Bergey?s Manual of Systematics of Archaea and

Tepidamorphus

Journal: Bergey’s Manual of Systematics of Archaea and Bacteria

Manuscript ID gbm01455.R1

Wiley - Manuscript type:For Genus Peer Paper Review

Date Submitted by the Author: n/a

Complete List of Authors: Albuquerque, Luciana; Center for Neuroscience and Cell Biology, University of Coimbra, Biotechnology Rainey, Fred; University of Alaska Anchorage, Biological Sciences da Costa, Milton; Center for Neuroscience and Cell Biology, University of Coimbra, Biotechnology

Alphaproteobacteria, Rhodobiaceae, Slightly Thermophilic, Aerobic, Keywords: Budding

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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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 16S RNA gene sequence neighbor joining tree demonstration the position of the genus Tepidamorphus within the radiation of genera of the order Rhizobiales. The scale bar represents one nucleotide substitution 46 per 100 nucleotides. Numbers at branching points represent bootstrap values from 1000 replications. The 47 tree was rooted using the sequence of parvum. 48 49 246x291mm (300 x 300 DPI) 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Bergey?s Manual of Systematics of Archaea and Bacteria Page 2 of 26

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 26 27 28 29 30 31 Morphology of type strain of Tepidamorphus gemmatus in liquid cultures. Differential interference 32 microscopy showing overall features and budding (a, b). Transmission electron microscopy showing irregular 33 shaped rod- and large ovoid-shaped cells, clear intracellular inclusions may represent polyhydroxyalkanoate 34 (PHA) deposits (c). Aggregates of rod-shaped cells viewed by negative staining (d). Rod shaped cell with 35 and elongated hyphal–like bud (e). Two attached cells with several flagella (f). Rod-shaped cells producing 36 long rod-shaped or ovoid-shaped buds (g, h). *Reprinted with permission from Albuquerque et al. (2010a), Systematic and Applied Microbiology, Elsevier. 37 38 39 254x190mm (96 x 96 DPI) 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Page 3 of 26 Bergey?s Manual of Systematics of Archaea and Bacteria

1 2 3 1 / / Rhizobiales / Rhodobiaceae 4 5 2 6 7 3 Tepidamorphus 8 9 10 4 11 VP 12 5 Albuquerque, Rainey, Pena, Tiago, Veríssimo, Nobre and da Costa 2010b, 1447 13 14 6 (Effective publication: Albuquerque, Rainey, Pena, Tiago, Veríssimo, Nobre and da 15 16 7 Costa 2010a, 65) 17 18 8 19 For Peer Review 20 9 Luciana Albuquerque, Center for Neuroscience and Cell Biology, University of 21 22 23 10 Coimbra, Coimbra, Portugal 24 25 11 Fred A. Rainey, Department of Biological Sciences, University of Alaska Anchorage, 26 27 12 Anchorage, AK, USA 28 29 13 Milton S. da Costa, Center for Neuroscience and Cell Biology, University of Coimbra, 30 31 14 Coimbra, Portugal 32 33 15 34 35 36 16 Te.pi.da.mor’phus. L. masc. adj. tepidus, moderately warm, lukewarm, tepid; Gr. masc. 37 38 17 adj. amorphos, without form, shapeless; N.L. masc. n. Tepidamorphus, an organism 39 40 18 without a distinctive morphology that grows at warm temperatures. 41 42 19 43 44 20 Abstract: 45 46 21 Irregular rodshaped cells, 0.5–2.0 µm in width and 1.0–1.5 µm in length. Motile. Cells 47 48 with long rodshaped structures; multiplies by budding. Endospores are not observed. 49 22 50 51 23 Stain Gramnegative. Colonies are nonpigmented. Slightly thermophilic. 52 53 24 Chemoorganotrophic. Strictly aerobic. Bacteriochlorophyll a and puf genes are not 54 55 25 present. Oxidase and catalase positive. Thiosulfate is not oxidized to sulfate. Sugars, 56 57 58 59 60 John Wiley & Sons Bergey?s Manual of Systematics of Archaea and Bacteria Page 4 of 26

1 2 3 26 organic acids and amino acids are used as carbon and energy sources. Major respiratory 4 5 27 quinone is ubiquinone 10 (U10). Major polar lipids are phosphatidylcholine (PC), 6 7 28 phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol 8 9 29 (DPG) and phosphatidylmonomethylethanolamine (PME). Major fatty acids are 10 11 30 primarily saturated and monounsaturated straightchained. Isolated from hydrothermal 12 13 14 31 areas. 15 16 32 DNA G+C content (mol%): 67 (HPLC). 17 18 33 Type : Tepidamorphus gemmatus Albuquerque, Rainey, Pena, Tiago, 19 For Peer Review 20 34 Veríssimo, Nobre and da Costa 2010b, 1447VP. (Effective publication: Albuquerque, 21 22 35 Rainey, Pena, Tiago, Veríssimo, Nobre and da Costa 2010a, 65). 23 24 36 25 26 27 37 Keywords: 28 29 38 Alphaproteobacteria, Rhodobiaceae, Slightly Thermophilic, Aerobic, Budding 30 31 39 32 33 40 Irregular rod-shaped cells, 0.5–2.0 µm in width and 1.0–1.5 µm in length. Motile. 34 35 41 Cells with long rodshaped structures; multiplies by budding. Endospores are not 36 37 42 observed. Stain Gramnegative. Colonies are nonpigmented. Slightly thermophilic. 38 39 Chemoorganotrophic. Strictly aerobic. Bacteriochlorophyll a and puf genes are not 40 43 41 42 44 present. Oxidase and catalase positive. Thiosulfate is not oxidized to sulfate. Sugars, 43 44 45 organic acids and amino acids are used as carbon and energy sources. Major 45 46 46 respiratory quinone is ubiquinone 10 (U-10). Major polar lipids are 47 48 47 phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol 49 50 48 (PG), diphosphatidylglycerol (DPG) and phosphatidylmonomethylethanolamine 51 52 49 (PME). Major fatty acids are primarily saturated and monounsaturated straight- 53 54 55 50 chained. Isolated from hydrothermal areas. 56 57 58 59 60 John Wiley & Sons Page 5 of 26 Bergey?s Manual of Systematics of Archaea and Bacteria

