Amphibacillus marinus sp. nov., a new member of the genus 1

Amphibacillus isolated from the South China Sea 2

Biao Ren1,3†, Na Yang1,3†, Jian Wang1, Xiao-Long Ma1, Qian Wang1,3, Feng Xie1,3, 3

Hui Guo1,3, Zhi-Heng Liu1, Benoît Pugin4, Li-Xin Zhang1,2* 4

5

(1) Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology, 6

Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic 7

of China. 8

(2) South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, 9

P. R. China 10

(3) Graduate School of Chinese Academy of Sciences, Beijing, 100049, P. R. China 11

(4) Laboratorio de Microbiología Molecular, Departamento de Biología, Facultad de Química y 12

Biología, Universidad de Santiago de Chile, Santiago, Chile 13

14

* Author for Correspondence: Li-Xin Zhang；lzhang03@gmail.com 15

16

Running title: Amphibacillus marinus sp. nov 17

Category: New taxa-Firmicutes and Related Organisms 18

† These authors contributed equally to this work. 19

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of 20

strain J1T is GU213062. 21

22

IJSEM Papers in Press. Published August 3, 2012 as doi:10.1099/ijs.0.045807-0

A Gram-positive, spore-forming, rod-shaped bacterium, designated J1T was 23

isolated from deep sea mud collected from the South China Sea, and subjected to 24

polyphasic taxonomic investigation. Phylogenetic analysis based on 16S rRNA 25

gene sequences revealed that J1T clustered with the type strains of Amphibacillus 26

cookii, Amphibacillus sediminis and Amphibacillus jilinensis，and exhibited the 27

range of similarity of 93.9%-97.0% to the species in genus Amphibacillus. The 28

DNA G+C content was 36.7%. Chemotaxonomic analysis showed no quinones, 29

and the cell wall contained meso-diaminopimelic acid as the diagnostic diamino 30

acid for strain J1T. The major cellular fatty acids were iso-C15:0 and anteiso-C15:0. 31

The strain J1T was positive for catalase activity and negative for oxidase activity. 32

On the basis of phylogenetic position and phenotypic properties, strain J1T 33

represents a new species of the genus Amphibacillus and the name Amphibacillus 34

marinus sp. nov. is proposed. The type strain is J1T (=CGMCC 1.10434T = JCM 35

17099T). 36

37

Exploration of the microbial diversity in marine environment showed an intriguing 38

picture for that many bioactive compounds isolated from marine-derived 39

microorganisms (Demain & Zhang, 2005). The main interest of our group is to 40

construct a high quality marine microbial natural products library to screen bioactive 41

metabolites by high throughput techniques (Bian et al., 2008). Based on the previous 42

microbial diversity research on the South China Sea sediments, many new species 43

have been isolated in our lab, such as Amycolatopsis marina (Bian et al., 2009), 44

Verrucosispora sediminis (Dai et al., 2010) and Prauserella marina (Wang et al., 45

2010) among which the Prauserella marina had the anti-BCG (Bacille 46

Calmette-Guérin) activity while Verrucosispora sediminis had the antifungal and 47

antibacterial activities. Recently, another new bio-surfactant-producing strain 48

belonging to the genus Amphibacillus was isolated by using an alkaline medium. 49

The genus Amphibacillus was established by Niimura et al. (1990) and to date, only 50

seven species have been described, namely, Amphibacillus xylanus (Niimura et al., 51

1990), Amphibacillus fermentum (Zhilina et al., 2001), Amphibacillus tropicus 52

(Zhilina et al., 2001), Amphibacillus sediminis (An et al., 2007), Amphibacillus 53

jilinensis (Wu et al., 2010), Amphibacillus cookii (Pugin et al., 2011) and 54

Amphibacillus indicireducens (Hirota et al., 2012). None of these species were 55

isolated from the marine environment as strain J1T was in this genus. The genus 56

