International Journal of Systematic and Evolutionary Microbiology (2016), 66, 2971–2979 DOI 10.1099/ijsem.0.001131

The first representative of the globally widespread subdivision 6 , Vicinamibacter silvestris gen. nov., sp. nov., isolated from subtropical savannah soil Katharina J. Huber,1 Alicia M. Geppert,1 Gerhard Wanner,2 Barbel€ U. Fösel,1 Pia K. Wüst1 and Jörg Overmann1,3

Correspondence 1Department of Microbial Ecology and Diversity Research, Leibniz Institute DSMZ – German Katharina J. Huber Collection of Microorganisms and Cell Cultures, Braunschweig, Germany [email protected] 2Department of Biology I, Biozentrum Ludwig Maximilian University of Munich, Planegg- Martinsried, Germany 3Technical University Braunschweig, Braunschweig, Germany

Members of the Acidobacteria are abundant in a wide variety of soil environments. Despite this, previous cultivation attempts have frequently failed to retrieve representative phylotypes of Acidobacteria, which have, therefore, been discovered by culture-independent methods (13175 acidobacterial sequences in the SILVA database version 123; NR99) and only 47 species have been described so far. Strain Ac_5_C6T represents the first isolate of the globally widespread and abundant subdivision 6 Acidobacteria and is described in the present study. Cells of strain Ac_5_C6T were Gram-stain-negative, immotile rods that divided by binary fission. They formed yellow, extremely cohesive colonies and stable aggregates even in rapidly shaken liquid cultures. Ac_5_C6T was tolerant of a wide range of temperatures (12–40 C) and pH values (4.7–9.0). It grew chemoorganoheterotrophically on a broad range of substrates including different sugars, organic acids, nucleic acids and complex proteinaceous compounds. T The major fatty acids of Ac_5_C6 were iso-C17 : 1 !9c,C18 : 1 !7c and iso-C15 : 0. Summed

feature 3 (C16 : 1 !7c/C16 : 1 !6c), iso-C17 : 0 and C16 : 0 were also detected. Phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and an unidentified glycolipid were identified as polar lipids. The major quinone was MK-8. The DNA G+C content of Ac_5_C6T was 65.9 mol%. With 16S rRNA gene sequence similarities of 83–84 %, the closest described relatives were borealis KA1T, Acidobacterium capsulatum 161T, Granulicella pectinovorans TPB6011T, Occallatibacter riparius 277T and Paludibaculum fermentans P105T. According to the morphological, physiological and molecular characteristics, the novel Vicinamibacter gen. nov., and the novel species, Vicinamibacter silvestris sp. nov. (type strain Ac_5_C6T = DSM 29464T = LMG 29035T) are proposed.

Acidobacteria make up to 5–70 % of the soil microbial com- Despite their high abundance, previous cultivation attempts munity and so constitute one of most abundant phyla in have frequently failed to retrieve representative phylotypes soils (Janssen, 2006; Jones et al., 2009; Lauber et al., 2009). of Acidobacteria, which have instead been discovered by cul- ture-independent methods (13175 acidobacterial sequences in the SILVA database version 123; NR99; Pruesse et al., Abbreviations: SE, Soil extract; SSE , Soil solution equivalent; OM, outer 2007). While around 8800 representatives of the Proteobac- membrane; P, periplasm; CM, cytoplasmic membrane; IB, Inclusion bod- teria, Firmicutes and have been isolated ies; LTP, Living Tree Project; R, Ribosomes. from soil (Hugenholtz et al., 1998; Janssen et al., 2002; The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene Söhngen et al., 2016), only 47 different species of sequence of strain Ac_5_C6 T is KP761690. Acidobacteria have been described so far (Parte, 2014). The phylum of the Acidobacteria is currently divided into 26 Five supplementary tables and one supplementary figure are available with the online Supplementary Material. subdivisions (sds) (Barns et al., 2007) of which only six

