This article is protected by copyright. All rights reserved. 1

Received Date:14-Oct-2013 1

Accepted Date:12-Mar-2014 2

Distribution and diversity of Verrucomicrobia 3

methanotrophs in geothermal and acidic environments 1

4

5

Christine E. Sharp,1 Angela V. Smirnova,

1 Jaime M. Graham,

1 Matthew B. Stott,

2 Roshan 6

Khadka,1 Tim R. Moore

3, Stephen E. Grasby,

4 Maria Strack

5 and Peter F. Dunfield

1* 7

8

1Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, AB, Canada 9

T2N 1N4 10

2GNS Science, Extremophile Research Group, Private Bag 2000, 3352 Taupo, New Zealand 11

3Department of Geography, McGill University, 805 Sherbrooke St. W, Montreal, QC, Canada, H3A 0B9 12

4Geological Survey of Canada, 3303 33

rd St. NW, Calgary, AB, Canada T2L 2A7 13

5Department of Geography, University of Calgary, 2500 University Dr. NW, Calgary, AB, Canada T2N 14

1N4 15

16

*Correspondence: 17 Dr. Peter F. Dunfield 18 Department of Biological Sciences 19 University of Calgary 20 Calgary, Alberta, T2N 1N4, Canada 21 pfdunfie@ucalgary.ca 22 23

Running title: Diversity of verrucomicrobial methanotrophs 24

25

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copyediting, typesetting, pagination and proofreading process, which may lead to differences between this

version and the Version of Record. Please cite this article as doi: 10.1111/1462-2920.12454 Acc

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Summary 26

Recently, methanotrophic members of the phylum Verrucomicrobia have been 27

described, but little is known about their distribution in nature. We surveyed 28

methanotrophic bacteria in geothermal springs and acidic wetlands via pyrosequencing of 29

16S rRNA gene amplicons. Putative methanotrophic Verrucomicrobia were found in 30

samples covering a broad temperature range (22.5-81.6°C), but only in acidic conditions 31

(pH 1.8-5.0), and only in geothermal environments, not in acidic bogs or fens. 32

Phylogenetically, three 16S rRNA gene sequence clusters of putative methanotrophic 33

Verrucomicrobia were observed. Those detected in high-temperature geothermal samples 34

(44.1-81.6°C) grouped with known thermoacidiphilic “Methylacidiphilum” isolates. A 35

second group dominated in moderate-temperature geothermal samples (22.5-40.1°C) and 36

a representative mesophilic methanotroph from this group was isolated (strain LP2A). 37

Genome sequencing verified that strain LP2A possessed particulate methane 38

monooxygenase, but its 16S rRNA gene sequence identity to “Methylacidiphilum” was 39

only 90.6%. A third group clustered distantly with known methanotrophic 40

Verrucomicrobia. Using pmoA-gene targeted quantitative-PCR, two geothermal soil 41

profiles showed a dominance of LP2A-like pmoA sequences in the cooler surface layers 42

and “Methylacidiphilum”-like pmoA sequences in deeper, hotter layers. Based on these 43

results there appears to be a thermophilic group and a mesophilic group of 44

methanotrophic Verrucomicrobia. However, both were detected only in acidic 45

geothermal environments. 46

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Introduction 47

Aerobic methanotrophs are a unique group of microorganisms that use methane 48

(CH4) as their sole energy source. Until recently, all known methanotrophs belonged to 49

the Alphaproteobacteria or Gammaproteobacteria and were found to obtain some or all 50

of their cell carbon by assimilating formaldehyde or formate produced through CH4 51

oxidation (Op den Camp et al., 2009; Semrau et al., 2010). In addition to the 52

Proteobacteria, aerobic methanotrophy has now been observed in members of the 53

Verrucomicrobia and candidate division NC10 (Op den Camp et al., 2009; Ettwig et al., 54

2010). Verrucomicrobial methanotrophs were first isolated from geothermal 55

environments in Italy, New Zealand and Russia (Dunfield et al., 2007; Pol et al., 2007; 56

Islam et al., 2008) and have been given the proposed genus name “Methylacidiphilum” 57

(Op den Camp et al., 2009). “Methylacidiphilum” strains exhibit growth at pH 1, making 58

them the most acidophilic methanotrophs currently known. A thermophilic phenotype is 59

also displayed, with an upper growth temperature of 65°C (Op den Camp et al., 2009). 60

Aerobic methanotrophs first oxidize methane to methanol by either a particulate 61

(pMMO) or soluble (sMMO) methane monooxygenase. Methanol is subsequently 62

oxidized to formaldehyde, formate and carbon dioxide (Stein et al., 2012; Semrau et al., 63

2010). Proteobacterial methanotrophs assimilate carbon from formaldehyde and/or 64

formate via the serine cycle or the ribulose monophosphate (RuMP) pathway 65

(Chistoserdova, 2011). Genome analysis of “M. infernorum” strain V4 and “M. 66

fumariolicum” strain SolV showed that key enzymes of both the RuMP pathway and 67

serine cycle were absent (Hou et al., 2008; Khadem et al., 2012b), but genes encoding the 68

Calvin-Benson-Bassham (CBB) cycle were present. Khadem et al. (2011) applied both 69 Acc

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13CH4 and

13CO2 during growth experiments and verified that CO2 is the only carbon 70

source for strain SolV. Genome and transcriptome analysis confirmed that all genes 71

necessary for the CBB cycle were present and expressed. Using stable isotope probing 72

(SIP) with 13

CH4 and 13

CO2, individually and in combination, Sharp et al. (2012) 73

demonstrated that strain V4 only assimilated 13

CO2, not 13

CH4. These studies verified the 74

autotrophic nature of the verrucomicrobial methanotrophs. Methanotrophy in 75

“Methylomirabilis oxyfera” (candidate division NC10) is also autotrophic (Wu et al., 76

2011). 77

Three complete pmoCAB operons were detected in all three studied 78

“Methylacidiphilum” strains. Strains V4 and SolV also had a fourth pmoC copy and 79

strain Kam1 had an additional pmoCA operon (Op den Camp et al., 2009). Degenerate 80

polymerase chain reaction (PCR) primers previously developed to detect the pmoA genes 81

in proteobacterial methanotrophs do not amplify “Methylacidiphilum” pmoA genes 82

because of multiple mismatches in the target sequences (Dunfield et al., 2007). New PCR 83

primers have been designed to target the pmoA genes in “Methylacidiphilum” strain V4 84

(Sharp et al., 2012) and strain Kam1 (Erikstad et al., 2012). 85

Because Verrucomicrobia methanotrophs cannot be detected with pmoA-targeted 86

PCR primers commonly used to detect methanotrophs in the environment, they have 87

possibly been overlooked in most ecological studies of methanotrophic communities. 88

Consequently, almost nothing is known about their distribution in nature. Their detection 89

to date is limited to a few cultivation-based studies performed on acidic, volcanic 90

environments. We do not know whether they occur in other habitats as well. Using 91

pyrosequencing of 16S rRNA gene amplicons we analyzed the distribution and diversity 92 Acc

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of verrucomicrobial methanotrophs in geothermal areas in Canada and New Zealand 93

covering a temperature range of 7.5 to 99°C and a pH range of 1.8 to 9.0, and in several 94

acidic bogs and fens in Canada covering a temperature range of 6.3 to 21°C and a pH 95

range of 3.3 to 4.9. We also examined the vertical distribution of the verrucomicrobial 96

methanotrophs within two soil depth profiles from geothermal areas in New Zealand via 97

pmoA gene-targeted quantitative PCR. 98

99

Results 100

Distribution and diversity of verrucomicrobial methanotrophic communities 101

A database of 16S rRNA gene sequences was generated by 454-pyrosequencing 102

of PCR amplicons (generated with PCR primers 926f and 1392r) from 165 geothermal 103

samples and 8 bog/fen samples. Putative methanotroph sequences were identified in 92 of 104

these samples, covering a temperature range of 6.3-81.6°C and a pH range of 1.8-8.6 105

(Supplementary Table S1). Putative methanotrophic Verrucomicrobia sequences were 106

found between 22.5-81.6°C, but only in samples with a pH at or below 5.0 (Fig. 1A). The 107

percent contribution of putative methanotroph reads to the total 16S rRNA gene reads 108

ranged from 0.01% to 16.5% (Supplementary Table S1). Interestingly, despite the acidic 109

pH of the 8 bog/fen samples (pH 3.3-4.9), no 16S rRNA gene sequences clustering 110

closely to the known methanotrophic Verrucomicrobia were identified in any of them 111

(Supplementary Table S1), although some proteobacterial methanotrophs were found, 112

indicating an active methane cycle. 113

The majority of putative proteobacterial methanotroph sequences were found in 114

samples with near neutral pH (Fig. 1A). Below pH 3, only one sample (LOR21, pH 2.6, a 115 Acc

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low-temperature geothermal feature) had putative proteobacterial methanotrophs, 116

although even here verrucomicrobial methanotrophs were far more abundant (Fig. 1A, 117

Supplementary Table S1). LOR21 was also the only sample in which we detected both 118

verrucomicrobial and proteobacterial methanotrophs. All bog/fen samples showed a 119

dominance of alphaproteobacterial methanotrophs over gammaproteobacterial 120

methanotrophs. Gammaproteobacterial methanotrophs were only identified in one of the 121

bog samples (Mer Bleue Hummock) (Supplementary Table S1). 122

Less distinct trends were observed when analysing methanotroph communities 123

versus temperature (Fig. 1B). All three groups of methanotrophs were observed over a 124

large temperature range. Putative verrucomicrobial methanotrophs were detected from 125

22.5 to 81.6°C, but not below 22.5°C. 126

Phylogenetic analysis of the 16S rRNA genes from putative methanotrophs 127

revealed three distinct groups within the subphylum 6 of the Verrucomicrobia (Fig. 2, 128

Supplementary Fig. S1). Each group was composed of several distinct OTUs and 129

therefore each may represent several closely related species or genera. One group showed 130

high similarity to the three previously cultured “Methylacidiphilum” species, “M. 131

kamchatkense” strain Kam1, “M. fumariolicum” strain SolV and “M. infernorum” strain 132

V4. The most abundant OTU in this group, OTU 4811 (2094 sequences), showed 100% 133

identity to “M. infernorum” strain V4. A second group of OTUs showed high identity (> 134

96.0%) to methanotrophic bacterium LP2A that we isolated from one site (see below for 135

a detailed description of the isolate LP2A). The third group of OTUs (OTUs 28998, 136

15743 and 59083) showed 90.6–92.8% identity to the 16S rRNA gene sequence of strain 137

LP2A. This last group of OTUs, unlike the other two, is not closely related to a 138 Acc

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demonstrably methanotrophic isolate. Therefore, its identity as a methanotroph is based 139

solely on its position within the subphylum 6 of the phylum Verrucomicrobia 140

(“Methylacidiphilales”), which to date is only known to contain methanotrophs (Hedlund, 141

2010; Sharp et al., 2013). 142

143

Isolate LP2A, physiology and genome 144

Strain LP2A (Loop Road isolate 2A) was isolated from a cool, acidic geothermal 145

mud (Sample LOR21 in Supplementary Table S1) in Reporoa, New Zealand, by 146

incubating soil on mineral salts medium with methane added to the incubation 147

atmosphere as the sole energy substrate. A full genome analysis (see below) verified that 148

the organism possessed the signature methanotrophy genes pmoCAB that encode pMMO. 149

The closest cultured organism to strain LP2A was “M. infernorum” strain V4 (accession 150

number NR_074583.1), however the 16S rRNA gene sequence identity was only 90.6%. 151

The 16S rRNA-based phylogeny shows that isolate LP2A belongs to a new phylogenetic 152

branch of the Verrucomicrobia subphylum 6 only distantly related to previously known 153

“Methylacidiphilum” strains (identified as “Group 2” in Fig. 2). 154

Like the previously described thermophilic “Methylacidiphilum” spp., isolate 155

LP2A is acidophilic. However unlike “Methylacidiphilum” spp., it is mesophilic. Growth 156

was observed from 17-37°C (optimum 30°C), with no growth observed at 5°C or 45°C 157

(Supplementary Fig. S2). The pH range for growth is difficult to determine exactly as the 158

strain rapidly acidifies neutral media, but growth was observed from pH 1.0 to 5.2 159

(optimum 3.1). 160 Acc

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As some rare earth metals have been reported to stimulate the growth of “M. 161

fumariolicum” SolV (Pol et al., 2013) we tested strain LP2A, as well as “M. infernorum” 162

V4, for their growth response to cerium and lanthanum. These metals strongly stimulated 163

growth of both strains in liquid medium (Supplementary Fig. S2). 164

A draft genome of strain LP2A was sequenced at the Joint Genome Institute using 165

long-read Pacific Biosystems technology, and was assembled into three contigs. The draft 166

genome is 2.47 Mbp in size with a GC content of 62.74%. Automated annotation 167

identified 2327 protein-encoding genes. A complete list of the key predicted 168

methylotrophy genes and pathways, and a comparison to “M. infernorum” strain V4 is 169

shown in Supplementary Table S2. In general, the methane and carbon processing 170

machinery in LP2A is very similar to that described in “M. infernorum” (Hou et al., 171

2008). Two complete pmoCAB operons encoding particulate methane monooxygenase 172

(pMMO) and a third orphan pmoC copy were identified. The two pmoCAB operons are 173

very similar, showing 94.6, 98.0, and 97.8% derived amino acid identities (or 98.2, 99.6 174

and 99.3% similarities) in the pmoC, pmoA and pmoB genes, respectively. Hence the 175

pmoA copies cluster closely in the phylogeny (Fig. 3). No genes encoding sMMO were 176

detected. LP2A lacks key enzymes for the RuMP pathway and the serine cycle, but 177

encodes a complete Calvin-Benson-Bassham cycle, suggesting that it obtains all of its 178

carbon autotrophically. The pmoA-based phylogeny agrees with the 16S rRNA-based 179

phylogeny about the relationship between strain LP2A and the other methanotrophic 180

bacteria, and again calculates “Methylacidiphilum” as its closest cultivated neighbour 181

(Fig. 3). The pmoA1 and pmoA2 genes of strain LP2A showed only 69.8-69.9% identity 182 Acc

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to the pmoA1 and pmoA2 genes of “Methylacidiphilum” strain V4 at the nucleic acid 183

level (74.0-74.4% at the amino acid level). 184

185

Methanotrophic bacteria in geothermal soil profiles: A. Tikitere 186

Soil characteristics and methane oxidation rates calculated for the Tikitere soil 187

depth profile have been published elsewhere (Fig. 4A, 4B) (Sharp et al., 2012). Applying 188

both “Methylacidiphilum”-specific and strain LP2A-specific pmoA gene-targeted qPCR 189

systems to the Tikitere depth profile showed the highest number of total verrucomicrobial 190

pmoA copies (Fig. 4B) and “Methylacidiphilum” pmoA copies (Fig. 4A) at a depth of 10–191

15 cm. This corresponds to the original sample from which “M. infernorum” strain V4 192

was isolated, and has an in situ temperature similar to the optimum growth temperature of 193

strain V4 (Fig. 4B). LP2A-like pmoA sequences were found in highest copy numbers at 194

depths of 1-10 cm and temperatures between 36.8-44.1°C; low copy numbers were 195

observed in all other depths (Fig. 4A). The overall distribution pattern of 196

verrucomicrobial pmoA genes with depth is congruent with the distribution of methane 197

oxidation rates (Fig. 4B). 198

All Verrucomicrobia sequences detected via 16S rRNA gene pyrosequencing of 199

soils from the Tikitere depth profile (TIK 3-8) belonged to OTU 4811, which is 100% 200

identical to “M. infernorum” strain V4 (Fig. 2, Supplementary Fig. S1, Supplementary 201

Table S3). However, there was a very low relative abundance of Verrucomicrobia 202

sequences (0.01-0.40% of total 16S rRNA gene sequences) (Supplementary Fig. S3A, 203

Supplementary Table S1) compared to Thaumarchaeota, Euryarchaeota and 204

Crenarchaeota sequences in these samples, which together comprised up to 98.5% of the 205 Acc

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total 16S rRNA gene sequences recovered (Supplementary Fig. S3A). The dominance of 206

Thaumarchaeota and Crenarchaeota is not unexpected due to the high levels of ammonia 207

and hydrogen sulfide in the geothermal gas that filters through this soil, estimated at 208

mixing ratios of 420 mmol mol-1

gas and 84 mmol mol-1

gas, respectively (Giggenbach, 209

1994). The phylum Thaumarchaeota comprises all known archaeal ammonia oxidizers 210

(Pester et al. 2011) and some members of the Crenarchaeota are acid-tolerant sulfur 211

oxidizers (Macur et al. 2013). 212

As many of the “universal” 16S rRNA gene PCR primers are biased against 213

amplification of the Verrucomicrobia, a second set of “universal” PCR primers (515f and 214

806r) was used to examine the diversity of verrucomicrobial methanotrophs at Tikitere. 215

