For Peer Review
Bergey?s Manual of Systematics of Archaea and Bacteria
Pullulanibacillus
Journal: Bergey’s Manual of Systematics of Archaea and Bacteria
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Wiley - Manuscript type: Genus Paper
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Complete List of Authors: Albuquerque, Luciana; Center for Neuroscience and Cell Biology, University of Coimbra, Biotechnology Tiago, Igor; Centre for Functional Ecology, Life Sciences Department, University of Coimbra Veríssimo, António; Centre for Functional Ecology, Life Sciences Department, University of Coimbra da Costa, Milton; Center for Neuroscience and Cell Biology, University of Coimbra, Biotechnology
Firmicutes, Sporolactobacillaceae, Mesophilic, Moderately acidophilic, Keywords: Aerobic
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1 2 3 1 Firmicutes / Bacilli or Firmibacteria / Bacillales / Sporolactobacillaceae 4 5 6 2 7 8 3 gbm014445 9 10 4 11 12 13 5 Pullulanibacillus 14 15 6 16 17 7 Hatayama, Shoun, Ueda, and Nakamura 2006, 2549VP. Emend Pereira, Albuquerque, 18 19 VP 20 8 Nobre, Tiago, Veríssimo, Pereira and da Costa 2013, 161 . 21 For Peer Review 22 9 23 24 10 Luciana Albuquerque, Center for Neuroscience and Cell Biology, University of Coimbra, 25 26 27 11 Coimbra, Portugal 28 29 12 Igor Tiago, Centre for Functional Ecology, Department of Life Sciences, University of 30 31 13 Coimbra, Coimbra, Portugal 32 33 António Verissimo, Centre for Functional Ecology, Department of Life Sciences, 34 14 35 36 15 University of Coimbra, Coimbra, Portugal 37 38 16 Milton S. da Costa, Department of Life Sciences, University of Coimbra, Coimbra, 39 40 17 Portugal 41 42 43 18 44 45 19 Pul.lu.la.ni.ba.cil’lus. N.L. n. pullulanum pullulan; L. masc. n. bacillus a small staff; N.L. 46 47 20 masc. n. Pullulanibacillus a small staff hydrolyzing pullulan. 48 49 50 21 51 52 22 The species of the genus Pullulanibacillus produce rod-shaped cells, 0.5–4.0 µm in width 53 54 23 and 2.1–60.0 µm in length that stain Gram-positive. The endospores are oval, sub- 55 56 terminal to terminal in swollen sporangia. Some species possess flagella and motility is 57 24 58 59 25 observed. Colonies are nonpigmented or yellow-pigmented. The species of the genus 60
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1 2 3 26 Pullulanibacillus are mesophilic and are moderately acidophilic. The species are strictly 4 5 6 27 aerobic, cytochrome c oxidase is negative, and catalase is positive. The species of the 7 8 28 genus are chemoorganotrophic utilizing carbohydrates, polyols, amino acids and organic 9 10 29 acids as single carbon sources. Yeast extract is not necessary for growth in minimal 11 12 30 defined medium. The peptidoglycan contains meso-diaminopimelic acid (meso-Dpm) 13 14 15 31 plus alanine and glutamic acid and is of type A1γ. Menaquinone 7 (MK-7) is the major 16 17 32 respiratory quinone. Polar lipids consist of phosphatidylglycerol (PG), 18 19 33 diphosphatidylglycerol (DPG), unidentified phospholipids, unidentified aminolipids and 20 21 For Peer Review 22 34 unidentified glycolipids; in some species DPG and glycolipids are not detected. The 23 24 35 predominant fatty acids are saturated branched- and straight-chain; some species contains 25 26 36 large amounts of unsaturated fatty acids. DNA G+C content is 39.3–45.0 (%) (HPLC) 27 28 The members of the genus Pullulanibacillus have been isolated from soil, tea and a 29 37 30 31 38 uranium mill tailling effluent. 32 33 39 Type species: Pullulanibacillus naganoensis Hatayama, Shoun, Ueda, and 34 35 40 Nakamura 2006, 2549VP. 36 37 38 41 39 40 41 42 Keywords: Firmicutes, Sporolactobacillaceae, mesophilic, moderately acidophilic, 42 43 43 aerobic 44 45 44 46 47 48 45 Rod-shaped cells, 0.5–4.0 µm in width and 2.1–60.0 µm in length. Endospores are oval, 49 50 51 46 sub-terminal to terminal in a swollen sporangium. Stain Gram-positive. Some species are 52 53 47 motile. Colonies are nonpigmented or yellow-pigmented. Mesophilic and moderately 54 55 48 acidophilic. Strictly aerobic. Cytochrome c oxidase negative and catalase positive. 56 57 49 Chemoorganotrophic. Yeast extract is not necessary for growth in minimal medium. 58 59 60 50 Carbohydrates, polyols, amino acids and organic acids serve as single carbon sources.
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1 2 3 51 The peptidoglycan contains meso-diaminopimelic acid (meso-Dpm) plus alanine and 4 5 6 52 glutamic acid and is of type A1γ. Menaquinone 7 (MK-7) is the major respiratory 7 8 53 quinone. Polar lipids consist of phosphatidylglycerol (PG), diphosphatidylglycerol 9 10 54 (DPG), unidentified phospholipids, unidentified aminolipids and unidentified 11 12 55 glycolipids; in some species DPG and glycolipids are not detected. Major fatty acids are 13 14 15 56 saturated branched- and straight-chain; some species contains large amounts of 16 17 57 unsaturated fatty acids. Isolated from soil, tea and a uranium mill tailling effluent. 18 19 58 DNA G+C content (%): 39.3–45.0 (HPLC). 20 21 For Peer Review 22 59 Type species: Pullulanibacillus naganoensis Hatayama, Shoun, Ueda, and Nakamura 23 24 60 2006, 2549VP 25 26 61 Number of species with validated names: 4. 27 28 Family classification: The genus Pullulanibacillus is classified within the family 29 62 30 31 63 Sporolactobacillaceae (fbm00117). 32 33 64 34 35 36 65 Further descriptive information 37 38 66 39 40 67 Cell morphology and colony characteristics 41 42 43 68 The species of Pullulanibacillus produces Gram-positive rod-shaped cells, 0.5–1.0 µm in 44 45 69 width and 1.0–60.0 µm in length and form terminal to subterminal spores within a swollen 46 47 70 sporangium. With the exceptions of the type strain of P. pueri, which is motile by means 48 49 50 71 of peritrichous flagella, motility has not been observed in other strains of this genus. The 51 52 72 colonies of species P. naganoensis and P. uraniitolerans are nonpigmented; P. camelliae 53 54 73 and P. pueri are yellow to pale-yellow pigmented. Colonies of these organisms are 55 56 circular and convex with 1 to 3 mm in diameter after 48h of growth. Colonies of P. 57 74 58 59 60