1 2 3 51 DNA G+C content (mol%): 67 (HPLC). 4 5 52 Type species: Tepidamorphus gemmatus Albuquerque, Rainey, Pena, Tiago, 6 7 53 Veríssimo, Nobre and da Costa 2010b, 1447VP. (Effective publication: Albuquerque, 8 9 54 Rainey, Pena, Tiago, Veríssimo, Nobre and da Costa 2010a, 65). 10 11 55 Number of validated species: 1. 12 13 14 56 Family classification: The genus Tepidamorphus is classified whithin the family 15 16 57 Rhodobiaceae (fbm00172). 17 18 58 19 For Peer Review 20 59 Further descriptive information 21 22 23 60 24 25 61 Phylogeny 26 27 62 The genus Tepidamorphus comprises, at this time, one species named T. gemmatus, 28 29 T 63 represented by two strains, CB27A and CB26A. 16S rRNA gene sequencebased 30 31 32 64 comparisons show T. gemmatus to fall within the radiation of the order Rhizobiales 33 34 65 (obm00071) (Figure 1). The Tepidamorphus lineage clusters with a clade comprising 35 36 66 the genera Microbaculum, Lutibaculum, and Butyratibacter, sharing 9496% similarity 37 38 67 with species of these genera (Kumar et al., 2012; Su et al., 2017; Wang et al., 2017). 39 40 68 These similarity values as well as other characteristics support the genus status of the 41 42 69 Tepidamorphus lineage. Genera peripheral to this clade include Afifella, Bauldia, 43 44 45 70 Dichotomicrobium (gbm00817), Methyloceanibacter, Methyloligella, and 46 47 71 (gbm00849) (Albuquerque et al., 2014; Doronina et al., 2013; Hirsch and Hoffmann 48 49 72 1989; Srinivas et al., 2007; Takeuchi et al., 2014; Urdiain et al., 2008; Yee et al., 2010). 50 51 73 The genus Rhodobium is the type genus of the family Rhodobiaceae (fbm00172) 52 53 74 (Garrity et al., 2005). Although the cluster of the genera listed above is not supported by 54 55 75 bootstrap analyses, for the purpose of this outline we include the genus Tepidamorphus 56 57 58 59 60 John Wiley & Sons Bergey?s Manual of Systematics of Archaea and Bacteria Page 6 of 26

1 2 3 76 within the family Rhodobiaceae and await further analysis of the phylogeny of the 4 5 77 Alphaproteobacteria (cbm00041) based on whole genome sequence comparisons. 6 7 78 8 9 79 Cell morphology and colony characteristics 10 11 80 Tepidamorphus gemmatus forms translucent nonpigmented colonies and the cells are 12 13 14 81 irregular rodshaped, 0.5–2.0 µm in width by 1.0–1.5 µm in length with ovoid and long 15 16 82 rodshaped budding structures (Figure 2a, 2b) and is motile by means of one or more 17 18 83 flagella (Albuquerque et al., 2010a). Negative staining electronmicroscopy of 19 For Peer Review 20 84 exponential phase cultures grown in liquid medium shows that the cells have irregular 21 22 85 morphologies (Figure 2c), with frequent branching and aggregation (Figure 2d). Some 23 24 86 branches originate from a lateral position of the cells while others are apical. However, 25 26 27 87 many of these branches resemble elongated buds (Figure 2e, 2f, 2g, 2h). A dark electron 28 29 88 dense band is frequently seen at the junction of separation of the cells. Electron dense 30 31 89 circular structures appear to correspond to polyphosphate granules. Lipid like 32 33 90 inclusions, corresponding to polyhydroxyalkanoate (PHA), are very common and these 34 35 91 frequently surround a small electrondense granule. Carotenoid pigments are not 36 37 92 detected. 38 39 40 93 41 42 94 Nutrition and growth conditions 43 44 95 This slightly thermophilic species has an optimum growth temperature of about 45– 45 46 96 50ºC and the temperature range for growth is between 30ºC and 50ºC. The optimum pH 47 48 97 for growth of the type strain of T. gemmatus is in the range of pH 7.5–8.5, but does not 49 50 98 grow below pH 6.5 or above pH 9.5 (Table 1). Growth occurs in Thermus medium and 51 52 99 Degryse medium 162, however, the growth rate increases in Degryse medium 162. 53 54 55 56 57 58 59 60 John Wiley & Sons Page 7 of 26 Bergey?s Manual of Systematics of Archaea and Bacteria

