International Journal of Systematic and Evolutionary Microbiology (2016), 66, 5529–5536 DOI 10.1099/ijsem.0.001552

Raineyella antarctica gen. nov., sp. nov., a psychrotolerant, D-amino-acid-utilizing anaerobe isolated from two geographic locations of the Southern Hemisphere Elena Vladimirovna Pikuta,1 Rodolfo Javier Menes,2 Alisa Michelle Bruce,3† Zhe Lyu,4 Nisha B. Patel,5 Yuchen Liu,6 Richard Brice Hoover,1 Hans-Jürgen Busse,7 Paul Alexander Lawson5 and William Barney Whitman4

Correspondence 1Department of Mathematical, Computer and Natural Sciences, Athens State University, Athens, Elena Vladimirovna Pikuta AL 35611, USA [email protected] 2Catedra de Microbiología, Facultad de Química y Facultad de Ciencias, UDELAR, 11800 or Montevideo, Uruguay [email protected] 3Biology Department, University of Alabama in Huntsville, Huntsville, AL 35899, USA 4Microbiology Department, University of Georgia in Athens, Athens, GA 30602, USA 5Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK 73019, USA 6Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA 7Institut für Mikrobiologie - Veterinarmedizinische€ Universitat€ Wien, A-1210 Wien, Austria

A Gram-stain-positive bacterium, strain LZ-22T, was isolated from a rhizosphere of moss Leptobryum sp. collected at the shore of Lake Zub in Antarctica. Cells were motile, straight or pleomorphic rods with sizes of 0.6–1.0Â3.5–10 µm. The novel isolate was a facultatively anaerobic, catalase-positive,   psychrotolerant mesophile. Growth was observed at 3–41 C (optimum 24–28 C), with 0–7 % (w/v) NaCl (optimum 0.25 %) and at pH 4.0–9.0 (optimum pH 7.8). The quinone system of strain LZ-22T

possessed predominately menaquinone MK-9(H4). The genomic G+C content was 70.2 mol%. Strain 10J was isolated from a biofilm of sediment microbial fuel cell, in Uruguay and had 99 % 16S rRNA gene sequence similarity to strain LZ-22T. DNA–DNA-hybridization values of 84 % confirmed that both strains belonged to the same . Both strains grew on sugars, proteinaceous compounds, and some amino- and organic acids. Strain LZ-22T uniquely grew on D-enantiomers of histidine and valine while neglecting growth on L-enantiomers. Both strains were sensitive to most of the tested antibiotics but resistant to tested nitrofurans and sulfanilamides. Phylogenetic analyses of the 16S rRNA gene sequences indicated that the strains were related to members of the family (~93–94 % 16S rRNA gene sequence similarity) with formation of a separate branch within the radiation of the genera Granulicoccus and Luteococcus. Based on phenotypic and genotypic characteristics, we propose the affiliation of both strains into a novel species of a new genus. The name Raineyella antarctica gen. nov., sp. nov. is proposed for the novel taxon with the type strain LZ- 22T (=ATCC TSD-18T=DSM 100494T=JCM 30886T).

†Present address: Clinical Laboratory Sciences Department, University of West Florida, Pensacola, FL 32514, USA. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains LZ-22T and 10J are KM406782 and LT560379, respec- tively. The GenBank/EMBL/DDBJ accession number for the draft genome sequence of strain LZ-22T is IMG OID 2651870304. Four supplementary figures and three supplementary tables are available with the online Supplementary Material.