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1 2 3 75 naganoensis have entire margins but P. uraniitolerans have undulate margins (Niu et al., 4 5 6 76 2016; Niu et al., 2015, Pereira et al., 2013; Tomimura et al., 1990). 7 8 77 9 10 78 Nutrition and growth conditions 11 12 79 The species of the genus Pullulanibacillus are mesophile; they have an optimum growth 13 14 15 80 temperature of about 28–37ºC and the temperature range for growth is between 15 to 16 17 81 50ºC. The species are moderately acidophilic, the optimum pH for growth is in the range 18 19 82 of 4.0–6.0 for P. naganoensis, P. pueri and P. uraniitolerans, but the optimum pH of P. 20 21 For Peer Review 22 83 camelliae is higher, between 6.5–7.5. The organisms do not grow below pH 3.0 or above 23 24 84 pH 8.0. The species of this genus do not require NaCl for growth; growth occurs in media 25 26 85 with NaCl up to 5.0% (w/v) for species P. naganoensis and P. camelliae, 6.0% (w/v) for 27 28 species P. uraniitolerans and 9.0% (w/v) for species P. pueri (Table 1). 29 86 30 31 87 The strains of Pullulanibacillus can utilize carbohydrates, polyols, amino acids and 32 33 88 organic acids as single carbon sources. Only lactate and fumarate were tested as possible 34 35 89 single carbon sources for P. camelliae but this organism did not use them for growth. 36 37 38 90 Of the polyols tested, P. uraniitolerans only uses glycerol for growth (Table 1). Yeast 39 40 91 extract is not necessary for growth in a minimal medium (Niu et al., 2016; Niu et al., 41 42 92 2015). 43 44 45 93 There have been conflicting results related to the production of acid from the type 46 47 94 strains of the species of this genus using the API 50CH system. For example, the type 48 49 95 strain of the species of P. naganoensis appears not to produce acid from the carbon 50 51 sources using the API 50CH test strips (Pereira et al., 2013). However, Tomimura et al. 52 96 53 54 97 (1990) found that P. naganoensis, produced acid from L-arabinose, D-xylose, D-glucose, 55 56 98 D-mannitol and lactose, by measuring the decrease of the pH in a minimal medium using 57 58 99 the method of Gordon et al. (1973). Also using the API 50CH, Prasirtsak et al., 2016, Yao 59 60
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1 2 3 100 et al., 2016 and Yan et al. 2018 found acid production from a large variety of carbon 4 5 6 101 sources by P. naganoensis. The type strains of the species P. camelliae and P. pueri 7 8 102 produce acid from several carbohydrates using the API 50CH (Yao et al., 2016; Yan et 9 10 103 al., 2018). However, the type strain of P. uraniitolerans does not appear to produce acid 11 12 104 from carbohydrates using the API 50CH (Pereira et al., 2013; Yan et al., 2018). The 13 14 15 105 conflicting results for the production of acid from carbohydrates appears to be due to 16 17 106 different methods used or different protocols. The species of genus Pullulanibacillus are 18 19 107 strictly aerobic and chemoorganotrophic. All organisms are catalase positive and 20 21 For Peer Review 22 108 cytochrome c oxidase negative. Only P. camelliae reduces nitrate to nitrite, in the other 23 24 109 species the reduction did not occur (Table 1). 25 26 110 The type strain of P. uraniitolerans as well as strain UG-3 were able to grow in media 27 28 containing up to 5000 p.p.m. U(VI), while P. naganoensis was only able to grow in media 29 111 30 31 112 containing up to 200 p.p.m. U(VI). The ability of the strains to use U(VI) as an electron 32 33 113 acceptor under anaerobic conditions was tested for 14 days on Alicyclobacillus solid 34 35 114 medium supplemented with 100, 200, 2000 and 5000 p.p.m. U(VI). Growth was not 36 37 38 115 observed, indicating that U(VI) was not used as an electron donor under anaerobic 39 40 116 conditions. 41 42 43 117 44 45 118 Chemotaxonomic characteristics 46 47 48 119 The polar lipid profile, on thin-layer chromatography, of the species of the genus 49 50 120 Pullulanibacillus consists of PG, DPG, unidentified phospholipids, unidentified 51 52 121 aminolipids and unidentified glycolipids (Table 1). Glycolipids are not detected in the 53 54 122 species P. camelliae (Niu et al., 2016) and DPG is not detected in P. pueri (Niu et al., 55 56 57 123 2015). Menaquinone 7 (MK-7) is the major respiratory quinone of the organisms of this 58 59 124 genus, the most common quinone in the family Sporolactobacillaceae (fbm00117). It has 60
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1 2 3 125 been reported by Hatayama et al., (2006) that P. naganoensis also possesses minor 4 5 6 126 amounts of menaquinone 5 (MK-5). The fatty acid profile of the species of genus 7 8 127 Pullulanibacillus are dominated by saturated branched chain fatty acids namely anteiso- 9 10 128 C15:0, anteiso-C17:0, iso-C15:0 and iso-C16:0; P. camelliae an unsaturated fatty acid, C18:1 11 12 129 ω7c, is the predominant acyl chain followed by anteiso-C and anteiso-C ; P. pueri 13 17:0 15:0 14 15 130 also possess large amounts of C18:1 ω7c (Table 2). 16 17 131 The genus Pullulanibacillus have a cell-wall type Alγ and the peptidoglycan contains 18 19 132 meso-Dpm as diagnostic diamino acid, which is common in the most of endospore- 20 21 For Peer Review 22 133 forming Gram-positive rods. The species P. naganoensis and P. uraniitolerans possess 23 24 134 peptidoglycan with meso-Dpm plus alanine and glutamic acid (Pereira et al., 2013). 25 26 135 27 28 Genome features 29 136 30 31 137 There is a plan by the Joint Genome Institute - JGI (Project ID: 1175327) to sequence the 32 33 138 genome of the type strain of Pullulanibacillus naganoensis DSM 10191T, which to date 34 35 139 is “In Progress”, and no information regarding the genome of this organism is provided. 36 37 38 140 To our knowledge, no genome data is available in other databases. 39 40 141 41 42 142 Ecology 43 44 45 143 The type strains of the species of genus Pullulanibacillus have been isolated from several 46 47 144 different environments such as soil (Tomimura et al., 1990, Hatayama et al., 2006), acid 48 49 145 uranium mill tailing effluent (Pereira et al., 2013) and fermented tea (Niu et al., 2016; Niu 50 51 et al., 2015). Several other sequences from strains closely related to the type strain 52 146 53 54 147 Pullulanibacillus naganoensis have been deposited in GenBank, but all represent 55 56 148 unpublished data, i.e. Pullulanibacillus sp. CA42 (AB520682) isolated from Chinese 57 58 149 water chestnut, Pullulanibacillus sp. D' C-1 (AB479193) and Pullulanibacillus sp. 59 60