Amphibacillus was characterized by spore-forming, rod-shaped, straight or slightly 57

curved and motile, cells which grew at pH 7.0 and 12.0, containing 58

meso-diaminopimelic acid in the cell wall, with anteiso- and iso- branched and 59

straight-chain acids as the major cellular fatty acids, lacking isoprenoid quinones and 60

oxidase activity, variable catalase activity and with the G+C mol% between 36-42% 61

(An et al., 2007; Hirota et al., 2012; Niimura et al., 1990). 62

Marine derived strain J1T was originally isolated from a deep sea mud [GPS 63

coordinates for the sample site are 114º34’58.314” E, 17º53’59.545” N, at a depth of 64

3601 meters], after 4 weeks incubation in Horikoshi medium (Horikoshi, 1999) 65

(glucose 10.0 g, yeast extract 5.0 g, polypeptone 5.0 g, KH2PO4 1.0 g, MgSO4 0.2 g, 66

Na2CO3 10.0 g, NaCl 50.0 g, distilled water 1000 mL, natural pH value) at 28 °C. The 67

isolate was maintained on Horikoshi or DSMZ medium 529 slants at 4 °C and as 68

suspensions of clones in glycerol (25%, v/v) at -70 °C. Biomass for chemical and 69

molecular studies was obtained by cultivation in shaking flasks (200 r.p.m) with 70

DSMZ medium 529 broth at 28 °C for 3 days. 71

Genomic DNA extraction, PCR amplification and 16S rRNA gene sequencing of 72

isolate J1T were carried out according to the procedures described by Kim et al. 73

(1998). The initial taxonomic classification of the 16S rRNA sequence was carried out 74

by using the IDENTIFY program of the online sever of the EzTaxon 75

(http;//147.47.212.35:8080/) (Chun et al., 2007). Sequences longer than 1300nt or 76

without ambiguous nucleotides of the most closely related strains were downloaded 77

from the DDBJ/EMBL/GenBank. Multiple alignments with all the cited 16S rRNA 78

sequences and calculations of levels of sequence similarity were carried out using 79

CLUSTAL W (Thompson et al., 1994). The phylogenetic tree was constructed using 80

three methods, including the neighbor-joining (NJ) tree (Saitou & Nei, 1987) using 81

the software package Mega version 4.0 (Tamura et al., 2007); the 82

maximum-parsimony (MP) tree using the software package PHYLIP version 3.6 83

(Felsenstein, 2002); the maximum-likelihood (ML) tree using the online version of 84

PhyML (Guindon et al., 2010). The topology of the phylogenetic trees was evaluated 85

by bootstrap re-sampling method with 1000 replicates (Felsenstein, 1985). The 86

phylogenetic tree shown in Fig. 1 indicated that strain J1T belonged to the genus 87

Amphibacillus. Genomic DNA for the determination of the G+C content was prepared 88

according to the method of Marmur (1961) and was determined by the thermal 89

denaturation (Tm) method (Mandel et al., 1968) with Escherichia coli K-12 (CGMCC 90

1.748) as the reference strain using the PerkinElmer’s LAMBDA 35 UV/Vis 91

spectrophotometer fitted with a thermal controller. 92

The highest degree of 16S rRNA gene sequence similarity of strain J1T (1490 nt) was 93

found with A. cookii (97.0%), A. sediminis (96.9%), followed by A. jilinensis (96.7%). 94

Phylogenetic analysis based on 16S rRNA sequence analysis revealed that strain J1T 95

formed a cluster with the three most closely related species A. cookii, A. jilinensis and 96

A. sediminis (Fig. 1). MP and ML trees were similar to the NJ tree. All the trees 97

supported that isolate J1T belonged to the genus Amphibacillus. The DNA G+C 98

content of strain J1T was 36.7%. 99

Chemosystematic studies were carried out to compare J1T chemical profile and that of 100

