Anaerobacillus isosaccharinicus sp. nov., an alkaliphilic bacterium which

degrades isosaccharinic acid

Running title: A. isosaccharinicus an alkaliphilic isosaccharinate degrader

Naji M. Bassil and Jonathan R. Lloyd

Research Centre for Radwaste Disposal & Williamson Research Centre for Molecular

Environmental Science, School of Earth and Environmental Sciences, The University

of Manchester, Oxford Road, Manchester M13 9PL, UK

Correspondence: Naji M. Bassil. E-mail: naji.bassil@manchester.ac.uk Phone:

+44(0)161 275 0309.

Key words: Anaerobacillus, Alkaliphiles, Isosaccharinic acid, Radioactive waste,

Geological disposal

Contents category: New Taxon (Firmicutes and Related Organisms)

Main text word count: 2838

*

*he GenBank accession number for the 16S rRNA gene sequence of strain NB2006T is KJ852652.1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

12

Abstract

Strain NB2006T was isolated from an isosaccharinate-degrading, nitrate-

reducing enrichment culture in minimal freshwater medium at pH 10. Analysis

of the 16S rRNA gene sequence indicated that this strain was most closely related

to species of the newly established genus Anaerobacillus. This was supported by

phenotypic and metabolic characterisation that showed that NB2006T is rod-

shaped, Gram-stain-positive, motile, and forms endospores. It is an aerotolerant

anaerobe, and an obligate alkaliphile that grows at pH 8.5-11, can tolerate up to

6% (w/v) NaCl, and grows between 10 and 40oC. In addition, it can utilise a

number of organic substrates, and is able to reduce nitrate and arsenate. The

predominant cellular fatty acids were C16:0, C16:111c, anteiso-C15:0, iso-C15:0,

C16:17c/iso-C15:02OH and C14:0. The cell wall peptidoglycan contained meso-

diaminopimelic acid and the DNA G+C content was 37.7 mol%. In silico DNA–

DNA hybridization with the four known species of the genus Anaerobacillus

showed 21.8, 21.9, 22.4, and 21.5% relatedness to A. arseniciselenatis DSM

15340T, A. alkalidiazotrophicus DSM-22531T, A. alkalilacustris DSM-18345T, and

A. macyae DSM-16346T, respectively. Strain NB2006T differed from other species

of the genus Anaerobacillus in its ability to metabolise isosaccharinate, an

alkaline hydrolysis product of cellulose. Based on the consensus of phylogenetic

and phenotypic analyses, this strain represents a novel species of the genus

Anaerobacillus, for which the name Anaerobacillus isosaccharinicus sp. nov. is

proposed. The type strain is NB2006T (=DSM 100644T =LMG 30032T).

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

The genus Anaerobacillus was proposed by Zavarzina et al. (2009) to contain strictly

anaerobic or aerotolerant, Gram-positive rods that are obligate or moderately

alkaliphilic, halotolerant or moderately halophilic, grow through fermentation or

anaerobic respiration, and are able to reduce arsenate. At the time of writing, the

genus Anaerobacillus comprises only four species with validly published names:

Anaerobacillus macyae [1], Anaerobacillus alkalidiazotrophicus [2], Anaerobacillus

arseniciselenatis [3], and Anaerobacillus alkalilacustris [4], with A. arseniciselenatis

as the type species.

Isosaccharinate is a cellulose alkali hydrolysis product that is found in paper pulp

effluent streams [5]. It is also expected to form under the anaerobic, hyperalkaline

conditions of a geological disposal facility for intermediate-level radioactive waste

[6], and has the potential to increase radionuclide mobility in the subsurface [7, 8].

For these reasons, the microbial degradation of isosaccharinate, has received

considerable recent attention [9–11]. Two aerobic Gram-negative bacteria, capable of

isosaccharinate degradation have been isolated previously [12, 13]. Here we describe

a novel species belonging to the newly established genus Anaerobacillus that utilises

isosaccharinate under anaerobic, alkaline conditions.