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1 2 3 150 09M123 (AB719201) isolated from soil. One uncultured bacterial clone 75B (KJ600972) 4 5 6 151 also closely related to Pullulanibacillus species was isolated from banana plant 7 8 152 rhizosphere (Xue et al., 2015). Considering the information available we observe that the 9 10 153 organisms belonging to this genus can inhabit several different environments, soil being 11 12 154 the more consensual habitat. 13 14 15 155 16 17 156 Enrichment and isolation procedures 18 19 20 157 The type strain of P. naganoensis was isolated from a soil sample collected in Nagano 21 For Peer Review 22 158 Prefecture in Japan (Tomimura et al., 1990). The sample was resuspended in sterile water 23 24 159 (2 g of soil per 10 ml of water). The suspension (0.1 ml) was spread on plate 1 medium 25 26 27 160 and incubated at 30ºC. Colonies with blue zones (capable of hydrolyzing pullulan) were 28 29 161 picked and transferred to plate 2 medium. Colonies surrounded by blue zones on exposure 30 31 162 to iodine vapors were selected and considered to be pullulanase producers. One of the 32 33 isolates was designated strain D39 and became the type strain of P. nagamoensis. The 34 163 35 36 164 organism is routinely grown on Alicyclobacillus medium 37 38 165 (https://www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium402.pdf) with pH 39 40 166 adjusted to 5.0 and incubated at 30ºC (Albuquerque et al., 2000; Pereira et al., 2013). 41 42 43 167 Plate 1 medium contains (per liter of water) 1.0 g yeast extract (Oxoid), 2.0 g tryptone 44 45 168 (Difco), 1.0 g (NH4)2SO4, 0.3 g KH2PO4, 0.2 g MgSO4.7H2O, 10 mg FeSO4.7H2O, 0.2 g 46 47 169 CaCl2.2H2O, 1.0 mg MnCl2.4H2O, 10.0 g soluble starch (Sigma), 3.0 g blue-colored 48 49 50 170 soluble starch, 7.5 g red-colored pullulan, 20.0 g agar, pH adjusted to 4.0 (Rinderknecht 51 52 171 et al., 1967; Tomimura et al., 1990). 53 54 172 Plate 2 medium contains the same ingredients as plate 1 medium but soluble starch 55 56 and colored pullulan was substituted by 10.0 g/l of amylopectin and the pH was adjusted 57 173 58 59 174 to 4.0 (Tomimura et al., 1990). 60
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1 2 3 175 The type strain of P. camelliae was isolated from a ripened Pu’er tea sample collected 4 5 6 176 in Anning city, Yunnan province, China (Niu et al., 2016). The spread-plate method, on 7 8 177 Luria-Bertani (LB) medium at 37ºC, was used to isolate the organism. The strain is 9 10 178 routinely grown on LB at 30ºC (Niu et al., 2016). 11 12 179 The type strain of P. pueri was isolated from a ripened Pu’er tea sample obtained 13 14 15 180 from a manufacturer in Puer City, Yunnan province, China (Niu et al., 2015). The sample 16 17 181 was stored (post-fermented) for six years. The same method and medium used to isolate 18 19 182 P. camelliae were used to isolate P. pueri. The organism is routinely grown on 20 21 For Peer Review 22 183 Alicyclobacillus medium with pH adjusted to 6.0–6.5 at 30ºC (Niu et al., 2015). 23 24 184 The type strain of P. uraniitolerans was isolated from a uranium mine tailing effluent, 25 26 185 Urgeiriça, Central Portugal (Pereira et al., 2013). The water sample was transported at 27 28 ambient temperature, processed within 1 h of sampling and heated at 70ºC for 10 minutes 29 186 30 31 187 to select spore-forming bacteria and filtered through membrane filters (Gelman type GN- 32 33 188 6; pore size 0.45 mm; diameter 47 mm). The filters were placed on the surface of 34 35 189 Alicyclobacillus medium, with pH adjusted to 4.0. Two isolates, designated UG-2T and 36 37 38 190 UG-3 were recovered on Alicyclobacillus plates after 5 days of incubation at 30ºC. The 39 40 191 organism is routinely grown on Alicyclobacillus medium with pH adjusted to 4.0 at 37ºC 41 42 192 (Pereira et al., 2013). 43 44 45 193 46 47 194 Maintenance procedures 48 49 50 195 Pullulanibacillus strains do not require special procedures for maintenance and long-term 51 52 196 storage. Generally, the organisms are maintained on Alicyclobacillus medium with pH 53 54 197 adjusted to 4.0–5.0 or 6.0–7.0, depending on the species, or at 4°C for a few days and can 55 56 be stored frozen at –70°C in the same liquid media containing 15% glycerol without loss 57 198 58 59 60
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1 2 3 199 of viability for several years. Long-term preservation is by freeze drying or storage in 4 5 6 200 liquid nitrogen. 7 8 201 9 10 202 Procedures for testing special characteristics 11 12 13 203 The species of the type strains of P. uraniitolerans, isolated from a uranium mine tailing, 14 15 204 and P. naganoensis isolated from soil were grown in the presence several concentrations 16 17 205 of U(VI)., The organisms are incubated for 5 days on Alicyclobacillus medium 18 19 20 206 (Albuquerque et al., 2000) supplemented with 2, 10, 50, 100, 150, 250, 300 and 500 ppm 21 For Peer Review 22 207 U(VI) obtained from pitchblende (uraninite, UO3, U2O5), using 80% (v/v) sulphuric acid 23 24 208 and 20% (v/v) H2O2 to provide the necessary redox conditions for the uranium mineral to 25 26 27 209 oxidize to U(VI) (Pereira et al., 2013). The level of U(VI) in these solutions were 28 29 210 measured using liquid scintillation spectrometry (Pereira et al., 2015), diluted and added 30 31 211 to liquid growth medium. The pH was adjusted to the optimum pH for growth. 32 33 Experiments were also performed using uranyl nitrate [UO (NO ) .6H O] at 34 212 2 3 2 2 35 36 213 concentrations of 1000, 2000, 4000 and 5000 ppm because U(VI) cannot be obtained 37 38 214 from pitchblende at levels higher that 500 ppm. A medium with a concentration of 200 39 40 215 ppm was used as a control. Simultaneously, the strains were cultivated on Alicyclobacillus 41 42 43 216 medium supplemented with 8% sodium nitrate to confirm that the growth inhibition is 44 45 217 caused by U(VI) and not by nitrate. 46 47 218 48 49 50 219 Differentiation of the genus Pullulanibacillus from other genera 51 52 53 220 The four validly published species of the genus Pullulanibacillus can be distinguished 54 55 221 from each other on the bases of phenotypic and chemotaxonomic parameters, notably the 56 57 222 fatty acid composition of P. camelliae where C18:1 ω7c is the major fatty acid while in the 58 59 60 223 other type strains anteiso-C15:0 is the major fatty acid (Table 2). The organisms can also
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1 2 3 224 be distinguished by the ability to grow on different single carbon sources (Table 1). It is 4 5 6 225 noteworthy that the two strains of P. uraniitolerans grow in medium with very high U(VI) 7 8 226 while the type strain of P. naganoensis does not. 9 10 227 The most obvious distinctive characteristics of the species of the genus 11 12 228 Pullulanibacillus with those of the closely related genera Scopulibacillus and Sinobaca 13 14 15 229 are the higher growth temperature range of the species of the genus Scopulibacillus, the 16 17 230 lower pH range of the species of genus Pulullanibacillus and the higher NaCl levels for 18 19 231 growth of the species Sinobaca qinghaiensis. Moreover, MK-5 is the only respiratory 20 21 For Peer Review 22 232 quinone identified in Sinobaca qinghaiensis, while MK-7 is the major respiratory quinone 23 24 233 of the species of the genera Pulullanibacillus and Scopulibacillus. In addition to these 25 26 234 distinguishing characteristics, the cells of Sinobaca qinghaiensis form cocci instead of 27 28 rod-shaped cells as in the other organisms discussed above (Li et al., 2006; Li et al., 2008) 29 235 30 31 236 (Table 1, Table 3). 