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Downloaded from www.microbiologyresearch.org by IP: 52.0.234.91 On: Fri, 26 Jul 2019 03:05:33 K. J. Huber and others contain described species. The eleven genera, of the DSMZ media number 12 and 1124 (instead of garden Acidobacterium (Kishimoto et al., 1991), Terriglobus (Baik soil a mixture of Namibian sandy savannah soils was used; et al., 2013; Eichorst et al., 2007; M€annistö et al., 2011; Pasc- Wüst et al., 2016; available in the online Supplementary ual et al., 2015; Whang et al., 2014;), Edaphobacter (Koch Material), which was supplemented with a 10-vitamin solu- et al., 2008; Wang et al., 2015), Acidicapsa (Kulichevskaya tion (Balch et al., 1979; Supplementary Material) and trace et al., 2012), Granulicella (M€annistö et al., 2012; Pankratov element solution SL-10 (Tschech & Pfennig, 1984; Supple- & Dedysh, 2010; Yamada et al., 2014), (Pan- mentary Material). After 9 weeks of incubation at 20 C in kratov et al., 2012), (Jiang et al., 2015; Okamura the dark, cultures were screened for acidobacterial growth et al., 2011), (Dedysh et al., 2012), Occallatibacter by phylum-specific PCR using the primer pair Acido31f (Foesel et al., 2015), ’Terracidiphilus’ (Garcia-Fraile et al., (Barns et al., 1999) and 1492r (Lane, 1991). Cultures that 2015) and ’Silvibacterium’ (Lladó et al., 2016) belong to sd1. yielded positive PCR products were plated on SE/HD 1:10 The two genera, Bryobacter (Kulichevskaya et al., 2010) and plates (pH 7.0) solidified with purified agar (15 g lÀ1; Paludibaculum (Kulichevskaya et al., 2014), fall into sd3. Oxoid). A pure culture of Ac_5_C6T was finally obtained by The eight genera Blastocatella (Foesel et al., 2013), subsequent restreaking on plates. Unless otherwise noted, Pyrinomonas (Crowe et al., 2013), Aridibacter (Huber et al., SSE/HD 1 : 10 (pH 7.0) was used in the following physiolog- 2014), (Tank & Bryant, 2015), ical tests and for biomass production (Supplementary Mate- Tellurimicrobium (Pascual et al., 2015), Stenotrophobacter rial). The SSE/HD 1 : 10 (pH 7.0) medium was modified (Pascual et al., 2015), Brevitalea (Wüst et al., 2016) and medium DSMZ number 1426, as it was buffered with Arenimicrobium (Wüst et al., 2016) into sd4 and the three HEPES instead of MES. The medium was based on the soil genera Holophaga (Liesack et al., 1994), Geothrix (Coates solution equivalent (SSE; Angle et al., 1991), supplemented et al., 1999) and Acanthopleuribacter (Fukunaga et al., 2008) with 10-vitamin solution (Balch et al., 1979), a trace ele- into sd8. Finally, the genus ’Thermotomaculum’ (Izumi ment solution SL-10 (Tschech & Pfennig, 1984) and solidi- et al., 2012) is a representative of sd10 and the genus fied with purified agar (Oxoid). Data used from the Thermoanaerobaculum (Losey et al., 2013) a member of literature had been obtained under comparable growth con- sd23. In particular, no isolate of sd6 is available, although ditions to those implemented here for strain Ac_5_C6T. All the latter represents the dominant subdivision in many soils media employed contained or glucuronic acid, and (Barns et al., 1999; Foesel et al., 2014; Jones et al., 2009). yeast or casamino acids (0.1–0.5 g lÀ1) as growth factors. 16S rRNA sequence analyses revealed that members of sd6 Proteinaceous substrates such as peptone (0.5 g lÀ1) were dominate in soils with a neutral or (slightly) basic pH, also present. together with of sd4, whereas members of sd 1 and T 3 prevail in soils which are (slightly) acidic (Foesel et al., Strain Ac_5_C6 formed solid colonies on agar plates with 2014; Jones et al., 2009; Naether et al., 2012). About half of a diameter of about 1 mm; they were yellow, circular, con- the soils on earth have a neutral or (slightly) basic pH vex-hemispherical and opaque with entire margins. These (www.fao.org/soils-portal/soil-survey/soil-properties/chem- features are comparable to the soil isolates described by ical-properties/en/). Thus, they would constitute suitable George et al. 2011. Even during shaking liquid cultures of sources for the cultivation of representatives of sd6. How- strain Ac_5_C6T formed yellow aggregates, which were very ever, no isolate of this subdivision has been described in solid and could not be disrupted without damaging the detail so far, but the successful isolation of 11 cells. Light microscopic observations (Zeiss Axio Lab.A1; Acidobacteria sd6 strains from Belgian soil samples has been Carl Zeiss, with AxioCam Mrm camera) confirmed that reported previously (George et al., 2011). the cells of strain Ac_5_C6T were rod-shaped, 1.3–2.0 µm – In the present study we describe the first representative of long, approximately 0.6 0.7 µm in diameter and that they T sd6 Acidobacteria, Vicinamibacter silvestris gen. nov., sp. divided by binary fission. Strain Ac_5_C6 formed aggre- nov., which was isolated from a semiarid subtropical savan- gates and single cells also occurred occasionally (Fig. 1a). nah soil. Consistent with all other Acidobacteria characterized so far, strain Ac_5_C6Twas Gram-stain-negative (Gerhardt et al., Strain Ac_5_C6T was isolated from a riparian woodland soil  1994). India ink and malachite green staining (Bast, 2011) collected from south of the Okavango river (17 53¢35.52†S;  confirmed, respectively, that capsules and spores were not 20 14¢57.52†E; 1061 m above sea level) near the village of formed in strain Ac_5_C6T. To dissolve the ultrastructure Mashare in the North-East of Namibia. The soil was a T clayey sand with a slightly basic pH (7.5 and 8.0 measured of strain Ac_5_C6 , cells were high-pressure frozen and cryosubstituted for electron microscopy to prevent artefact in 10 mM CaCl2 and in distilled water, respectively). Total bacterial cell numbers were determined in the clayey soil formation due to cell shrinkage as a reaction to chemical with fluorescent microscopy, as described in Lunau et al., fixation (Wanner et al., 2008). Like other representatives of T 2005. For the enrichment of Acidobacteria the soil was the Acidobacteria, strain Ac_5_C6 revealed a Gram- diluted with 10 mM HEPES (pH 7.0) and 20 µl of the negative structure of the cell wall and intracellular inclusion diluted soil suspension containing approximately 10 cells bodies (Fig. 1b). As shown by Foesel et al. (2015) and Wüst were inoculated in 180 µl of liquid SE (soil extract)/HD et al. (2016), high numbers of ribosomes could be detected 1 : 10 medium (pH 7.0). This medium is based on a mixture in strain Ac_5_C6T.

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The almost full-length 16S rRNA gene fragment of strain isolates within sd6 Acidobacteria where they constituted a Ac_5_C6T was amplified by colony-PCR using primer pair stable cluster together with strains IGE-001–IGE-009 iso- 8f (Turner et al., 1999) and 1492r (Lane, 1991). The result- lated from a Belgian soil (George et al., 2011) (Fig. 2). The ing sequence comprised 1476 unambiguous nucleotides pairwise 16S rRNA gene sequence similarities (ARB neigh- between Escherichia coli positions 28 and 1491 (Brosius et al., bour-joining tool without the use of an evolutionary substi- 1978). The sequence was added to the 16S rRNA-based ‘All- tution model) between strain Ac_5_C6T and the former Species Living Tree’ Project (LTP) database (Yarza et al., isolates were 97.2–99.2 %. Strains IGE-010 (91.7 %) and 2008) release 123 (September 2015) using the program IGE-011 (94.5 %) were more distantly related. The closest package ARB version 6.0 (Ludwig et al., 2004). After auto- relatives of strain Ac_5_C6T were representatives of mated alignment with the Fast aligner tool implemented in Acidobacteria sds 1 and 3, namely Acidicapsa borealis KA1T, ARB, the alignment was manually refined based on second- Granulicella pectinovorans TPB6011T, Acidobacterium capsu- T T ary structure information. Phylogenetic trees were con- latum 161 , Occallatibacter riparius 277 and Paludibaculum T structed using neighbour-joining, maximum parsimony and fermentans P105 with 16S rRNA nucleotide similarities of maximum likelihood algorithms (40 % maximum frequency 83–84 %. filter, resulting in 1418 valid columns between positions 64 To determine the G+C content of strain Ac_5_C6T, cells and 1439 of the E. coli 16S rRNA reference gene; 1000 boot- were disrupted in a French press and the DNA was subse- strap resamplings). All three methods placed the three novel quently purified using hydroxyapatite (Cashion et al., 1977). The sample was treated with P1 nuclease and bovine alkaline phosphatase (Mesbah et al., 1989) and the resulting desoxy- ribonucleosides were analysed by HPLC (Shimadzu), employing conditions adapted from Tamaoka & Komagata (1984). The G+C content of strain Ac_5_C6T was 65.9 mol % (calculated by the method introduced by Mesbah et al., 1989); this exceeds the G+C content of 46.5–62.7 mol% of the other described species of Acidobacteria sds 1, 3, 4, 8, 10 and 23 including the thermophilic representatives Pyrinomo- nas methylaliphatogenes K22T, Chloracidobacterium thermo- philum BT, ‘Thermotomaculum hydrothermale’ AC55T and Thermoanaerobaculum aquaticum MP-01T (Table S1, avail- able in the online Supplementary Material). Only Geothrix fermentans H-5T and Arenimicrobium luteum Ac_12_G8T exhibit higher G+C contents with 68.9 mol% and 66.9 mol %, respectively (Table S1). Isoprenoid quinones were extracted from dried biomass with chloroform/methanol (2 : 1, v/v) as introduced by Col- lins & Jones (1981). The subsequent analysis by HPLC (Tindall, 1990) identified MK-8 as the major quinone of strain Ac_5_C6T, which is in congruence with the majority of the described representatives of the Acidobacteria sds 1, 3 and 4 (Table S1). In addition, minor amounts of MK-9 were detected in strain Ac_5_C6T. The polar lipid composition was determined by two-dimensional TLC, as described previously (modified after Bligh & Dyer, 1959; Tindall et al., 2007; Fig. S1). Phos- phatidylcholine, phosphatidylglycerol, diphosphatidylgly- cerol, phosphatidylethanolamine and an unidentified glycolipid were identified as the polar lipids of strain Ac_5_C6T. This polar lipid composition matched that reported for the sd4 member Tellurimicrobium multivorans Ac_18_E7T (Pascual et al., 2015), except for the presence of an unidentified phosphoglycolipid in Ac_18_E7T and the T Fig. 1. a) Phase-contrast photomicrograph of strain Ac_5_C6T. presence of an unidentified glycolipid in Ac_5_C6 . Also, Bar, 10 µm. b) Transmission electron micrograph of an ultrathin apart from the additional presence of an unidentified glyco- T section of strain Ac_5_C6T. Bar, 200 nm. OM = outer membrane, lipid in Ac_5_C6 , the polar lipid composition of strain P = periplasm, CM = cytoplasmic membrane, IB = inclusion body, Ac_5_C6T also corresponded with the polar lipid composi- R = ribosomes. tion of the savannah strains, Aridibacter spp. (Huber et al., http://ijs.microbiologyresearch.org 2973

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2014) and Stenotrophobacter spp. (Pascual et al., 2015). hydrothermale’ AC55T and in Thermoanaerobaculum aqua- Phosphatidylethanolamine and phosphatidylglycerol were ticum MP-01T (Table S1). also present in the two sd1 species Acidobacterium capsula- T T The temperature and pH ranges and optima for growth tum 161 and Telmatobacter bradus TPB6017 (Pankratov were determined between 10 and 56 C and between pH 1.0 et al., 2012) and in the sd23 member Thermoanaerobaculum T and 11.0 under oxic conditions in liquid SSE/HD 1 : 10 aquaticum MP-01 (Losey et al., 2013). Additionally, strain medium in duplicate and triplicate, respectively. Depending Ac_5_C6T contains phosphatidylcholine, as do T on the desired pH range, MES, HEPES, HEPPS or CHES other Acidobacteria except i.e. Bryobacter aggregatus MPL3 (Sigma-Aldrich; 10 mM each) were used as buffers. Growth and ‘Thermotomaculum hydrothermale’ AC55T. Apart from T was determined by measuring the optical density at 660 nm. strain Ac_5_C6 , diphosphatidylglycerol was also deter- Growth of strain Ac_5_C6T was detected between 12 and mined in the sd23 member Thermoanaerobaculum aquati-  T 40 C and between pH 4.7 and 9.0. Optimal growth (defined cum MP-01 (Losey et al., 2013) and in the savannah soil as 75 % of the highest growth rate achieved) was reached representatives of sd4 Aridibacter spp., Tellurimicrobium at 26–31 C and pH 6.5–8.1. The absolute optimum was T multivorans Ac_18_E7 , Stenotrophobacter spp., Brevitalea observed at 28 C and pH 7.0. Hence, strain Ac_5_C6T toler- T spp. and Arenimicrobium luteum Ac_12_G8 (Huber et al., ates a broad range of temperatures and pH values, a feature 2014; Pascual et al., 2015; Wüst et al., 2016). shared with the representatives of sd4 Acidobacteria Blastoca- tella fastidiosa A2-16T, Aridibacter spp., P. methylaliphato- For fatty acid analysis about 40 mg wet weight of cells were T T harvested and extracted according to the standard protocol genes K22 , Tellurimicrobium multivorans Ac_18_E7 , Stenotrophobacter spp., Brevitalea spp. and Arenimicrobium (Sasser, 1990) of the Microbial Identification System (MIDI T Inc.; version 6.1). The fatty acid composition was identified luteum Ac_12_G8 (Crowe et al., 2014; Foesel et al., 2013; Huber et al., 2014; Pascual et al., 2015; Wüst et al., 2016). by the comparison to the TSBA40 peak naming table data- À1 À1 base. Strain Ac_5_C6T possessed straight chain, methyl HD 1 : 10 medium [0.25 g l yeast extract, 0.5 g l peptone, 0.1 g lÀ1 glucose, 0.1 ml lÀ1 10-vitamin solution (Balch et al., and/or hydroxyl-branched unsaturated and monosaturated À1 fatty acids. The major components were iso-C !9c 1979; Supplementary Material) and 1 ml l trace element 17 : 1 solution SL-10 (Tschech & Pfennig, 1984; Supplementary (30.1 %), C !7c (28.6 %) and iso-C (13.4 %). Fur- 18 : 1 15 : 0 Material)] with varying NaCl concentrations [0–10 % (w/v)] thermore, summed feature 3 (C !7c/C !6c, 5.6 %), 16 : 1 16 : 1 was used to determine the salt tolerance of strain Ac_5_C6T. iso-C (5.4 %) and C (4.4 %) occurred in notable 17 : 0 16 : 0 Strain Ac_5_C6T tolerated NaCl concentrations of 0–1 % amounts. The full profile is given in Table S2. (w/v), which is comparable to the preferences of other Thus, more than half (58.7 %) of the fatty acids of strain savannah soil isolates (Table S1). However, strain Ac_5_C6T Ac_5_C6T consisted of the two unsaturated (methyl- grew best at a NaCl concentration of 0 % (w/v). branched) fatty acids C !7c and iso-C !9c. This fea- 18 : 1 17 : 1 Six parallels of liquid oxic SSE/HD 1 : 10 medium without ture clearly distinguishes the sd6 strain Ac_5_C6T from all peptone, yeast extract and glucose were used to determine Acidobacteria described so far. In contrast, iso-C which T 15 : 0 the growth substrates utilized by strain Ac_5_C6 . The is the major component (>35 %) in most of the described was supplemented with different sugars, Acidobacteria only constituted the third most abundant – T organic acids, keto acids, alcohols, amino acids (0.5 10 mM compound in strain Ac_5_C6 . The presence of C16 : 0 and each), casamino acids, casein hydrolysate, laminarin, pep- ! ! ! summed feature 3 (C16 : 1 7c/C16 : 1 6c) or C16 : 1 7c are a tone, yeast extract (0.05 % w/v each) and Tween 80 feature that strain Ac_5_C6T and some members of sd1 and T (0.001 % w/v) (Table S3), accordingly. Unlike most of the sd4 such as Granulicella spp., Bryocella elongata SN10 , T T subtropical savannah strains of sd4, strain Ac_5_C6 grew Terriglobus roseus KBS 63 , Edaphobacter spp., Aridibacter on various sugars (Table S4), a trait common to the sd1 spe- spp. have in common (Dedysh et al., 2012; Eichorst et al., cies Acidobacterium capsulatum 161T, Terriglobus roseus KBS 2007; Huber et al., 2014); Koch et al., 2008; Pankratov & 63T, Edaphobacter spp. and Bryocella elongata SN10T as well T Dedysh, 2010), although strain Ac_5_C6 showed lower rel- as the sd4 representative Tellurimicrobium multivorans ative contents compared to the other strains. No summed Ac_18_E7T (Table S4). However, strain Ac_5_C6T also uti- feature 1 (iso-C15 : 1 H/C13 : 0 3-OH) was detected in strain lized proteinaceous substrates like casamino acids, yeast T Ac_5_C6 as opposed to the sd4 Acidobacteria such as Blas- extract and peptone as do the sd4 representatives from semi- T tocatella fastidiosa A2-16 , Aridibacter spp., Tellurimicrobium arid savannah soils Blastocatella fastidiosa A2-16T (Foesel T multivorans Ac_18_E7 , Stenotrophobacter spp., Brevitalea et al., 2013), Aridibacter spp. (Huber et al., 2014), Tellurimi- T spp. and Arenimicrobium luteum Ac_12_G8 , which were crobium multivorans Ac_18_E7T (Pascual et al., 2015), two also isolated from subtropical soils. Iso-C17 : 0 was detected Stenotrophobacter spp. (Pascual et al., 2015), Brevitalea spp. in strain Ac_5_C6Tas in the sd1 Acidobacteria Acidicapsa and Arenimicrobium luteum Ac_12_G8T (Wüst et al., 2016). spp., Occallatibacter riparius 277Tand Telmatobacter bradus Additionally, the ability of strain Ac_5_C6T to degrade com- TPB6017T, in the sd4 Acidobacteria, Pyrinomonas methylali- plex substrates like cellulose, chitin, starch, xylan, pectin phatogenes, K22T, Tellurimicrobium multivorans Ac_18_E7T, and lignin was tested on solidified SSE medium supple- Brevitalea spp. and Arenimicrobium luteum Ac_12_G8T, in mented with 0.005 % (w/v) yeast extract and a final concen- Acanthopleuribacter pedis FYK2218T, in ‘Thermotomaculum tration of 0.5 g lÀ1 of the respective polymer. Strain

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Fig. 2. Rooted neighbour-joining phylogenetic tree (Felsenstein correction) based on almost full-length 16S rRNA gene sequences showing the relationship of strain Ac_5_C6T to related taxa. Bootstrap values (expressed as a percentage of 1000 replicates) are indicated at the respective branching points. The following sequences were used as outgroups: Planctopirus limnophila DSM 3776T (X62911), Gimesia maris DSM 8797T (AJ231184) and Blastopirellula marina DSM 3645T (X62912). Bar, 10 % nucleotide divergence.

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Ac_5_C6T could not degrade any of the polymeric sub- fission. Cytochrome c-oxidase positive. -positive. strates when tested for the appearance of clearing zones with No capsule formation. Major fatty acids are iso-C17 : 1 !9c, suitable aqueous staining solutions (Pascual et al., 2015). C18 : 1 !7c and iso-C15 : 0; minor components are C16 : 0, iso- C and summed feature 3 (C !7c/C !6c). The The API ZYM and API 20NE test systems (Biomerieux) 17 : 0 16 : 1 16 : 1 major quinone is MK-8. Phosphatidylcholine, phosphati- were employed to test exoenzyme activity and the fermenta- dylglycerol, diphosphatidylglycerol, phosphatidylethanol- tion of sugars. While different exoenzyme activities were amine and an unidentified glycolipid are the polar lipids. determined, the of sugars could not be dem- Tolerant to a wide range of pH values. Aerobic, chemoorga- onstrated (Table S5). Cytochrome c-oxidase and catalase notrophic mesophile that grows on different sugars, organic activities were determined as described in Cowan et al., acids and complex proteinaceous substrates. (1993) and Gerhardt (1994). In addition, the activity of cytochrome c-oxidase was verified using Bactident Oxidase The type species is Vicinamibacter silvestris. (Merck). The resistance or susceptibility of strain Ac_5_C6Tto com- mon antibiotics was tested on SSE/HD 1 : 10 agar plates Description of Vicinamibacter silvestris, supplemented with the following antibiotics (concentra- sp. nov. tions used in parenthesis): ampicillin (100 µg mlÀ1), carbenicillin (100 µg mlÀ1), kanamycin (50 µg mlÀ1), chlor- Vicinamibacter silvestris (sil.ves’tris. L. masc. adj. silvestris amphenicol (34 µg mlÀ1), erythromycin (25 µg mlÀ1), spec- pertaining to the forest soil from which the type strain was tinomycin (25 µg mlÀ1), streptomycin (25 µg mlÀ1), isolated). À À tetracycline (12.5 µg ml 1) and gentamicin (16 µg ml 1). – – T Cells are 1.3 2.0 µm long and 0.6 0.7 µm in diameter. Liq- Strain Ac_5_C6 showed resistance to ampicillin, carbeni- uid cultures and colonies on agar plates are yellow. Tight cillin, kanamycin, chloramphenicol, erythromycin, tetracy- aggregates are formed even in shaken cultures and cannot cline and gentamicin and susceptibility to spectinomycin be disrupted. Colonies on agar plates are 1–2 mm in size and streptomycin (Table S1). and are circular, convex-hemispherical and opaque with T entire margins. Grows at temperatures of 12–40 C (best at In summary, strain Ac_5_C6 is an aerobic, chemoorgano-  heterotrophic, yellow pigmented mesophile with a tolerance 25.5–31.1 C) and pH 4.7–9.0 (best at 6.5–8.1). Under opti- to a broad range of pH values and temperatures, as well as mal growth conditions the doubling time is 17.4 h. The forming strong aggregates in liquid cultures. The high G+C strain grows at NaCl concentrations of up to 1 % (w/v; content exceeds that of known thermophilic Acidobacteria optimum, 0 % w/v). and the high amounts of the methyl-branched and unsatu- Grows on arabinose, , galactose, glucose, lactose, ! ! rated fatty acids C18 : 1 7c and iso-C17 : 1 9c distinguishes lyxose, maltose, rhamnose, xylose, cellobiose, melezitose, this first described representative of sd6 Acidobacteria from raffinose, sucrose, trehalose, hydroxy-proline, N-acetylga- all other Acidobacteria characterised so far. Furthermore, lactosamine, N-acetylglucosamine, fumarate, gluconate, glu- 16S rRNA gene sequence analysis unambiguously places T tamate, casamino acids, casein hydrolysate, peptone, yeast strain Ac_5_C6 in sd6 Acidobacteria and with similarities extract and fermented rumen extract. Weak growth is – T of 83 84 % clearly distinguishes strain Ac_5_C6 from its observed on fucose, mannose, acetoin, ascorbate, citrate, phylogenetically closest described relatives with validly pub- glycerin, heptanoic acid, isovaleric acid, maleic acid, 2-oxo- lished names: Acidicapsa borealis KA1T, Granulicella pectini- T T glutarate, pyruvate, alanine, asparagine, aspartate, proline, vorans TPB6011 , Acidobacterium capsulatum 161 , protocatechuate, shikimate, trimethoxybenzoate and lami- Occallatibacter riparius 277T and Paludibaculum fermentans T narin. No growth is observed on erythrose, erythrulose, sor- P105 of sds 1 and 3. bose, glucosamine, adonitol, arabitol, dulcitol, lyxitol, Based on the phylogeny, morphology and physiology of mannitol, myo-inositol, sorbitol, xylitol, arginine, cysteine, strain Ac_5_C6T, the novel genus Vicinamibacter gen. nov., glutamine, glycine, histidine, isoleucine, leucine, lysine, including the novel species, Vicinamibacter silvestris sp. methionine, ornithine, phenylalanine, serine, threonine, nov., is proposed. tryptophan, tyrosine, valine, adipate, acetate, benzoate, butyrate, a-hydroxybutyrate, b-hydroxybutyrate, g-hydrox- ybutyrate, isobutyrate, caproate, caprylate, isocitrate, croto- Description of Vicinamibacter, gen. nov. nate, formate, 2-oxogluconate, glucuronate, glycolate, glyoxylate, laevulinate, lactate, malate, malonate, nicotinic Vicinamibacter (Vi.cin.a.mi.bac’ter. L. masc. n. vicinus acid, oxaloacetate, propionate, succinate, tartrate, 2-oxoval- neighbour; L. v. amare love; N. L. masc. n. bacter a short erate, butanol, 1,2-butandiol, 2,3-butandiol, ethanol, ethyl- rod; rod-shaped bacterium, that loves its neighbour refer- ene glycol, methanol, propanol, 1,2-propandiol and ring to the strong formation of aggregates even during Tween 80. growth in rapidly shaken liquid cultures). Positive for alkaline and acid phosphatase, naphtol-AS-BI- Gram-stain-negative, non-spore-forming, non-motile rods phosphohydrolase, a-chymotrypsin, trypsin, esterase C4, that occur as single cells or aggregates and divide by binary leucine arylaminidase and valine arylaminidase. Cysteine

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