A. jilinensis Y1T (=CGMCC 1.5123T), A. sediminis Shu-P-Ggiii25-2T (=JCM 23213T), 101

A. xylanus Ep01T (=DSM 6626T), A. fermentum Z-7984T (=DSM 13869T), A. tropicus 102

Z-7792T (=DSM 13870T), A. cookii JW/BP-GSL-QDT(=DSM 23721T) which were 103

supplied by Xufen Zhu from Zhejiang University, the Japan Collection of 104

Microorganisms (JCM), and the German Resource Centre for Biological Material 105

(DSMZ). Fatty acids were extracted, purified, methylated and quantified by gas 106

chromatography (Sasser, 1990) using the standard Microbial Identification System 107

(MIDI Inc; Microbial ID) after cultivation in TSB (tryptic soy broth, BD BactoTM, pH 108

9.0) for 2 days at 28 °C, and were identified by TSBA6 database /peak naming table. 109

Polar lipids were extracted and examined by two-dimensional TLC (Solvent system I: 110

Chloroform: methanol: water=65: 25: 4 (v/v); Solvent system II: Chloroform: acetic 111

acid: methanol: water=80:18:12:5 (v/v); stained with Molybdenum blue reagent, 112

anisaldehyde reagent, ninhydrin reagent, and draggendorff reagent) and identified by 113

using published procedures of Minnikin et al. (1980). Halolactibacillus alkaliphilus 114

CGMCC 1.6843T (Cao et al., 2008), H. miurensis DSM 17074T and H. halophilus 115

JCM 21694T (Ishikawa et al., 2005) were also used as reference strains in polar lipids 116

analysis. The analysis of cell-wall peptidoglycan was modified from the methods of 117

Schleifer & Kandler (1972) and Hasegawa et al. (1983). Generally, about two loops 118

of strains were put in an ampoule tube, and then 0.2 mL 6 M HCl was added. Sealed 119

the tube by alcohol torch and incubated at 120 oC for about 4 h until the color of the 120

hydrolysate turn to dark brown. After cooling, 5 μL hydrolysate was directly spotted 121

on a thin cellulose plate (microcrystalline powder, Merck). 1μL standard solution 122

contained DD-, meso- and LL-diaminopimelic acid were spotted on the same plate. 123

Spread the plate twice with the solvent solution (methanol : pyridine : acetic acid : 124

water = 10 : 1 : 0.25 : 5, v/v) after air dried the TLC plates, 0.4% ninhydrin solution 125

was sprayed on and heated at 110 oC for 2-3 min. Isoprenoid quinones were isolated 126

and extracted according to Minnikin et al. (1984), and separated by HPLC 127

(Kroppenstedt, 1982). 128

Strain J1T contained iso-C15:0 (29.6%) and anteiso-C15:0 (36.2%) as the major cellular 129

fatty acid, which were characteristically different to that of other species in the genus 130

Amphibacillus (Table 1). Four polar lipids including diphosphatidylglycerol (DPG), 131

phosphatidylglycerol (PG), one middle Rf value unknown phospholipid (PL7) and 132

one low Rf value unknown phospholipid (PL4) were detected in strain J1T and other 133

six Amphibacillus species. Other unknown polar lipids detected in trace amount on the 134

TLC plates were obviously different among these seven strains (Fig. S3). The 135

presence of the different types of the unknown polar lipids can clearly separate the 136

genus Amphibacillus from the genus Halolactibacillus though the later genus also 137

contained DPG and PG (Fig. S3). The genus Paraliobacillus contained PME and PC 138

as major polar lipid (Chen et al., 2009)，which was quite different from the genus 139

Amphibacillus. The diagnostic cell wall diamino acid in the peptidoglycan layer of the 140

isolate J1T and the five reference strains was meso-diaminopimelic acid except strain 141

A. xylanus possessing DD-diaminopimelic acid as well (Fig. S4). In congruence with 142

the other species of Amphibacillus, strain J1T did not contain isoprenoid quinone. 143