32 33 237 34 35 36 238 Taxonomic comments 37 38 239 Based on the 16S rRNA gene sequence phylogeny, species of genus Pullulanibacillus 39 40 240 (Niu et al., 2016; Niu et al., 2015, Pereira et al., 2013; Tomimura et al., 1990) are 41 42 43 241 separated into two groups comprising two species each namely Pullulanibacillus 44 45 242 naganoensis with P. uraniitolerans, and P. camelliae with P. pueri, respectively. 46 47 243 The type species of genus Pullulanibacillus, P. naganoensis and P. uraniitolerans 48 49 50 244 share 98.2% 16S rRNA gene sequence similarity, while P. pueri and P. camelliae share 51 52 245 97.5% between them. The type strains of P. pueri and P. camelliae share 95.9% and 53 54 246 95.0% 16S rRNA gene similarity with the type strain P. naganoensis, respectively. 55 56 Moreover, the type strains of P. naganoensis and P. uraniitolerans are phylogenetically 57 247 58 59 248 related to the type strain of genus Sinobaca (Li et al. 2006; Li et al. 2008) sharing low 60
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1 2 3 249 16S rRNA gene sequence similarity, namely 91.0 and 90.7%, respectively. The group 4 5 6 250 formed by P. pueri and P. camelliae is phylogenetically distant from the P. naganoensis 7 8 251 and P. uraniitolerans and is phylogenetically more closely related to the type strains of 9 10 252 genus Scopulibacillus (Lee and Lee 2009; Yan et al., 2018; Yao et al. 2016) with whom 11 12 253 they share between 94.8 and 96.6% 16S rRNA gene sequence similarity (Figure 1). The 13 14 15 254 phylogenetic data suggest that the two groups of the genus Pullulanibacillus may 16 17 255 represent distinct genera, but additional phenotypic and genomic results are necessary to 18 19 256 support this hypothesis. 20 21 For Peer Review 22 257 23 24 258 List of the species of the genus Pullulanibacillus 25 26 27 259 28 29 260 1. Pullulanibacillus camelliae 30 31 261 Niu, Xiong, Zhu, Song, Tang and Li 2016, 4764VP 32 33 34 262 35 36 263 ca.mel’li.ae. N.L. gen. n. camelliae of Camellia, referring to the isolation of the type strain 37 38 264 from fermented green tea, Camellia sinensis. 39 40 265 Forms rod-shaped cells, straight or slightly curved rods, 0.7 to 0.9 m in width and 41 42 43 266 2.5 to 4.4 m in length. The cells stain Gram-positive and are nonmotile. Oval endospores 44 45 267 are formed at a subterminal position in a slightly swollen sporangium. Colonies on LB 46 47 48 268 are circular, smooth, glossy, convex, translucent and pale yellow, with a diameter of 1.0 49 50 269 mm after 48h of growth. Strictly aerobic. The optimum growth temperature is about 30– 51 52 270 37ºC; growth occurs in the range of 20–50ºC. The optimum pH for growth is about 6.5– 53 54 7.5; the pH range for growth is 6.0–8.0. The organism does not require additional NaCl 55 271 56 57 272 for growth; growth occurs in media with NaCl up to 5.0% (w/v). Catalase positive. 58 59 273 Cytochrome c oxidase and DNase negative. Methyl-red test is positive. Nitrate reduction 60
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1 2 3 274 test positive in the API 20E, but nitrite reduction, H2S and indole production test negative. 4 5 6 275 Alkaline phosphatase test positive in the API ZYM but esterase (C4), valine arylamidase, 7 8 276 cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, N-acetyl-β- 9 10 277 glucosaminidase, α-mannosidase and α-fucosidase test negative. Gelatin is hydrolyzed. 11 12 278 Casein, starch, aesculin, xylan, hippurate, Tween 20, 60 and 80, CM-cellulose, pullulan 13 14 15 279 and pectin are not hydrolyzed. Yeast extract is not necessary for growth in minimal 16 17 280 medium. Carbohydrates, polyols and amino acids serve as single carbon sources (Table 18 19 281 1). Acid is produced from carbohydrates using API 50CH system. The peptidoglycan 20 21 For Peer Review 22 282 contains meso-Dpm as diagnostic diamino acid. The peptidoglycan type is A1γ. Major 23 24 283 respiratory quinone is menaquinone 7 (MK-7). The lipid profile consists of 25 26 284 phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), five unknown phospholipids 27 28 and one unknown aminolipid. The predominant fatty acids are C ω7c, anteiso-C and 29 285 18:1 17:0 30 31 286 anteiso-C15:0. This bacterium was isolated from a ripened Pu’er tea sample obtained from 32 33 287 a manufacturer in Anning City, China. 34 35 288 Source: Pu’er tea 36 37 38 289 DNA G+C content (%): 45.2 (HPLC). 39 40 290 Type strain: 7578-24, CGMCC 1.15371, JCM 31236. 41 42 291 GenBank/EMBL/DDBJ/SILVA SSU accession number (16S rRNA gene): KT438833. 43 44 45 292 46 47 293 2. Pullulanibacillus naganoensis 48 49 294 Hatayama, Shoun, Ueda and Nakamura 2006, 2549VP (Bacillus naganoensis Tomimura, 50 51 Zeman, Frankiewicz, and Teague 1990, 124) 52 295 53 54 296 55 56 297 na.ga.no.en’sis. N.L. gen. n. naganoensis of Nagano, a Japanese Prefecture. 57 58 59 60
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1 2 3 298 Forms rod-shaped cells 0.5 to 1.0 m in width and 2.1 to 10.0 m in length. The cells 4 5 6 299 stain Gram-positive and are nonmotile. Oval endospores are formed at a terminal position 7 8 300 in a swollen sporangium. Colonies are opaque, smooth, glistening, circular with entire 9 10 301 margins, convex and nonpigmented, with about 2 to 3 mm in diameter after 72h of growth 11 12 13 302 on plate 2 medium. Strictly aerobic. The optimum growth temperature is about 28–33ºC; 14 15 303 growth occurs in the range of 25–40ºC. Moderate acidophilic; the optimum pH for growth 16 17 304 is around 5.0; the pH range for growth is 4.0–6.0. The organism does not require 18 19 305 additional NaCl for growth; growth occurs in media with NaCl up to 5.0% (w/v). Able to 20 21 For Peer Review 22 306 grow in media containing up to 200 p.p.m. U(VI). Nitrate and nitrite are not reduced. 23 24 307 Methylene blue is not reduced. Catalase positive. Cytochrome c oxidase and DNase 25 26 308 negative. Voges-Proskauer, H S production and indole production test negative. Esterase 27 2 28 29 309 lipase (C 8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, 30 31 310 α-glucosidase, β-glucosidase, α-galactosidase and β-galactosidase test positive in the API 32 33 311 ZYM, but alkaline phosphatase, lipase (C14), valine arylamidase, cystine arylamidase, 34 35 36 312 trypin, α-chymotrypsin, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase 37 38 313 test negative. Starch, aesculin, arbutin, xylan, pullulan, CM-cellulose and pectin are 39 40 314 hydrolyzed. Hippurate, salicin, tyrosine, elastin, tributyrin, Tween 20, 40, 60 and 80 are 41 42 315 not hydrolyzed. Yeast extract is not necessary for growth in minimal medium. 43 44 45 316 Carbohydrates, polyols, organic acids and amino acids serve as single carbon sources 46 47 317 (Table 1). Acid is produced from carbohydrates. The peptidoglycan contains meso-Dpm 48 49 318 as diagnostic diamino acid plus alanine and glutamic acid. The peptidoglycan type is A1γ. 50 51 52 319 Major respiratory quinone is menaquinone 7 (MK-7); presence of menaquinone 5 (MK- 53 54 320 5) in minor amounts. The polar lipid profile consists of phosphatidylglycerol (PG), 55 56 321 diphosphatidylglycerol (DPG), one unidentified phospholipid, one unidentified 57 58 59 322 aminolipid and three unidentified glycolipids. The predominant fatty acids are anteiso- 60