An isosaccharinate-degrading, nitrate-reducing enrichment culture grown on minimal

freshwater medium at pH 10 had been set up previously and maintained at 20oC under

anaerobic conditions [14]. The freshwater medium contained per litre 2.5 g NaHCO3,

0.25 g NH4Cl, 0.6 g NaH2PO4.H2O, 0.1 g KCl, 2 g NaNO3, 0.8 g calcium

isosaccharinate and 10 ml of mineral and vitamin mixes [15], and was pH adjusted to

pH 10 using NaOH, and made anaerobic by flushing with N2 for 1 hour [14]. The

enrichment culture was inoculated with 5% (w/v) of a sediment sample from a legacy

lime-kiln in Harpur Hill, Buxton, United Kingdom [16, 17], and was enriched for

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

isosaccharinate-degrading bacteria at 20oC by transferring 1% inoculum into fresh

medium every month [14]. At the end of the fourth subculture of this enrichment

culture, 50 l samples were plated on agar plates as previously described [14]. Only

one colony morphology was observed, and showed protruding, small, round and

transparent colonies after 3 days of incubation under anaerobic conditions at 20oC.

Five representative colonies were selected as described previously [14], and were all

found to degrade isosaccharinate under nitrate-reducing conditions at pH 10.

The 16S rRNA genes of these isolates, were amplified by PCR and sequenced using

the following universal primers: 8F (5’-AGAGTTTGATCCTGGCTCA-3’), 530F (5’-

GTGCCAGCMGCCGCGG-3’), 943R (5’-ACCGCTTGTGCGGGCCC-3’), 1492R

(5’-TACGGYTACCTTACGACTT-3’) [18, 19], as described previously [14]. The

almost complete (1520 bp) 16S rRNA gene sequences of these strains showed more

than 99% similarity, indicating that they belong to the same species, therefore, strain

NB2006T was selected as a representative strain. Phylogenetic neighbours with valid

names based on the 16S rRNA gene sequence similarities were identified using the

EZBioCloud identification service (http://www.ezbiocloud.net/; [20]). The 16S rRNA

gene sequence of NB2006T was aligned to available sequences of all species with

valid names belonging to the Anaerobacillus genus, and some related species of the

genus Bacillus that showed more than 96% identity, using Clustal W [21] in the

software package MEGA version 7 [22]. Phylogenetic trees were then reconstructed

with the neighbour-joining algorithm [23] using maximum composite likelihood

distance [24], and the maximum-likelihood algorithm using Kimura's two-parameter

model [25], with the complete deletion option, gamma distributed with invariant sites

value of 5, and bootstrap [26] of 1000 replications.

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

Phylogenetic analysis based on the 16S rRNA gene sequence showed that NB2006T

was most closely related to species of the newly established genus Anaerobacillus, A.

macyae JMM-4T (97.96%), A. alkalidiazotrophicus MS6T (97.56%), A.

arseniciselenatis DSM 15340T (97.45%), and A. alkalilacustris Z-0521T (97.01%).

Both the neighbour-joining and maximum-likelihood phylogenetic trees revealed that

strain NB2006T clustered with other members of the genus Anaerobacillus with a

bootstrap value of 99 %, and clearly separated from the aerobic, alkaliphilic bacilli

(Figure S1 available in the online Supplementary Material).

Strain NB2006T was characterised using a polyphasic taxonomic approach and

compared to reference Anaerobacillus strains obtained from the Deutsche Sammlung

von Mikroorganismen und Zellkulturen (DSMZ) culture collection (Braunschweig,

Germany): A. arseniciselenatis DSM-15340T (=E1HT =ATCC 700614T =KCTC

5192T), A. alkalidiazotrophicus DSM-22531T (=MS 6T =NCCB 100213T =UNIQEM

U377T), and A. alkalilacustris DSM-18345T (=Z-0521T =VKM B-2403T). These

strains grew well in the same medium as NB2006T (for A. arseniciselenatis DSM-

15340T, 0.9% NaCl was added). A. macyae DSM-16346T (=JMM-4T = CIP 108766T

=JCM 12340T) did not grow in the same medium, therefore, it was not included in the

phenotypic analysis.