1 2 3 100 Tepidamorphus gemmatus is unable to grow on any of the polyols examined, 4 5 101 except glycerol, but this organism grows on single carbon and energy sources such as 6 7 102 sugars, organic acids and amino acids (Table 1). Yeast extract is necessary for growth in 8 9 103 a minimal medium (Albuquerque et al., 2010a). 10 11 104 12 13 14 105 Metabolism and metabolic pathways 15 16 106 Cytochrome oxidase and catalase are present. The organism reduces nitrate to nitrite but 17 18 107 anaerobic growth with nitrate as electron acceptor is not observed. The addition of 19 For Peer Review 20 108 thiosulfate to modified medium 27 lacking sulfate, vitamin B12 solution, L 21 22 109 cysteiniumchloride and resazurin, and containing yeast extract, succinate and acetate 23 24 110 (http://www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium27.pdf) does not 25 26 27 111 lead to an increase in the biomass of T. gemmatus indicating that thiosulfate was not 28 29 112 used as an energy source in the presence of organic substrates. Moreover, the levels of 30 31 113 sulfate and thiosulfate remaining in the medium after growth of the organism indicate 32 33 114 that thiosulfate is not oxidized to sulfate. Phototrophic growth is not observed. 34 35 115 Bacteriochlorophyll a is not detected in T. gemmatus under aerobic or anaerobic growth 36 37 116 conditions and the presence of pufL and pufM genes are also not detected. 38 39 Tepidamorphus gemmatus appears to be exclusively organotrophic (Albuquerque et al., 40 117 41 42 118 2010a). 43 44 119 45 46 120 Polar lipids, respiratory lipoquinones and fatty acids 47 48 121 The polar lipid pattern of T. gemmatus on twodimensional thinlayer chromatography 49 50 122 revealed the presence of phosphatidylcholine (PC), phosphatidylethanolamine (PE), 51 52 123 phosphatidylglycerol (PG), diphosphatidylglycerol (DPG) and 53 54 55 124 phosphatidylmonomethylethanolamine (PME) in addition to one unidentified 56 57 58 59 60 John Wiley & Sons Bergey?s Manual of Systematics of Archaea and Bacteria Page 8 of 26

1 2 3 125 phospholipid, two unidentified aminolipids and two unidentified glycolipids. 4 5 126 Ubiquinone 10 (U10) is the major respiratory quinone. The fatty acid composition of T. 6 7 127 gemmatus was dominated by C19:0 cyclo ω8c and C18:0 (Table 2) (Albuquerque et al., 8 9 128 2010a). 10 11 129 12 13 14 130 Ecology and habitats 15 16 131 This bacterium has only been isolated from a runoff of the hot spring known as Caldeira 17 18 132 da Barrela, Furnas, on the Island of São Miguel in the Azores with a temperature of 19 For Peer Review 20 133 40ºC and a pH of 8.5. Uncultured clones closely related to the type strain of T. 21 22 134 gemmatus have been recovered from subsurface water in Kalahari Shield, South Africa 23 24 135 (DQ230971), an aquarium coral collected near Isla San Cristobal at Bocas del Toro, 25 26 27 136 Panama (FJ202923) (Sunagawa et al., 2009), a pine tree (KJ410602) and a red pine 28 29 137 weevil gut (KF116011). 30 31 138 32 33 139 Enrichment and isolation procedures 34 35 T 36 140 The two T. gemmatus isolates, CB27A and CB26A, were recovered from water 37 38 141 samples collected in screw cap bottles, transported and maintained without temperature 39 40 142 control for 6 days, and then filtered through membrane filters. These filters were placed 41 42 143 on the surface of Thermus medium agar plates (Albuquerque and da Costa, 2014), 43 44 144 wrapped in plastic bags and incubated at 50°C until colonies appeared on the filters. 45 46 145 Later it was observed that the organism had a higher growth rate in Degryse medium 47 48 162. 49 146 50 51 147 Thermus medium 52 53 148 (https://www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium1033.pdf) contains 54 55 149 (per liter of water) 1 g yeast extract (Difco), 1 g tryptone (Difco), 100 ml of a 56 57 58 59 60 John Wiley & Sons Page 9 of 26 Bergey?s Manual of Systematics of Archaea and Bacteria

1 2 3 150 macroelements solution (10x concentrated), 10 ml of a trace elements solution (100x 4 5 151 concentrated) and 10 ml of 0.17 mM FeCl3.6H2O, pH adjusted to 8.2 before 6 7 152 autoclaving. The 10x concentrated macroelements solution contains per liter of water: 1 8 9 153 g nitrilotriacetic acid, 0.6 g CaSO4.2H2O, 1 g MgSO4.7H2O, 0.08 g NaCl, 1.03 g KNO3, 10 11 154 6.89 g NaNO , 1.11 g Na HPO . The 100x concentrated trace elements solution 12 3 2 4 13 14 155 contains per liter of water: 0.22 g MnSO4.H2O, 0.05 g ZnSO4.7H2O, 0.05 g H3BO3, 15 16 156 0.0025 g CuSO4.5H2O, 0.0025 g Na2MoO4.2H2O, 0.0046 g CoCl2.6H2O (Albuquerque 17 18 157 and da Costa 2014; Castenholz 1969; Williams and da Costa, 1992). 19 For Peer Review 20 158 Degryse medium 162 contains (per liter of water) 2.5 g yeast extract (Difco), 2.5 g 21 22 159 tryptone (Difco), 100 ml of a macroelements solution (10x concentrated), 5 ml of a trace 23 24 160 elements solution (identical to the trace elements of Thermus medium), 15 ml of 0.2 M 25 26 27 161 Na2HPO4.12H2O, 10 ml of 0.2 M KH2PO4 and 0.5 ml of 0.01 M ferric citrate, pH 28 29 162 adjusted to 7.5 before autoclaving. The 10x concentrated macroelements solution 30 31 163 contained per liter of water: 1 g nitrilotriacetic acid, 0.4 g CaSO4.2H2O and 2 g 32 33 164 MgCl2.6H2O (Degryse et al., 1978, Kristjánsson et al., 1986; Williams and da Costa, 34