Primers 515f and 806r have been shown to be relatively unbiased against 216

Verrucomicrobia (Bergmann et al., 2011). Indeed, the relative abundance of 217

Verrucomicrobia in the Tikitere depth profile increased up to 11.1% when using these 218

primers in PCR (Supplementary Fig. S3B). Although the dominant OTU (28) was 100% 219

identical to “M. infernorum” strain V4, other sequences grouping in groups 1 and 2 were 220

also identified. Sequences from TIK4 and TIK5 clustered with OTU 1415 221

(Supplementary Table S4), which was identical to the methanotrophic isolate LP2A 222

(Supplementary Fig. S4) and supported our qPCR results. The overall microbial 223

community amplified with primers 515f and 806r was more diverse than that detected 224

with primers 926f and 1392r (Supplementary Fig. S3). 225

226

Methanotrophic bacteria in geothermal soil profiles: B. Rotokawa 227 Acc

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Temperatures of the Rotokawa soil samples ranged from 35.2°C at the surface to 228

68.7°C at the deepest point sampled (40 cm), and pH values ranged from 1.8 to 2.4 229

(Table 1). Methane oxidation was detected through the entire soil depth profile. Initial 230

methane oxidation rates for all samples were linear and ranged from 1.0 to 20.4 μmol 231

CH4 g-1

day-1

(Table 1, Fig. 4C). Oxidation rates were highest at the surface (incubation 232

temperature 37°C) and lowest in the deepest samples (65°C) (Fig. 4C). 233

The surface layers of the Rotokawa soil depth profile had the highest number of 234

total verrucomicrobial pmoA copies (Fig. 4D) and were dominated by LP2A-like pmoA 235

sequences (Fig. 4C). Very few “Methylacidiphilum”-like pmoA copies were detected 236

(Fig. 4C). The in situ temperatures of the upper two samples (35.2 and 40.1°C, Table 1) 237

fall within the growth temperature range of isolate LP2A. Conversely, 238

“Methylacidiphilum”-like pmoA sequences dominated at depths of 10-30 cm, and 239

temperatures of 46.9-64.5°C (Fig. 4C), consistent with the growth temperature range of 240

the characterized “Methylacidiphilum” isolates. The trend of methane oxidation rates for 241

the Rotokawa depth profile is consistent with the qPCR-results on the distribution of 242

verrucomicrobial-pmoA genes. 243

Unlike the Tikitere soil profile, verrucomicrobial 16S rRNA gene sequences were 244

obtained from all depth samples of the Rotokawa soil, ranging from 1.0 to 13.9% of total 245

16S rRNA gene reads (Supplementary Fig. S5, Supplementary Table S1). The low 246

numbers again reflect the dominance of Archaea. When archaeal sequences are removed, 247

verrucomicrobial methanotrophs account for up to 55% of the total bacterial 16S rRNA 248

gene reads, mainly in depths of 5-30 cm (data not shown). The verrucomicrobial-pmoA 249

pyrosequencing results were consistent with the 16S rRNA pyrosequencing results. The 250 Acc

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LP2A-like OTU (OTU 73267) was most prevalent in the two surface samples (1-10 cm), 251

and declined to low numbers throughout the rest of the depth profile (Fig. 2, 252

Supplementary Table S3). Similar to Tikitere, OTU 4811 dominated at depths of 10-30 253

cm. The third most dominant OTU, 55164, was found in all Rotokawa samples with the 254

exception of the surface layer (Supplementary Table 3). This OTU is distinct from LP2A 255

but part of the larger LP2A cluster of OTUs (Group 2) (Fig. 2). 256

257

Sequencing of pmoA genes retrieved from Rotokawa 258

Many of the unique methanotrophic Verrucomicrobia 16S rRNA gene OTUs 259

were predominant in sequences from a depth of 10-30 cm. Although gas data was not 260

collected at the Rotokawa geothermal spring, data from the Tikitere geothermal area 261

suggests that O2 is present down to a depth of 60 cm (Dunfield et al., 2007). In order to 262

complement the 16S rRNA gene sequence diversity studies, “Methylacidiphilum”-like 263

pmoA genes were amplified from the Rotokawa soil using primers V170f and V613b to 264

examine if there was detectable diversity amongst pmoA sequences. Unfortunately, not 265

enough DNA was obtained from a depth of 15-25 cm, but samples at depths of 10-15 and 266

25-30 cm were successfully amplified. Phylogenetic analyses of pmoA sequences from 267

10-15 cm and 25-30 cm showed two main groups of sequences, which grouped closely 268

with the pmoA1 and pmoA2 gene copies of “M. infernorum” strain V4 (Fig. 3). The two 269

dominant OTUs, numbered 38 and 3, showed 98.8% and 99.8% respective similarity to 270

pmoA1 and pmoA2 of V4. Several other OTUs were observed ranging from 94.9-97.7% 271

sequence identity to the genes from Methylacidiphilum isolates. Therefore the pmoA 272

sequencing analysis showed some environmental diversity of this gene, but the diversity 273 Acc

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was very limited compared to the 16S rRNA diversity detected. It is possible that some of 274

the 16S rRNA gene sequences do not represent methanotrophs; however, it is more likely 275

that the limitation in pmoA recovery was due to the primer design. The design of 276

universal pmoA primers for Verrucomicrobia will require the characterization of pmoA 277

genes from more new bacteria such as strain LP2A described in this study. 278

279

Discussion 280

No trends were observed in the temperature ranges of the verrucomicrobial versus 281

proteobacterial methanotrophs detected in the sample set, except that the 282

Verrucomicrobia were detected only above 22.5°C. This is consistent with previously 283

reported overlapping growth temperature ranges of the verrucomicrobial and 284

proteobacterial methanotrophs (Dunfield, 2009; Op den Camp et al., 2009). Putative 285

Verrucomicrobia methanotroph sequences were found in sites with a wide temperature 286

range from 22.5 to 81.6°C. However, putative Verrucomicrobia methanotrophs were only 287

detected in acidic samples with a pH at or below 5.0, and only in the geothermal sites 288

rather than the bogs and fens. No 16S rRNA gene sequences closely related to 289

verrucomicrobial methanotrophs were observed in the bog/fen 16S rRNA gene sequence 290

datasets and no verrucomicrobial pmoA sequences could be amplified from these samples 291