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1 2 3 323 C15:0, anteiso-C17:0, iso-C15:0 and iso-C16:0. This bacterium was isolated from a soil sample 4 5 6 324 in Nagano Prefecture in Japan. 7 8 325 Source: Soil. 9 10 326 DNA G+C content (%): 45.0 (HPLC) (Tomimura et al., 1990). 11 12 327 DNA G+C content (%): 39.3 (HPLC) (Pereira et al., 2013). 13 14 15 328 DNA G+C content (%): 40.8 (HPLC) (Niu et al., 2006). 16 17 329 Type strain: D39, ATCC 53909, DSM 10191, LMG 12887. 18 19 330 GenBank/EMBL/DDBJ/SILVA SSU accession number (16S rRNA gene): AB021193. 20 21 For Peer Review 22 331 23 24 332 3. Pullulanibacillus pueri 25 26 333 Niu, Tang, Song, Xiong, Tian, Zhang, Hu and Zhu 2015, 2170VP 27 28 334 29 30 335 pu’e.ri. N.L. n. puerum Pu’er, a tea from China; N.L. gen. n. pueri of Pu’er. 31 32 33 336 Forms rod-shaped cells 0.5 to 0.6 m in width and 1.0 to 2.6 m in length. The cells 34 35 337 stain Gram-positive and are motile by peritrichous flagella. Oval endospores are formed 36 37 338 at a sub-terminal position. Colonies on CYC medium are yellow-pigmented, circular with 38 39 40 339 entire margins, slightly convex with a diameter of 3.0 mm after 48h of growth. Strictly 41 42 340 aerobic. The optimum growth temperature is about 30–37ºC; growth occurs in the range 43 44 341 of 20–50ºC. Moderate acidophilic; the optimum pH for growth is between 5.0 and 6.0; 45 46 47 342 the pH range for growth is 4.0–8.0. The organism does not require additional NaCl for 48 49 343 growth, but optimum growth occurs by the addition of 2% NaCl to the medium; growth 50 51 344 occurs in media with NaCl up to 8.0–9.0% (w/v). Nitrate is reduced to nitrite. Catalase 52 53 54 345 positive. Cytochrome c oxidase and DNase negative. Voges-Proskauer, H2S production 55 56 346 and indole production test negative. Alkaline phosphatase, esterase (C4), esterase lipase 57 58 347 (C 8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α- 59 60
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1 2 3 348 chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-galactosidase, β- 4 5 6 349 glucuronidase, α-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α- 7 8 350 fucosidase test positive in the API ZYM but lipase (C14) and β- glucosidase test negative. 9 10 351 Gelatin, casein, salicin, amygdalin and Tween 60 are hydrolyzed. Starch, xylan, 11 12 352 hippurate, pullulan, CM-cellulose, pectin and Tween 20 are not hydrolyzed. Yeast extract 13 14 15 353 is not necessary for growth in minimal medium. Carbohydrates, polyols, organic acids 16 17 354 and amino acids serve as single carbon sources (Table 1). Acid is produced from 18 19 355 carbohydrates using API 50CH system. The peptidoglycan contains meso-Dpm as 20 21 For Peer Review 22 356 diagnostic diamino acid. The peptidoglycan type is A1γ. Major respiratory quinone is 23 24 357 menaquinone 7 (MK-7). The polar lipid profile consists of phosphatidylglycerol (PG), 25 26 358 two unidentified phospholipids, three unidentified aminolipids and two unidentified 27 28 glycolipids. The predominant fatty acids are anteiso-C , anteiso-C and C ω7c. 29 359 17:0 15:0, 18:1 30 31 360 This bacterium was isolated from a ripened Pu’er tea sample obtained from a 32 33 361 manufacturer in Puer City, China. 34 35 362 Source: Pu’er tea 36 37 38 363 DNA G+C content (%): 38.7 (HPLC). 39 40 364 Type strain: YN3, CGMCC 1.12777, JCM 30075, DSM 100927. 41 42 365 GenBank/EMBL/DDBJ/SILVA SSU accession number (16S rRNA gene): KF733796. 43 44 45 366 46 47 367 4. Pullulanibacillus uraniitolerans 48 49 368 Pereira, Albuquerque, Nobre, Tiago, Veríssimo, Pereira and da Costa 2013, 161VP 50 51 369 52 53 370 u.ra.ni.i.tole.rans. N.L. n. uranium uranium; L. part. adj. tolerans tolerating; N.L. part. 54 55 56 371 adj. uraniitolerans uranium tolerating. 57 58 59 60
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1 2 3 372 Forms rod-shaped cells 1.0 m in width and 4.0 to 60.0 m in length. The cells stain 4 5 6 373 Gram-positive and are nonmotile. Endospores are formed at a terminal position in a 7 8 374 swollen sporangium. Colonies are circular with undulate margins, low, convex, opaque 9 10 375 and nonpigmented on Alycyclobacillus medium. Strictly aerobic. The optimum growth 11 12 13 376 temperature is about 37ºC; growth occurs in the range of 15–45ºC. The optimum pH for 14 15 377 growth is about 4.0; the pH range for growth is 3.0–6.5. The organism does not require 16 17 378 additional NaCl for growth, but optimum growth occurs by the addition of 2% NaCl to 18 19 379 the medium; growth occurs in media with NaCl up to 6.0% (w/v). Able to grow in media 20 21 For Peer Review 22 380 containing up to 5000 p.p.m. U(VI). Nitrate is reduced to nitrite. Nitrate is not reduced to 23 24 381 nitrite. Catalase positive. Cytochrome c oxidase and DNase negative. Voges-Proskauer, 25 26 382 H S production and indole production test negative. Esterase lipase (C 8), leucine 27 2 28 29 383 arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, acid 30 31 384 phosphatase, naphthol-AS-BI-phosphohydrolase, α-glucosidase, β-glucosidase and N- 32 33 385 acetyl-β-glucosaminidase test positive in the API ZYM but Alkaline phosphatase, lipase 34 35 36 386 (C14), α-galactosidase, β-galactosidase, β-glucuronidase, α-mannosidase and α- 37 38 387 fucosidase test negative. Gelatin, aesculin, arbutin, hippurate, CM-cellulose, pectin, 39 40 388 salicin are hydrolyzed. Starch, casein, elastin, xylan, pullulan, tributyrin, Tween 20, 40, 41 42 389 60 and 80 are not hydrolyzed. Yeast extract is not necessary for growth in minimal 43 44 45 390 medium. Carbohydrates, organic acids and amino acids serve as single carbon sources 46 47 391 (Table 1). Acid is not produced from any substrates using API 50CH system. The 48 49 392 peptidoglycan contains meso-Dpm as diagnostic diamino acid plus alanine and glutamic 50 51 52 393 acid. The peptidoglycan type is A1γ. Major respiratory quinone is menaquinone 7 (MK- 53 54 394 7). The polar lipid profile consists of phosphatidylglycerol (PG), diphosphatidylglycerol 55 56 395 (DPG), one unknow phospholipid, one unknow aminolipid and three glycolipids; two 57 58 59 396 additional minors slowly migrating glycolipids are detected. The predominant fatty acids 60