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The 2008 Tawani International Schirmacher Oasis/Lake was checked in AM medium in which sodium salts were Untersee, Antarctica Expeditions were designated official US/ replaced by equimolar concentrations of potassium salts, Russia/Austria International Antarctic Expeditions con- and then NaCl was added. The test for poly-b-hydroxybu- ducted as a part of the 2008 International Polar Year. The tyrate was performed according to Smibert & Krieg (1994) goal of these expeditions was to study the microbial biodi- except that ether was replaced by chloroform, pellet was versity and search for extremophiles in the Schirmacher only washed once with acetone and then chloroform, and Oasis (Fig. S1, available in the online Supplementary Mate- the volumes of each were reduced five-fold. All experiments rial) and the perennially (for millennia) ice-covered Lake were performed in triplicate. Staining with Nile blue A, Untersee in East Antarctica. Several anaerobic psychrotoler- Loeffler methylene blue and toluidine blue was also per- ant and psychrophilic were isolated from samples formed to verify composition of inclusion bodies (Smibert collected during these expeditions (Hoover, 2008; Guisler & Krieg, 1994). et al., 2009; Townsend et al., 2009; Hoover & Pikuta, 2009). Here, we present the taxonomic description of one of these Strain 10J (Fig. S3) was isolated from the anode biofilm of a anaerobes, strain LZ-22T isolated from a rhizosphere of Lep- sediment microbial fuel cell inoculated with the sludge of tobryum sp. moss (Fig. S2) collected near Lake Zub in the an artificial wetland from winery wastewater. A piece of the Schirmacher Oasis of Central Dronning Queen Maud Land, anode was transferred to anaerobic solution, homogenized,   East Antarctica (70 44¢ 32¢¢ S 11 37¢ 39¢¢ E) at an elevation and serially diluted. Aliquots were spread on agar plates ~ containing acetate as the electron donor and ferric citrate as of 1 km (Fig. S1).  the electron acceptor, and incubated anaerobically at 30 C T During characterization of strain LZ-22 , a second strain for 14 days. Purity of colonies was confirmed by micro- (10J) was isolated from the anode biofilm of a sediment scopic examination. Strain 10J wasmaintained on tryptic microbial fuel cell which previously had been inoculated soy agar (Difco) and stored as 20 % (v/v) glycerol suspen- by the sludge of an artificial wetland formed with a winery À    sions in tryptic soy broth (Difco) at 80 C. Strain 10J was wastewater in Juanicó, Canelones, Uruguay (34.6 S 56.3 deposited in the Indian Microbial Culture Collection under W). Subsequent investigations suggested that both strains accession number MCC 3126. were closely related and represented a novel species of a new genus in the family Propionibacteriaceae. Morphology and growth observations were performed with a T phase contrast microscope (Micromaster) at 1000 magnifica- After collection, the sample which yielded strain LZ-22 tion. High resolution collimated darkfield microscopy was was returned to the laboratory frozen with dry ice and À  performed as previously described (Pikuta et al., 2006b) with then stored at 80 C. This sample gave good growth of the following modifications: using a proprietary illumination enrichment cultures that led to isolation of several high numerical aperture cardioid immersion condenser with strains of anaerobic psychrophilic and psychrotolerant variable internal aperture that provided high obliquity colli- bacteria. mated light to cells in the focal plane of 0.95 numerical aper- For cultivation, anaerobic synthetic medium with D-glucose ture. SPlan Apo 40 Olympus oil immersion objective and 16X À1 (AM) was applied which contained (l ): 3.0 g D-glucose, Leitz eyepiece with digital image amplification were applied. 5.0 g NaCl, 0.3 g KCl, 0.3 g KH PO , 0.1 g MgSO . 7H O, T 2 4 4 2 The cellular morphology and ultrastructure of strain LZ-22 1.0 g NH Cl, 0.0125 g CaSO . 7H O, 0.3 g NaHCO 0.4 g 4 4 2 3, was checked with negative staining of thin sections. cell sus- Na S . 