium in 2008 [ pHH20: 7,40 ; % sand-silt-clay: 24-64-12 ; % humidity: 8.7 ; % C 50

(dry soil) 3.22 ± 0.03 ; % N (dry soil) 0.24 ± 0.01 ]. A bacterial 16S rRNA gene library of one 51

hundred clones was built from soil UCL and screened for Acidobacteria by PCR following the 52

protocol of (8) (except that we used the pDrive vector (Qiagen, Venlo, NL) for library 53

construction). Acidobacterial 16S rRNA gene sequences represented 31% of that inventory. 54


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

4

Sixty-seven percent of Acidobacterial 16S rRNA gene sequences were affiliated with 55

Acidobacteria subdivision 6. These relative abundances were in the range of what is usually 56

observed in soils (7, 10, 34) and more particularly in soils with a comparable pH (12). 57

Phylogenetic inference of Acidobacteria 16S rRNA gene sequences was performed using an 58

updated version of our ARB (http://www.arb-home.de) database (8) including all 59

acidobacterial 16S rRNA gene sequences (>1300 bp) available in February 2010 in the SILVA 60

database and the sequences generated in this study. Sequences were integrated in the existing 61

ARB alignment using the automated alignment tool of the software complemented by a manual 62

alignment. The optimized topology of the Acidobacteria subtree (8) (in which the subdivisions 63

were delineated according to Hugenholtz et al. (9)) was not changed. Sequences were 64

incorporated into this tree using the ARB parsimony interactive tool. 65

66

The cultivation protocol of soil bacteria was based on a very dilute medium and long 67

incubation times similar to (11). Serial dilutions of soil UCL suspensions were plated on (i) 68

dilute nutrient broth supplemented with 15 g /L agar (Sigma-Aldrich) and 0.065 g/L nutrient 69

broth (NB) (Oxoïd) (i.e., 200 times less than the manufacturer’s instructions) (“ANB 1/200”) 70

(ii) the same medium supplemented with soil extract (“ANB 1/200 + SE”). The soil extract was 71

prepared by mixing 300g of dried soil UCL with 1 L of sterile distilled water and stirring the 72

slurry for 1 h. The slurry was then centrifuged and the 0.2 µm-filtered supernatant was added 73

in a 1:1 (vol/vol) ratio to autoclaved ANB 1/200. Soil suspensions were prepared by mixing 1 g 74

of soil UCL with 100 mL of sterile dH2O, stirring for 2 h at 300 rpm, and settling for 1h. The 75

supernatant was then serially diluted and plated onto ANB 1/200 and ANB 1/200 + SE plates. 76

Plates were incubated for up to 3 months in the dark at room temperature (19 to 24 °C). 77

78


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

5

The numbers of colonies that were visible on plates progressively increased over three months 79

of incubation. After 91 days of incubation, CFU numbers had reached 1.55 x 107 ± 0.14 x 107 80

and 1.82 x 107 ± 0.16 x 107 CFU per gram of dry soil on ANB and ANB+SE plates, 81

respectively. Cultured Acidobacteria were not detected on the plates by plate wash PCR (using 82

primers Acid31F (1) and 518R-GC (20)) after 21 days of incubation, whereas they were 83

detected on all the plates analyzed after 35, 63 and 91 days of incubation. This delay (one 84

month) needed to detect cultured Acidobacteria illustrates their slow growth, as reported in 85

another study (4). DGGE analysis of the PCR products and subsequent identification of the 86

DGGE bands revealed an increase in cultured Acidobacteria diversity over the incubation time 87

and the predominance of subdivision 6 members in the cultured Acidobacteria community 88

(data not shown). The PCR mix, PCR cycle and DGGE conditions were those described in (8). 89

90

A protocol similar to (31) was used to localize Acidobacteria on the plates after 3 months of 91

incubation. Three thousand nine hundred colonies from ANB 1/200 or ANB 1/200 +SE plates 92

were subcultured onto fresh ANB 1/200 plates using toothpicks. After one month of 93

incubation, an Acidobacteria colony PCR (with an annealing temperature at 58°C) was 94

performed on each individual colony using primers Acid31F and 1492R. Thirty-six colonies 95

gave a positive PCR amplicon (0.9%), among which eighteen could be recovered as pure 96

cultures and repeatedly subcultured. The medium ANB 1/200 + SE proved to be superior to 97