35 165 1992). The concentration of Na2HPO4.12H2O and KH2PO4 has been reduced in this 36 37 166 medium because the growth of some isolates was inhibited by the level of phosphate 38 39 described in the original composition (Degryse et al., 1978). All concentrated solutions 40 167 41 42 168 of the both media can be stored at 4ºC. A minimal medium derived from Degryse 43 44 169 medium 162 can be used to assess assimilation of organic compounds by replacing 45 46 170 tryptone by ammonium sulfate (0.5 g/l) and decreasing the amount of yeast extract to 47 48 171 0.2 g/l. 49 50 172 51 52 53 173 Maintenance procedures 54 55 56 57 58 59 60 John Wiley & Sons Bergey?s Manual of Systematics of Archaea and Bacteria Page 10 of 26

1 2 3 174 Strains of T. gemmatus do not require special procedures for maintenance and longterm 4 5 175 storage. Generally, the organisms are maintained on Degryse medium 162 at 4°C for a 6 7 176 few days and can be stored frozen at –70°C in Thermus or Degryse medium 162 8 9 177 containing 15% glycerol without loss of viability for several years. Longterm 10 11 178 preservation is by freeze drying or storage in liquid nitrogen. 12 13 14 179 15 16 180 Taxonomic comments 17 18 19 181 The family RhodobiaceaeFor comprises Peer species Review of the genera Afifella (Urdiain et al., 2008), 20 21 182 Bauldia (Albuquerque et al., 2014; Yee et al., 2010), Lutibaculum (Kumar et al., 2012), 22 23 183 Microbaculum (Su et al., 2017), Rhodobium (Srinivas et al., 2007; Urdiain et al., 2008), 24 25 184 Tepidamorphus (Albuquerque et al., 2010a), Butyratibacter (Wang et al., 2017), 26 27 185 Methyloligella (Doronina et al., 2013), Methyloceanibacter (Takeuchi et al., 2014) and 28 29 186 Dichotomicrobium (Hirsch and Hoffmann, 1989) but there are few phenotypic 30 31 32 187 characteristics that define this family (Urdiain et al., 2008). The species of the genera 33 34 188 Rhodobium and Afifella are phototrophic under anaerobic laboratory conditions. 35 36 189 Moreover, bacteriochlorophyll a (Bchl a), or puf genes that encode the photosynthetic 37 38 190 reaction center proteins and the core lightharvesting complexes, have not been detected 39 40 191 in the species of other genera examined. 41 42 192 The only known species of Tepidamorphus, T. gemmatus has a peculiar 43 44 45 193 pleomorphic morphology, based upon rodshaped cells that appear to reproduce by 46 47 194 budding and the formation of long irregular cells. The closest relatives, namely 48 49 195 Microbaculum marinum, Lutibaculum barantangense and Butyratibacter algicola 50 51 196 produce short or ovalshaped rods, but buds have not been reported (Kumar et al., 2012; 52 53 197 Su et al., 2017; Wang et al., 2017). The distantly related species of the genera Afifella, 54 55 198 Dichotomicrobium and Rhodobium (Hirsch and Hoffmann, 1989; Srinivas et al., 2007; 56 57 58 59 60 John Wiley & Sons Page 11 of 26 Bergey?s Manual of Systematics of Archaea and Bacteria

1 2 3 199 Urdiain et al., 2008) also produce buds. Tepidamorphus gemmatus was isolated from an 4 5 200 inland hot spring with very low NaCl content and grows in media with only up to about 6 7 201 3% NaCl. The most closely related genera, on the other hand, have been recovered from 8 9 202 marine environments such as deep oceanic water and from a mud volcano and are 10 11 203 halophilic or halotolerant. Therefore, at present, the cell morphology represents the most 12 13 14 204 distinctive characteristic of T. gemmatus. 15 16 205 17 18 206 Characteristics of the species of the genus Tepidamorphus 19 For Peer Review 20 21 207 22 23 208 Tepidamorphus gemmatus 24 25 209 Albuquerque, Rainey, Pena, Tiago, Veríssimo, Nobre and da Costa 2010b, 1447VP 26 27 210 (Effective publication: Albuquerque, Rainey, Pena, Tiago, Veríssimo, Nobre and da 28 29 211 Costa 2010a, 65) 30 31 32 212 gem.ma'tus. L. masc. adj. gemmatus, provided with buds. 33 34 213 Forms irregular rodshaped cells 0.5–2.0 µm in width by 1.0–1.5 µm in length with 35 36 214 ovoid and long rodshaped budding structures and are motile by means of one or more 37 38 215 flagella. The cells stain Gramnegative. Endospores are not formed. Colonies on 39 40 216 Thermus and Degryse medium 162 are nonpigmented. Carotenoid pigments are not 41 42 217 detected. The optimum growth temperature is about 45–50ºC; growth occurs in the 43 44 45 218 range of 30–50ºC; the optimum pH is between 7.5 and 8.5; the pH range for growth is 46 47 219 6.5–9.5. Grows in media without added salt; the optimum NaCl concentration for 48 49 220 growth is 0–1%; growth occurs in media with NaCl up to 3% (w/v). 50 51 221 Chemoorganotrophic. Aerobic with a strictly respiratory type of metabolism. 52 53 222 Bacteriochlorophyll a is not present; presence of pufL and pufM genes are not detected. 54 55 223 Reduced sulfur compounds are not oxidized to sulfate in the presence of a carbon 56 57 58 59 60 John Wiley & Sons Bergey?s Manual of Systematics of Archaea and Bacteria Page 12 of 26