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1 2 3 397 are anteiso-C17:0 and anteiso-C15:0. This bacterium was isolated from a uranium mill tailing 4 5 6 398 effluent in Urgeiriça, Central Portugal. 7 8 399 Source: Water from a uranium mine. 9 10 400 DNA G+C content (%): 39.5 (HPLC). 11 12 401 Type strain: UG-2, DSM 19429, LMG 24205. 13 14 15 402 GenBank/EMBL/DDBJ/SILVA SSU accession number (16S rRNA gene): 16 17 403 AM931441. 18 19 404 Additional Remarks: Strain UG-3 is an additional strain of this species. DNA G+C 20 21 For Peer Review 22 405 content for the additional strain UG-3 is 39.7% (HPLC). This bacterium was isolated from 23 24 406 the same water sample from a uranium mine as the type strain. 25 26 407 27 28 Acknowledgements 29 408 30 31 409 This research was supported by the European Union’s Horizon 2020 Research and 32 33 410 Innovation programme under Metafluidics Grant Agreement No 685474. This work was 34 35 411 also supported by FEDER funds through the Operational Programme Competitiveness 36 37 38 412 Factors - COMPETE 2020 and national funds by FCT - Foundation for Science and 39 40 413 Technology under the strategic project UID/NEU/04539/2013 and the exploratory project 41 42 414 IF/01061/2014. Igor Tiago acknowledges an Investigator contract, reference 43 44 45 415 IF/01061/2014. 46 47 416 48 49 417 References 50 51 52 418 53 54 419 Albuquerque L, Rainey FA, Chung AP, Sunna A, Nobre MF, Grote R et al. (2000) 55 56 420 Alycyclobacillus hesperidum sp. nov. and related genomic species from solfataric soils of 57 58 421 São Miguel in the Azores. Int J Syst Evol Microbiol 50: 451–457. 59 60
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1 2 3 422 4 5 6 423 Felsenstein, J (1985) Confidence limits on phylogenies: An approach using the bootstrap. 7 8 424 Evolution 39: 783–791. 9 10 425 11 12 426 Gordon RE, Haynes WC, & Pang CH-N (1973) The genus Bacillus. In Agriculture 13 14 15 427 Handbook No 427, U.S. Department of Agriculture, Washington, D.C; pp 9–10. 16 17 428 18 19 429 Hatayama K, Shoun H, Ueda Y, & Nakamura A (2006) Tuberibacillus calius gen. nov., 20 21 For Peer Review 22 430 sp. nov., isolated from a compost pile and reclassification of Bacillus naganoensis 23 24 431 Tomimura et al., 1990 as Pullulanibacillus naganoensis gen. nov., comb. nov. and 25 26 432 Bacillus laevolacticus comb. nov. Int J Syst Evol Microbiol 56: 2545–2551. 27 28 29 433 30 31 434 Jukes TH & Cantor CR (1969) Evolution of protein molecules. In Mammalian Protein 32 33 435 Metabolism, HN Munro (ed). Academic Press, New York; pp 21–132. 34 35 436 36 37 38 437 Lee SD & Lee DW (2009) Scopulibacillus darangshiensis gen. nov., sp. nov., isolated 39 40 438 from rock. J Microbiol 47: 710–715. 41 42 439 43 44 45 440 Li WJ, Zhi XY, & Euzéby JP (2008) Proposal of Yaniellaceae fam. nov., Yaniella gen. 46 47 441 nov. and Sinobaca gen. nov. as replacements for the illegitimate prokaryotic names 48 49 442 Yaniaceae Li et al. 2005, Yania Li et al. 2004, emend Li et al. 2005, and Sinococcus Li et 50 51 al. 2006, respectively. Int J Syst Evol Microbiol 58: 525–527. 52 443 53 54 444 55 56 57 58 59 60
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1 2 3 445 Li WJ, Zhang YQ, Schumann P, Tian XP, Zhang YQ, Xu LH Zhi XY, & Jiang CL (2006) 4 5 6 446 Sinococcus qinghaiensis gen. nov., sp. nov., a novel member of the order Bacillales from 7 8 447 a saline soil in China. Int J Syst Evol Microbiol 56: 1189–1192. 9 10 448 11 12 449 Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar et al. (2004) ARB: 13 14 15 450 A Software Environment for Sequence Data. Nucl Acids Res 32: 1363–1371. 16 17 451 18 19 452 Niu L, Xiong M, Zhu D, Song L, Tang T, & Li WS (2016) Pullulanibacillus camelliae 20 21 For Peer Review 22 453 sp. nov., isolated from Pu’er tea. Int J Syst Evol Microbiol 66: 4760–4765. 23 24 454 25 26 455 Niu L, Tang T, Song L, Xiong M, Tian J, Zhang K et al. (2015) Pullulanibacillus pueri 27 28 sp. nov., isolated from Pu’er tea. Int J Syst Evol Microbiol 65: 2167–2171. 29 456 30 31 457 32 33 458 Pereira AJSC, Pereira MD, Neves LJPF, Azevedo JMM, & Campos ABA (2015) 34 35 459 Evaluation of groundwater quality based on radiological and hydrochemical data from 36 37 38 460 two uraniferous regions of Western Iberia: Nisa (Portugal) and Ciudad Rodrigo (Spain). 39 40 461 J Environ Earth Sciences 73: 2717–2731. 41 42 462 43 44 45 463 Pereira SG, Albuquerque L, Nobre MF, Tiago I, Veríssimo A, Pereira A et al. (2013) 46 47 464 Pullulanibacillus uraniitolerans sp. nov., na acidophilic, U(VI)-resistant species isolated 48 49 465 from na acid uranium mill tailing efluente and emended description of the genus 50 51 Pullulanibacillus. Int J Syst Evol Microbiol 63: 158–162. 52 466 53 54 467 55 56 468 Rinderknecht, H, Wilding P, & Haverback B J (1967) A new method for the 57 58 469 determination of α-amylase. Experientia 23: 805. 59 60
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1 2 3 470 4 5 6 471 Saitou N & Nei M (1987) The neighbor-joining method: A new method for reconstructing 7 8 472 phylogenetic trees. Mol Biol Evol 4: 406–425. 9 10 473 11 12 474 Tomimura E, Zeman NW, Frankiewicz JR, & Teague WM (1990) Description of Bacillus 13 14 15 475 naganoensis sp. nov. Int J Syst Bacteriol 40: 123–125. 16 17 476 18 19 477 Yan ZF, Lin P, Li CT, Liu Y, Kook MC, & Yi TH (2018) Scopulibacillus cellulosilyticus 20 21 For Peer Review 22 478 sp. nov., a cellulose-degrading bacterium isolated from tea. Antonie van Leeuwenhoek 23 24 479 111: 2087–2094. 25 26 480 27 28 Yao S, Zhai L, Xin C, Liu Y, Xu L, Zhang X et al. (2016) Scopulibacillus daqui sp. nov., 29 481 30 31 482 a thermophilic bacterium isolated from high temperature daqu. Int J Syst Evol Microbiol 32 33 483 66: 4723–4728. 34 35 484 36 37 38 485 Xue C, Penton CR, Shen Z, Zhang R, Huang Q, Li R et al. (2015) Manipulating the 39 40 486 banana rhizosphere microbiome for biological control of Panama disease. Scientific 41 42 487 Reports 5: 11124. 43 44 45 488 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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1 2 3 4 489 TABLE 1. Characteristics of the species of the genus Pullulanibacillus 5 6 7 Pullulanibacillus Pullulanibacillus Pullulanibacillus Pullulanibacillus 8 9 Characteristic naganoensisa,b,c,d,e,f,g camelliaec,f pueric,d,f,g uraniitoleransc,d,e,g 10 T T T T 11 ATCC 53909 7578-24 YN3 UG-2 12 13 Morphology Rods Rods Rods Rods 14 For Peer Review 15 Gram reaction Positive Positive Positive Positive 16 Colonial morphology (margins) Entire nd Entire undulate 17 18 Pigmentation Nonpigmented Pale yellow Yellow Nonpigmented 19 20 0.5–1.0 x 2.1–10.0a 0.7–0.9 x 2.5–4.4 21 Cell size (m) 0.5–0.6 x 1.0–2.6 1.0 x 4.0–60.0 e 22 3.0–22.0 in lenght 23 Motility Nonmotile Nonmotile Peritrichous flagella Nonmotile 24 25 Temperature for growth (ºC) 26 27 Range 25–40a 20–50 20–50 15–45e 28 g g 29 20–37 25–40 30 Optimum 28–33 30–37 30–37 37 31 32 pH for growth 33 a,g e 34 Range 4.0–6.0 6.0–8.0 4.0–8.0 3.0–6.5 35 3.5–7.5e 3.0–6.0g 36 37 Optimum 5.0 6.5–7.5 5.0–6.0 4.0 38 39 40 41 42 21 43 John Wiley & Sons 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Bergey?s Manual of Systematics of Archaea and Bacteria Page 22 of 34