ANB 1/200 for isolation of Acidobacteria from the soil environment, as twelve out of the 98

eighteen isolates (IGE002, IGE003, IGE004, IGE006, IGE007, IGE008, IGE009, IGE011, 99

IGE013, IGE014, IGE017, IGE018) were initially isolated on the former. Three isolates 100

belonged to subdivision 1, two to subdivision 3, two to subdivision 4 and eleven (61%) to 101

subdivision 6 (Fig. 1). Clearly, subdivision 6 predominated within these isolates, which was 102

remarkable considering that this was the first time that members of this subdivision had been 103


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

6

isolated under laboratory conditions. A neighbour joining (NJ) distance matrix was generated 104

for the sequences presented in Fig. 1 using ARB and it was used as input file for the program 105

DOTUR (28). DOTUR grouped isolates IGE012 to IGE014 (subdivision1), Edaphobacter 106

modestus Jbg-1 and Edaphobacter aggregans Wbg-1 as a single operational taxonomic unit 107

(OTU) at the 0.97 identity level (OTU0.03). All the other strains represented novel 108

Acidobacteria strains. Isolates IGE001 to IGE009 (subdivision 6) were grouped as a single 109

OTU0.03. Other isolates (IGE010, IGE011, IGE015, IGE016, IGE017, IGE018) represented 110

unique OTU0.03. Therefore, our isolates represented multiple OTU0.03 in the subdivisions 3, 4 111

and 6. 112 113

On ANB 1/200 plates, colonies of Acidobacteria took 3-4 days (subdivision 1), 1-2 weeks 114

(subdivision 3 and 4), or 3-4 weeks (subdivision 6) to form colonies with a diameter > 20 µm. 115

After 3 months of incubation, colonies generally reached 1-2 mm diameter, except subdivision 116

1 and subdivision 4 colonies that were bigger (3-5 mm diameter). Colony size kept increasing 117

slowly with increasing incubation times (up to 1.5 year) except for subdivision 1 isolates. 118

Colonies were white except subdivision 1 colonies that were all pale pink, subdivision 6 119

colonies that were all yellow and strain IGE017 (subdivision 4) that was pink. Subdivision 1 120

and 4 isolates formed smooth, circular, semi-transparent colonies, whereas subdivision 3 and 6 121

isolates formed dense, rubbery colonies, generally with an irregular margin at a later stage of 122

development (> 2 months of incubation) (Fig. 2). Subdivision 6 colonies were notably cohesive 123

and partly grown into the agar after three months of incubation (except on ANB 1/200 + SE), 124

which hampered the collection of colony material using toothpicks or even a scalpel. This 125

feature might reflect a competitive advantage in the soil environment via the production of as-126

yet uncharacterized extrapolymeric substances that promote adherence to soil particles, 127

resistance to dessication, and/or help to retain nutrients (25, 33). Their production has 128


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

7

previously been observed for subdivision 1 and 3 isolates (5, 15, 16, 21), and full or incomplete 129

cellulose synthesis operons have been identified in the genomes of strains belonging to those 130

subdivisions (33). 131 132

In order to improve the growth of Acidobacteria in laboratory conditions, we tested different 133

growth media, i.e. agar (15 g/L, pH 5.5) supplemented with increasing concentrations of NB 134

[0.065 g/L (“ANB 1/200”), 0.130 g/L (“ANB 1/100”), 0.260 g/L (“ANB 1/50”), 0.510 g/L 135

(“ANB 1/25”), 1.300 g/L (“ANB 1/10”), or 6.500 g/L (“ANB ½”)] , “ANB + SE”, and “ANB 136

1/200 + FC” (same medium as ANB 1/200, where a solution of 2000 U of catalase [bovine 137

liver; Sigma-Aldrich] was 0.2 µm-filtered and spread onto each plate). Aliquots of the same 138

suspension of strains IGE012 (subdivision 1), IGE015 (subdivision 3), IGE017 (subdivision 4) 139

or IGE001 (subdivision 6) were plated on each medium. After 3 months of incubation, cells 140

were collected from triplicate plates using a glass spreader and/or a razor blade and sterile 141

dH20. They were stained with 4’,6-diaminido-2-phenylindole (DAPI) (1 µg/mL) for 142

enumeration by microscopy. 5.72 x 108 ± 1.62 x 108, 8.42 x 107 ± 1.88 x 107, 2.68 x 107 ± 0.42 143

x 107, and 1.90 x 108 ± 0.30 x 108 cells were recovered per plate of standard medium for each 144

strain, respectively. None of the strains could grow on agar without nutrients (data not shown). 145