NB2006T was maintained in a liquid medium containing, per litre 5 g Na2CO3, 2.5 g

NaHCO3, 0.25 g NH4Cl, 0.6 g NaH2PO4.H2O, 0.1 g KCl, 2 g NaNO3, 3 g yeast extract

and 10 ml of the same mineral mix [15], at pH 9.8 and 30oC. It was also maintained at

4oC on solid agar plates containing the same medium, or in 20% (v/v)

glycerol:medium at -80oC. Colony appearance was examined after incubation on solid

medium at 30°C for 3 days. Growth at different pH values (pH 7.5–11.5, at intervals

of 0.5 pH units, adjusted by varying the ratio of NaHCO3 and Na2CO3), temperatures

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

(4, 10, 20, 30, 35, 40°C), NaCl (w/v) concentrations (0, 0.5, 1.0, 2.0, 3.0, 3.5, 4.0, 4.5,

5.0, 6.0, 9.0 and 12.0%) and NaHCO3 (w/v) concentrations (0, 0.5, 1.0, 1.5, 2.0, 2.5,

3.0, 3.5, 4.0, 4.5 and 5.0%) was tested by measuring the increase in optical density at

a wavelength of 600 nm (OD600nm) after incubation of liquid cultures for 3 days at

30oC (except for the temperature test). The relationship to oxygen was assessed by

observing bacterial growth in vertical agar tubes containing resazurin solution, and by

observing bacterial growth in liquid cultures flushed with different ratios of O2:N2 (0-

20% O2, in 5% increments). The morphology of the cells and spores was examined by

light and electron microscopy. Gram-staining was done on heat-fixed slides using a

Gram-staining kit (Sigma). Flagella were stained on air-dried slides using Flagella

Stain Droppers (BD). Endospores were stained on heat-fixed slides with malachite

green (Pro-Lab) over steam for 5 min and then counterstained with Safranin T

solution (Fluka) for 1 min. Motility was observed in wet mounts, and by growth on

semisolid culture medium (by adding 0.1% (w/v) agar). Catalase activity was

determined by production of bubbles after the addition of a drop of 3% (v/v) H2O2.

Oxidase activity was determined by the oxidation of tetramethyl-p-phenylenediamine.

Casein, gelatin and starch hydrolysis were tested on skimmed milk agar plates

(Sigma, by checking for discolouration of the media), gelatin tubes (Sigma, by

checking for solidification of the media at 4oC), and soluble starch agar plates (Fisher,

by checking for colouration after the addition of Gram’s iodine solution), incubated

for 3 days at 30oC. All experiments were prepared in duplicate, and the reference

strains were tested alongside the novel isolate (NB2006T).

The utilisation of different electron donors and acceptors was tested in 10 ml butyl

stoppered serum bottles containing the same anaerobic freshwater minimal medium,

and measuring the OD600nm at the beginning of the experiment, and after 3 days of

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

incubation at 30oC. The concentrations of the carboxylic acids, nitrate, nitrite,

selenate, and selenite were measured by ion exchange chromatography as previously

described [14]. The concentrations of arsenate and arsenite in solution were

determined by coupled ion chromatography-inductively coupled plasma-mass

spectrometry using the method developed previously [27]. For the electron donor

tests, the yeast extract was removed from the growth medium, and 5 mM of the Na

salt of carboxylic acids (formate, acetate, lactate, pyruvate, succinate, malate,

fumarate, gluconate, isosaccharinate, citrate, malonate), sugars and sugar alcohols

(glucose, fructose, sucrose, lactose, sorbitol, mannitol, myo-inositol), and amino acids

(glycine, serine, cysteine, glutamate, histidine, asparagine, arginine) were added. For

the utilisation of electron acceptor tests, nitrate was removed and the medium was

supplemented with 10 mM Na2SO4, 2 mM Na2SeO4, or 2 mM Na3AsO4. All

experiments were prepared in duplicate, and the reference strains were tested

alongside strain NB2006T.