1 2 3 224 source. Nitrate is reduced to nitrite. Cytochrome oxidase and catalase positive. DNAse 4 5 225 negative. Urease test positive but indole production, methylred and VogesProskaur 6 7 226 test negative with the API 20 NE. Alkaline phosphatase, esterase (C 4), esterase lipase 8 9 227 (C 8), leucine arylamidase, trypsin, acid phosphatase and naphtholASBI 10 11 228 phosphohydrolase test positive in API ZYM; other activities are negative. Gelatin, 12 13 14 229 casein, hippurate, tween 40 and 60 are hydrolyzed. Starch, aesculin, arbutin, elastin, 15 16 230 xylan, Ltyrosine, CMcellulose, filter paper, tween 20 and 80 are not hydrolyzed. Yeast 17 18 231 extract is required for growth. Sugars, organic acids and amino acids are used as single 19 For Peer Review 20 232 carbon sources, but is unable to grow on any of the polyols examined, except glycerol 21 22 233 (Table 1). Acid is produced from the following carbohydrates using API 50 CH: 23 24 234 glycerol, Dribose, Dxylose, Lxylose, Dglucose, Dfructose, Lsorbose, Dturanose, 25 26 27 235 Dlyxose and Dtagatose. Major respiratory quinone is ubiquinone 10 (U10). Major 28 29 236 polar lipids are phosphatidylcholine (PC), phosphatidylethanolamine (PE), 30 31 237 phosphatidylglycerol (PG), diphosphatidylglycerol (DPG) and 32 33 238 phosphatidylmonomethylethanolamine (PME). The major fatty acids are C19:0 cyclo ω8c 34

35 239 and C18:0 (Table 2). This bacterium was isolated from the runoff of a hot spring known 36 37 240 as Caldeira da Barrela, in the Furnas geothermal area on the Island of São Miguel, 38 39 Portugal. 40 241 41 42 242 Source: Hydrothermal water. 43 44 243 DNA G+G content (mol%): 67.0 (HPLC). 45 46 244 Type strain: CB27A, DSM 19345, LMG 24113. 47 48 245 GenBank/EMBL/DDBJ/SILVA SSU accession number (16S rRNA gene): 49 50 246 GU187912. 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Page 13 of 26 Bergey?s Manual of Systematics of Archaea and Bacteria

1 2 3 247 Additional Remarks: CB26A (DSM 19344, LMG 24114) is an additional strain of 4 5 248 this species. DNA G+C content of strain CB26A is 66.0 mol%. This bacterium was 6 7 249 isolated from the same hot spring as the type strain. 8 9 250 10 11 251 Acknowledgements 12 13 14 252 This research was supported by the European Union’s Horizon 2020 Research and 15 16 253 Innovation programme under Metafluidics Grant Agreement No 685474. This work was 17 18 254 also supported by FEDER funds through the Operational Programme Competitiveness 19 For Peer Review 20 255 Factors COMPETE 2020 and national funds by FCT Foundation for Science and 21 22 256 Technology under the strategic project UID/NEU/04539/2013. 23 24 257 25 26 27 258 References 28 29 259 30 31 260 Albuquerque L & da Costa MS (2014) Family Thermaceae In The Prokaryotes-Other 32 33 261 Major Lineages of Bacteria and The Archaea, 4th ed., E Rosenberg, EF DeLong, S 34 35 36 262 Lory, E Stackebrandt, & F Thompson (eds). SpringerVerlag Berlin Heidelberg; pp 37 38 263 955–987. 39 40 264 41 42 265 Albuquerque L, RossellóMora R, & da Costa MS (2014) Family Rhodobiaceae In The 43 44 266 Prokaryotes-Other Major Lineages of Bacteria and The Archaea, 4th ed., E Rosenberg, 45 46 267 EF DeLong, S Lory, E Stackebrandt, & F Thompson (eds). SpringerVerlag Berlin 47 48 268 Heidelberg; pp 513–531. 49 50 51 269 52 53 54 55 56 57 58 59 60 John Wiley & Sons Bergey?s Manual of Systematics of Archaea and Bacteria Page 14 of 26