The soil extract-amended medium was the best growth medium among the media tested for the 146

four Acidobacteria strains and resulted in cell number increase of 7.6 to 15.6-fold (Fig. 3). This 147

result corroborates the beneficial effect of this undefined medium amendment on the initial 148

isolation of Acidobacteria from the soil environment and on the growth of fastidious soil 149

bacteria in general (4). However, it is probable that this positive effect was overestimated for 150

subdivision 6 isolates, as collection of colony material was notably easier on that medium. The 151

addition of filtered catalase resulted in an increase in cell numbers as well, although more 152

moderate (1.2 to 9.5-fold). This result confirms the general sensitivity of Acidobacteria to 153


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

8

damage by reactive oxygen species (5, 31), a phenomenon well studied in E. coli (30). Finally, 154

the four strains exhibited a different response to increasing nutrient concentrations which likely 155

reflected different lifestyle strategies among members of this broad phylum. Acidobacteria are 156

known to be more abundant in environments with low C availability (7) and seem to prefer 157

bulk soil to nutrient-“rich” rhizosphere (3, 14, 19), but such intra-phylum differences regarding 158

nutrient concentrations have not been previously reported. Strain IGE012 (subdivision 1) 159

showed a constant increase in cell numbers with increasing nutrient concentration, to reach a 160

13.5-fold increase on ANB ½. This response was not surprising considering that subdivision 1 161

is the most readily cultured group of Acidobacteria (4, 23, 26, 27). For the 3 other strains, the 162

medium ANB ½ inhibited growth, as no colonies were observed on the plates. The nutrient 163

concentrations at which the largest number of bacterial cells was achieved for each 164

Acidobacteria strain, relative to ANB 1/200, was: ANB 1/10 for strain IGE015 (2-fold increase 165

in cell numbers), ANB 1/25 for strain IGE017 (5.9-fold increase), and ANB 1/50 for strain 166

IGE001 (1.5-fold increase), respectively. The optimal nutrient concentration for strain IGE001 167

could actually be lower, as we have no data on ANB 1/100 due to a problem in the preparation 168

of the medium. For each strain, cell numbers on the medium with the highest cell count 169

(ANB+SE and ANB+FC excluded) were statistically different from those on standard medium 170

(one-tailed Student’s t-test, P = 0 x 10-6 for strains IGE012, IGE015 or IGE017, and P = 12 x 171

10-6 for strain IGE001). To isolate Acidobacteria strains, some other research groups used 172

richer media than our standard 200-fold diluted NB, e.g. 10-fold diluted TSA (4, 13). However, 173

in light of our results, such media are suboptimal for the recovery of subdivision 6 strains, as 174

the number of cells collected on ANB 1/10 represented only 2% of the cells collected on ANB 175

1/200 (Fig.3). Assessment of the nutrient concentrations based on colony diameter measured 176

after 3 months of incubation led to similar conclusions (data not shown). The sole exception 177

was strain IGE017, which developed very large diameter (but scarce) bright pink-coloured 178


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

9

colonies on ANB 1/10 (data not shown). This strong coloration is reminiscent of the pink 179

coloration observed for some Acidobacteria subdivision 1 strains under higher oxygen 180

concentration (5) and may be due to a stress response linked to higher nutrient levels. We are 181

presently characterizing the physiology of our strains. Moreover, some of these isolates have 182

been found to contain domains predicted to function within polyketide synthase pathways (24), 183

and it will be of interest to establish the diversity of secondary metabolites expressed by these 184

Acidobacteria strains. 185

186

Due to their small size, slow growth, adaptation to very low nutrient concentrations, and 187

attachment to the agar substrate, it is likely that subdivision 6 Acidobacteria were overlooked 188

in previous cultivation attempts, or that the alkaline soil used in this study was more conducive 189

to subdivision 6 cultivation. This study illustrates that there is still great potential to isolate 190

novel environmental strains using simple and inexpensive cultivation strategies. 191