1 2 3 4 NaCl concentration for growth (%) 5 6 Optimum 0 nd 2 2 7 8 Range 0–5.0 0–9.0d,f 0–6.0 0–5.0 9 g 10 0–8.0 11 Resistance to U(VI) (ppm) 200 nd nd 5000 12 13 Nitrate reduction – + – – 14 For Peer Review 15 Nitrite reduction – – nd nd 16 Voges-Proskauer test – +c,–f – – 17 18 Enzymes (API ZYM) 19 20 Alkaline phosphatase – + + – 21 e,f c,d e c,d 22 Esterase (C 4) + ,– – + + ,– 23 Esterase lipase (C 8) + – +d,–f + 24 25 Lipase (C14) – – – – 26 27 Leucine arylamidase +e,–f – + + 28 29 Valine arylamidase – – + + 30 Cystine arylamidase – – + + 31 32 Trypsin – – + + 33 34 α-chymotrypsin – – + + 35 Acid phosphatase + – +d,–f + 36 37 Naphthol-AS-BI-phosphohydrolase + – + + 38 39 40 41 42 22 43 John Wiley & Sons 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 23 of 34 Bergey?s Manual of Systematics of Archaea and Bacteria
1 2 3 4 α-galactosidase + – +c,f,–d – 5 6 β-galactosidase + + + – 7 8 β-glucuronidase + + + – 9 10 β-glucosidase + – – + 11 N-acetyl-β-glucosaminidase – – + + 12 13 α-mannosidase – – + – 14 For Peer Review 15 α-fucosidase – – + – 16 Hydrolysis of 17 18 Gelatin +a,e,f,–c,d + + + 19 20 Starch + – – – 21 a e,f,g 22 Casein + ,– – + – 23 Aesculin +e,–f – +d,–f +e,–f 24 25 Xylan + – – – 26 27 Hippurate – – – + 28 29 Tween 20 – – – – 30 Tween 60 – – + – 31 32 Tween 80 – – – – 33 34 Pullulan + – – – 35 CM-cellulose +c,–f – – + 36 37 Pectin w – – + 38 39 40 41 42 23 43 John Wiley & Sons 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Bergey?s Manual of Systematics of Archaea and Bacteria Page 24 of 34
1 2 3 4 Salicin – nd + + 5 6 Chitin + – – nd 7 8 Assimilation of – 9 10 D-glucose + + + + 11 D-fructose +e,–c,d + + + 12 13 D-galactose + nd + + 14 For Peer Review e c,d 15 D-mannose + + + + ,– 16 L-rhamnose +e,–c,d + + – 17 18 D-ribose + + + +e,–c,d 19 20 D-xylose +e,–c,d + + + 21 22 D-arabinose – nd + – 23 L-arabinose + – + + 24 25 Sucrose + nd + + 26 27 Maltose + – + + 28 29 Lactose + nd + + 30 D-cellobiose + – – + 31 32 D-trehalose +e,–c,d – + + 33 34 Salicin – + + + 35 Glycerol – + – + 36 37 Xylitol + + – – 38 39 40 41 42 24 43 John Wiley & Sons 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 25 of 34 Bergey?s Manual of Systematics of Archaea and Bacteria
1 2 3 4 Sorbitol +c,d,–e + + – 5 6 Mannitol + + + – 7 8 Myo-inositol + nd + – 9 10 L-arabitol + + + – 11 Methanol – nd – – 12 13 α-ketoglutarate + nd – – 14 For Peer Review 15 Lactate – – + – 16 Acetate – nd – – 17 18 Succinate – nd – – 19 20 Citrate – nd – + 21 22 Fumarate + – – – 23 Formate – nd – – 24 25 D-gluconate + nd + + 26 27 D-asparagine +c,d,–e – – +e,–c,d 28 29 Glycine – nd – – 30 L-histidine + – – – 31 32 L-proline + nd + + 33 34 L-glutamine nd nd – – 35 L-serine – + + – 36 37 L-valine – – + – 38 39 40 41 42 25 43 John Wiley & Sons 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Bergey?s Manual of Systematics of Archaea and Bacteria Page 26 of 34
1 2 3 4 L-phenylalanine – nd – – 5 6 L-leucine – nd – – 7 8 L-ornithine – + – + 9 10 Methionine – nd – – 11 Acid production from carbohydrates + + + – 12 13 Peptidoglycan type A1γ A1γ A1γ A1γ 14 For Peer Review 15 Diagnostic peptidoglycan amino acid meso-Dpm plus alanine meso-Dpm plus alanine meso-Dpm meso-Dpm 16 and glutamic acid and glutamic acid 17 18 Major respiratory lipoquinone MK-7 MK-7 MK-7 MK-7 19 20 Polar lipids PG, DPG, PL, AL, GLs PG, DPG, AL, PLs PG, PL, ALs, GLs PG, DPG, PL, AL, GLs 21 22 Major fatty acids anteiso-C15:0, anteiso- C18:1 ω7c, anteiso-C17:0, anteiso-C17:0, anteiso- anteiso-C17:0, anteiso-C15:0 23 C , iso-C iso-C anteiso-C C C ω7c 24 17:0 15:0, 16:0 15:0 15:0, 18:1 25 G+C content (mol%) (HPLC) 45.0a; 40.8b; 39.3e 45.2 38.7 39.5 26 27 Habitat Soil Pu’er tea Pu’er tea Uranium mine 28 490 29 30 491 All strains are catalase and α-glucosidase positive. All strains are negative for cytochrome c oxidase, DNase, urease, arginine dihydrolase, H2S and indole production. P. naganoensis, P. 31 492 camelliae and P. pueri test positive for methyl-red, but negative for hydrolize of tyrosine. P. naganoensis, P. uraniitolerans and P. pueri test negative for lecithinase, lysine decarboxylase, 32
33 493 ornithine decarboxylase and tryptophan decarboxylase. P. pueri hydrolize amygdalin. P. naganoensis and P. uraniitolerans hydrolize arbutin, but do not hydrolize elastin, Tween 40 and 34 494 tributirin. P. naganoensis does not assimilate propionate. P. pueri assimilate glycogen, but does not assimilate L-lysine, L-arginine, L-isoleucine, L-threonine, L-cysteine and L-tyrosine. P. 35
36 495 naganoensis and P. uraniitolerans assimilate L-glutamate, but do not assimilate L-sorbose, ribitol, erythritol, D-arabitol, ethanol, aspartate and L-alanine. 37 496 +, positive; –, negative; nd, not determined; MK, menaquinone; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; PL, unidentified phospholipid; PLs, unidentified phospholipids; 38 AL, unidentified aminolipid; ALs, unidentified aminolipids; GLs, unidentified glycolipids. 39 497 40 41 42 26 43 John Wiley & Sons 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 27 of 34 Bergey?s Manual of Systematics of Archaea and Bacteria
1 2 3 4 498 aTomimura et al. (1990). 5 b 6 499 Hatayama et al. (2006). 7 500 cNiu et al (2016). 8 d 9 501 Niu et al (2015). 10 502 ePereira et al (2013). 11 503 fYan et al (2018). 12 13 504 gYao et al (2016). 14 505 For Peer Review 15 16 506 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 27 43 John Wiley & Sons 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Bergey?s Manual of Systematics of Archaea and Bacteria Page 28 of 34