9H O, 0.005 % resazurin, 0.1 g yeast extract (Fisher- 2 2 pension was collected by filtration and fixed in mixture of 1 % Biotech), 2 ml vitamin solution (Wolin et al., 1963), and 1 ml modified trace elements solution (Kevbrin & Zavarzin, formaldehyde, 2 % glutaraldehyde, 1 % OsO4 in 0.1 mM 1992). The trace elements solution contain.ed: 49.5 mg sodium cacodylate buffer (pH 7.2) for 15 min. The solution was extracted into a 10 ml syringe with a Swinney (Fisher Sci- MnCl2 . 4H2O, 392 mg FeSO4(NH4)2SO4 . 6H2O, 119 mg entific) polycarbonate filter (0.4 µm pore diameter), and then CoCl2 . 6H2O, 71.8 mg ZnSO4 . 7H2O, 100 mg NiCl2, 0.85 mg CuCl2 . 2H2O, 16.5 mg Na2WO4 . 2H2O, 47.3 mg the solution was filtered and fixed with the same fixative for Na2SeO4, 3.1 mg H3BO3, 10.3 mg Na2MoO4 . 2H2O and another 15 min. The samples were rinsed with distilled water 5 ml HCl conc. in 500 ml distill water (the only modifica- three times, 5 min each, and en bloc stained with 0.5 % uranyl tion was 2Â amount of the selenium salt). High-purity acetate in the dark for 60 min. The samples were then dehy- nitrogen was used as a gas phase. The final pH was adjusted drated using an ethyl alcohol series, and the polycarbonate fil- to 7.4 with NaOH or H2SO4. To obtain a pure culture, ters were taken out from the syringe and infiltrated with 1 : 1 serial dilutions and the roll-tube technique were used with ethanol : LR white embedding resin for 1 h, and then infil-  2 % (w/v) agar AM medium incubated at 3 C. Growth sub- trated with 100 % resin for another 1 h. The cells were embed- À1 strates were tested in final concentrations of 3 g l by ded in 100 % LR white resin (Electron Microscopy  injecting them into Hungate tubes with 10 ml AM medium Sciences, USA) at 60 C overnight. Ultra-thin sections for without glucose. The growth was monitored for 14 days at  transmission electron microscopy were cut on a Leica EM 24 C. UC7 microtome. Sections 90 nm thick were stained with lead The optimal pH for growth was determined by titrating AM citrate and observed and photographed with a JEOL JEM- + medium with H2SO4 or NaOH. Dependence upon Na ion 1400 transmission electron microscope. 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Anaerobic growth was assessed on solid medium incubated Genomics Facility in the University of Georgia, Athens GA in anaerobic chambers (GENbox anaer bioMerieux). (USA), and of strain 10J was performed at Macrogen (Korea) Tests for biochemical characters were performed according as described previously (Menes et al., 2011). to standard methods (Barrow & Feltham, 1993; Smibert &  DNA–DNA hybridization experiments were carried out at Krieg, 1994) incubated for up to 10 days at 30 C. Reduction Microbial Culture Collection, National Centre for Cell Sci- of nitrate was performed in AM medium and potassium nitrate (5 mM). Respiration of ferric iron was performed in ence (Pune, India). Genomic DNA was extracted and puri- AM medium supplemented with ferric citrate (50 mM) and fied according to the protocol of Marmur (1961). The acetate or malate (10 mM) as electron donors. Ferrous iron analysis was done by fluorimetry using a Step One Plus production was measured with the ferrozine method Real-Time PCR system (Applied Biosystems) fitted with 96- (Stookey, 1970). The measurements of acid production well thermal cycling block, a 96-well plate as described by from D-glucose were performed in AM medium, and gas Loveland-Curtze et al. (2011). DNA suspended in 2 saline was detected with a Durham tube. Fatty acids were analysed sodium citrate buffer was used for the analysis. The reasso- by HPLC in an Aminex HPX 87H column (BioRad) accord- ciation of DNA was carried out at an optimum temperature  ing to Menes & Muxí (2002). Other physiological and bio- of 80 C according to De Ley et al. (1970) and Gillis et al. chemical tests were conducted using the API ZYM, API (1970). The genome of strain LZ-22T was sequenced as 20NE and API50CH strips according to the manufacturer’s  described in the Supplementary Materials. instructions (bioMerieux), and incubated for 72 h at 30 C. Cells of strain LZ-22T had a width of 0.6–1 µm and length Antimicrobial susceptibility tests were determined with of 3.5–10 µm (Fig. 1a, d, e). The rods stained Gram-positive MicroScan Pos combo panel type 33 and Neg combo panel and were rounded at the ends. Soon after isolation, cells type 50 (Siemens, Healthcare Diagnostics Inc). The panels possessed a gliding snake-like motility that was seldom were inoculated according to the manufacturer’s protocol  observed in later cultures. Morphology similar to strain 10J using the turbidity method, incubated for 72 h at 30 C and (Fig. S3) was common in later cultures of strain LZ-22T, read on a MicroScan auto SCAN-4 System instrument. The sometimes with characteristic mini-cells (Fig. 1d) formed at interpretation was performed according to Clinical and Labo- uneven and many planes of cell division that resulted in ratory Standards Institute (2015). The strains were subcul-  dichotomic branching. Control 16S rRNA gene sequencing tured onto trypticase soy agar and incubated for 72 h at 30 C was performed in later cultures to confirm the purity and prior to inoculation. In addition, strain LZ-22T was checked À identity to the original culture. Later cultures also demon- in AM medium with chloramphenicol (100 µg ml 1) and strated a tendency to clump (Fig. 1b) and possessed a thick gentamicin, tetracycline, rifampicin, ampicillin and kanamy- À1 exomuco-polysaccharide layer on cell surface (Fig. 1c). cin (all at 250 µg ml ). Also nitrofurans and sulfanilamides were checked in both strains (Table S2). Round or oval terminally located structures reminiscent of spores that refracted light during phase contrast microscopy Fatty acid methyl esters were extracted from cells exponen-  were present. These large inclusions occupied up to two- tially grown on AM medium for 48 h at 25 C using the thirds of a cell volume following growth in AM medium Sherlock Microbial Identification System (MIDI) version (Fig. 1c). These structures were not stained with Nile blue € 6.1 as described previously (Kampfer & Kroppenstedt, A, and the absence of poly-b-hydroxybutyrate was con- 1996; Kuykendall et al., 1988; Miller, 1982). Analysis was firmed by the method of extraction and poly-b- carried out with an Agilent Technologies 6890N gas chro- hydroxybutyrate hydrolysis (Smibert & Krieg, 1994). Stain- matograph equipped with a phenyl methyl silicone fused   ing with Loeffler methylene blue and toluidine blue con- silica capillary column (HP- 2.25 m 0.2 mm 0.33 µm firmed their identity as volutin or polyphosphate granules. film thickness) and flame ionization detector. Hydrogen was the carrier gas. The temperature program was initiated Colonies of strain LZ-22T grown on 3 % agar by the roll-   À at 170 C and increased at 5 C min 1 to a final temperature tube technique had a light cream colour, circular concave  of 270 C. The relative amount of each fatty acid was shape, and a smooth and shiny surface with a transparent expressed in terms of the percentage of total fatty acids circle area on the edges. The size of colonies varied from 0.5 using the QTSA1 peak naming library. to 4.0 mm in diameter. The older colonies had denser con- sistency in the cent compared to perimeter. DNA was extracted according to the protocol of the E.Z.N.A. Bacterial DNA kit (OMEGA bio-tek GA). The purity of DNA Strain LZ-22T grew within the temperature range of 3–   was checked by gel electrophoresis, and additional RNAase 41 C with optimum growth at 24–30 C (Fig. S4a). The pH treatment was conducted. The 16S rRNA gene was PCR- range for growth was 4–9, with optimum growth at pH 7.8 amplified with the following primers: B16S-R, 5¢-TACGG (Fig. S4b). The optimum salinity for growth was 0.25 % (w/ YTACCTTGTTACGAC-3¢ and B16S-F, 5¢-AGAGTTTGATC v) NaCl, with the growth range of 0–7 % (w/v) NaCl (Fig.  MTGGCTCAG-3¢ The PCR products were purified with S4c). The strain survived freezing at À80 C without cryo- ZYMO DNA Clean & Concentrator-5 column following the protectants, and biomass could be revived after being frozen  manufacturer’s instructions. Sequencing of the 16S rRNA at À20 C for 6 years. The strain also survived pasteuriza-  gene amplicon of strain LZ-22T was performed at Georgia tion for 30 min at 70 C.