192

Isabelle George benefited from a grant of “Chargée de Recherches” of the National Fund for 193

Scientific Research of Belgium (FNRS). Her visits to the National Oceanography Centre, 194

Southampton, UK were supported by travels grants from the FNRS and the British 195

Council/FNRS-CWBI Partnership Programme in Science. Laurence Pesché and Benjamin 196

Vidick are acknowledged for their help. 197

198

Nucleotide sequence accession numbers. 199

Sequence data have been deposited in the GenBank database under the following accession 200

numbers: (i) JF521744 to JF521838 for the Acidobacteria 16S rRNA gene sequences of soil 201

UCL (ii) GU187023 to GU187040 for the 16S rRNA gene sequences of the Acidobacteria 202

isolates. 203

204


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

10

REFERENCES 205

References 206

1. Barns, S. M., S. L. Takala, and C. R. Kuske. 1999. Wide distribution and diversity 207

of members of the bacterial kingdom Acidobacterium in the environment. Appl 208

Environ Microbiol 65:1731-7. 209

2. Coates, J. D., D. J. Ellis, C. V. Gaw, and D. R. Lovley. 1999. Geothrix fermentans 210

gen. nov., sp. nov., a novel Fe(III)-reducing bacterium from a hydrocarbon-211

contaminated aquifer. Int J Syst Bacteriol 49 Pt 4:1615-22. 212

3. da Rocha, U. N., L. van Overbeek, and J. D. van Elsas. 2009. Exploration of 213

hitherto-uncultured bacteria from the rhizosphere. FEMS Microbiol Ecol 69:313-28. 214

4. Davis, K. E., S. J. Joseph, and P. H. Janssen. 2005. Effects of growth medium, 215

inoculum size, and incubation time on culturability and isolation of soil bacteria. Appl 216


5. Eichorst, S. A., J. A. Breznak, and T. M. Schmidt. 2007. Isolation and 218

characterization of soil bacteria that define Terriglobus gen. nov., in the phylum 219

Acidobacteria. Appl Environ Microbiol 73:2708-17. 220

6. Epstein, S. S. 2009. Uncultivated Microorganisms. Springer-Verlag, New York. 221

7. Fierer, N., M. A. Bradford, and R. B. Jackson. 2007. Toward an ecological 222

classification of soil bacteria. Ecology 88:1354-64. 223

8. George, I. F., M. R. Liles, M. Hartmann, W. Ludwig, R. M. Goodman, and S. N. 224

Agathos. 2009. Changes in soil Acidobacteria communities after 2,4,6-trinitrotoluene 225

contamination. FEMS Microbiol Lett 296:159-66. 226

9. Hugenholtz, P., B. M. Goebel, and N. R. Pace. 1998. Impact of culture-independent 227

studies on the emerging phylogenetic view of bacterial diversity. J Bacteriol 228

180:4765-74. 229


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

11

10. Janssen, P. H. 2006. Identifying the dominant soil bacterial taxa in libraries of 16S 230

rRNA and 16S rRNA genes. Appl Environ Microbiol 72:1719-28. 231

11. Janssen, P. H., P. S. Yates, B. E. Grinton, P. M. Taylor, and M. Sait. 2002. 232

Improved culturability of soil bacteria and isolation in pure culture of novel members 233

of the divisions Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia. 234

Appl Environ Microbiol 68:2391-6. 235

12. Jones, R. T., M. S. Robeson, C. L. Lauber, M. Hamady, R. Knight, and N. Fierer. 236

2009. A comprehensive survey of soil acidobacterial diversity using pyrosequencing 237

and clone library analyses. ISME J 3:442-53. 238

13. Joseph, S. J., P. Hugenholtz, P. Sangwan, C. A. Osborne, and P. H. Janssen. 2003. 239

Laboratory cultivation of widespread and previously uncultured soil bacteria. Appl 240


14. Kielak, A., A. S. Pijl, J. A. van Veen, and G. A. Kowalchuk. 2008. Differences in 242

vegetation composition and plant species identity lead to only minor changes in soil-243

borne microbial communities in a former arable field. FEMS Microbiol Ecol 63:372-244