1 2 3 270 Albuquerque L, Rainey FA, Pena A, Tiago I, Verissímo A, Nobre MF, & da Costa MS 4 5 271 (2010a) Tepidamorphus gemmatus gen. nov., sp. nov., a slightly thermophilic member 6 7 272 of the Alphaproteobacteria. Syst Appl Microbiol 33: 60–66. 8 9 273 10 11 274 Albuquerque L, Rainey FA, Pena A, Tiago I, Verissímo A, Nobre MF, & da Costa MS 12 13 (2010b) Validation List no. 134. Int J Syst Evol Microbiol 60: 1477–1479. 14 275 15 16 276 17 18 277 Doronina NV, Poroshina MN, Kaparullina EN, Ezhov VA, & Trotsenko YA (2013) 19 For Peer Review 20 278 Methyloligella halotolerans gen. nov., sp. nov., and Methyloligella solikamskensis sp. 21 22 279 nov., two nonpigmented halotolerant obligately methylotrophic bacteria isolated from 23 24 280 the Ural saline environments. Syst Appl Microbiol 36: 148–154. 25 26 281 27 28 282 Castenholz RW (1969) Thermophilic bluegreen algae and the thermal environment. 29 30 31 283 Bacteriol Rev 33: 476–504. 32 33 284 34 35 285 Degryse E, Glansdorff N, & Pierard A (1978) A comparative analysis of extreme 36 37 286 thermophilic bacteria belonging to the genus Thermus. Arch Microbiol 117: 189–196. 38 39 287 40 41 288 Garrity GM, Bell JA, & Lilburn T (2005) Family X. Rhodobiaceae fam. nov. In 42 43 44 289 Bergey's Manual of Systematic Bacteriology, 2nd ed., vol. 2 (The Proteobacteria), part 45 46 290 C (The Alpha, Beta, Delta, and Epsilonproteobacteria), DJ Brenner, NR Krieg, JT 47 48 291 Staley, & M Garrity (eds). Springer, New York; p. 571. 49 50 292 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons Page 15 of 26 Bergey?s Manual of Systematics of Archaea and Bacteria

1 2 3 293 Hirsch P & Hoffman B (1989) Dichotomicrobium thermohalophilum gen. nov., spec. 4 5 294 nov., budding prothecate bacteria from the solar lake (Sinai) and some related strains. 6 7 295 Syst Appl Microbiol 11: 291–301. 8 9 296 10 11 297 Kristjánsson JK, Hreggvidsson GO, & Alfredsson GA (1986) Isolation of halotolerant 12 13 14 298 Thermus scotoductus spp. from submarine hot springs in Iceland. Appl Environ 15 16 299 Microbiol 52: 1313–1316. 17 18 300 19 For Peer Review 20 301 Kumar PA, Srinivas TNR, Manasa P, Madhu S, & Shivaji S (2012) Lutibaculum 21 22 302 baratangense gen. nov., sp. nov., a proteobacterium isolated from a mud volcano. Int J 23 24 303 Syst Evol Microbiol 62: 2025–2031. 25 26 27 304 28 29 305 Srinivas TNR, Kumar PA, Sasikala Ch, Ramana ChV, & Imhoff JF (2007) Rhodobium 30 31 306 gokarnense sp. nov., a novel phototrophic alphaproteobacterium from a saltern. Int J 32 33 307 Syst Evol Microbiol 57: 932–935. 34 35 308 36 37 309 Su Y, Han S, Wang R, Yu X, Fu G, Chen C et al (2017) Microbaculum marinum gen. 38 39 nov., sp., nov., isolated from deep seawater. Int J Syst Evol Microbiol 67: 812–817. 40 310 41 42 311 43 44 312 Sunagawa S, DeSAntis TZ, Piceno YM, Brodie EL, DeSalvo MK, Voolstra CR et al 45 46 313 (2009) Bacterial diversity and white plague diseaseassociated community changes in 47 48 314 the Caribbean coral Montastraea faveolata. ISME J 3: 512–521. 49 50 315 51 52 316 Takeuchi M, Katayama T, Yamagishi T, Hanada S, Tamaki H, Kamagata Y et al (2014) 53 54 55 317 Methyloceanibacter caenitepidi gen. nov., sp. nov., a facultatively methylotrophic 56 57 58 59 60 John Wiley & Sons Bergey?s Manual of Systematics of Archaea and Bacteria Page 16 of 26

1 2 3 318 bacterium isolated from marine sediments near a hydrothermal vent. Int J Syst Evol 4 5 319 Microbiol 64: 462–468. 6 7 320 8 9 321 Urdiain M, LópezLópez A, Gonzalo C, Busse HJ, Langer S, Kämpfer P et al (2008) 10 11 322 Reclassification of Rhodobium marinum and Rhodobium pfennigii as Afifella marina 12 13 14 323 gen. nov. comb. nov. and Afifella pfennigii comb. nov., a new genus of 15 16 324 photoheterotrophic Alphaproteobacteria and emended descriptions of Rhodobium, 17 18 325 Rhodobium orientis and Rhodobium gokarnense. Syst Appl Microbiol 31: 339–351. 19 For Peer Review 20 326 21 22 327 Wang G, Wang Y, Su H, Yu X, Wu H, Li T et al (2017) Butryratibacter algicola gen. 23 24 328 nov., sp., nov., a marine bacterium from the culture broth of Picochlorum sp. 122. Int J 25 26 27 329 Syst Evol Microbiol 67: 3209–3213. 28 29 330 30 31 331 Williams RAD & da Costa MS (1992) The genus Thermus and related microorganisms. 32 33 332 In The Prokaryotes, 2nd ed., A Balows, HG Trüper, M Dworkin, W Harder, & KH 34 35 333 Schleifer (eds). Springer, New York; pp 3745–3753. 36 37 334 38 39 Yee B, Oertli GE, Fuerst JA, & Staley JT (2010) Reclassification of the polyphyletic 40 335 41 42 336 genus Prosthecomicrobium to form two novel genera, Vasilyevaea gen. nov. and 43 44 337 Bauldia gen. nov. with four new combinations: Vasilyevaea enhydra comb. nov., 45 46 338 Vasilyevaea mishustinii comb. nov., Bauldia consociate comb. nov. and Bauldia 47 48 339 litoralis comb. nov. Int J Syst Evol Microbiol 60: 2960–2966. 49 50 340 51 52