No-isoprenoid quinone can also indicate that strain J1T did not belong to genus 144

Paraliobacillus for which contained menaquinone-7 as the major isoprenoid quinine 145

(Chen et al., 2009; Ishikawa et al., 2002). 146

Gram staining of J1T was carried out by the standard Gram reaction and was 147

confirmed by using the non-staining method (Buck, 1982). Cell morphology was 148

examined using a light microscopy (model BH2; Olympus) and a transmission 149

electron microscopy (JEM 1400). For transmission electron microscopy observation, 150

cells were negatively stained with 1% (w/v) phosphotungstic acid after air-drying. 151

Motility was observed at 12 and 36 h with the light microscope. Anaerobic growth 152

was tested in fluid tetrathionate medium. Colony morphology was observed on DSMZ 153

medium 529 plate after incubation at 28 °C for 3 days. DSMZ medium 529 broth was 154

used for testing the growth at various temperatures (4-57 °C), different pHs (6.0-11.0) 155

and NaCl (0-24%, w/v) concentrations. Appropriate biological buffers were used for 156

different pH values: Na2HPO4/ NaH2PO4 buffer, glycine/ NaOH buffer, Na2HPO4/ 157

NaOH buffer for pH below 8.0, pH 8.0-11.0 and pH above 11.0, respectively. Catalase 158

and oxidase activities, hydrolysis of casein, gelatin, Tween-20, 40 and 80, nitrate 159

reduction, H2S production were determined as reported by Barrow & Feltham (1993). 160

Hydrolysis of starch was tested using starch agar with 95% ethanol and Gram’s iodine 161

(Claus & Berkeley, 1986). Metabolism of citrate was tested by using Simmon’s citrate 162

medium (Smibert, 1981). Sole carbon source utilization tests were performed by using 163

a minimal medium ((NH4)2SO4 2.0 g, NaH2PO4 0.5 g, K2HPO4 0.5 g, MgSO4 0.2 g, 164

CaCl2 0.1 g, distilled water 1000 mL). The corresponding filtration-sterilized sugar 165

(1%, w/v), alcohol and glycerol (0.1%, w/v), organic acid and amino acids (0.1%, 166

w/v), casein hydrolysate, yeast extract, tryptone, starch (each at 10 g/L) were added to 167

the minimal medium. 168

Isolate J1T formed circular, convex and semi-opaque white colonies on DSMZ 169

medium 529 plate after 3 days cultivation at 28 °C. Good growth occurred at 7-55 °C 170

(optimal, 28 °C), pH 7.5-10.0 (optimal 9.0). NaCl was not required for growth and 171

could be tolerated at concentrations up to 12 % (w/v) NaCl (optimal, 5-11). The 172

isolate J1T was Gram-positive, catalase-positive, oxidase-negative, weak positive to 173

Tween-40. Fresh J1T cells were rod-shaped (0.2-0.5 μm wide and 1.0-4.0 μm long), 174

motile with flagella (Fig. S1). Ellipsoidal endospores were observed after 3 days 175

cultivation on ten times diluted DSMZ medium 529 broth (Fig. S2). Forming 176

endospores indicated that strain J1T did not belong to genus Halolactibacillus since 177

non-spore-form is one of the obvious phenotypic character of genus Halolactibacillus. 178

Results obtained from carbon utilization pattern revealed that the isolate had the 179

ability to utilize following substrates for growth: D-fructose, D-trehalose, -lactose, 180

L-rhamnose, maltose, D-cellobiose, D-xylose, D-galactose, D-arabinose, methanol, 181

D-sorbitol, D-mannitol, glycerol, inositol, starch, and casein hydrolysate. Other 182

phenotypic characters and comparisons with reference strains were listed in Table 2. 183

Thus, based on the phylogenetic analysis, chemotaxonomic properties and the profile 184

of metabolic properties revealed that strain J1T belonged to the genus Amphibacillus. 185