82. 245

15. Kishimoto, N., Y. Kosako, and T. Tano. 1991. Acidobacterium capsulatum gen. 246

nov., sp. nov.: an Acidophilic chemoorganotrophic bacterium containing menaquinone 247

from acidic mineral environment. Curr Microbiol 22:1-7. 248

16. Koch, I. H., F. Gich, P. F. Dunfield, and J. Overmann. 2008. Edaphobacter 249

modestus gen. nov., sp. nov., and Edaphobacter aggregans sp. nov., acidobacteria 250

isolated from alpine and forest soils. Int J Syst Evol Microbiol 58:1114-22. 251

17. Liesack, W., F. Bak, J. U. Kreft, and E. Stackebrandt. 1994. Holophaga foetida 252

gen. nov., sp. nov., a new, homoacetogenic bacterium degrading methoxylated 253

aromatic compounds. Arch Microbiol 162:85-90. 254


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

12

18. Mannisto, M. K., S. Rawat, V. Starovoytov, and M. M. Haggblom. 2011. 255

Terriglobus saanensis sp. nov., a novel Acidobacterium isolated from tundra soil of 256

Northern Finland. Int J Syst Evol Microbiol. 257

19. Marilley, L., and M. Aragno. 1999. Phylogenetic diversity of bacterial communities 258

differing in degree of proximity of Lolium perenne and Trifolium repens roots. Appl 259

Soil Ecol 13:127-136. 260

20. Muyzer, G., E. C. de Waal, and A. G. Uitterlinden. 1993. Profiling of complex 261

microbial populations by denaturing gradient gel electrophoresis analysis of 262

polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ 263

Microbiol 59:695-700. 264

21. Pankratov, T. A., and S. N. Dedysh. 2010. Granulicella paludicola gen. nov., sp. 265

nov., G. pectinivorans sp. nov., G. aggregans sp. nov. and G. rosea sp. nov., novel 266

acidophilic, polymer-degrading Acidobacteria from Sphagnum peat bogs. Int J Syst 267

Evol Microbiol. 268

22. Pankratov, T. A., L. A. Kirsanova, E. N. Kaparullina, V. V. Kevbrin, and S. N. 269

Dedysh. 2011. Telmatobacter bradus gen. nov., sp. nov., a cellulolytic facultative 270

anaerobe from subdivision 1 of the Acidobacteria and emended description of 271

Acidobacterium capsulatum Kishimoto et al. 1991. Int J Syst Evol Microbiol. 272

23. Pankratov, T. A., Y. M. Serkebaeva, I. S. Kulichevskaya, W. Liesack, and S. N. 273

Dedysh. 2008. Substrate-induced growth and isolation of Acidobacteria from acidic 274

Sphagnum peat. ISME J 2:551-60. 275

24. Parsley, L. C., J. Linneman, A. M. Goode, K. Becklund, I. George, R. M. 276

Goodman, N. B. Lopanik, and M. R. Liles. 2011. Polyketide synthase pathways 277

identified from a metagenomic library are derived from soil Acidobacteria. FEMS 278

Microbiol Ecol:in press, DOI:10.1111/j.1574-6941.2011.01122.x. 279


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

13

25. Roberts, I. S. 1996. The biochemistry and genetics of capsular polysaccharide 280

production in bacteria. Annu Rev Microbiol 50:285-315. 281

26. Sait, M., K. E. Davis, and P. H. Janssen. 2006. Effect of pH on isolation and 282

distribution of members of subdivision 1 of the phylum Acidobacteria occurring in 283

soil. Appl Environ Microbiol 72:1852-7. 284

27. Sait, M., P. Hugenholtz, and P. H. Janssen. 2002. Cultivation of globally distributed 285

soil bacteria from phylogenetic lineages previously only detected in cultivation-286

independent surveys. Environ Microbiol 4:654-66. 287

28. Schloss, P. D., and J. Handelsman. 2005. Introducing DOTUR, a computer program 288

for defining operational taxonomic units and estimating species richness. Appl 289


29. Schoenborn, L., P. S. Yates, B. E. Grinton, P. Hugenholtz, and P. H. Janssen. 291

2004. Liquid serial dilution is inferior to solid media for isolation of cultures 292

representative of the phylum-level diversity of soil bacteria. Appl Environ Microbiol 293