(data not shown). This agrees with other pyrosequencing surveys of 16S rRNA genes in 292

acidic peats, which have also failed to detect methanotrophic Verrucomicrobia (Kip et al., 293

2012, Serkebaeva et al., 2013). Kip et al. (2012) also designed pmoA PCR primers 294

universal to the three “Methylacidiphilum” strains, and failed to amplify the gene in 295

Patagonian peat bogs. Although it is impossible to prove that the Verrucomicrobia 296 Acc

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methanotrophs were absent from bog and fen samples, our pyrosequencing pipeline 297

analysed 2448-7510 16S rRNA gene sequences from each sample without detecting any 298

putative Verrucomicrobia methanotrophs, although Proteobacteria methanotrophs were 299

abundant (Supplementary Table S1). The data therefore suggest that aerobic 300

methanotrophy in these bog and fen environments is highly dominated by Proteobacteria. 301

Methanotrophic activity in acidic peats generally peaks at pH 5-5.5 (Dunfield et al., 302

1993). As all the verrucomicrobial methanotrophs described to date (including LP2A) 303

grow optimally at pH levels between 2.0 and 3.5, this argues against a strong activity of 304

methanotrophic Verrucomicrobia in peat bogs. 305

It has recently been reported that “M. fumariolicum” SolV requires the rare earth 306

element cerium for growth (Pol et al., 2013). We confirmed a strong stimulatory effect of 307

cerium and lanthanum on the growth of strains LP2A and V4 (Supplementary Fig. S2). 308

Very slow growth rates were still observed in media without rare earth metals added, but 309

these may have been supported by trace metal contamination of glassware. The rare earth 310

elements have been found to especially occur in metamorphic and magmatic rocks, which 311

are commonly found in volcanic areas (Tyler, 2004). Geochemical studies of peat bogs 312

have only found the rare earth elements in very low concentrations (Efremova et al., 313

1999; Vodyanitskii et al., 2012). This provides an attractive hypothesis for why the 314

Verrucomicrobia methanotrophs were detected only in geothermal environments and 315

largely absent from ombrotrophic bogs. To compare minerotrophic and ombrotrophic 316

wetlands we did include one wetland fen, the Ochre Beds, which is fed by a cold 317

“thermal” spring that runs through a pyrite deposit, making this site both very acidic and 318

minerotrophic (Grasby et al., 2013). Unexpectedly, only Alphaproteobacteria 319 Acc

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methanotrophs were observed in the surface layers of this site, with no putative 320

Verrucomicrobia methanotrophs. There could also be a temperature limitation on the 321

growth of methanotrophs in these temperate zone environments, as we have detected the 322

Verrucomicrobia methanotrophs only at temperatures above 22.5°C. 323

“Methylacidiphilum” isolates are thermophiles growing between 37 and 65°C 324

(Op den Camp et al., 2009), and predominant OTUs detected in high-temperature 325

samples grouped closely with the sequences from known “Methylacidiphilum” isolates 326

(Supplementary Table S3, Fig. 2, Supplementary Fig. S1). These have been shown via 327

13CO2-SIP to be active at 55°C in one soil (Sharp et al., 2012). However, 328

Verrucomicrobia 16S rRNA gene sequences obtained from the more moderate-329

temperature (< 40°C) geothermal environments were dominated by a unique group of 330

OTUs that showed low (90.6-95.2%) 16S rRNA gene sequence identity to the cultured 331

thermophilic “Methylacidiphilum” strains. Previous studies have referred to enrichment 332

of mesophilic verrucomicrobial methanotrophs, suggesting the methanotrophic 333

Verrucomicrobia are not limited to a single genus-level group (Dunfield et al., 2007; Op 334

den Camp et al., 2009; Khadem et al., 2011). However, here we report for the first time 335

on the isolation and the genomic analysis of such a mesophile. Methanotrophic isolate 336

LP2A was obtained from a cool acidic geothermal mud in Reporoa, New Zealand. It 337

displayed an acidophilic (optimum pH 3.1), mesophilic (optimum 30°C) phenotype. A 338

draft genome for strain LP2A (Accession number PRJNA185245) verified its 339

methanotrophic lifestyle and suggested that its core metabolic pathways are very similar 340

to the thermophilic “Methylacidiphilum” spp. (Hou et al., 2008; Khadem et al., 2012b). 341

It lacks common proteobacterial methanotrophic pathways such as the 342 Acc

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tetrahydromethanopterin pathway, the serine cycle, and the RuMP pathway. Carbon 343

fixation is predicted to occur from CO2 rather than CH4, via the CBB cycle. There are 344

two closely related pmoCAB operons encoding pMMO, but no sMMO encoded in the 345

genome. 346

The overall 16S rRNA gene sequence diversity detected in the various studies 347

suggests that there may be three main clusters of putative methanotrophic 348

Verrucomicrobia. Two of these clusters now include known methanotrophic isolates 349

(strain LP2A or the three “Methylacidiphilum” spp.) and are dominant in either low-350

temperature or high-temperature environments, respectively. Quantitative PCR analyses 351

of the soil profiles from Rotokawa and Tikitere provided good in situ support for the 352

existence of a thermophilic group and a mesophilic group. The cooler surface soil 353

samples (0-10 cm; 35.2-44.1°C) in each site were dominated by the mesophilic LP2A-354

like pmoA sequences (Fig. 4). The deeper samples (10-30 cm; 46.9-64.5°C) displayed a 355

dominance of pmoA sequences related to the thermophilic “Methylacidiphilum” isolates 356

(Fig. 4). Methanotrophy in the third group is only hypothetical based on its phylogenetic 357

position within the Verrucomicrobia close to the two verifiable methanotrophic groups. 358