Colonies were protruding, small, round and transparent after 3 days of incubation

under anaerobic conditions at 30oC. Growth was observed after 3 days of incubation

at pH values between 8.5 and 11, with optimal growth at pH 9.8-10. Temperatures

between 10 and 40oC supported growth, and 30oC was optimal. NB2006T grew in the

same minimal medium supplemented with 0-6.0% (w/v) NaCl (optimal at 2.0%), and

0-5.0% (w/v) NaHCO3 (optimal at 0.5%). NB2006T showed growth in the anaerobic

zone in vertical agar tubes, and grew in liquid cultures flushed with 0-10% O2. Light

and electron microscopy showed that NB2006T cells are Gram-positive rods, that are

2-5x0.5-0.7 m in size (Figure S2 available in the online Supplementary Material),

motile using 4 peritricus flagella, can live singly or in chains of up to 6 connected

rods, and can form circular, subterminal spores. NB2006T was catalase and oxidase

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

positive, hydrolysed gelatin and starch, and was able to utilise lactate, pyruvate,

succinate, malate, fumarate, gluconate, isosaccharinate, glucose, fructose, sucrose,

serine, asparagine, and arginine as electron donors. It grew by anaerobic respiration

where it reduced nitrate to nitrite, and arsenate to arsenite. It also grew by

fermentation of yeast extract or gluconate, which led to the production of acetate and

formate as fermentation products. A phenotypic and biochemical comparison between

strain NB2006T and the selected type strains of species of the genus Anaerobacillus

showed metabolic differences between the tested strains (Table S1 available in the

online Supplementary Material).

Cellular fatty acid composition, cell-wall peptidoglycan analysis, and determination of

the DNA G+C content of strain NB2006T were performed by the DSMZ Identification

Service (Braunschweig, Germany).

The predominant cellular fatty acids (>5%) of strain NB2006T were C16:0 (23.35%),

C16:111c (22.96%), anteiso-C15:0 (15.33%), iso-C15:0 (8.30%), C16:17c/iso-C15:02OH

(6.28%), and C14:0 (5.48%) which is consistent with other members of the genus

Anaerobacillus [2, 4]. The cell-wall diagnostic diamino acid was meso-

diaminopimelic acid, and the peptidoglycan type was A1γ. The DNA G+C content of

strain NB2006T, determined by HPLC, was 37.7 mol%.

Draft genomes of the Anaerobacillus species used in this study had been published

recently [28, 29]. The genome sequences with accession numbers MLQQ01,

MLQS01, MLQR01, LQXD01, and LELK01 were retrieved from the NCBI database.

Original average nucleotide identity, and average nucleotide identity of orthologous

genes were calculated using the Orthologous Average Nucleotide Identity Tool

version 0.93 [30]. Digital DNA–DNA hybridization values were determined using the

Genome-to-Genome Distance Calculator version 2.1 using formula 2 [31]. The

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

genomes were re-annotated using Prokka version 1.11 [32], and the core genome that

contains the genes shared by all species of the Anaerobacillus genus was identified

using the software package Roary version 3.8.0 [33], with a minimum sequence

identity of 70% [34]. A phylogenetic tree was reconstructed using the neighbour-

joining algorithm [23] with maximum composite likelihood distance [24], and the

maximum-likelihood algorithm using Kimura's two-parameter model [25] and a

gamma correction value of 0.5 using MEGA version 7 [22]. Functional annotation

was performed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG)

database (http://www.genome.jp/kegg/), and metabolic pathways for several

phenotypic features were mapped using KEGG Mapper

(http://www.genome.jp/kegg/). Key enzymes were identified from the metabolic

pathway graphs (Figures S3-S12 available in the online Supplementary Material), and

the presence of genes coding for these enzymes in the tested genome was determined

(Table S5 available in the online Supplementary Material).

The genome of NB2006T was 4.95 Mbp, and contained 4984 genes [29]. It is

important to note that the calculated G+C mol% was slightly lower (35.8 mol%) than

that acquired by direct HPLC analysis (Table S2 available in the online

Supplementary Material), which may be due to the genome sequence being

incomplete [29]. The original average nucleotide identity, and the average nucleotide

identity of orthologous genes between NB2006T and the other species of the genus

Anaerobacillus were 66.60-74.92%, and 67.24-75.62% (Table S3 available in the

online Supplementary Material), respectively, well below the threshold of 95-96% for

species delineation [35, 36]. Furthermore, the digital DNA–DNA hybridization values

were 21.5-22.4% (Table S3 available in the online Supplementary Material), well

below the threshold of 70% for species delineation [35]. Phylogenetic analysis of the

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

core genes (294 genes) showed a different tree topology than that based on the 16S

rRNA gene sequence, which is more consistent with phenotypic observations;

NB2006T clustered more closely with the alkaliphilic species of the Anaerobacillus

genus (A. arseniciselenatis, A. alkalilacustris, and A. alkalidiazotrophicus), rather

than with the slightly alkalitolerant species (A. macyae). The strictly fermentative

species (A. alkalilacustris, and A. alkalidiazotrophicus) also clustered together with

this analysis (Figure 1).

Genomic comparisons revealed that NB2006T shared 294 genes with all other

members of the Anaerobacillus genus (core genes), and it shared 1745 genes with at

least 1 species of the genus (accessory genes), while 2932 genes were unique to

NB2006T (Table S4 available in the online Supplementary Material). Generally, the

metabolic functions that were identified from the genome (Table 1) agreed well with

those observed in the growth experiments (Table S1 available in the online

Supplementary Material), except in the dissimilatory nitrate reduction pathway, where

the A. alkalilacustris DSM-18345T genome codes for the periplasmic nitrate reductase

napA (Table S5 and Figure S11 available in the online Supplementary Material), but

the bacterium does not reduce nitrate; and in the sorbitol utilisation pathway, where A.

alkalidiazotrophicus DSM-22531T genome does not code for the L-iditol 2-

dehydrogenase gene (Table S5 and Figure S5 available in the online Supplementary

Material), but the biochemical tests showed that it can degrade sorbitol. It is possible

that these inconsistencies are due to the incomplete nature of the genomes being

compared, and may be resolved when complete genomes are sequenced and

annotated.

Based on the consensus of phylogenetic and phenotypic analyses, strain NB2006T

represents a novel species of the genus Anaerobacillus, for which the name

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

Anaerobacillus isosaccharinicus sp. nov. is proposed. The ability to utilise

isosaccharinic acid has not been demonstrated previously for this genus, and indeed

has not been demonstrated under anaerobic conditions for any isolate in pure culture

before.

Description of Anaerobacillus isosaccharinicus sp. nov.

Anaerobacillus isosaccharinicus [i.so.sac.cha.ri'ni.cus. N.L. neut. n. acidum

isosaccharinicum isosaccharinic acid; N.L. masc. adj. isosaccharinicus pertaining to

isosaccharinic acid].

Cells are Gram-stain-positive, aerotolerant anaerobes, motile using 4 peritrichous

flagella, rods of 0.5–0.7 µm diameter, and 2–5 µm length, and form circular

subterminal endospores with unswollen sporangia. Colonies were protruding, small,

round and transparent after 3 days of incubation under anaerobic conditions at 30oC.

Bacterial growth was observed at pH values between 8.5 and 11 (optimal at 9.8-10),

temperature between 10 and 40oC (optimal at 30oC), 0-6.0% (w/v) NaCl (optimal at

2.0%), and 0-5.0% (w/v) NaHCO3 (optimal at 0.5%). Cells were catalase- and

oxidase-positive. The cell-wall peptidoglycan contains meso-diaminopimelic acid.

The primary fatty acids were C16:0, C16:111c, anteiso-C15:0, iso-C15:0, C16:17c/iso-

C15:02OH, and C14:0. The strain hydrolyses starch and gelatin, and utilises lactate,

pyruvate, succinate, malate, fumarate, gluconate, isosaccharinate, glucose, fructose,

sucrose, serine, asparagine, and arginine as electron donors. It reduces nitrate to

nitrite, and arsenate to arsenite, and ferments yeast extract and gluconate, releasing

acetate and formate as fermentation products.

The type strain is NB2006T (=DSM 100644T, =LMG 30032T), which was isolated

from a legacy limekiln effluent in Harpur Hill, Buxton, UK. The DNA G+C content

of the type strain is 37.7 mol%.

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

Funding Information

This work was supported by the BIGRAD consortium under the UK Natural

Environmental Research Council, (NE/H007768/1). This project has also received

funding from the Euratom research and training programme 2014-2018 under grant

agreement No 661880.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

References

1. Santini JM, Streimann ICA, vanden Hoven RN. Bacillus macyae sp. nov., an arsenate-respiring bacterium isolated from an Australian gold mine. Int J Syst Evol Microbiol 2004;54:2241–4.

2. Sorokin ID, Kravchenko IK, Tourova TP, Kolganova TV, Boulygina ES, et al. Bacillus alkalidiazotrophicus sp. nov., a diazotrophic, low salt-tolerant alkaliphile isolated from Mongolian soda soil. Int J Syst Evol Microbiol 2008;58:2459–64.

3. Blum J, Bindi A, Buzzelli J, Stolz J, Oremland R. Bacillus arsenicoselenatis, sp. nov., and Bacillus selenitireducens, sp. nov.: two haloalkaliphiles from Mono Lake, California that respire oxyanions of selenium and arsenic. Arch Microbiol 1998;171:19–30.

4. Zavarzina DG, Tourova TP, Kolganova TV, Boulygina ES, Zhilina TN. Description of Anaerobacillus alkalilacustre gen. nov., sp. nov.—Strictly anaerobic diazotrophic Bacillus isolated from soda lake and transfer of Bacillus arseniciselenatis, Bacillus macyae, and Bacillus alkalidiazotrophicus to Anaerobacillus as the new combinations A. arseniciselenatis comb. nov., A. macyae comb. nov., and A. alkalidiazotrophicus comb. nov. Microbiology 2009;78:723–731.

5. Sjöström E. Wood chemistry, fundamentals and applications. 1993. Epub ahead of print 1993. DOI: 10.1016/0008-6215(94)90030-2.

6. Glaus MA, van Loon LR. Degradation of cellulose under alkaline conditions: New insights from a 12 years degradation study. Environ Sci Technol 2008;42:2906–2911.

7. Warwick P, Evans N, Vines S. Studies on some divalent metal α-isosaccharinic acid complexes. Radiochim Acta 2006;94:363–368.

8. Keith-Roach MJ. The speciation, stability, solubility and biodegradation of organic co-contaminant radionuclide complexes: A review. Sci Total Environ 2008;396:1–11.

9. Kuippers G, Bassil NM, Boothman C, Bryan N, Lloyd JR. Microbial degradation of isosaccharinic acid under conditions representative for the far field of radioactive waste disposal facilities. Mineral Mag 2015;79:1443–1454.

262

263

264

265

266

267

268

269

270271272

273274275276

277278279280

281282283284285286287

288289

290291292

293294

295296297

298299300

10. Kyeremeh IA, Charles CJ, Rout SP, Laws AP, Humphreys PN. Microbial community evolution is significantly impacted by the use of calcium isosaccharinic acid as an analogue for the products of alkaline cellulose degradation. PLoS One 2016;11:1–10.

11. Charles CJ, Rout SP, Garratt EJ, Patel K, Laws AP, et al. The enrichment of an alkaliphilic biofilm consortia capable of the anaerobic degradation of isosaccharinic acid from cellulosic materials incubated within an anthropogenic, hyperalkaline environment. FEMS Microbiol Ecol;91. Epub ahead of print 2015.

12. Strand S, Dykes J, Chiang V. Aerobic microbial degradation of glucoisosaccharinic acid. Appl Environ Microbiol 1984;47:268–271.

13. Bailey M. Utilization of glucoisosaccharinic acid by a bacterial isolate unable to metabolize glucose. Appl Microbiol Biotechnol 1986;1206:493–498.

14. Bassil NM, Bryan N, Lloyd JR. Microbial degradation of isosaccharinic acid at high pH. ISME J 2015;9:310–320.

15. Lovley D, Greening R, Ferry J. Rapidly growing rumen methanogenic organism that synthesizes coenzyme M and has a high affinity for formate. Appl Environ Microbiol 1984;48:81–87.

16. Rout SP, Charles CJ, Garratt EJ, Laws AP, Gunn J, et al. Evidence of the generation of isosaccharinic acids and their subsequent degradation by local microbial consortia within hyper-alkaline contaminated soils, with relevance to intermediate level radioactive waste disposal. PLoS One 2015;10:1–13.

17. Rizoulis A, Steele HM, Morris K, Lloyd JR. The potential impact of anaerobic microbial metabolism during the geological disposal of intermediate-level waste. Mineral Mag 2012;76:3261–3270.

18. Lane DJ. 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (editors). Nucleic acid techniques in bacterial systematics. London: John Wiley & Sons Ltd. pp. 115–174.