However, the phylogenetic distances from known Amphibacillus species and unique 186

phenotypic character represented that strain J1T was a novel species within the genus 187

Amphibacillus, for which the name Amphibacillus marinus sp. nov. was proposed. 188

Emended description of the genus Amphibacillus Niimura et al. (1990), An et al. (2007) and 189

Hirota et al. (2012) 190

Amphibacillus (Am.phi.ba.cil′lus. Gr. Pref. Amphi both sides or double ; L. Dim. N. 191

bacillus a small rod; N.L. masc. n. Amphibacillus rod capable of both aerobic and 192

anaerobic growth). 193

The description is based on that given by Niimura et al. (1990), An et al. (2007) and 194

Hirota et al. (2012) with the following additions. Cell wall contains 195

meso-diaminopimelic acid or contains both meso-diaminopimelic and 196

DD-diaminopimelic acid. Characteristic cellular polar lipids are 197

diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and some other unknown 198

polar lipids. The type species of the genus is Amphibacillus xylanus. 199

Description of Amphibacillus marinus sp. nov 200

Amphibacillus marinus (ma.ri′nus. L. masc. adj. marinus of the sea, marine). 201

Cells are rod-shaped, approximately 0.2-0.5 μm wide and 1.0-4.0 μm long, facultative 202

aerobic, Gram-positive, and motile by means of several flagella. Ellipsoidal 203

endospores are produced within a swollen sporangium, and situated terminal. 204

Colonies grown on DSMZ medium 529 plate are circular, convex and semi-opaque 205

white. The optimal temperature is 28 °C; growth occurs at 7-55 °C. Optimal pH for 206

growth is 9.0 with growth occurring at the pH range of 7.5-10.0. NaCl is not required 207

for growth and can be tolerated at concentrations up to 12% NaCl (w/v). Catalase is 208

positive and oxidase is negative. H2S and indole are not produced. Nitrate is not 209

reduced. Urease and gelatinase activities and utilization of citrate are negative. Starch 210

and casein can not be hydrolyzed. The following substrates can be used as the sole 211

carbon sources: D-fructose, D-trehalose, -lactose, L-rhamnose, maltose, 212

D-cellobiose, D-xylose, D-galactose, D-arabinose, methanol, D-sorbitol, D-mannitol, 213

glycerol, inositol, starch and casein hydrolysate. Cell walls contain peptidoglycans of 214

the meso-diaminopimelic acid type. Isoprenoid quinones are not detected but little 215

pigment can be observed. The major cellular fatty acids of strain J1T are iso-C15:0, 216

anteiso-C15:0 and major cellular polar lipids are diphosphatidylglycerol (DPG), 217

phosphatidylglycerol (PG). The genomic DNA G+C content of the type strain is 218

36.7%. 219

The type strain, J1T (=CGMCC 1.10434T = JCM 17099T), was isolated from deep sea 220

mud from the South China Sea. 221

Acknowledgement 222

The authors thank Xufen Zhu, Takuji Kudo, Hans-Peter Klenk, Juergen Wiegel and 223

Yuguang Zhou for kindly providing the reference strains. The authors are very 224

grateful for the technical support provided by Yuguang Zhou, Yuqin Zhang, Jingnan 225

Liang, Yuhua Xin, Wenjun Li and Michael Goodfellow. They also would like to 226

thank Elizabeth Ashforth, Sarah A Stanley, Hong Gao, Krishna Bolla and Wenjun Li 227

for their critical reading of the manuscript and helpful discussions. This work was 228

supported in part by grants from the Ministry of Science and Technology of China 229

(2011ZX11102-011-11, 2012CB725203, 2007DFB31620), National Natural Science 230

Foundation of China (31100075, 81102362, 31170095, 31000004) and the CAS Pillar 231

Program (XDA04074000) and. L. Z. is an Awardee for National Distinguished Young 232