1 2 3 4 507 TABLE 2. Fatty acid composition of the species Pullulanibacillus naganoensis DSM 10191T, Pullulanibacillus 5 6 T T T 7 508 camelliae 7578-24 , Pullulanibacillus pueri YN3 and Pullulanibacillus uraniitolerans UG-2 grown on CYC* agar 8 9 509 plates at 30ºC for 24 h 10 11 12 Fatty acidsa ECL Pullulanibacillus Pullulanibacillus Pullulanibacillus Pullulanibacillus 13 14 Fornaganoensis Peercamelliae Reviewpueri uraniitolerans 15 DSM 10191T 7578-24T YN3T UG-2T 16 17 18 iso-C14:0 13.619 1.4 – – 0.4 19 C14:0 14.000 0.6 – 1.0 1.1 20 21 iso-C15:0 14.623 18.4 5.4 6.9 5.7 22 23 anteiso-C15:0 14.713 31.6 10.1 14.6 35.3 24 iso-C 15.627 11.5 3.2 1.7 5.2 25 16:0 26 C16:0 16.000 1.3 1.5 3.0 4.5 27 28 iso-C17:0 16.630 4.4 3.8 4.4 2.1 29 30 anteiso-C17:0 16.723 23.7 30.6 42.1 41.2 31 C 18.000 0.8 – 2.4 1.1 32 18:0 33 C18:1 ω9c 17.769 – – 1.2 0.7 34 35 C18:1 ω7c 17.823 1.4 45.4 17.1 0.4 36 510 Results are the percentage of the total fatty acids; –, not detected. ECL, equivalent chain length. 37 38 39 40 41 42 28 43 John Wiley & Sons 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 29 of 34 Bergey?s Manual of Systematics of Archaea and Bacteria
1 2 3 4 511 *CYC agar plates contains per liter of water 33.4 g Czapek-Dox liquid medium, modified (Oxoid), 2.0 g yeast extract (Difco), 6.0 g vitamin 5 6 512 assay casamino acids (Difco) (Niu et al., 2016). 7 513 aNiu et al. (2016). 8 9 10 11 12 13 14 For Peer Review 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 29 43 John Wiley & Sons 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Bergey?s Manual of Systematics of Archaea and Bacteria Page 30 of 34
1 2 3 514 4 5 6 515 TABLE 3. Characteristics that distinguish Pullulanibacillus from the closely related genera, 7 516 Scopulibacillus and Sinobaca 8 9 Characteristic Pullulanibacillusa,b,c,d,e Scopulibacillusf,g,h Sinobacai,j 10 11 12 Morphology Rods Rods Cocci 13 14 Cell size (m) 0.5–1.0 x 1.0–60.0 0.7–1.2 x 1.4–7.0 0.8–1.0 15 16 Motility Motile / Nonmotile Nonmotile Motile 17 Temperature for growth (ºC) 18 19 Range 15–50 20–60 nd 20 21 Optimum For Peer28–37 Review 30–50 28 22 23 pH for growth 24 Range 3.0–8.0 4.0–9.0 nd 25 26 Optimum 5.0–7.5 8.0 8.0–9.5 27 28 NaCl concentration for growth (%) 29 Range 0–9.0 0–10 1–25 30 31 Optimum 0–2.0 2.0 10 32 - - 33 Reduction of NO3 to NO2 + / – + – 34 35 Presence of 36 Cytochrome c oxidase + + / – – 37 38 Major respiratory lipoquinone MK-7 MK-7 MK-5 39 40 Polar Lipids PG, DPG, PL, AL, GLs PG, DPG, PE, PL, PG, DPG, PL, GL, 41 42 AL, APL, GL ALs 43 Major fatty acids anteiso-C15:0, anteiso- anteiso-C17:0, iso-C16:0, anteiso-C15:0, 44 45 C17:0, iso-C15:0, iso-C16:0, iso-C17:0, C18:1 ω7c anteiso-C17:0 46 47 C18:1 ω7c 48 G+C content (mol%) (HPLC) 39.3–45.2 40.5–50.8 47.0 49 50 Habitat Soil / Tea / Uranium Tea / Rock / High- Saline soil 51 52 mine temperature Daqu* 53 54 517 55 518 +, positive; –, negative; nd, not determined. MK, menaquinone; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; PE, 56 519 phosphatidylethanolamine; PL, unidentified phospholipid; AL, unidentified aminolipid; ALs, unidentified aminolipids; APL, 57 520 unidentified aminophospholipid; GL, glycolipid; GLs, glycolipids. 58 59 521 *Daqu is a traditional saccharifying and fermenting agent used to flavor a liquor. 60 522 aTomimura et al. (1990).
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1 2 3 523 bHatayama et al. (2006). 4 cNiu et al (2016). 5 524 6 525 dNiu et al (2015). 7 526 ePereira et al (2013). 8 9 527 fLee and Lee (2009). 10 528 gYao et al (2016). 11 h 12 529 Yan et al (2018). 13 530 iLi et al (2006). 14 i 15 531 Li et al (2008). 16 532 17 18 533 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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1 2 3 534 4 5 6 535 Figure 1. The phylogenetic dendrogram obtained by comparison of the 16S rRNA gene 7 8 536 sequences of all the type strains of the genera belonging to the family 9 10 537 Sporolactobacillaceae is shown. This dendrogram shows the closest phylogenetic 11 12 538 relatives to the four type strains of genus Pullulanibacillus and their phylogenetic 13 14 15 539 relationships. The phylogenetic dendrograms were constructed using ARB software 16 17 540 package (Ludwig et al., 2004) with neighbor-joining (Saitou & Nei, 1987) and Jukes– 18 19 541 Cantor correction (Jukes & Cantor, 1969) and bootstrap analysis (Felsenstein, 1985) of 20 21 For Peer Review 22 542 1000 resampling’s of the dataset. The numbers on the tree branches indicate the 23 24 543 percentages of bootstrap sampling, derived from 1000 replications. The type strains of 25 26 544 genus Pullulanibacillus are indicated in bold. Scale bar, 1 inferred nucleotide 27 28 substitutions per 100 nucleotides. 29 545 30 31 546 32 33 34 547 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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1 2 3 548 4 5 6 549
7 T 65 Sporolactobacillus kofuensis JCM 341 (AB374517) T 8 94 Sporolactobacillus laevolacticus IAM 12321 (D16270) 9 Sporolactobacillus nakayamae DSM 16324T (AJ698860) T 62 Sporolactobacillus nakayamae subsp nakayamae DSM 11696 (AJ634663) 10 T 71 Sporolactobacillus shoreicorticis MK21-7 (LC062596) 11 Sporolactobacillus inulinus IFO 13595T (AB101595) 12 Sporolactobacillus terrae M-116T (AJ634662) Sporolactobacillus pectinivorans GD201205T (KP340805) T 13 52 Sporolactobacillus vineae SL153 (EF581819) T 14 97 Sporolactobacillus shoreae BK92 (AB911245) Sporolactobacillus putidus QC81-06T (AB374522) 15 T 67 Sporolactobacillus spathodeae BK117-1 (AB911246) 16 “Scopulibacillus cellulosilyticus” THG-NT9T (MF919603) T 17 95 Scopulibacillus daqui ZQ18-1 (KJ136650) Scopulibacillus darangshiensis DLS-06T (AM711118) T 18 99 Pullulanibacillus camelliae 7578-24 (KT438833) T 19 50 Pullulanibacillus pueri YN3 (KF733796) T 60 Caenibacillus caldisaponilyticus B157 (LC062711) 20 T 95 Camelliibacillus cellulosilyticus THG-YT1 (MG786604) 21 TForuberibacillus cPeeralidus 607T (AB2 3Review1786) Pullulanibacillus uraniitolerans UG-2T (AM931441) 22 T 67 Pullulanibacillus naganoensis ATCC 53909 (AB021193) 23 Sinobaca qinghaiensis YIM 70212T (DQ168584) 24 0.01 25 550 26 551 27 28 552 29 30 553 31 32 554 33 34 555 Figure 1. 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 Figure 1. The phylogenetic dendrogram obtained by comparison of the 16S rRNA gene sequences of all the 26 type strains of the genera belonging to the family Sporolactobacillaceae is shown. This dendrogram shows 27 the closest phylogenetic relatives to the four type strains of genus Pullulanibacillus and their phylogenetic 28 relationships. The phylogenetic dendrograms were constructed using ARB software package (Ludwig et al., 2004) with neighbor-joining (Saitou & Nei, 1987) and Jukes–Cantor correction (Jukes & Cantor, 1969) and 29 bootstrap analysis (Felsenstein, 1985) of 1000 resampling’s of the dataset. The numbers on the tree 30 branches indicate the percentages of bootstrap sampling, derived from 1000 replications. The type strains of 31 genus Pullulanibacillus are indicated in bold. Scale bar, 1 inferred nucleotide substitutions per 100 32 nucleotides. 33 34 279x157mm (300 x 300 DPI) 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons