1 Parviterribacter Kavangonensis and Parviterribacter Multiflagellatus a Novel Genus and 1 Two Novel Species Within the Order S

Parviterribacter kavangonensis gen. nov., sp. nov. and Parviterribacter multiflagellatus sp. nov., novel members of Parviterribacteraceae fam. nov. within the order Solirubrobacterales, and emended descriptions of the classes Thermoleophilia and Rubrobacteria and their orders and families

Item Type Article

Authors Overmann, Jörg; Foesel, Bärbel U.; Geppert, Alicia; Rohde, Manfred

Citation Parviterribacter kavangonensis gen. nov., sp. nov. and Parviterribacter multiflagellatus sp. nov., novel members of Parviterribacteraceae fam. nov. within the order Solirubrobacterales, and emended descriptions of the classes Thermoleophilia and Rubrobacteria and their orders and families 2016, 66 (2):652 International Journal of Systematic and Evolutionary Microbiology

DOI 10.1099/ijsem.0.000770

Journal International Journal of Systematic and Evolutionary Microbiology

Download date 27/09/2021 05:53:20

Link to Item http://hdl.handle.net/10033/608787 1 Parviterribacter kavangonensis and Parviterribacter multiflagellatus a novel genus and 2 two novel species within the order Solirubrobacterales and emended description of the 3 classes Thermoleophilia and Rubrobacteria and its orders and families

4 Bärbel U. Foesel1*, Alicia Geppert1, Manfred Rohde2, and Jörg Overmann1, 3

5 1 Department of Microbial Ecology and Diversity Research, Leibniz Institut DSMZ - 6 Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany 7 2 Central Facility for Microscopy, Helmholtz Centre for Infection Research, Braunschweig, 8 Germany 9 3 Technische Universität Braunschweig, Braunschweig, Germany

10 Running title: Parviterribacter kavangonensis gen. nov., sp. nov. and P. multiflagellatus, sp. 11 nov.

12 Subject category: New Taxa, subsection Actinobacteria

13 Footnote 14 The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of 15 Parviterribacter kavangonensis D16/0/H6T and P. multiflagellatus A22/0/F9_1T are 16 KP981370 and KP981371, respectively.

17 18 * Correspondence: B. U. Foesel, Leibniz Institut DSMZ - Deutsche Sammlung von 19 Mikroorganismen und Zellkulturen, Inhoffenstraße 7B, 38124 Braunschweig, Germany. 20 Tel: +49-531-2616-364. Fax: +49-531-2616-418. Email: [email protected]. 21

1 22 ABSTRACT

23 Two Gram-positive, non-spore-forming bacteria (strains D16/0/H6T and A22/0/F9_1T) 24 were isolated from Namibian semiarid savanna soils. 16S rRNA gene sequence analysis 25 revealed an identity of 96.6% of the two strains to each other and placed them within the 26 order Solirubrobacterales of the class Thermoleophilia. Closest validly described 27 phylogenetic relatives were several strains of the genus Solirubrobacter and the species 28 Conexibacter arvalis with pairwise sequence similarities of ≤ 94.0%. Cells of strain 29 D16/0/H6T were ovoid to rod shaped whereas strain A22/0/F9_1T formed regular rods. Cells 30 of both strains were motile and divided by binary fission. Colonies were pink and white to 31 pale yellowish/brownish in color, respectively. Strains D16/0/H6T and A22/0/F9_1T were 32 aerobic chemoheterotrophic mesophiles with a broad temperature (13 - 43°C and 17 - 43°C, 33 respectively) and pH (4.5 - 8.5 and 5.0 - 9.5, respectively) range of growth. Complex, 34 proteinaceous substrates and glucose were the preferred carbon and energy source. Strain 35 A22/0/F9_1T also grew on various carboxylic acids. For both strains the peptidoglycan 36 diamino acid was meso-2,6-diaminopimelic acid. The major quinone was MK-8. As minor 37 compound, MK-7 occurred in strain D16/0/H6T; strain A22/0F9_1T also contained MK-7, T 38 MK-7(H2), and MK-8(H2). Major fatty acids of strain D16/0/H6 were C17:0 10-methyl, iso- T 39 C16:0 and C18:1 9c. Strain A22/0F9_1 contained C18:1 9c, C17:1 8c, C17:1 6c and iso-C16:0 as 40 major components. The DNA G+C contents of strains D16/0/H6T and A22/0/F9_1T were 72.8 41 and 74.0 mol%, respectively. Based on these characteristics, the two isolates are described as 42 two novel species of the novel genus Parviterribacter gen. nov., P. kavangonensis sp. nov. 43 strain D16/0/H6T (= DSM 25205T = LMG 26950T) and P. multiflagellatus sp. nov. strain 44 A22/0/F9_1T (= DSM 25204T = LMG 26949T). As the novel genus and species cannot be 45 clearly assigned to an established family within the order Solirubrobacterales, the novel 46 family Parviterribacteraceae fam. nov. is proposed.

2 47 Recently, the former subclass Rubrobacteridae within the class Actinobacteria 48 (Stackebrandt et al., 1997) has been dissected and its members assigned to the two newly 49 established classes Rubrobacteria and Thermoleophilia (Ludwig et al., 2012b). The new 50 class Rubrobacteria (Suzuki, 2012) encompasses the established order Rubrobacterales with 51 the family Rubrobacteraceae (Stackebrandt et al., 1997) and the genus Rubrobacter (Suzuki 52 et al., 1988). The second class Thermoleophilia (Suzuki & Whitman, 2012) comprises the two 53 orders Thermoleophilales (Reddy & Garcia-Pichel, 2009) and Solirubrobacterales (Reddy & 54 Garcia-Pichel, 2009). The order Thermoleophilales harbors the single family 55 Thermoleophilaceae (Stackebrandt, 2004; Zhi et al., 2009) with the genus Thermoleophilum 56 (Zarilla & Perry, 1984) including the two thermophilic species Thermoleophilum album 57 (Zarilla & Perry, 1984) and T. minutum (Zarilla & Perry, 1986). The Solirubrobacterales 58 comprise three families of mesophilic, mostly soil derived bacteria, namely the 59 Solirubrobacteraceae, Conexibacteraceae, and Patulibacteraceae (Stackebrandt, 2004; Zhi et 60 al., 2009). The Solirubrobacteraceae harbor the genus Solirubrobacter (Singleton et al., 61 2003) with its currently five species S. pauli (Singleton et al., 2003), S. soli (Kim et al., 62 2007), S. ginsenosidimutans (An et al., 2011), S. phytolaccae (Wei et al., 2014), and S. 63 taibaiensis (Zhang et al., 2014). The Conexibacteraceae contain the genus Conexibacter 64 (Monciardini et al., 2003) with its two members C. woesei (Monciardini et al., 2003) and C. 65 arvalis (Seki et al., 2012). The Patulibacteraceae comprise the genus Patulibacter (Takahashi 66 et al., 2006) that so far includes the four species P. minatonensis (Takahashi et al., 2006), P. 67 americanus (Reddy & Garcia-Pichel, 2009), P. ginsengiterrae (Kim et al., 2012), and P. 68 medicamentivorans (Almeida et al., 2013). In addition, there is a species, Bactoderma rosea 69 (Tepper & Korshunova, 1973; Yarza et al., 2013), whose 16S rRNA gene sequence was 70 obtained only recently (Yarza et al., 2013) and that phylogenetically is affiliated to the genus 71 Patulibacter (99.7% sequence similarity to P. minatonensis). Finally, the order Gaiellales 72 (Albuquerque et al., 2011) so far has not been included in the reorganization of Ludwig et al. 73 (2012b). It encompasses the family Gaiellaceae (Albuquerque et al., 2011) with the single 74 genus Gaiella (Albuquerque et al., 2011) and the mesophilic species Gaiella occulta 75 (Albuquerque et al., 2011) established for an isolate from a deep mineral water aquifer. 76 In the present study, two novel isolates are described that extend the series of soil 77 isolates of the order Solirubrobacterales and represent a new family with a novel genus and 78 two novel species. 79 Strain D16/0/H6T originates from a pristine dark loam with neutral pH (6.8 and 7.2

80 measured in 2 mM CaCl2 and in distilled water, respectively) sampled in spring 2008 at Mile

3 81 46, Northern Namibia. Strain A22/0/F9_1T was isolated from a clayey sand soil with slightly

82 basic pH (7.4 and 8.2 measured in 2 mM CaCl2 and in distilled water, respectively). The latter 83 soil sample was collected in spring 2009 from a pasture at the farm Erichsfelde in central 84 Namibia. Soil suspensions were prepared in 10 mM MES, pH 5.5 for the 2008 sample and 85 HEPPS, pH 8.0 for the 2009 sample, respectively; 100 µl aliquots were plated on agar- 86 solidified SSE/HD 1:10 medium (Foesel et al., 2013; Supplementary Material and Methods) 87 buffered at pH 5.5 or 8.0 with 10 mM MES or HEPPS, respectively. Pure cultures were 88 obtained by restreaking. Unless otherwise noted SSE/HD 1:10 of the respective pH was also 89 used for further physiological tests and biomass production. 90 On solid media strain D16/0/H6T formed small colonies of ≤ 0.1 mm in diameter that 91 were light pink in color, translucent, smooth, shiny, and convex with entire margins. The 92 colonies of strain A22/0/F9_1T had a size of up to 1 mm, were whitish to pale 93 yellowish/brownish in color, translucent, smooth, shiny, and convex with entire margins. In 94 liquid culture no particularities such as intensive coloring or aggregation were observed. 95 Strain D16/0/H6T formed ovoid cells to short rods of an average size of (1.2 ± 0.2) x 96 (0.6 ± 0.04) µm (Fig. 1 a). In older cultures also longer rods of up to 3 µm occurred. Cells of 97 strain A22/0/F9_1T were regular rods with a length of (1.8 ± 0.3) and a width of (0.6 ± 0.03) 98 µm (Fig. 1 d). Both strains were motile. They divided by binary fission and stained Gram- 99 positive in standard procedures (Smibert & Krieg, 1994). This Gram-type was verified by the 100 KOH-test (Buck, 1982) and corresponds to the Gram-type of all Solirubrobacterales, while 101 the Thermoleophilales and Gaiellales stain Gram-negative (Table 1). Similar to the related 102 taxa, capsule or spore formation assessed by India ink and malachite green staining 103 (Beveridge et al., 2007), respectively, was not detected. For transmission electron microscopy 104 cells were prepared as described earlier (Foesel et al., 2013). Cells of strain D16/0/H6T had a 105 single, lateral flagellum whereas cells of strain A22/0/F9_1T carried multiple flagella (Fig. 1 106 b, e). Pili or fimbria were absent. Although flagella of strain A22/0/F9_1T sometimes seemed 107 to interlink cells, the formation of well organized network-like structures due to self- 108 aggregation of flagella as shown for Conexibacter woesei (Monciardini et al., 2003) was not 109 observed. Ultrastructural analyses confirmed the Gram-positive cell wall structure (Fig. 1 c, 110 f). Cells of strain D16/0/H6T contained conspicuous intracytoplasmic inclusion bodies. 111 Furthermore a surface layer could be detected which was missing for strain A22/0/F9_1T. 112 Almost full-length 16S rRNA gene fragments of strains D16/0/H6T and A22/0F9_1T 113 were amplified and sequenced as described before (Foesel et al., 2013). The resulting 114 sequences comprised 1456 (D16/0/H6T) and 1455 (A22/0F9_1T) unambiguous nucleotides

4 115 between E. coli positions 28 and 1491 (Brosius et al., 1978). Together with not yet included 116 reference sequences they were added to the 16S rRNA-based ‘All-Species Living Tree’ 117 Project (LTP) database (Yarza et al., 2008) release 119 (November 2014) using the program 118 package ARB version 6.0 (Ludwig et al., 2004). After automated alignment with the Fast 119 aligner tool implemented in ARB, the alignment was manually refined based on secondary 120 structure information. Phylogenetic trees were calculated using neighbor-joining, maximum- 121 parsimony, and maximum-likelihood algorithms (40% maximum frequency filter calculated 122 for the considered sequences resulting in 1366 valid columns between position 83 and 1406 of 123 the E. coli 16S rRNA reference gene; 1000 bootstrap resamplings). All three methods 124 assigned the two novel strains D16/0/H6T and A22/0F9_1T to the order Solirubrobacterales. 125 In the maximum-likelyhood (Fig. 2) and the neighbor-joining trees (Supplementary Fig. S1a), 126 the two species constitute a separate, deeply branching lineage, while the maximum- 127 parsimony algorithm (Supplementary Fig. S1b) clustered the sequences together with the 128 family Patulibacteraceae within the order Solirubrobacterales. Based on nucleotide identity 129 alone (ARB neighbor-joining tool without the use of an evolutionary substitution model), the 130 closest validly described relatives of D16/0/H6T were Solirubrobacter ginsenosidimutans and 131 S. phytolaccae (94.0%) followed by Conexibacter arvalis (93.8%) and S. soli (93.6%). The 132 next relatives of A22/0F9_1T were S. phytolaccae (93.5%), S. ginsenosidimutans (93.1%), 133 and S. soli (93.3%). Pairwise 16S rRNA gene sequence identity between the two novel strains 134 was 96.6%. 135 For DNA-DNA hybridization cells were disrupted in a Constant Systems TS 0.75 KW 136 (IUL Instruments, Koenigswinter, Germany), DNA was purified from the crude lysate by 137 chromatography on hydroxyapatite (Cashion et al., 1977). Hybridization was carried out 138 according to established protocols (De Ley et al., 1970; Huss et al., 1983) using a model Cary 139 100 Bio UV/VIS-spectrophotometer equipped with a Peltier-thermostatted 6x6 multicell 140 changer and a temperature controller with in situ temperature probe (Varian). Duplicate 141 measurements yielded hybridization levels of 30.2% and 28.4% between strain D16/0/H6T 142 and strain A22/0F9_1T, which is far below the threshold value of 70% DNA-DNA relatedness 143 generally accepted for the definition of a novel species (Wayne et al., 1987). 144 Signature nucleotide patterns originally established for the former subclass 145 Rubrobacteridae and its subordinate taxonomic ranks (Stackebrandt, 2004; Stackebrandt et 146 al., 1997) were adopted from the most recent revisions (Reddy & Garcia-Pichel, 2009; Zhi et 147 al., 2009) and reevaluated considering the recent taxonomic conversions within this group

5 148 (see above) and the addition of several novel species. The updated signature nucleotides are 149 given in the respective taxon descriptions. An overview is provided in Table 2. 150 For determination of the peptidoglycan composition whole cells of strains D16/0/H6T 151 and A22/0F9_1T were hydrolyzed (4N HCl, 100 °C, 15 hours) and the hydrolysates subjected 152 to thin-layer chromatography on cellulose plates according to Protocol 1 of Schumann (2011). 153 In both strains meso-2,6-diaminopimelic acid (meso-Dpm) was found as diagnostic diamino 154 acid of the peptidoglycan which is a common feature of all members of the class 155 Thermoleophilia (Table 1). 156 For G+C content determination cells were disrupted in a French press. The DNA was 157 purified using hydroxyapatite (Cashion et al., 1977). After sample treatment with P1 nuclease 158 and bovine alkaline phosphatase (Mesbah et al., 1989) the resulting deoxyribonucleosides 159 were analyzed by high performance liquid chromatography (HPLC, Shimadzu Corporation, 160 Kyoto, Japan) with conditions adapted from Tamaoka & Komagata (1984). The molar G+C 161 content was calculated from the ratio of deoxyguanosine (dG) and thymidine (dT) (Mesbah et 162 al., 1989) to be 72.8 and 74.0 mol% for strains D16/0/H6T and A22/0F9_1T, respectively. 163 These values range within the G+C contents reported for the members of the orders 164 Solirubrobacterales and Gaiellales, while the G+C content of the Themoleophilales and the 165 Rubrobacterales is lower (Tab.1). 166 Isoprenoid quinones were extracted from dried biomass with chloroform/methanol (2:1, 167 v/v) (Collins & Jones, 1981) and analyzed via HPLC (Tindall, 1990). The quinone system of 168 strains D16/0/H6T and A22/0F9_1T comprised menaquinone MK-8 (87 and 67%, 169 respectively) as major component. In addition, strain D16/0/H6T contained MK-7 (11%). 170 Strain A22/0F9_1T possessed MK-7 (12%), too, but also the partially hydrogenated

171 menaquinones MK-7(H2) (8%), MK-8(H2) (11%) and MK-9(H2) (3%). With respect to 172 cellular quinones, the two novel strains thus resemble the Rubrobacterales, which contain 173 MK-8 as predominant quinone (Tab.1), rather than the three families of the order

174 Solirubrobacterales that are characterized by the dominance of MK-7(H4)

175 (Solirubrobacteraceae, Conexibacteraceae) and DMK-7 or MK-7(H2) (Patulibacteraceae). 176 Cultures for fatty acid analysis were grown under identical conditions (SSE/HD 1:10 177 agar plates, pH 5.5, 18 d at room temperature). About 40 mg wet weight of fresh cells were 178 harvested and extracted according to the standard protocol (Sasser, 1990) of the Microbial 179 Identification System (MIDI Inc.; version 6.1) for fatty acids analysis. Compounds were 180 identified by comparison to the TSBA40 peak naming table database. Both strains possessed 181 straight chain, methyl or hydroxyl-branched saturated and monounsaturated fatty acids, but

6 182 differed largely in their major components and their full profiles (Supplementary Table S1). T 183 Strain D16/0/H6 contained C17:0 10-methyl (36.0%), iso-C16:0 (21.6%) and C18:1 9c (10.0%) 184 as major components. In addition, the fatty acid profile of strain D16/0/H6T showed a variety 185 of minor peaks below 4% including several peaks potentially not identified or misidentified 186 by the standard methods of the MIDI System (Albuquerque et al., 2011, 2014; Carreto et al., 187 1996). Those were indicated accordingly (Supplementary Table S1). Major fatty acids of T 188 strain A22/0F9_1 were C18:1 9c (35.1%), C17:1 8c (16.0%), C17:1 6c (11.9%) and iso-C16:0

189 (11.7%). In addition, summed feature 6 (C19:1 ω9c and/or C19:1 ω11c) and C17:0 10-methyl

190 occurred in significant amounts (8.6% and 7.2%, respectively). The occurrence of C17:0 10- 191 methyl as major component is a trait that strain D16/0/H6T shares with the Rubrobacterales 192 (Albuquerque et al., 2014), while members of the order Solirubrobacterales contain these 193 unusual fatty acids in low relative proportions at best (Almeida et al., 2013; Kim et al., 2012; 194 Reddy & Garcia-Pichel, 2009; Sasser, 1990; Seki et al., 2012; Wei et al., 2014; Zhang et al.,

195 2014). The high amounts of the methyl-branched fatty acid iso-C16:0 and the presence of

196 certain monounsaturated fatty acids of different length (C18:1 9c, C17:1 8c, C17:1 6c) in 197 strains D16/0/H6T and A22/0F9_1T are shared with different members of the 198 Solirubrobacterales (Table 1). 199 Growth ranges and optima of temperature and pH were determined under oxic 200 conditions in liquid SSE/HD 1:10 media. Temperature ranges of 10 - 56°C and pH values of 201 2.5 - 10.0 were tested. Depending on the respective pH value MES, HEPES, HEPPS, or 202 CHES (Sigma-Aldrich, Steinheim, Germany or Applichem, Darmstadt, Germany) were used 203 as buffer system at a final concentration of 10 mM. Since SSE/HD1:10 already contains high 204 amounts of different salts, salt tolerance was tested in liquid DSMZ medium 830 205 (http://www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium830.pdf) amended with 206 NaCl to final concentrations of 0 - 10% (w/v). Growth was determined by following the 207 optical density at 660 nm. Strain D16/0/H6T grew at temperatures of 13 - 43°C and pH 4.5 - 208 8.5. Optimal growth (defined as ≥ 75% of highest growth rate) was determined at 35 - 43°C 209 (highest rate at 40°C) and pH 5.0 - 8.0 (highest rate at pH 6.0). Growth of strain A22/0/F9_1T 210 occurred at 17 - 43°C and pH 5.0 - 9.5. It grew optimally at 32 - 43°C (highest rate at 35°C) 211 and pH 6.0 - 7.5 (highest rate at pH 6.5). Minimal doubling times were 9.0 and 9.2 h for strain 212 D16/0/H6T and A22/0/F9_1T, respectively. Strain D16/0/H6T grew best at NaCl 213 concentrations of 0%, but only with reduced growth rate at 0.25% and 0.5%, while at ≥ 1.0% 214 NaCl no growth at all occurred. Strain A22/0F9_1T tolerated NaCl concentrations of 0 - 1.0%, 215 the highest growth rate was observed at 0.25% NaCl. In summary, both strains exhibit broad

7 216 ranges of tolerance towards temperature and pH, achieve comparable doubling times, but 217 show different, individual minima, maxima and optima. In comparison to most of the 218 mesophilic soil isolates within the order Solirubrobacterales the two novel isolates show 219 elevated temperature optima and maxima, but by far do not reach the preferred growth 220 temperatures of the Thermoleophilales and the thermophilic strains of the Rubrobacterales 221 (Table 1). 222 Anaerobic growth with alternative electron acceptors was evaluated in liquid medium

223 830 (see above) with N2 as the gas phase and 10 mM NaNO3, 10 mM Na2SO4, 10 mM iron

224 pyrophosphate (Fe4(P2O7)3·x H2O), or 2 mM NaNO2 as electron acceptor. Fermentative 225 growth was assessed using the API20E test system (Biomérieux, Marcy L’Etoile, France). 226 Concentrations of electron acceptors were determined colorimetrically (Cataldo et al., 1975; 227 Gadkari, 1984; Harrigan & Mc Cance, 1966; Tabatabai, 1992; Tamura et al., 1974). However, 228 strains D16/0/H6T and strain A22/0F9_1T neither reduced nitrate, nitrite, sulfate or iron (III), 229 nor showed fermentative growth. 230 The range of growth substrates utilized by strains D16/0/H6T and strain A22/0F9_1T 231 was determined in microtiter plates in two parallels using soil solution equivalent (SSE) 232 (Angle et al., 1991) amended with 50 mg L-1 of yeast extract as basal medium. In total 106 233 single substrates were tested including sugars, organic acids, keto acids, alcohols, amino acids 234 (0.5 to 10 mM), casamino acids, casein hydrolysate, laminarin, peptone, yeast extract (0.05% 235 w/v each), and Tween 80 (0.001% w/v). Substrate utilization was recorded as positive if the

236 final OD660 surpassed the control value without addition of substrate by 1.5 fold. Growth on 237 the polymers cellulose, chitin and starch was tested on solidified media as described before 238 (Huber et al., 2014) with the additional staining of chitin plates with Congo Red (Thiagarajan 239 et al., 2011). Cytochrome c-oxidase and catalase activities were monitored employing 240 standard protocols (Cowan, 1974; Smibert & Krieg, 1994). Cytochrome c-oxidase 241 additionally was tested by Bactident® Oxidase test strips (Merck, Darmstadt, Germany). 242 Further physiological features (e. g. indol formation, urease activity, activity of different 243 exoenzymes) were determined using the API20E and the API ZYM test systems 244 (Biomérieux). Strains D16/0/H6T and A22/0F9_1T both grew well on glucose and protein 245 containing, complex substrates such as casamino acids, peptone, or yeast extract (Table 1; 246 Supplementary Table S2). Strain A22/0F9_1T also grew on several longer-chain organic 247 acids. In contrast, sugars were the preferred growth substrate of many of the related species 248 (Table 1). Exoenzyme profiles of strains D16/0/H6T and strain A22/0/F9_1T (Supplementary 249 Table S3) on the whole resemble those known from species of the related families. Only the

8 250 Solirubrobacteraceae and Rubrobacteraceae show additional activity of several carbohydrate 251 hydrolyzing enzymes, which largely is in congruence with the observed substrate ranges of 252 the respective species. Additional results of the physiological tests are compiled in 253 Supplementary Tables S2 and S3, and in the species descriptions below. 254 In summary, strains D16/0/H6T and strain A22/0/F9_1T are affiliated with the order 255 Solirubrobacterales, but cannot be unambiguously attributed to one of the established 256 families Solirubrobacteraceae, Conexibacteraceae or Patulibacteraceae. Their distinct 257 phylogentic position and the specific combination of phenotypic traits differentiate the two 258 isolates from the members of all existing families. Although several distinguishing features 259 were observed between the two strains, their close phylogenetic relationship indicates that 260 they belong to the same genus. Consequently, strains D16/0/H6T and strain A22/0/F9_1T are 261 proposed to form the novel genus Parviterribacter, including the two novel species, P. 262 kavangonensis and P. multiflagellatus constituting the novel family Parviterribacteraceae. 263 Furthermore, we propose the order Gaiellales as a further, deep-branching order of the 264 class Thermoleophilia based on its phylogenetic position, the observed signature nucleotide 265 pattern and several phenotypic properties shared with other members of the class. 266 Based on its phylogenetic position the species Bactoderma rosea is proposed to be a 267 member of the genus Patulibacter. Whether it should be assigned to its phylogenetically 268 closest relative, the species P. minatonensis, or remain an autonomous species needs to be 269 clarified by future physico-chemical characterization.

270 Description of Parviterribacter, gen. nov. 271 Parviterribacter (Par.vi.ter.ri.bac’ter. L. adj. parvus, small; L. fem. n. terra, earth; N.L. 272 masc. n. bacter, a rod; N.L. masc. n. Parviterribacter small, rod-shaped bacterium isolated 273 from soil). 274 Gram-positive, non-spore-forming, motile, ovoid to rod shaped cells that divide by 275 binary fission. No capsule formation. Strictly aerobic, chemoorganotrophic mesophiles. 276 Catalase positive, cytochrome c-oxidase variable. The peptidoglycan contains meso-Dpm as 277 diagnostic diamino acid. Major fatty acids consist of mono-unsaturated and iso- and/or 10- 278 methyl-branched fatty acids. Major quinone is MK-8. The DNA G+C content is 73 - 74%. 279 The type species is Parviterribacter kavangonensis. 280

281 Description of Parviterribacter kavangonensis, sp. nov.

9 282 Parviterribacter kavangonensis (ka.van.go.nen´sis. N.L. masc. adj. kavangonensis 283 deriving from the Kavango region, Namibia). 284 Displays the following characteristics in addition to those given in the genus description. 285 Cells are 1.0 - 1.5 µm long and 0.6 - 0.7 µm in diameter. On agar plates forms small colonies 286 of ≤ 0.1 mm in diameter that are light pink in color, translucent, smooth, shiny, and convex 287 with entire margins. Grows at 13 - 43°C and pH 4.5 - 8.5; optimal growth at 35 - 43°C and pH 288 5.0 - 8.0. Minimal doubling time is 9.0 h. Tolerates NaCl concentrations of up to 0.5% (w/v); 289 optimal growth occurs in the absence of NaCl. 290 Grows on glucose, mannose, lyxitol, aspartate, lysine, hydroxy-proline, acetate, 291 butyrate, β-hydroxybutyrate, propionate, protocatechuate, pyruvate, succinate, casamino 292 acids, casein hydrolysate, peptone, and yeast extract. No growth is observed on arabinose, 293 cellobiose, erythrose, erythrulose, fructose, fucose, galactose, lactose, lyxose, maltose, 294 melizitose, raffinose, rhamnose, sorbose, sucrose, trehalose, xylose, glucosamine, N- 295 acetylglucosamine, N-acetylgalactosamine, acetoin, adonitol, arabitol, dulcitol, mannitol, 296 myo-inositol, sorbitol, xylitol, alanine, arginine, cysteine, glutamate, glycine, histidine, 297 isoleucine, leucine, methionine, ornithine, phenylalanine, proline, threonine, tryptophan, 298 tyrosine, valine, adipate, ascorbate, benzoate, trimethoxybenzoate, -hydroxybutyrate, - 299 hydroxybutyrate, isobutyrate, caproate, caprylate, citrate, isocitrate, crotonate, formate, 300 fumarate, gluconate, 2-oxogluconate, glucuronate, 2-oxoglutarate, glycolate, glyoxylate, 301 heptanoate, isovalerate, laevulinate, lactate, malate, malonate, nicotinic acid, oxaloacetate, 302 shikimate, tartrate, 2-oxovalerate, butanol, 1,2-butandiol, 2,3-butandiol, ethanol, ethylene 303 glycol, glycerol, methanol, propanol, 1,2-propandiol, fermented rumen extract, chitin, 304 cellulose, laminarin, starch, and Tween 80. 305 Aesculin and gelatine are hydrolyzed, albeit gelatin only very slowly. Indol and acetoin 306 formation is not observed. Urease and arginine dihydrolase negative. The following additional 307 enzyme activities are present: acidic phosphatase, alkaline phosphatase, naphtol-AS-BI- 308 phosphohydrolase, esterase (C4), esterase lipase (C8), leucine-arylamidase, cystine- 309 arylamidase (weak),-glucuronidase, -mannosidase, and -fucosidase (weak). Lipase 310 (C14), valine-arylamidase, trypsin, -chymotrypsin, - and -galactosidase, - and - 311 glucosidase, and N-acetyl--glucosaminidase activity is absent. 312 Oxygen is the sole electron acceptor. The major quinone is MK-8, in addition MK-7

313 occurs. The fatty acid profile is dominated by C17:0 10-methyl, iso-C16:0, and C18:1 9c. 314 The type strain is D16/0/H6T (= DSM 25205T = LMG 26950T), and was isolated from a 315 loamy subtropical savanna soil at Mile 46, Kavango region, Northern Namibia (18°17’14.0´´S

10 316 19°15’32.3´´E, 1163 m height above sea level). The DNA G+C content of the type strain is 317 72.8 mol%.

318 Description of Parviterribacter multiflagellatus, sp. nov. 319 Parviterribacter multiflagellatus (mul.ti.fla.gel.la’tus. L. adj. multus, many, numerous; 320 L. n. flagellum, a whip; L. masc. suff. -atus, suffix denoting provided with; N.L. masc. adj. 321 multiflagellatus, provided with numerous flagella). 322 In addition to those given in the genus description shows the following features. Cells 323 are 1.3 - 2.3 µm long and 0.6 - 0.7 µm wide. Colonies attain a size of up to 1 mm, are whitish 324 to pale yellowish/brownish in color, translucent, smooth, shiny, and convex with entire 325 margins. Grows at 17 - 43°C and pH 5.0 - 9.5; optimal growth occurs at 32 - 43°C and pH 6.0 326 - 7.5. Minimum doubling time is 9.2 h. Tolerates NaCl concentrations of up to 1.0% (w/v); 327 optimal growth occurs at 0.25%. 328 Grows on glucose, cellobiose, N-acetylglucosamine, adonitol, glutamate, butyrate, 329 isobutyrate, caproate, caprylate, crotonate, gluconate, heptanoate, laevulinate, glycerol, 330 protocatechuate, pyruvate, succinate, casamino acids, casein hydrolysate, peptone, yeast 331 extract, and starch. No growth is observed on arabinose, erythrose, erythrulose, fructose, 332 fucose, galactose, lactose, lyxose, maltose, mannose, melizitose, raffinose, rhamnose, sorbose, 333 sucrose, trehalose, xylose, glucosamine, N-acetylgalactosamine, acetoin, arabitol, dulcitol, 334 lyxitol, mannitol, myo-inositol, sorbitol, xylitol, alanine, arginine, aspartate, cysteine, glycine, 335 histidine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, hydroxy- 336 proline, threonine, tryptophan, tyrosine, valine, acetate, adipate, ascorbate, benzoate, 337 trimethoxybenzoate, α-hydroxybutyrate, β-hydroxybutyrate, -hydroxybutyrate, citrate, 338 isocitrate, formate, fumarate, 2-oxogluconate, glucuronate, 2-oxoglutarate, glycolate, 339 glyoxylate, isovalerate, lactate, malate, malonate, nicotinic acid, oxaloacetate, propionate, 340 shikimate, tartrate, 2-oxovalerate, butanol, 1,2-butandiol, 2,3-butandiol, ethanol, ethylene 341 glycol, methanol, propanol, 1,2-propandiol, fermented rumen extract, chitin, cellulose, 342 laminarin, and Tween 80. 343 Aesculin and gelatine are hydrolyzed. Indol and acetoin are not formed. Urease and 344 arginine dihydrolase negative. The following additional enzyme activities are present: acidic 345 phosphatase (weak), alkaline phosphatase (weak), naphtol-AS-BI-phosphohydrolase, esterase 346 (C4), esterase lipase (C8), lipase (C14; weak), leucine-arylamidase (weak), cystine- 347 arylamidase (weak),-glucosidase, and N-acetyl--glucosaminidase (weak). Valine-

11 348 arylamidase, trypsin, -chymotrypsin, - and -galactosidase,-glucosidase, - 349 glucuronidase, -mannosidase, and -fucosidase activity is absent. 350 Oxygen is the sole electron acceptor. The major quinone is MK-8, as further notable

351 constituents MK-7, MK-7(H2), and MK-8(H2) occur. The fatty acid profile is dominated by

352 C18:1 9c, C17:1 8c, C17:1 6c, and iso-C16:0. 353 The type strain is A22/0/F9_1T (= DSM 25204T = LMG 26949T), which was isolated 354 from a sandy subtropical savanna soil in Erichsfelde, central Namibia (21°38´15.8´´S 355 16°52´03.9´´E, 1497 m height above sea level). The DNA G+C content of the type strain is 356 74.0 mol%.

357 Description of Parviterribacteriaceae, fam. nov. 358 Parviterribacteriaceae (Par.vi.ter.ri.bac.te.ra.ce’ae. N.L. masc. n. Parviterribacter type 359 genus of the family; suff. -aceae, ending denoting a family; N.L. fem. pl. n. 360 Parviterribacteraceae, the Parviterribacter family). 361 The family Parviterribacteraceae is a member of the order Solirubrobacterales. 362 Currently, the family comprises the sole genus Parviterribacter. The delineation of the family 363 is determined by its phylogenetic position of the 16S rRNA gene sequences of its members 364 and the quinone system (MK-8 as major quinone). The detailed description is the same as that 365 given for the genus Parviterribacter. Signature nucleotides in the 16S rRNA gene sequence 366 (Escherichia coli reference gene; Brosius et al. (1978)) partially shared with other families 367 within the order Solirubrobacterales are: 52:359 (C-G), 408:434 (G-C), 590:649 (U-A), 368 600:638 (C-G), 823:877 (A-U), and 999:1041 (U-A). Signatures differentiating the 369 Parviterribacteriaceae from all other Solirubrobacterales families are C-G and U-G at 370 positions 681:709 and 1115:1185, respectively. Signatures shared with all members of the 371 order Solirubrobacterales or even the class Themoleophilia are listed in the respective taxon 372 descriptions. The type genus of this family is Parviterribacter.

373 Emended description of the family Solirubrobacteraceae Stackebrandt 2005 (effective 374 publication Stackebrandt, 2004) emendet Zhi et al. 2009 375 The description is as given by Zhi et al. (2009) with the following amendments. Gram- 376 positive, non motile rods. Aerobic mesophiles with a preference for sugars as sole carbon

377 source. The sole or predominant quinone is MK-7(H4). Dominating fatty acids are C18:1 9c

378 and C16:0 iso. Updated signature nucleotides in the 16S rRNA gene sequence partially shared 379 with other Solirubrobacterales families are: 52:359 (C-G), 144:178 (C-G), 408:434 (G-C), 380 600:638 (C-G), 681:709 (U-A), 823:877 (G-C), 999:1041 (U-A), and 1115:1185 (C-G). The

12 381 signature C-G at positions 145:177 and 590:649 differentiates the Solirubrobacteriaceae from 382 any other family of the order. Signatures shared with all members of the order 383 Solirubrobacterales and the class Themoleophilia are listed in the respective taxon 384 descriptions.

385 Emended description of the family Conexibacteraceae Stackebrandt 2005 (effective 386 publication Stackebrandt, 2004) emendet Zhi et al. 2009 387 The description is as given by Zhi et al. (2009) with the following amendments. Gram- 388 positive, motile rods. Aerobic, psychrotolerant mesophiles. The diagnostic amino acid is

389 meso-Dpm, the major respiratory quinone is MK-7(H4). Shared, dominating fatty acids are

390 C17:1 6c and C18:1 9c. Signature nucleotides in the 16S rRNA gene sequence partially 391 shared with other Solirubrobacterales are: 590:649 (U-A), 600:638 (C-G), 681:709 (U-A), 392 823:877 (G-C), and 1115:1185 (C-G). Signatures differentiating the Conexibacteriaceae from 393 any other family of the order are: 52:359 (U-A), 144:178 (U-A), 145:177 (U-A), 408:434 (A- 394 U), and 999:1041 (G-U). Signatures shared with all members of the order Solirubrobacterales 395 or even the class Themoleophilia are listed in the respective taxon descriptions.

396 Emended description of the family Patulibacteraceae Takahashi et al. 2006 emendet Zhi 397 et al. 2009 398 The description is as given by Takahashi et al. (2006) and Zhi et al. (2009) with the

399 following amendments. The main isoprenoid quinone is DMK-7 or MK-7(H2). Updated 400 signature nucleotides in the 16S rRNA gene sequence partially shared with other 401 Solirubrobacterales families are: 52:359 (C-G), 144:178 (C-G), 408:434 (G-C), 590:649 (U- 402 A), 600:638 (U-G), 681:709 (U-A), 823:877 (A-U), and 1115:1185 (C-G). Signatures shared 403 with all members of the order Solirubrobacterales or even the class Themoleophilia are listed 404 in the respective taxon descriptions.

405 Emended description of the order Solirubrobacterales Reddy and Garcia-Pichel 2009 406 The description is as given by Reddy and Garcia-Pichel (2009) with the following 407 amendments. Contains the families Solirubrobacteraceae, Conexibacteraceae, 408 Patulibacteraceae, and Parviterribacteraceae. Updated 16S rRNA gene sequence signature 409 nucleotides are: 63:104 (G-C), 70:98 (G-C), 293:304 (G-C), 370:391 (C-G), 580:761 (U-A), 410 670:736 (A-U), 722:733 (U-A), 941:1342 (A-U), 1118:1155 (U-A), and 1311:1326 (A-U). 411 Signatures shared with the other orders of the class Thermoleophilia are given in the class 412 description.

13 413 Emended description of the family Thermoleophilaceae Stackebrandt 2005 (effective 414 publication Stackebrandt, 2004) emendet Zhi et al. 2009 415 The description is as given by Zhi et al. (2009) with the following amendments. Gram- 416 negative (although typical Gram-positive cell wall structure as demonstrated by TEM), non 417 motile rods. Aerobic, moderately thermophilic strains that utilize n-alkanes as sole carbon 418 source. Cell wall peptidoglycan is based on meso-Dpm. The updated pattern of 16S rRNA 419 gene sequence signature nucleotides partially shared with families of the neighboring orders 420 Solirubrobacterales and Gaiellales consists of: 52:359 (C-G), 63:104 (G-C), 70:98 (G-C), 421 139:224 (G-C), 144:178 (C-G), 145:177 (C-G), 377:386 (C-G), 408:434 (G-C), 590:649 (C- 422 G), 600:638 (C-G), 681:709 (C-G), 722:733 (U-A), 823:877 (G-C), 999:1041 (A-U), and 423 1115:1185 (C-G). Signatures differentiating the Thermoleophilaceae from any other family 424 within the Thermoleophilia are 293:304 (G-U), 370:391 (G-C), 580:761 (C-G), 670:736 (G- 425 C), 941:1342 (G-C), 1118:1155 (C-G), and 1311:1326 (G-C). On order level no further 426 signatures can be added to the above given profile. Signatures shared with all members of the 427 class Thermoleophilia can be seen from the class description.

428 Emended description of the order Thermoleophilales Reddy and Garcia-Pichel 2009 429 The description is as given by Reddy and Garcia-Pichel (2009) with the following 430 amendments. Updated 16S rRNA gene sequence signature nucleotides largely correspond to 431 those given in the description of the family Thermoleophilaceae. Positions 52:359, 144:178, 432 408:434, 823:877, 999:1041, and 1115:1185 become meaningless on order level, positions 433 139:224, 145:177, 377:386, 590:649, 600:638, and 681:709 become unique features within 434 the class Thermoleophilia. Signatures shared within the whole class Thermoleophilia are 435 listed in the respective description.

436 Emended description of the family Gaiellaceae Albuquerque et al. 2012 (effective 437 publication Albuquerque et al., 2011) 438 The description is as given by Albuquerque et al. (2011) with the following 439 amendments. The family is assigned to the class Thermoleophilia as own, deep-branching 440 order (see below). 16S rRNA gene sequence signature nucleotides partially shared with 441 families of the neighboring orders Solirubrobacterales and Thermoleophilales consists of: 442 52:359 (C-G), 144:178 (C-G), 293:304 (G-C), 370:391 (C-G), 408:434 (G-C), 580:761 (U- 443 A), 670:736 (A-U), 681:709 (U-A), 823:877 (G-C), 941:1342 (A-U), 999:1041 (A-U), 444 1115:1185 (C-G), 1118:1155 (U-A), and 1311:1326 (A-U). Signatures differentiating the 445 Gaiellaceae from any other family within the Thermoleophilia are 63:104 (C-G), 70:98 (A-

14 446 G), 139:224 (C-G), 145:177 (U-G), 377:386 (G-C), 590:649 (G-C), 600:638 (G-C), and 447 722:733 (U-G). On order level no further signatures can be added. Signatures shared with all 448 members of the class Thermoleophilia can be seen from the class description.

449 Emended description of the order Gaiellales Albuquerque et al. 2012 (effective 450 publication Albuquerque et al., 2011) 451 The description is as given by Albuquerque et al. (2011) with the following 452 amendments. Phylogenetically the order is a deep branching member of the class 453 Thermoleophilia. 16S rRNA gene sequence signature nucleotide patterns are those given in 454 the description of the family Gaiellaceae with the following changes. Signature positions 455 52:359, 144:178, 408:434, 823:877, 999:1041, and 1115:1185 are insignificant on order level, 456 positions 139:224 and 681:709 are unique. Signatures shared with all members of the class 457 Thermoleophilia are given in the class description.

458 Emended description of the class Thermoleophilia Suzuki and Whitman 2013 459 The description is as given by Suzuki and Whitman (2012) with the following 460 amendments. In addition, contains the order Gailellales. 16S rRNA gene sequence signature 461 nucleotide patterns of the class partially shared with other classes of the phylum 462 Actinobacteria are: 127:234 (G-C), 242:284 (C-G), 291:309 (U-A), 316:337 (C-G), 409:433 463 (G-C), 438:496 (U-A), 657:749 (U-A), 819 (A), 953:1228 (G-C), 954:1226 (G-C), 955:1225 464 (U-A), 1051:1207 (G-C), 1313:1324 (G-C), and 1410:1490 (A-U). Signatures differentiating 465 the Thermoleophilia from all other classes of the phylum Actinobacteria are G-C and G-A at 466 positions 66:103 and 661:744, respectively.

467 Emended description of the family Rubrobacteraceae Rainey et al. 1997 emend. 468 Stackebrandt 2004 emend. Zhi et al. 2009 469 The description is as given by Zhi et al. (2009) with the following amendments. 470 Currently is the only family within the class Rubrobacteria. 16S rRNA gene sequence 471 signature nucleotides partially shared with families of the neighboring class Thermoleophila 472 consist of: 63:104 (C-G), 293:304 (G-U), 370:391 (C-G), 377:386 (G-C), 408:434 (G-C), 473 670:736 (A-U), 681:709 (C-G), 941:1342 (A-U), and 1115:1185 (C-G). Signatures 474 differentiating the Rubrobacteraceae from any other family compared with are: 52:359 (G-C), 475 66:103 (A-U), 70:98 (A-U), 139:224 (U-A), 144:178 (G-C), 145:177 (G-C), 409:433 (C-G), 476 438:496 (G-G), 657:749 (G-C), 661:744 (G-C), 953:1228 (U-A), 954:1226 (C-G), 1051:1207 477 (C-G), and 1313:1324 (U-A). Comparison on order level does not add further signature

15 478 positons. Additonal signatures uniform within the family but only meaningful on class level 479 are listed in the description of the Rubrobacteria.

480 Emended description of the order Rubrobacterales Rainey et al. 1997 emend. Reddy and 481 Garcia-Pichel 2009 and Zhi et al. 2009 482 The description is as given by Reddy and Garcia-Pichel (2009) and Zhi et al. (2009) 483 with the following amendments. Known members are mesophilic or moderately thermophilic 484 to thermophilic. Updated 16S rRNA gene sequence signature nucleotides are those given in 485 the description of the family Rubrobacteraceae. Sole exception is position 408:434 that 486 becomes insignificant on order level. Further signatures acting on class level can be seen from 487 the description of the Rubrobacteria.

488 Emended description of the class Rubrobacteria Suzuki 2013 489 The description is as given by Suzuki (2012) with the following amendments. 16S 490 rRNA gene sequence signature nucleotide patterns of the class partially shared with other 491 classes of the phylum Actinobacteria are: 66:103 (A-U), 127:234 (G-C), 242:284 (C:G), 492 291:309 (U-A), 316:337 (C-G), 661:744 (G-C), 670:736 (A-U), 819 (A), 941:1342 (A-U), 493 955:1225 (U-A), 1313:1324 (U-A), and 1410:1490 (A-U). 494 Signatures differentiating the Rubrobacteria from all other classes of the phylum 495 Actinobacteria are: 52:359 (G-C), 70:98 (A-U), 139:224 (U-A), 144:178 (G-C), 145:177 (G- 496 C), 293:304 (G-U), 409:433 (C-G), 438:496 (G-G), 502:543 (C-G), 657:749 (G-C), 681:709 497 (C-G), 953:1228 (U-A), 954:1226 (C-G), 1051:1207 (C-G), and 1115:1185 (C-G). 498

16 499 ACKNOWLEDGEMENTS

500 We thank Dr. Peter Schumann, Dr. Cathrin Spröer, Gabi Pötter, Anika Wasner, Bettina 501 Sträubler and Birgit Grün (all DSMZ) for analysis of cellular fatty acid composition, 502 respiratory quinones, peptidogycan amino acid composition, and G+C-content as well as for 503 performing DNA-DNA-hybridization. Ina Schleicher (HZI) assisted with electron 504 microscopic preparations. Dr. Peter Schumann is also acknowledged for advice regarding 505 peptidoglycan structure and fatty acid composition, Prof. Dr. Erko Stackebrandt for helpful 506 thoughts on signature nucleotides. Dr. Pia Wuest is acknowledged for critically reading the 507 manuscript. 508 Soils in Namibia were sampled under collection permits 1245/2008 and 1358/2009, and 509 exported under permits ES 23744 (of March 27, 2008) and ES 24478 (of April 6, 2009). 510 Isolation and deposit of strains D16/0/H6T and A22/0/F9_1T is under the MTA of the NBRI 511 (National Botanical Research Institute, Namibia) of April 05, 2012. This work was supported 512 by grants of German Federal Ministry of Science and Education to Jörg Overmann 513 (BIOLOG/BIOTA project 01LC0621C and TFO project 01LL0912M).

514

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636 Stackebrandt, E. (2005b). Conexibacteraceae fam. nov. In Validation of publication of new 637 names and new combinations previously effectively published outside the IJSEM, List 638 no. 102. Int J Syst Evol Microbiol 55, 547-549.

639 Stackebrandt, E. (2005c). Thermoleophilaceae fam. nov. In Validation of publication of new 640 names and new combinations previously effectively published outside the IJSEM, List 641 no. 102. Int J Syst Evol Microbiol 55, 547-549.

642 Stackebrandt, E., Rainey, F. A. & Ward-Rainey, N. L. (1997). Proposal for a New 643 Hierarchic Classification System, Actinobacteria classis nov. Int J Syst Bacteriol 47, 644 479-491.

645 Suzuki, K.-I. (2012). Class V. Rubrobacteria class. nov. In Bergey's Manual of Systematic 646 Bacteriology, second edn, vol. 5 (The Actinobacteria), part B, pp. 2004-2009. Edited 647 by M. Goodfellow, Kämpfer, P., Busse, H.-J., Trujillo, M. E., Suzuki, K.-I., Ludwig, 648 W., Whitman, W. B. New York: Springer.

649 Suzuki, K.-I. & Whitman, W. B. (2012). Class VI. Thermoleophilia class. nov. In Bergey's 650 Manual of Systematic Bacteriology, second edn, vol. 5 (The Actinobacteria), part B, 651 pp. 2010-2028. Edited by M. Goodfellow, Kämpfer, P., Busse, H.-J., Trujillo, M. E., 652 Suzuki, K.-I., Ludwig, W., Whitman, W. B. . New York: Springer.

653 Suzuki, K.-I. & Whitman, W. B. (2013). Thermoleophilia class. nov. In List of new names 654 and new combinations previously effectively, but not validly, published, List no. 151. 655 Int J Syst Evol Microbiol 63, 1577-1580.

656 Suzuki, K.-I., Collins, M. D., Iijima, E. & Komagata, K. (1988). Chemotaxonomic 657 characterization of a radiotolerant bacterium, Arthrobacter radiotolerans: Description 658 of Rubrobacter radiotolerans gen. nov., comb. nov. FEMS Microbiol Lett 52, 33-39.

659 Suzuki, K. I. (2013). Rubrobacteria class. nov. In List of new names and new combinations 660 previously effectively, but not validly, published, List no. 151. Int J Syst Evol 661 Microbiol 63, 1577-1580.

662 Tabatabai, M. A. (1992). Appendix: Methods of measurements of sulfur in soils, plants, 663 materials and water. In Sulfur cycling on the continents: wetlands, terrestrial 664 ecosystems and associated water bodies (Scope 48), pp. 307-344. Edited by R. W. 665 Howarth, J. W. B. Stewart & M. V. Ivanov. West Sussex, England: John Wiley & 666 Sons Ltd.

667 Takahashi, Y., Matsumoto, A., Morisaki, K. & Ōmura, S. (2006). Patulibacter 668 minatonensis gen. nov., sp. nov., a novel actinobacterium isolated using an agar 669 medium supplemented with superoxide dismutase, and proposal of Patulibacteraceae 670 fam. nov. Int J Syst Evol Microbiol 56, 401-406.

21 671 Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed- 672 phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125-128.

673 Tamura, H., Goto, K., Yotsuyanagi, T. & Nagayama, M. (1974). Spectrophotometric 674 determination of iron(II) with 1,10-phenanthroline in presence of large amounts of 675 iron(III). Talanta 21, 314-318.

676 Tepper, E. Z. & Korshunova, G. F. (1973). Taxonomic position of microorganisms of the 677 Bactoderma group and their role in soil. Microbiology 42, 430-434.

678 Thiagarajan, V., Revathi, R., Aparanjini, K., Sivamani, P., Girilal, M., Priya, C. S. & 679 Kalaichelvan, P. T. (2011). Extra cellular chitinase production by Streptomyces sp. 680 PTK19 in submerged fermentation and its lytic activity on Fusarium oxysporum PTK2 681 cell wall. Int J Current Sci, 30-44.

682 Tindall, B. J. (1990). Lipid composition of Halobacterium lacusprofundi. FEMS Microbiol 683 Lett 66, 199-202.

684 Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., 685 Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other 686 authors (1987). Report of the ad hoc committee on reconciliation of approaches to 687 bacterial systematics. Int J Syst Bacteriol 37, 463-464.

688 Wei, L., Ouyang, S., Wang, Y., Shen, X. & Zhang, L. (2014). Solirubrobacter phytolaccae 689 sp. nov., an endophytic bacterium isolated from roots of Phytolacca acinosa Roxb. Int 690 J Syst Evol Microbiol 64, 858-862.

691 Yarza, P., Richter, M., Peplies, J., Euzeby, J., Amann, R., Schleifer, K.-H., Ludwig, W., 692 Glöckner, F. O. & Rosselló-Móra, R. (2008). The All-Species Living Tree project: 693 A 16S rRNA-based phylogenetic tree of all sequenced type strains. Syst Appl 694 Microbiol 31, 241-250.

695 Yarza, P., Spröer, C., Swiderski, J., Mrotzek, N., Spring, S., Tindall, B. J., Gronow, S., 696 Pukall, R., Klenk, H.-P. & other authors (2013). Sequencing orphan species 697 initiative (SOS): Filling the gaps in the 16S rRNA gene sequence database for all 698 species with validly published names. Syst Appl Microbiol 36, 69-73.

699 Zarilla, K. A. & Perry, J. J. (1984). Thermoleophilum album gen. nov. and sp. nov., a 700 bacterium obligate for thermophily and n-alkane substrates. Arch Microbiol 137, 286- 701 290.

702 Zarilla, K. A. & Perry, J. J. (1986). Deoxyribonucleic acid homology and other 703 comparisons among obligately thermophilic hydrocarbonoclastic bacteria, with a 704 proposal for Thermoleophilum minutum sp. nov.. Int J Syst Bacteriol 36, 13-16.

705 Zhang, L., Zhu, L., Si, M., Li, C., Zhao, L., Wei, Y. & Shen, X. (2014). Solirubrobacter 706 taibaiensis sp. nov., isolated from a stem of Phytolacca acinosa Roxb.. Antonie van 707 Leeuwenhoek 106, 279-285.

708 Zhi, X.-Y., Li, W.-J. & Stackebrandt, E. (2009). An update of the structure and 16S rRNA 709 gene sequence-based definition of higher ranks of the class Actinobacteria, with the

22 710 proposal of two new suborders and four new families and emended descriptions of the 711 existing higher taxa. Int J Syst Evol Microbiol 59, 589-608.

23 712 TABLES

24 713 Table 1: Characteristics of D16/0/H6T and strain A22/0/F9_1T compared those of at the time of writing all validly described strains within related 714 families of the orders Solirubrobacterales, Thermoleophilales, Gaiellales and Rubrobacterales. 715 Strains/families: 1, D16/0/H6T; 2, A22/0/F9_1T; 3, Solirubrobacteraceae, comprising summarized features of Solirubrobacter pauli B33D1T 716 (Singleton et al., 2003), S. soli Gsoil 355T (Kim et al., 2007), S. ginsenosidimutans BXN5-15T (An et al., 2011), S. phytolaccae GTGR-8T (Wei et 717 al., 2014), S. taibaiensis GTJR-20T (Zhang et al., 2014); 4, Conexibacteracae, including Conexibacter woesei ID131577T (Monciardini et al., 718 2003), C. arvalis KV-962T (Seki et al., 2012); 5, Patulibacteraceae, including Bactoderma rosea (Tepper & Korshunova, 1973), Patulibacter 719 minatonensis KV-614T (Takahashi et al., 2006), P. americanus CP1777-2T (Reddy & Garcia-Pichel, 2009), P. ginsengiterrae P4-5T (Kim et al., 720 2012), P. medicamentivorans I11T (Almeida et al., 2013); 6, Thermoleophilacae, including Thermoleophilum album HS-5T (Zarilla & Perry, 1984), 721 T. minutum YS-4T (Zarilla & Perry, 1986); 7, Gaiellaceae, including Gaiella occulta F2-233T (Albuquerque et al., 2011); 8, Rubrobacteraceae, 722 including Rubrobacter radiotolerans IAM 12072T (Suzuki et al., 1988), R. xylanophilus PRD-1T (Carreto et al., 1996), R. taiwanensis LS-293T 723 (Chen et al., 2004), R. bracarensis VF70612_S1T (Jurado et al., 2012), R. aplysinae RV113T (Kämpfer et al., 2014), R. calidifluminis RG-1T and R. 724 naiadicus RG-3T (Albuquerque et al., 2014). 725 All strains are strictly aerobic, formation of spores or capsules is reported for none of them. 726 +, positive; -, negative; (+), weak growth detected, v, variable; ND, no data available. 727 When characteristics differ among strains, numbers in brackets give the number of strains showing the respective feature (first number) compared to 728 the number of all strains considered (second number).

25 Thermoleophilia Rubrobacteria

Solirubrobacterales Thermoleophilales Gaiellales Rubrobacterales Characteristic 1 2 3 4 5 6 7 8

Isolation source Terrestric Terrestric Terrestric (3/5), Terrestric Terrestric (3/5), Aquatic Aquatic Aquatic (5/7) , plant tissue (2/5) aquatic (1/5), ND (1/5) biofilm (1/7), marine sponge (1/7) Cell shape Ovoid cells to rods Rods Rods Rods Rods Rods Rods Cocci (2/7), rods (3/7), cocci-rods (2/7) Gram-staining Positive Positive Positive Positive Positive Negative Negative Positive Motility + + - + +(3/5), v(1/5), - (1/5) - - - (5/7), ND (2/7) Pigmentation Pink White-yellowish/ White (3/5), yellow White-yellowish White-yellowish Translucent-white Non-pigmented Pink-orange color brownish (1/5), pink (1/5) (3/5), pink (2/5) tones (6/7), non-pigmented (1/7) Oxidase + - + (2/5), (+) (1/5), - (2/5) + (1/2), - (1/2) + (2/5), - (3/5) ND + + (5/7), - (2/7) Catalase + + + + + + (1/2), ND (1/2) + + (5/7), ND (2/7) NaCl tolerance (%) ≤ 0.5 ≤ 1.0 ≤ 0.5-≤ 1.5 < 2-≤ 4 ≤ 1.5-≤ 3 ND <1 ≤ 2-≤ 30 Temperature range 13-43 17-43 7-38 5-46 5-39 45-70 20-42.5 10-45(2/7), (°C) 30-70(5/7)a Temperature optimum 35-43 32-43 25-30 28-38 24-32 58-62 35-37 28-37/ ND (2/7), (°C) 45-60 (5/7) a pH range 4.5-8.5 5.0-9.5 6.0-10.0 5.0-10.0 5.0-9.0 6.0-7.5 5.5-8.5 6.0-10.0 (5/7), ND (2/7) pH optimum 5.0-8.0 6.0-7.5 6.5-8.0 7.0-9.0 7.0 (2/5), ND (3/5) 6.8-7.0 6.5-7.5 6.0-8.0 (5/7), ND (2/7) DNA G+C-content 72.8 74.0 71.0-71.8 71-75 72-74.6 70.0-70.4 71.6±0.2 65.2-68.5 (6/7), (mol%) ND (1/7) Diamino acid meso-Dpm meso-Dpm ND meso-Dpm meso-Dpm meso-Dpm meso-Dpm L-Lys (3/7), ND (4/7)

Major quinone MK-8 MK-8 MK-7(H4) MK-7(H4) MK-7(H2)(1/5), DMK- ND MK-7 MK-8 (6/7), ND (1/7) 7(3/5), ND (1/5)

b c Major fatty acids C17:0 10-methyl, C17:1 c, C17:1 c(1/5), C16:0 (1/2), C18:1 9c (4/4 ), ND C15:0 iso 8-methyl, C18:0 (1/6 ), (≥ 10%) C18:1 9c, C17:1 8c, C18:1 9c(5/5) C17:1 c (2/2), C15:0 anteiso (3/4), C17:0 iso, C18:0 alcohol (1/6), C 12-methyl (4/6), C16:0 iso C18:1 9c, C18:3 6,9,12c (1/5), C18:1 9c (2/2), C17:0 anteiso (1/4) C17:0 iso 10-methyl 16:0 C17:0 12-methyl (1/6), C16:0 iso C16:0 iso (5/5) C16:0 iso (1/2) C18:0 12-methyl (1/6), C18:0 14-methyl (2/6), C17:0 iso (2/6), C18:0 iso (2/6), C18:0 iso 10(11)-methyl (1/6) Preferred carbon Complex protein- Complex protein- Sugars (3/5) Sugars (1/2) Sugars(4/4 b), n-alkanes Sugars, amino Sugars(5/7), sources d aceous substrates, aceous substrates, sugar alcohols (3/4), acids sugar alcohols (4/7), glucose glucose, carbocxlic acids (3/4) amino acids (2/7), carboxylic acids carboxylic acids (3/7)

26 729 a Given separately for the mesophilic strains R. bracarensis and R. aplysinae (first value) and the thermophilic species R. radiotolerans, R. 730 xylanophilus, R. taiwanensis, R. calidifluminis and R. naiadicus (second value). 731 b No or only few values exist for Bactoderma rosea for fatty acid profile and substrate range, respectively. 732 c Fatty acid profile of R. aplysinae was not considered, as probably misinterpretation of major components occurred (see text). 733 d Only most striking patterns observed within groups given; additionally strain-specific substrate usage from different substance classes occurred.