To confirm that this third group includes methanotrophs, environmental detection of 359

Verrucomicrobia pmoA genes would be useful. However there are currently too few 360

reference pmoA sequences from Verrucomicrobia to design practical universal pmoA 361

primers. 362

This study represents the first detailed ecological survey on the environmental 363

distribution and diversity of the methanotrophic Verrucomicrobia. Verrucomicrobia 364

methanotrophs were only found in geothermal springs at or below pH 5.0, but not in any 365 Acc

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of the acidic bogs or fens tested. 16S rRNA gene OTUs obtained from geothermal sites 366

grouped into three main phylogenetic clusters. Based on cultures, two of these groups can 367

be confirmed to include methanotrophs. The two groups appear to be dominant in 368

different temperature ranges, one is thermophilic and one mesophilic. 369

370

Experimental Procedures 371

Sampling sites 372

Geothermal samples were selected from a previous 16S rRNA gene 373

environmental survey of 165 soil, sediment and biomat communities (Sharp et al. 2014) 374

(SRA study accession number SRP028305). Samples were generally taken from surface 375

features with expected oxic/anoxic boundaries. Two soil depth profiles in the Rotokawa 376

geothermal area, Waikato, New Zealand (RTK 2-7) and the Hell’s Gate geothermal area, 377

Tikitere, New Zealand (TIK 3-9) were also sampled. In both sites, samples were collected 378

at various depths from the surface to 40 cm below the surface (Table 1, Sharp et al., 379

2012). Of all the sites analysed (Supplementary Table S1), methanotrophic 380

Verrucomicrobia have only been previously reported based on cultivation studies in 381

TIK5-2012 and LOR21-2010 (Dunfield et al., 2007) 382

Surface samples (0-15 cm) were collected from both hummock and hollow 383

microforms at Mer Bleue, Ontario (Frolking et al., 2002) in the summer of 2008 and 384

Wandering River, Alberta (Vitt et al., 2003) in the summer of 2010. A surface sample (0-385

10 cm) from the Ochre Beds wetland, BC was collected in the fall of 2009 (Grasby et al., 386

2013). Temperature was measured in the field using a handheld temperature probe model 387

HI9060 (Hanna Instruments). The pH was measured (1:1 peat:water) using an Accumet 388 Acc

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Basic AB15 pH meter (Fisher Scientific). Soils were frozen at -80°C within 2 d of 389

collection for DNA extraction. 390

391

Analysis of 16S rRNA genes and verrucomicrobial pmoA genes 392

16S rRNA gene amplicon pyrosequencing data for the 165 geothermal samples 393

were obtained from the survey of geothermal environments. Samples from Mer Bleue, 394

Wandering River and the Ochre Beds were processed for 16S rRNA gene 395

pyrosequencing by the methods of Sharp et al. (2014). All communities were therefore 396

characterized using 16S rRNA gene primers 926fw and 1392r. Sequence read sets were 397

analysed using the QIIME software platform (Caporaso et al., 2010) as per Sharp et al. 398

(2014), including a chimera check via Chimera Slayer. OTUs were identified 399

taxonomically via BLAST (Altschul et al., 1990) against the Silva release 111 database 400

(Pruesse et al., 2007). 401

The identity of all OTUs classified as members of the orders Rhizobiales (families 402

Methylocystaceae and Beijerinckiaceae), Methylococcales (family Methylococcaceae), 403

and Candidatus “Methylacidiphilum” were selected from the identification tables and 404

confirmed via comparison of the OTU reference sequences with GenBank sequences 405

using the NCBI BLAST software package (http://blast.ncbi.nlm.nih.gov/Blast.cgi). 406

Putative methanotroph OTUs were retained for further analysis only if the top cultured hit 407

identified using BLAST was a methanotroph. Representative methanotroph 16S rRNA 408

gene sequences were then aligned using the SINA aligner (www.arb-silva.de/aligner) 409

(Pruesse et al., 2012). Sequences were manually checked for homopolymer errors and 410

chimeric segments. All methanotroph OTUs represented by at least two reads from the 411 Acc

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pyrosequencing datasets were first added to a 16S rRNA gene phylogeny of the entire 412

Silva release 111 database (240809 sequences) using the parsimony-add function of ARB. 413

Only OTUs that clustered within previously described methanotroph clades were retained 414

in our analyses. 415

16S rRNA gene phylogenies for this study were constructed by first building a 416

skeleton tree of nearly complete sequences (> 1300 bp) using Neighbor-joining with a 417

Jukes-Cantor correction (Fig. 1). The shorter pyrosequences (400-450 bp) were then 418

added using the parsimony-add function of ARB. The phylogeny was verified via a 419

maximum likelihood quartet puzzling tree (Supplementary Fig. S1). 16S rRNA gene 420

sequences obtained from wetland samples in this study have been deposited in the SRA 421

database under accession number SRP032820. 422

Additional analyses were made of the Tikitere soil profile using 16S rRNA gene 423

primers 515f and 806r, which improve the detection of Verrucomicrobia (Bergmann et 424

al., 2011). Roche Titanium chemistry adapters and a 10-nt barcode were included on 425

primer 806r and products were sequenced and analyzed as previously described for 426

primers 926fw and 1392r. OTUs identified as “Methylacidiphilum” were confirmed and 427

phylogenies constructed as previously described. 16S rRNA sequences obtained in this 428

study have been deposited in the SRA database under accession number SRP032820. 429

pmoA gene amplicons for pyrosequencing were obtained via PCR using 430

verrucomicrobial-specific pmoA primers V170f and V613b (Sharp et al., 2012). These 431

primers were previously designed to target pmoA1 and pmoA2 of the 432

“Methylacidiphilum” isolates. The verrucomicrobial-pmoA primers were modified to 433

include the Roche Titanium chemistry adapters and a 10-nt barcode on primer V613b. 434 Acc

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pmoA sequences were quality filtered and clustered at 97% identity using QIIME 435

(Caporaso et al., 2010) and representative OTUs were identified via BLAST. pmoA 436

OTUs were aligned against a database of publically available pmoA sequences using 437

ARB (Ludwig et al., 2004). Sequences were manually checked for homopolymer errors 438

and chimeric segments. pmoA phylogenies were constructed using TREE_PUZZLE using 439

a Schoeniger-von Hasseler distance correction (Schmidt et al., 2002), 10,000 iterations 440

and a length filter of 450 bp covering only the length of the shorter pyrosequences. 441

Representative pmoA sequences obtained in this study have been deposited in the SRA 442

database under accession number SRP032820. 443

444

LP2A culturing and genome sequencing 445

Strain LP2A was isolated from a soil sample collected in the spring of 2010 from 446

an acidic mud pool at the Loop Road geothermal area (sample LOR21 in Supplementary 447

Table 1) in Reporoa, New Zealand (Sharp et al., 2014). The soil sample had a pH of 2.6 448

and in situ temperature of 22.5°C. Soil crumbs were spread aseptically onto solid medium 449

3.9C10.2 (pH 3.9) and incubated at 22°C. Medium 3.9C10.2 contained (l-1

): 0.2 g NH4Cl, 450

0.05 g KH2PO4, 0.02 g MgSO47H2O, 0.01 g CaCl26H2O, 0.005 g FeEDTA powder, 3 451

ml of FeEDTA solution (Stott et al., 2008), 3 ml of trace elements solution (Stott et al., 452

2008) and 1 ml of Wolin trace metals solution (Wolin et al., 1963). The medium was 453

solidified by adding 15 g l-1

Phytagel plus 1 g l-1

MgSO46H2O. After autoclaving, 10 mg 454

of yeast extract and 10 mg of vitamin solution (Stott et al., 2008) was added through a 455

sterile 0.2-μm filter. Incubations were carried out in glass desiccators supplemented with 456 Acc

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5% v/v CO2 and 20% v/v CH4. After > 20 weeks of incubation colony growth was 457

observed, and samples were subcultured until a single morphotype was observed. 458

Growth experiments were performed in liquid medium 3.9C10.2 in glass bottles 459

sealed with butyl rubber stoppers and injected with 20% CH4 and 10% CO2. Either 120-460

mL serum bottles containing 20 ml of medium or 300-mL bottles containing 50-mL of 461

medium were used. Bottles were incubated shaken at 120 revolutions per minute (r.p.m). 462

The strain LP2A inoculum was obtained from a plate culture to assure sterility. Cells 463

were first dispersed in a small amount of the liquid medium and then dispensed in equal 464

aliquots to the individual bottles containing medium. Growth was determined by 465

monitoring OD600 on an Ultraspec 10 Cell Density Meter (Amersham Biosciences). To 466

test the temperature range for growth, duplicate vials were incubated at 5°C, 17°C, 22°C, 467

30°C, 37°C, 45°C and 55°C. To test the pH range for growth, duplicate vials were 468

adjusted to pHs from 1.0 to 8.0 (in steps of 1 pH unit). The effect of the rare earth metals 469

cerium and lanthanum on the growth of strain LP2A and “M. infernorum” strain V4 was 470

determined by adding 320-380 nM of lanthanum (III) and cerium (III) chloride 471

heptahydrates and incubating at 30°C (LP2A) or 55°C (V4). 472

Genomic DNA from strain LP2A was extracted using a modified CTAB method 473

provided by the US Department of Energy (DoE) Joint Genome Institute (JGI). The draft 474

genome was generated using the Pacific Biosciences (PacBio) technology. A PacBio 475

SMRTbell library was constructed and sequenced by the JGI on the PacBio RS platform, 476

generating 153,830 filtered subreads totalling 374.4 Mbp. All general aspects of library 477

construction and sequencing performed at the JGI can be found at http://www.jgi.doe.gov. 478

Raw sequence reads were assembled using HGAP v. 2.0.0 (Chin et al., 2013), yielding 479 Acc

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three contigs in three scaffolds. The final draft genome was 2.5 Mbp with an average read 480

coverage of 194×. Genes in the draft genome were identified using Prodigal (Hyatt et al., 481

2010), followed by manual curation using GenePRIMP (Pati et al., 2010). Predicted CDs 482

were searched against the National Center for Biotechnology Information (NCBI) 483

nonredundant database, UniProt, TIGRFam, Pfam, KEGG, COG, and InterPro databases. 484

Additional gene prediction and annotation was performed using the Integrated Microbial 485

Genomes (IMG) platform (Markowitz et al., 2012). 486

487

pmoA primer design 488

pmoA primers were designed from a database of public-domain pmoA and amoA 489

sequences (total 3131 sequences) using the ARB software package (Ludwig et al., 2004). 490

Primers were designed to specifically target pmoA copies 1 and 2 from verrucomicrobial 491

methanotroph isolate LP2A. These were primers LVpmoAf (53 GGR TKG ACT 492

GGA AAG AYC G) and LVpmoAb (53 GCG AAR CTY CGC ATC GTT CC). PCR 493

parameters were first optimized on a pure culture of isolate LP2A via gradient PCR (60-494

70°C), optimal conditions are described in the Real-time pmoA PCR Quantification 495

section (see below). PCR products were verified via methods of Sharp et al. (2012). 496

Sequenced products displayed 100% similarity to pmoA1 and pmoA2 genes from strain 497

LP2A. pmoA primers specific for “Methylacidiphilum”-like pmoA genes (V170f and 498

V613b) were previously developed by Sharp et al. (2012). 499

500

Soil methane oxidation from depth profiles 501 Acc

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Soil methane oxidation rates from the Tikitere depth profile have previously been 502

published (Sharp et al., 2012). Methane oxidation rates were calculated for the Rotokawa 503

depth profile samples (RTK 2-7) exactly as described by Sharp et al. (2012). Samples 504

were incubated at near in situ temperatures (Table 1). Methane oxidation rates were 505

calculated using linear regression of mixing ratios versus time over five days. 506