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1 2 3 342 4 5 343 TABLE 1. Characteristics of Tepidamorphus gemmatus, strains CB27AT and CB26A 6 7 a, b 8 Tepidamorphus gemmatus Characteristic 9 CB-27AT / CB-26A 10 11 Morphology Rods 12 13 Cell size (m) 0.5–2.0 x 1.0–1.5 14 15 Motility Motile 16 Gram reaction Negative 17 18 Pigmentation Nonpigmented 19 For Peer Review 20 Temperature for growth (ºC) 21 Range 30–50 22 23 Optimum 45–50 24 pH for growth 25 26 Range 6.5–9.5 27 28 Optimum 7.5–8.5 29 NaCl concentration for growth (%) 30 31 Range 0–3 32 33 Optimum 0–1 34 Reduction of NO3 to NO2 + 35 36 Anaerobic growth – 37 Presence of 38 39 Catalase + 40 41 Oxidase + 42 DNAse – 43 44 API 20 NE 45 46 Urease + 47 Indol production – 48 49 Methylred – 50 VogesProskaur – 51 52 Enzymes (API ZYM) 53 54 Alkaline phosphatase + 55 Esterase (C 4) + 56 57 58 59 60 John Wiley & Sons Bergey?s Manual of Systematics of Archaea and Bacteria Page 18 of 26

1 2 3 Esterase lipase (C 8) + 4 Lipase (C14) – 5 6 Leucine arylamidase + 7 a b 8 Valine arylamidase + ,w 9 Cystine arylamidase – 10 11 Trypsin + 12 13 αchymotrypsin – 14 Acid phosphatase + 15 16 NaphtholASBIphosphohydrolase + 17 α-galactosidase – 18 19 βgalactosidaseFor Peer Review – 20 21 βglucuronidase – 22 αglucosidase – 23 24 βglucosidase – 25 Nacetylβglucosaminidase – 26 27 αmannosidase – 28 29 αfucosidase – 30 Hydrolysis of 31 32 Gelatin + 33 34 Starch – 35 Casein + 36 37 Aesculin – 38 Arbutin – 39 40 Elastin – 41 42 Xylan – 43 Hippurate + 44 45 Ltyrosine – 46 47 CMcellulose – 48 Filter paper – 49 50 Tween 20 – 51 Tween 40 + 52 53 Tween 60 + 54 55 Tween 80 – 56 57 58 59 60 John Wiley & Sons Page 19 of 26 Bergey?s Manual of Systematics of Archaea and Bacteria

1 2 3 Assimilation of 4 D–glucose +/– 5 6 D–fructose + 7 8 D–galactose +/– 9 D–mannose – 10 11 L–rhamnose +/– 12 13 L–fucose – 14 L–sorbose – 15 16 D–ribose + 17 D–xylose +a,–b 18 19 D–arabinoseFor Peer Review – 20 21 L–arabinose +/– 22 Sucrose – 23 24 Maltose – 25 Lactose – 26 27 D–cellobiose – 28 29 D–trehalose – 30 D–raffinose – 31 32 D–melibiose – 33 34 Glycerol + 35 Ribitol – 36 37 Xylitol – 38 39 Sorbitol – 40 D–mannitol – 41 42 myoinositol – 43 L–erythritol – 44 45 D–arabitol – 46 47 L–arabitol – 48 α–ketoglutarate + 49 50 DL–lactate + 51 a b 52 Acetate + ,w 53 Pyruvate + 54 55 Succinate +a,–b/– 56 57 58 59 60 John Wiley & Sons Bergey?s Manual of Systematics of Archaea and Bacteria Page 20 of 26

1 2 3 Malate – 4 Citrate – 5 6 Benzoate – 7 8 Fumarate – 9 Dgluconate + 10 11 Dglucoronate – 12 13 L–glutamate + 14 Aspartate – 15 b 16 Malonate w 17 L–alanine +/– 18 19 L–asparagineFor Peer Review +/– 20 21 Glycine – 22 L–histidine +/– 23 24 L–lysine + 25 Lproline + 26 27 L–glutamine + 28 29 L–arginine – 30 L–serine + 31 32 L–valine + 33 34 Acid production from 35 Glycerol + 36 37 Erythritol – 38 Darabinose – 39 40 Larabinose – 41 42 Dribose + 43 Dxylose + 44 45 Lxylose + 46 47 Dadonitol – 48 MethylβDxylopyranoside – 49 50 Dgalactose –a,+b 51 52 Dglucose + 53 Dfructose + 54 55 Dmannose – 56 57 58 59 60 John Wiley & Sons Page 21 of 26 Bergey?s Manual of Systematics of Archaea and Bacteria