70:4363-6. 294

30. Speck, M. L., B. Ray, and R. B. Read, Jr. 1975. Repair and enumeration of injured 295

coliforms by a plating procedure. Appl Microbiol 29:549-50. 296

31. Stevenson, B. S., S. A. Eichorst, J. T. Wertz, T. M. Schmidt, and J. A. Breznak. 297

2004. New strategies for cultivation and detection of previously uncultured microbes. 298

Appl Environ Microbiol 70:4748-55. 299

32. Stott, M. B., M. A. Crowe, B. W. Mountain, A. V. Smirnova, S. Hou, M. Alam, 300

and P. F. Dunfield. 2008. Isolation of novel bacteria, including a candidate division, 301

from geothermal soils in New Zealand. Environ Microbiol 10:2030-41. 302

33. Ward, N. L., J. F. Challacombe, P. H. Janssen, B. Henrissat, P. M. Coutinho, M. 303

Wu, G. Xie, D. H. Haft, M. Sait, J. Badger, R. D. Barabote, B. Bradley, T. S. 304


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

14

Brettin, L. M. Brinkac, D. Bruce, T. Creasy, S. C. Daugherty, T. M. Davidsen, R. 305

T. DeBoy, J. C. Detter, R. J. Dodson, A. S. Durkin, A. Ganapathy, M. Gwinn-306

Giglio, C. S. Han, H. Khouri, H. Kiss, S. P. Kothari, R. Madupu, K. E. Nelson, W. 307

C. Nelson, I. Paulsen, K. Penn, Q. Ren, M. J. Rosovitz, J. D. Selengut, S. 308

Shrivastava, S. A. Sullivan, R. Tapia, L. S. Thompson, K. L. Watkins, Q. Yang, 309

C. Yu, N. Zafar, L. Zhou, and C. R. Kuske. 2009. Three genomes from the phylum 310

Acidobacteria provide insight into the lifestyles of these microorganisms in soils. Appl 311


34. Youssef, N. H., and M. S. Elshahed. 2009. Diversity rankings among bacterial 313

lineages in soil. ISME J 3:305-13. 314

315

FIGURE LEGENDS 316

Figure 1: Phylogenetic affiliation of Acidobacteria isolates recovered from soil UCL and all 317

other published terrestrial isolates based on comparative analysis of 16S rRNA gene sequence 318

data. Isolates obtained in this study are in boldface. Sequences were inserted into the 319

Maximum Parsimony dendrogram using the parsimony insertion tool of ARB. Boostrap 320

values > 90% and > 70% are indicated in the figure as filled and open circles, respectively. 321

The scale bar represents 0.10 change per nucleotide. 322

323

Figure 2: Examples of Acidobacteria colonies recovered from soil UCL after 3 months of 324

incubation (2 months for strain IGE012) on ANB. A = strain IGE012 (subdivision 1), B = 325

strain IGE015 (subdivision 3), C = strain IGE016 (subdivision 3), D = strain IGE017 326

(subdivision 4), E = strain IGE001 (subdivision 6), F = strain IGE007 (subdivision 6), G = 327

strain IGE005 (subdivision 6), H = strain IGE010 (subdivision 6). Bar = 100 µm. All pictures 328


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

15

were taken from the bottom of the plate with a 10x objective, except picture A that was taken 329

with a 2.5 x objective. 330

331

Figure 3: Cell numbers recovered by plate washing after incubation of four Acidobacteria 332

strains for 91 days. Cell suspensions were plated on agar with increasing concentrations of 333

nutrient broth (ANB 1/200, ANB 1/100, ANB 1/50, ANB 1/25, ANB 1/10, ANB ½) or 334

supplemented with soil extract (ANB + SE) or filtered catalase (ANB + FC). Each solid bar 335

represents standard errors of triplicates. n.m.= not measured; n.c. = no colony. 336

337


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

16

Figure 1 338

339

340

Subdivision 3

Soil isolate IGE014 (GU187037)Soil isolate IGE013 (GU187038)Soil isolate IGE012 (GU187028)Edaphobacter modestus Jbg-1 (DQ528760

Edaphobacter aggregans Wbg-1(DQ528761)Isolate Ellin337(AF498719)

Isolate Ellin351(AF498733)Granulicella aggregans TPB6028 (AM887756)

Granulicella tundricola MP5ACTX9 (CP002480)Granulicella pectinivorans TPB6011 (AM887757)

Granulicella rosea T4 (AM887759)Granulicella paludicola OB1010 (AM887758)