27 734 Table 2: 16S rRNA gene signature nucleotide patterns for the classes Thermoleophilia Suzuki 735 and Whitman 2013 and Rubrobacteria Suzuki 2013 and subordinate orders and families. 736 Adapted from Reddy and Garcia-Pichel (2009) and Zhi et al. (2009) and revised considering 737 the recent taxonomic conversions and the addition of novel species. 738 Taxonomic units were taken from Ludwig et al. (2012a). Sequence patterns were compared 739 based on the ‘All-Species Living Tree’ Project (LTP) database (Yarza et al., 2008) release 740 119 (November 2014) using the program package ARB version 6.0 (Ludwig et al., 2004). 741 Class level: I, Actinobacteria; II, Acidimicrobiia; III, Coriobacteria; IV, Nitriliruptoria; V, 742 Rubrobacteria; VI, Thermoleophilia; classes I-IV used for validation of class signatures from 743 V and VI, but not further considered. 744 Order level: 1, Rubrobacterales; 2, Thermoleophilales; 3, Solirubrobacterales; 4, Gaiellales. 745 Family level: A, Rubrobacteraceae; B, Thermoleophilaceae; C, Solirubrobacteraceae; D, 746 Conexibacteraceae; E, Patulibacteraceae; F, Parviterribacteraceae; G, Gaiellaceae. 747 Bold letters indicate signatures characteristic of the order, grey background indicates 748 signatures characteristic of the class. Underlined letters highlight unique signatures among the 749 families of the Thermoleophilia, dotted lines represent uniqueness within the 750 Solirubrobacterales. A ‘v’ denotes variable signatures within the respective taxonomic unit, 751 grey font indicates meaningless signatures within the respective taxonomic unit. 752 Note that validity of signatures may change from one taxon level to the other which cannot 753 always be indicated in the table. 754

28 I II III IV V VI 1 2 3 4 Position(s) A B C D E F G 52:359 v v C-G v G-C C-G C-G U-A C-G C-G C-G 63:104 C-G C-G C-G C-G C-G G-C G-C G-C G-C G-C C-G 66:103 v A-U A-U v A-U G-C G-C G-C G-C G-C G-C 70:98 v v v v A-U G-C G-C G-C G-C G-C A-G 127:234 v A-U v A-U G-C G-C G-C G-C G-C G-C G-C 139:224 v v v v U-A G-C v G-C v G-C C-G 144:178 v v v v G-C C-G C-G U-A C-G v C-G 145:177 v v v C-G G-C C-G C-G U-A v v U-G 242:284 U-G U-G v U-G C-G C-G C-G C-G C-G C-G C-G 291:309 C-G U-A v U-A U-A U-A U-A U-A U-A U-A U-A 293:304 v v v G-C G-U G-U G-C G-C G-C G-C G-C 316:337 v C-G U-G C-G C-G C-G C-G C-G C-G C-G C-G 370:391 C-G C-G C-G C-G C-G G-C C-G C-G C-G C-G C-G 377:386 G-C G-C G-C G-C G-C C-G v C-G C-G C-G G-C 408:434 G-C v G-C G-C G-C G-C G-C A-U G-C G-C G-C 409:433 G-C G-C G-C G-C C-G G-C G-C G-C G-C G-C G-C 438:496 U-A U-A U-A U-A G-G U-A U-A U-A U-A U-A U-A 502:543 v v v v C-G C-G C-G C-G v C-G C-G 580:761 v C-G C-G v v C-G U-A U-A U-A U-A U-A 590:649 v U-G v C-G v C-G C-G U-A U-A U-A G-C 600:638 v v G-C G-C v C-G C-G U-G U-G C-G G-C 657:749 v U-A U-A v G-C U-A U-A U-A U-A U-A U-A 661:744 v G-C G-C G-C G-C G-A G-A G-A G-A G-A G-A 670:736 A-U C-G v A-U A-U G-C A-U A-U A-U A-U A-U 681:709 v U-A v v C-G C-G U-A U-A U-A C-G U-A 722:733 v v v v v U-A U-A U-A U-A U-A U-G 819 v U A A A A A A A A A 823:877 G-C G-C v G-C v G-C G-C G-C A-U A-U G-C 941:1342 v v A-U G-C A-U G-C A-U A-U A-U A-U A-U 953:1228 v v G-C G-C U-A G-C G-C G-C G-C G-C G-C 954:1226 G-C G-C G-C G-C C-G G-C G-C G-C G-C G-C G-C 955:1225 v C-G v C-G U-A U-A U-A U-A U-A U-A U-A 999:1041 v A-U v A-U v A-U U-A G-U v U-A A-U 1051:1207 G-C G-C G-C G-C C-G G-C G-C G-C G-C G-C G-C 1115:1185 U-G U-G v v C-G C-G C-G C-G C-G U-G C-G 1118:1155 U-A v v U-A v C-G U-A U-A U-A U-A U-A 1311:1326 v A-U v A-U v G-C A-U A-U A-U A-U A-U 1313:1324 U-A U-A G-C U-A U-A G-C G-C G-C G-C G-C G-C 1410:1490 G-C A-U A-U G-C A-U A-U A-U A-U A-U A-U A-U

29 FIGURE LEGENDS

Fig. 1. Phase-contrast photomicrographs of strains D16/0/H6T (a) and A22/0/F9_1T (d), transmission electron micrographs of strains D16/0/H6T (b, c) and A22/0/F9_1T (e, f). Scale bars, 10 µm (a, d), 1 µm (b), 0.5 µm (e) and 0.2 µm (c, f). Arrow heads indicate inclusion bodies, cm = cytoplasmic membrane, cw = cell wall, sf = surface layer.

Fig. 2. Rooted maximum-likelyhood phylogenetic tree based on almost full-length 16S rRNA gene sequences showing the relationship of strains D16/0/H6T and A22/0/F9_1T to each other and to related taxa. Bootstrap values (expressed as percentages of 1000 replicates) are indicated at the respective branching points. The following sequences were used as outgroup: Chloroflexus aggregans DSM 9485 (CP001337), Chloroflexus aurantiacus J-10-fl (D38365) and Roseiflexus castenholzii DSM 13941 (CP000804). Bar indicates 10% nucleotide divergence.

30 Figure 1.

31 Figure 2.

100 2 Nitriliruptoria 89 100 9 Acidimicrobiia 74

100 32 Coriobacteriia 61 100 Bactoderma rosea CECT 7959T (HE795127) 71 Patulibacter minatonensis KV-614T (AB193261) 99 Patulibacter americanus CP1777-2T (AJ871306) 100 Patulibacter ginsengiterrae P4-5T (EU710748) 41 Patulibacter medicamentivorans I11 T(AGUD01000068)

Conexibacter arvalis KV-962T (AB597950) Thermoleophilia T

47 100 Conexibacter woesei ID131577 (CP001854) Solirubrobacterales

95 Solirubrobacter pauli B33D1T(AY039806) T 88 Solirubrobacter phytolaccae GTGR-8 (KF459924) 79 Solirubrobacter taibaiensis GTJR-20T (KF551107) Solirubrobacter soli 355T (AB245334) 100 97 Solirubrobacter ginsenosidimutans BXN5-15T (EU332825) 45 strain A22/0/F9_1T 92 environmental sequence (KC554359) 68 environmental sequence (JN177852) environmental sequence (JN177847) 100 97 environmental sequence (EU132917) 88 strain D16/0/H6T 100 Thermoleophilum minutum YS-4T (AJ458464) Thermoleophilales Thermoleophilum album HS-5T (AJ458462) 80 Gaiella occulta F2-233T (JF423906) Gaiellales 95 Rubrobacter bracarensis VF70612_S1T (HE672086) 100 Rubrobacter aplysinae RV113T (GU318365) 71 Rubrobacter radiotolerans IAM 12072T (U65647)

100 Rubrobacter taiwanensis LS-293T (AF465803) obacteria

91 Rubrobacter calidifluminis RG-1T (KF494338)

brobacterales ubr

T u

Rubrobacter naiadicus RG-3 (KF494339) R

R 100 Rubrobacter xylanophilus PRD-1T (CP000386)

0.10