1 2 3 Lsorbose + 4 Lrhamnose – 5 6 Dulcitol – 7 8 Inositol – 9 Dmannitol – 10 11 Dsorbitol – 12 13 MethylαDmannopyranoside – 14 MethylαDglucopyranoside – 15 16 Nacetylglucosamine – 17 18 Amygdalin – 19 Arbutin For Peer Review – 20 21 Aesculin ferric citrate – 22 Salicin – 23 24 Dcellobiose – 25 26 Dmaltose – 27 Dlactose – 28 29 Dmelibiose –a,+b 30 31 Dsucrose – 32 Dtrehalose – 33 34 Inulin – 35 Dmelezitose – 36 37 Draffinose – 38 39 Starch – 40 Glycogen – 41 42 Xylitol – 43 44 Gentiobiose – 45 Dturanose + 46 47 Dlyxose + 48 Dtagatose + 49 50 Dfucose –a,+b 51 52 Lfucose – 53 Darabitol – 54 55 Larabitol – 56 57 58 59 60 John Wiley & Sons Bergey?s Manual of Systematics of Archaea and Bacteria Page 22 of 26

1 2 3 Potassium gluconate – 4 Potassium 2ketogluconate – 5 6 Potassium 5ketogluconate +a,–b 7 8 Major respiratory lipoquinone U10 9 Major polar lipids PC, PE, DPG, PG, PME 10 11 Major fatty acids C19:0 cyclo ω8c, C18:0 12 13 G+C content (mol%) (HPLC) 67.0 / 66.0 14 Habitat Hot spring 15

16 344 17 345 +, positive; –, negative; w, weakly positive. U, ubiquinone; PC, phosphatidylcholine; PE, 18 346 phosphatidylethanolamine; DPG, diphosphatidylglycerol, PG, phosphatidylglycerol, PME, 19 347 phosphatidylmonomethylethanolamine.For Peer Review 20 a 21 348 Albuquerque et al. (2010a). 22 349 bSu et al. (2017). 23 350 24 25 351 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 John Wiley & Sons Page 23 of 26 Bergey?s Manual of Systematics of Archaea and Bacteria

1 2 3 352 4 T 5 353 TABLE 2. Fatty acid composition of Tepidamorphus gemmatus, strains CB27A 6 354 and CB26A, grown on Degryse medium 162 agar plates at 45ºC for 48h 7 8 a 9 Fatty acids ECL Tepidamorphus gemmatus 10 T 11 CB-27A CB-26A 12 13 C14:0 3OH 15.488 1.6 ± 0.1 1.4 ± 0.1 14 C 16.000 7.4 ± 0.2 10.7± 0.1 15 16:0 16 C18:1 ω7c 17.823 3.7 ± 0.5 3.2 ± 0.1 17 18 C18:0 18.000 16.2 ± 0.2 23.8 ± 0.1 19 C ω7c 11methylFor Peer 18.081 Review8.3 ± 0.1 8.5 ± 0.1 20 18:1 21 C19:0 cyclo ω8c 18.902 58.2 ± 1.2 49.0 ± 0.2 22 23 C18:0 3OH 19.550 2.3 ± 0.2 1.9 ± 0.2 24 355 25 356 Results are the percentage of the total fatty acids. ±, results are the mean plus the standard deviation 26 of two to four analyses; values for fatty acids present at less than 1.0% are not shown. ECL, 27 357 28 358 equivalent chain length. 29 359 aAlbuquerque et al., 2010a. 30 31 360 32 361 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 John Wiley & Sons Bergey?s Manual of Systematics of Archaea and Bacteria Page 24 of 26

1 2 3 362 4 5 363 Figure 1. 16S RNA gene sequence neighbor joining tree demonstration the position of 6 7 364 the genus Tepidamorphus within the radiation of genera of the order Rhizobiales. The 8 9 365 scale bar represents one nucleotide substitution per 100 nucleotides. Numbers at 10 11 366 branching points represent bootstrap values from 1000 replications. The tree was rooted 12 13 14 367 using the sequence of Roseospirillum parvum. 15 16 368 17 18 369 Figure 2. Morphology of type strain of Tepidamorphus gemmatus in liquid cultures. 19 For Peer Review 20 370 Differential interference microscopy showing overall features and budding (a, b). 21 22 371 Transmission electron microscopy showing irregular shaped rod and large ovoidshaped 23 24 372 cells, clear intracellular inclusions may represent polyhydroxyalkanoate (PHA) deposits 25 26 27 373 (c). Aggregates of rodshaped cells viewed by negative staining (d). Rod shaped cell 28 29 374 with and elongated hyphal–like bud (e). Two attached cells with several flagella (f). 30 31 375 Rodshaped cells producing long rodshaped or ovoidshaped buds (g, h). 32 33 376 *Reprinted with permission from Albuquerque et al. (2010a), Systematic and Applied 34 35 377 Microbiology, Elsevier. 36 37 378 38 39

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1 2 3 381 4 5 382 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 383 47 48 384 Figure 1. 49 50 385 51 52 386 53 54 55 56 57 58 59 60 John Wiley & Sons Bergey?s Manual of Systematics of Archaea and Bacteria Page 26 of 26

1 2 3 387 4 5 388 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 389 26 27 390 28 29 391 30 31 32 392 Figure 2. 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 John Wiley & Sons