507

Real-time pmoA PCR Quantification 508

Quantitative PCR was performed on soil depth profiles from both the Rotokawa 509

and Tikitere geothermal area, using primers V170f and V613b (Sharp et al., 2012) and 510

LVpmoAf and LVpmoAb (this study). PCR assays for both primer pairs were prepared as 511

per Sharp et al. (2012). Cycling conditions for primers V170f and V613b were as per 512

Sharp et al. (2012). For primers LVpmoAf and LVpmoAb, assays were performed with a 513

three-step thermoprofile: an initial denaturation of 5 min at 94°C; 35 cycles of 94°C for 514

60 s, 66°C for 45 s and 72°C for 45 s; and a final elongation step of 72°C for 10 min. 515

Fluorescence data acquisition occurred during the last step of each cycle. Serial dilutions 516

of PCR-amplified pmoA from strain LP2A were used as calibration standards for the 517

LVpmoAf and LVpmoAb real-time assays. Standards were prepared as per Sharp et al. 518

(2012). The measured DNA amount could be converted to target molecules per microliter 519

and pmoA standards were adjusted to 108, 10

6, 10

4 and 10

2 target molecules μl

-1 for 520

storage at -20°C. Efficiencies for standard curves used to calculate qPCR values ranged 521

from 86-94%. 522

523

Acknowledgements 524 Acc

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The work was supported by a Natural Sciences and Engineering Research Council of 525

Canada (NSERC) Discovery Grant to PD, as well as by a Geothermal Resources of New 526

Zealand (GRN) funding to MBS. CES was supported by doctoral fellowships from 527

NSERC and Alberta Innovates Technology Futures (AITF). The work conducted by the 528

U.S. Department of Energy Joint Genome Institute is supported by the Office of Science 529

of the U.S. Department of Energy under contract no. DE-AC02-05CH11231. The authors 530

wish to thank Tikitere Trust at Hell’s Gate, as well as BC Parks (the British Columbia 531

Ministry of the Environment) and Parks Canada for permission to sample some sites. 532

533

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Table 1. Summary of methane oxidation rates and physiochemical properties of the 671 Rotokawa soil profile samples RTK 2-7. 672 673

Sample Site pH In situ

Temperature (°C)

Incubation

Temperature (°C) Depth (cm)

Methane Oxidation Rate

(μmol CH4 g-1

day-1

)

RTK2 2.3 35.2 37 1-5 17.5-20.4

RTK3 2.4 40.1 37 5-10 10.9-12.4

RTK4 2.4 46.9 45 10-15 9.5-12.1

RTK5 2.1 54.5 55 15-25 6.1-6.3

RTK6 1.8 64.5 65 25-30 11.8-12.7

RTK7 2.1 68.7 65 30-40 1.0-2.7

674 675

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Figure Legends 677

Figure 1. Scatter plots of pH (A) and temperature (B) versus percent methanotrophs of 678

total 16S rRNA gene reads retrieved from diverse samples. Symbol shape indicates the 679

methanotroph phylogenetic affiliation: Verrucomicrobia (), Gammaproteobacteria () 680

or Alphaproteobacteria (). Temperatures plotted were taken at the time of sampling. 681

Mean annual temperatures for the bog/fen environments are also reported in 682

Supplementary Table S1. 683

684

Figure 2. 16S rRNA gene-based phylogenetic tree of methanotrophic Verrucomicrobia. 685

A skeleton tree was constructed using nearly complete reference sequences (> 1300 bp) 686

via Neighbor-joining with a Jukes-Cantor correction and 10,000 bootstrap steps. Shorter 687

pyrosequences from this study (in bold) plus additional OTUs from a previous SIP study 688

(Sharp et al., 2012) were added to the skeleton tree using the parsimony function in ARB 689

(Ludwig et al., 2004). The total number of reads of each OTU in all samples is shown in 690

parentheses. Bootstrap values > 50% are shown. The scale bar represents 0.1 changes per 691

nucleotide position. A maximum-likelihood tree showed the same major groups and 692

branching pattern (Supplementary Fig. S1). 693

694

Figure 3. pmoA gene-based phylogenetic tree showing the position of genes from the 695

methanotrophic Verrucomicrobia in relationship to other pmoA and amoA genes. The tree 696

was constructed using TREE_PUZZLE, a quartet maximum-likelihood method, using a 697

Schoeniger-von Hasseler distance calculation (Schmidt et al., 2002), 10,000 iterations 698

and a length filter of 450 bases corresponding to the length of the shorter pyrosequences. 699 Acc

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pmoA sequences obtained from the draft genome of methanotroph strain LP2A are 700

included. Pyrosequences recovered from Rotokawa samples at depths of 10-15 cm and 701

25-30 cm are indicated in bold. The total number of reads of each OTU in all samples are 702

shown in parentheses. Support values > 50% are shown. The scale bar represents 0.1 703

changes per nucleotide position. 704

705

Figure 4. Methane oxidation rates (open circles), verrucomicrobial-pmoA copies (bars) 706

and in situ temperatures (closed circles) through soil depth profiles from Tikitere (A,B) 707

and Rotokawa (C,D) geothermal areas. Methane oxidation rates for Tikitere were 708

obtained from Sharp et al. (2012). Error bars represent the standard deviation of replicate 709

incubations and duplicate qPCR measurements of the same sample. The abundances of 710

“Methylacidiphilum”-like pmoA gene copies (black bars), isolate LP2A-like pmoA gene 711

copies (grey bars) and the sum of the two groups (shaded dark grey bars) per g soil are 712

shown on the upper x-axis. 713

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719 emi_12454_f2 720

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