19. Handley KM, Héry M, Lloyd JR. Marinobacter santoriniensis sp. nov., an arsenate-respiring and arsenite-oxidizing bacterium isolated from hydrothermal sediment. Int J Syst Evol Microbiol 2009;59:886–92.

20. Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, et al. Introducing EzBioCloud: A taxonomically united database of 16S rRNA and whole genome assemblies. Int J Syst Evol Microbiol. Epub ahead of print 22 December 2016. DOI: 10.1099/ijsem.0.001755.

21. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:4673–4680.

22. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol 2016;33:1870–1874.

23. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406–25.

24. Tamura K, Nei M, Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci 2004;101:11030–

301302303304

305306307308309

310311

312313

314315

316317318

319320321322

323324325

326327328

329330331

332333334335

336337338339

340341

342343

344345

11035.25. Kimura M. A simple method for estimating evolutionary rate of base

substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980;16:111–120.

26. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985;39:783–791.

27. Gault AG, Jana J, Chakraborty S, Mukherjee P, Sarkar M, et al. Preservation strategies for inorganic arsenic species in high iron, low-Eh groundwater from West Bengal, India. Anal Bioanal Chem 2005;381:347–353.

28. Wang J, Liu B, Liu G, Ge C, Chen Q, et al. Genome Sequence of Anaerobacillus macyae JMM-4 T (DSM 16346), the first genomic information of the newly established genus Anaerobacillus. Genome Announc 2015;3:e00922-15.

29. Bassil NM, Lloyd JR. draft genome sequences of four alkaliphilic bacteria belonging to the Anaerobacillus genus. Genome Announc 2017;5:e01493-16.

30. Lee I, Kim YO, Park SC, Chun J. OrthoANI: An improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol 2016;66:1100–1103.

31. Meier-Kolthoff JP, Auch AF, Klenk HP, Göker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 2013;14:60.

32. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 2014;30:2068–2069.

33. Page AJ, Cummins CA, Hunt M, Wong VK, Reuter S, et al. Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics 2015;31:3691–3693.

34. Chan JZM, Halachev MR, Loman NJ, Constantinidou C, Pallen MJ. Defining bacterial species in the genomic era: insights from the genus Acinetobacter. BMC Microbiol 2012;12:302.

35. Wayne LG, Brenner DJ, Colwell RR, Grimont PAD, Kandler O, et al. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 1987;37:463–464.

36. Meier-Kolthoff JP, Göker M, Spröer C, Klenk H-P. When should a DDH experiment be mandatory in microbial taxonomy? Arch Microbiol 2013;195:413–8.

346

347348349

350351

352353354

355356357358

359360

361362363

364365366

367368

369370371

372373374

375376377

378379380

381

382

Figure legends

Figure 1: Neighbour-joining phylogenetic tree reconstructed from the core genes

(281 genes) of species closely related to NB2006T. Bootstrap values >50 %, based on

1000 replicates, are indicated on branch points. Bacillus subtilis was used as an

outgroup. Bar, 0.05 nucleotide substitutions per site. A maximum-likelihood

phylogenetic tree showed the same results as the Neighbour-Joining phylogenetic tree.

383

384

385

386

387

388

389

Tables with legends

Table 1: Comparison of phenotypic features captured from the genomes of 1) A.

isosaccharinicus NB2006T and related strains, 2) A. arseniciselenatis DSM-15340T, 3)

A. alkalidiazotrophicus DSM-22531T, and 4) A. alkalilacustris DSM-18345T. *

represents features that show differences between the observed phenotype and that

captured from the genomes. ** represents features that show agreement between the

observed phenotype and that captured from the genomes.

1 2 3 4Catalase** + + + +Oxidase** + + - -

Reduction ofNitrate* + + - +Sulfate** - - - -

Utilisation ofFructose** + + + +Galactose** - - + +Sucrose** + + + +Cellobiose - - + +Trehalose - - + +Arabinose - - - -Mannose - - - -Xylose + - + -Sorbitol* - + - +Mannitol** - - + +myo-Inositol** - - - -L-leucine - - - -

Production ofIndol + + - -Acetoin (Voges-Proskauer test)

- - - -

390

391

392

393

394

395

396

397