Peat bog isolate AL (AM887754)Peat bog isolate KMR (AM887755)Terriglobus roseus KBS 63 (DQ660892)Terriglobus saanensis SP1PR5 (HM214538)Terriglobus saanensis SP1PR4 (HM214537)

Peat bog isolate TPB6017 (AM887760)

Isolate Ellin310 (AF498692)Acidobacterium capsulatum (D26171)

Koribacter versatilis Ellin345 (AF498727)Isolate Ellin323 (AF498705)

Bryobacter aggregatus MPL1011 (AM887761)Bryobacter aggregatus MOB76 (AM887762)Bryobacter aggregatus MPL3 (AM162405)

Soil isolate IGE016 (GU187035)Soil isolate IGE015 (GU187034)

Isolate Ellin342 (AF498724)Solibacter usitatus Ellin6076 (CP000473)

Soil isolate IGE001 (GU187036)Soil isolate IGE002 (GU187040)Soil isolate IGE009 (GU187024)Soil isolate IGE004 (GU187029)Soil isolate IGE003 (GU187030)Soil isolate IGE005 (GU187033)

Soil isolate IGE007 (GU187023)Soil isolate IGE008 (GU187025)Soil isolate IGE006 (GU187026)


Geothrix fermentans ATCC700665 (U41563)Holophaga foetida DSM 6591 (X77215 )


Isolate Ellin6075 (AY234727)

0.10

Subdivision 1

Subdivision 8

Subdivision 6

Subdivision 4

Subdivision 3

Soil isolate IGE014 (GU187037)Soil isolate IGE013 (GU187038)Soil isolate IGE012 (GU187028)Edaphobacter modestus Jbg-1 (DQ528760

Edaphobacter aggregans Wbg-1(DQ528761)Isolate Ellin337(AF498719)

Isolate Ellin351(AF498733)Granulicella aggregans TPB6028 (AM887756)

Granulicella tundricola MP5ACTX9 (CP002480)Granulicella pectinivorans TPB6011 (AM887757)

Granulicella rosea T4 (AM887759)Granulicella paludicola OB1010 (AM887758)

Peat bog isolate AL (AM887754)Peat bog isolate KMR (AM887755)Terriglobus roseus KBS 63 (DQ660892)Terriglobus saanensis SP1PR5 (HM214538)Terriglobus saanensis SP1PR4 (HM214537)

Peat bog isolate TPB6017 (AM887760)

Isolate Ellin310 (AF498692)Acidobacterium capsulatum (D26171)

Koribacter versatilis Ellin345 (AF498727)Isolate Ellin323 (AF498705)

Bryobacter aggregatus MPL1011 (AM887761)Bryobacter aggregatus MOB76 (AM887762)Bryobacter aggregatus MPL3 (AM162405)


Isolate Ellin342 (AF498724)Solibacter usitatus Ellin6076 (CP000473)

Soil isolate IGE001 (GU187036)Soil isolate IGE002 (GU187040)Soil isolate IGE009 (GU187024)Soil isolate IGE004 (GU187029)Soil isolate IGE003 (GU187030)Soil isolate IGE005 (GU187033)

Soil isolate IGE007 (GU187023)Soil isolate IGE008 (GU187025)Soil isolate IGE006 (GU187026)


Geothrix fermentans ATCC700665 (U41563)Holophaga foetida DSM 6591 (X77215 )


Isolate Ellin6075 (AY234727)

0.10

Subdivision 1

Subdivision 8

Subdivision 6

Subdivision 4


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

17

Figure 2 341

342

A B C D

E F G H

A B C D

E F G H


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

18

Figure 3 343

344

345

0

200

400

600

800

1000

1200

1400

1600ANB 1

/200

ANB 1/1

00ANB 1

/50

ANB 1/2

5ANB 1

/10

ANB 1/2

ANB 1/2

00 +

SE

ANB 1/2

00 +

FC

Cel

l num

bers

/ p

late

(a

s %

of

AN

B 1

/200

-num

bers

)

IGE012 (subdiv.1)

IGE015 (subdiv.3)

IGE017 (subdiv.4)

IGE001 (subdiv.6)

n. c

.n.

c.

n. c

.

n. m

.


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

2 downloaded from 3 //aem.asm.org/content/aem/early/2011/09/23/aem.05956-11.full.pdf · 62 arb...

Documents