Scholar Program in China. 233

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334

Figure legends: 335

336

Fig. 1. Neighbor-joining tree derived from 16S rRNA gene sequences for strain J1T 337

and related members with Bacillus subtilis as an outgroup. Only bootstrap values 338

greater than 50% are shown (1000 resamplings) at nodes. Solid circles indicate that 339

the corresponding nodes were also recovered in maximum-likelihood and 340

maximum-parsimony trees. Bar, 0.005 nucleotide substitutions per site. 341

342

Natronobacillus azotifigens 24KS-1T(EU143681)

Amphibacillus tropicus Z-7792T (AF418602)

Amphibacillus jilinensis Y1T(FJ169626)

Amphibacillus cookii DSM 23721T(HM057160)

Amphibacillus marinus J1T (GU213062)

Amphibacillus sediminis Shu-P-Ggiii25-2T (AB243866)

Amphibacillus fermentum Z-7984T (AF418603)

Amphibacillus xylanus JCM7361T (D82065)

Amphibacillus indicireducens C40T (AB665218)

Paraliobacillus ryukyuensis DSM 15140T (AB087828)

Paraliobacillus quinghaiensis YIM C158T(EU135728)

Halolactibacillus alkaliphilus H-5T (EF554593)

Halolactibacillus miurensis M23-1T (AB196784)

Halolactibacillus halophilus M2-2T (AB196783)

Gracilibacillus halotolerans NNT (AF036922)

Gracilibacillus dipsosauri DSM11125T (AB101591)

Gracilibacillus halophilus YIM C55.5T (EU135704)

Gracilibacillus orientalis XH-63T (AM040716)

Thalassobacillus devorans G-19.1T (AJ717299)

Halobacillus trueperi DSM 10404T (AJ310149)

Bacillus subtilis subsp. subtilis DSM10T (AJ276351)

100

100

90

100

100

99

58

80

67

66

53

50

94

0.005 343

344

Table1. Fatty acid content of strain J1T (A. marinus) and other type species from 345

genus Amphibacillus. 346

347

1, strain J1T; 2, A. jilinensis CGMCC 1.5123T; 3, A. sediminis JCM 23213T; 4, A. xylanus DSM 348

6626T; 5, A. fermentum DSM 13869T; 6, A. tropicus DSM 13870T; 7, A. cookii DSM 23721T. –, 349

not detected. Data for all strains were tested concurrently. 350

351

Fatty acid 1 2 3 4 5 6 7 Saturated

straight-chain

C10:0 0.3 0.8 – – 1.4 0.5 0.6

C11:0 – – – – 0.3 – 0.4

C12:0 1.0 0.4 1.5 0.4 6.7 0.4 0.2

C13:0 0.1 – 0.1 – 1.5 0.2 –

C14:0 7.1 3.7 49.0 6.9 28.1 6.5 4.9

C15:0 – – – – – – –

C16:0 4.9 5.0 27.3 10.4 8.5 6.8 4.4

C17:0 – – – – – – –

C18:0 0.3 – 0.2 – 0.3 – 0.4

Saturated

iso-branched

iso-C11:0 0.3 – – – 0.8 – –

iso-C12:0 – – 0.4 0.3 1.2 0.6 –

iso-C13:0 9.4 1.5 3.3 3.1 11.5 16.6 1.0

iso-C14:0 4.0 2.6 6.8 11.0 2.4 4.4 6.1

iso-C15:0 29.6 40.2 2.7 21.3 6.4 22.3 32.1

iso-C16:0 1.4 2.0 0.6 9.8 0.3 1.0 3.7

iso-C17:0 0.4 0.7 – 1.0 0.2 0.3 0.4

Saturated

anteiso-branched

anteiso-C11:0 – – – – 0.7 – –

anteiso-C13:0 3.7 0.8 1.7 1.5 16.7 5.7 0.5

anteiso-C15:0 36.2 39.5 6.3 31.6 11.8 33.4 43.0

anteiso-C17:0 1.0 2.6 – 2.8 0.4 0.5 1.8

Hydroxy

C9:0 3OH – – – – – – 0.1

C15:0 iso 3OH – 0.1 – – – 0.5 –

C15:0 2OH – – – – – 0.2 –

Summed

features*

1 0.2 – – – 0.4 – –

3 – – – – – – 0.3

352

*Data for all the strains are based on the Microbial Identification System. Summed 353

feature 1 consists of C18:1 ω9c and summed feature 3 consists of 16:1ω7c/16:1ω6c. 354

355

Table2. Differential characteristics of strain J1T (A. marinus) and other species from 356

the genus Amphibacillus. 357

358

1, strain J1T; 2, A. jilinensis CGMCC 1.5123T (Wu et al, 2010); 3, A. sediminis JCM 23213T (An 359

et al, 2007); 4, A. xylanus DSM 6626T (Niimura et al, 1990); 5, A. fermentum DSM 13869T 360

(Zhilina et al, 2001); 6, A. tropicus DSM 13870T(Zhilina et al, 2001); 7, A. cookii DSM 23721T 361

(Pugin et al., 2011). 362

＋, Positive reaction; －, negative reaction; weak, weak positive reaction; ND, not detected; *, 363

G+C content data from literature. 364

365

All strains were negative for oxidase, hydrolysis of Tween-20 and 80, cellulose, citrate, gelatin, 366

urea. All strains were negative for nitrate reduction, H2S production, indole test. Glucose, tryptone 367

and yeast extract can be used as sole carbon source by all strains but chitin can’t be used. 368

369

Characteristic 1 2 3 4 5 6 7

Motility ＋ ＋ － － ＋ ＋ ＋

Colony colour white white white yellowish yellowish white white

Spore formation ＋ ＋ ＋ ＋ － ＋ ＋

Anaerobic growth ＋ ＋ ＋ ＋ ＋ ＋ ＋

Catalase ＋ weak － － ＋ ＋ －

Growth at/in:

NaCl % (w/v) 0-12 0-16 0-12 0-5 1-17 1-12 1-14

Temperature (oC) 7-55 15-55 17-57 17-55 17-57 17-55 12-48

pH 7.5-10.0 7.5-10.5 7.0-10.5 9.0-10.5 8.0-10.5 8.0-11.0 6.5-10.0

Utilization of:

D-Fructose ＋ ＋ － ＋ － － ＋

D-Trehalose ＋ ＋ ＋ － ＋ ＋ ＋

Lactose ＋ － ＋ － ＋ － ＋

L-Rhamnose ＋ ＋ － － － － ＋

L-Sorbose － － － ＋ － － －

D-Raffinose － － ＋ ＋ － － ＋

D-Mannose － － － ＋ － － ＋

Maltose ＋ ＋ ＋ － ＋ － ＋

Sucrose － ＋ － ＋ － － －

D-Cellobiose ＋ ＋ － － ＋ ＋ ＋

D-Xylose weak － ＋ ＋ ＋ weak ＋

D-Galactose ＋ ＋ － － － ＋ ＋

D-Ribose － ＋ ＋ ＋ － － ＋

D-Arabinose ＋ ＋ － ＋ ＋ － ＋

Xylan － ＋ － － － － －

Methanol ＋ ＋ － － ＋ ＋ ＋

D-Sorbitol ＋ ＋ － ＋ － － ＋

D-Mannitol ＋ ＋ － － － － ＋

Glycerol ＋ ＋ － ＋ － － ND

Inositol ＋ ＋ ＋ ＋ ＋ － ＋

Ethanol － ＋ － ＋ － － ＋

Lactate － ＋ － ＋ ＋ － ND

L-Glycine － ＋ － ＋ － － －

L-Methionine － ＋ － － ＋ ＋ －

L-Histidine － ＋ － ＋ ＋ － ＋

L-Alanine － ＋ － ＋ ＋ － －

Starch ＋ ＋ ＋ ＋ － － ＋

Succinate － ＋ － ＋ ＋ － －

Casein hydrolysate ＋ ＋ － ＋ ＋ ＋ ND

Hydrolysis of:

Tween 40 weak ＋ weak ＋ － － ND

Starch － ＋ ＋ － ＋ ＋ ND

Casein － ＋ － － － － ND

DNA G+C content

(mol%) 36.7 37.7* 42.3* 36.0* 41.5* 39.2* 35.4*

370

371

Supplementary figures: 372

Fig. S1. Transmission electron micrograph showing general morphology and flagella 373

of a negatively stained cell of strain J1T cultured on medium Horikoshi agar at 28°C 374

for 3 days. Scale bar = 500.0 nm. 375

376

377

Fig. S2. (a) Light micrograph of the sporulating cell of strain J1T. (b) Transmission 378

electron micrograph of the sporulating cell, bar 1 μm. Arrows show the terminal 379

endospores. Cells of J1T were cultured for 3 days at 28 °C on ten times diluted DSMZ 380

medium 529. 381

382

383

384

385

Fig. S3. Two-dimensional TLC of the polar lipids of the strain J1T (a), A. jilinensis 386

CGMCC 1.5123T (b), A. sediminis JCM 23213T (c), A. xylanus DSM 6626T (d), A. 387

fermentum DSM 13869T (e), A. tropicus DSM 13870T (f), A. cookii DSM 23721T(g), 388

Halolactibacillus alkaliphilus CGMCC 1.6843T (h), H. miurensis DSM 17074T (i) 389

and H. halophilus JCM 21694T (j). 390

DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PL1-12, unknown 391

phospholipids; L1-20, unknown polar lipids; GL1-2, unknown glycolipids. 392

Polar lipids of the genus Amphibacillus: 393

L1

DPGPG

PL7

L2

PL2PL4

PL6PL5

DPG

PGPL7

PL2PL3PL4

L3

L4

DPG

PG

PL7

PL3PL4

L3

L9PL6

PG

PL7

PL4

DPG

×

DPG

PGGL1

L8

PL7

PL4

PL5

DPGPG

L10PL7PL2

PL4

PL8

PL1

L5

(a) (b) (c)

(d) (e) (f)

L6 L7

DPG

PG

PL7

PL2PL4

(g)

394

395

Polar lipids of the genus Halolactibacillus: 396

DPG

PG

GL2PL9

PL12

PL10

DPG

PG

PL9

L12

L11

DPG

PG

PL9

PL5

L11

L18L19

L17

L20

PL11

PL8

(h) (i) (j)

PL10

L12

L13L14

L15L16

L15

397

398

Fig. S4. TLC showing the DAP types of all the Amphibacillus strains. The cellulose 399

microcrystalline powder (Merck, 1023300500) was suspended in water (1:5.5, v/v) 400

and blended in a mortar for 30min. And then spread the mixture on the glass plate 401

(2010 cm) and air dried the plate overnight. 5 L samples were spotted and spread 402

twice with the solvent solution (methanol : pyridine : acetic acid : water = 10 : 1 : 403

0.25 : 5, v/v) after air dried the TLC plates, 0.4% ninhydrin solution was sprayed on 404

and heated at 110 oC for 2-3 min. 405

S, standard sample (Sigma D1377, CAS No. 583-93-7); J1T, strain J1T; Y1T, A. 406

jilinensis CGMCC 1.5123T; Shu-P-Ggiii25-2T, A. sediminis JCM 23213T; Ep01T, A. 407

xylanus DSM 6626T; Z-7984T, A. fermentum DSM 13869T; Z-7792T, A. tropicus 408

DSM 13870T, A. cookii DSM 23721T; DD, DD- diaminopimelic acid; meso, meso- 409

diaminopimelic acid; LL, LL- diaminopimelic acid. 410

Arrow shows the meso-DAP of strain J1T. 411

412

LLmesoDD

DSM 23721T

413 414

415