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(a) (b) (c)

(d) (e)

Fig. 1. Cell morphology of strain LZ-22T. (a) High-resolution collimated darkfield microscopy image of strain LZ-22T grown on AM medium with peptone. (b) Phase contrast micrograph of pleomorphic rods of strain LZ-22T grown on AM medium with 3 % NaCl. (c) Dividing cells of strain LZ-22T in thin sections by TEM: cells possess large inclusion bodies with polyphosphate granules (volutin); thick murein layer and small periplasmic space are typical attributes of Gram-positive cell wall. (d) TEM of negative-stained cells of strain LZ-22T demonstrating uneven cell division with formation of miniature spherical cells. (e) TEM of negative-stained cells of strain LZ-22T showing a clump of cells. Bars, 5 µm (a), 10 µm (b), 1 µm (c, d), 2 µm (e).

T Both strains were facultatively anaerobic. While the isolate The fatty acid profile of strain L-22 is shown in Table S3. The LZ-22T was catalase-positive, strain 10J was catalase- major fatty acids were anteiso-pentadecanoic acid (anteiso-  negative. Neither strain grew at 0 or 45 C. Both strains had C15 : 0, 50 %), iso-tetradecanoic acid (iso-C14:0, 7 %), pentade- a chemoheterotrophic fermentative metabolism. Growth canoic acid (C15 : 0, 5 %), and hexadecanoic acid (C16 : 0, 4 %). was observed on tryptic soy agar (Difco), plate count agar Biomass subjected to quinone analysis was grown aerobically (Difco), nutrient agar (Difco) and reinforced Clostridial  in AM medium and harvested at the exponential growth agar (Difco), aerobically or anaerobically after 48 h at 30 C. phase. Quinones were extracted following the protocol of In Triple Sugar Iron agar, acid butt and acid slant growth was observed but no gas formation after 96 h. Both Tindall (1990a, b) and Altenburger et al. (1996). HPLC appa- ratus and conditions were described by Stolz et al. (2007). strains produced acids from a variety of hexoses, disacchar- T ides and a few other sugars used in the API 50CH panel The quinone system of strain LZ-22 consisted of 95 % MK-9 (Table S1). Both strains hydrolysed starch but not gelatin, (H4) and 5 % MK-8(H4). This quinone system is similar to casein and Tweens 20 and 80 (Table S1). Strain LZ-22T but that reported for the close phylogenetic relatives Granulicoccus not 10J hydrolysed urea and aesculin. The other API 20NE phenolivorans and Propioniferax innocua (Maszenan et al., and API ZYM reactions are shown in Table S1. For the sub- 2007; Yokota et al., 1994). T strates listed in the species description, strain LZ-22 grew The DNA–DNA relatedness values for strains LZ-22T and both aerobically and anaerobically in AM medium within 10J were 84±4 %, above the 70 % threshold established for – 2 14 days. species delineation (Wayne et al., 1987), indicating that Under anaerobic conditions, the major fermentation prod- both strains should be affiliated to the same species. ucts of D-glucose were: acetate, lactate, propionate and suc- T The partial sequences of 16S rRNA genes of strains LZ-22 cinate. Hydrogen gas was not produced, but CO was 2 and 10J possessed 99 % sequence identity, consistent with measured at ~5 % of total gas phase. their assignment to a single species. Phylogenetic analysis The MICs for tested antibiotics are shown in Table S2, and further suggested that the isolates represent a new genus their sensitivities are summarized in the species description. within the family Propionibacteriaceae of the order

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Propionibacteriales (Fig. 2). At the time of writing, this fam- Luteococcus, Tessaracoccus, Microlunatus, Brooklawnia, Aur- ily includes 17 genera, all of which are distantly related to anticoccus, Friedmanniella, Propionibacterium, Nocardioides the isolates. For instance, the 16S rRNA gene of strain LZ- and Kribbella. Table 1 shows differential features of the 22T had sequence similarities of 91.9 and 93.2 % with those novel genus as compared to phylogenetically related genera of its closest relatives Granulicoccus phenolivorans PG- of the family Propionibacteriaceae. 02T and Propioniferax innocua ATCC 49929T and was dis- The draft genome sequence of strain LZ-22T was prepared tant enough to be separated into a new lineage on the genus level (Yarza et al., 2014). by the DOE Joint Genomes Institute (see Supplementary Materials). It comprised 3.76 mbp in 36 scaffolds. The cod- Large inclusions of volutin or polyphosphate granules in ing density was 90.4 %, and the G+C content was 70.22 %. T cytology of strain LZ-22 are not unique among Propioni- A total of 3416 protein-encoding and 64 RNA-encoding bacteriaceae, the same accumulations were described in genes were identified. The genome possessed two 16S rRNA T Microlunatus phosphovorus NM-1 (Nakamura et al., 1995). genes. Consistent with the 16S rRNA gene phylogeny, the On the basis of cytology, biochemical and physiological closest BLAST hits for individual genes were largely to other properties, both novel strains were distinguished from genomes from the and Propionibacteria- members of the eleven related genera: Granulicoccus, ceae. However, no particular genus within this family

Propioniferax innocua ATCC 49929 T (AF227165) 0.99 Granulicoccus phenolivorans PG-02T (AY566575) 0.05 1 Raineyella antarctica 10J (LT560379) Raineyella antarctica LZ-22T (KM406782) Luteococcus peritonei CCUG 38120 T (AJ132334) 0.96 0.99 T 0.99 Luteococcus sanguinis CCUG 33897 (AJ416758) Luteococcus japonicus NBRC 12422T (D85487)

T 1 Propionibacterium freudenreichii DSM 20271 (X53217) 1 Propionibacterium acidifaciens C3M_31 T (EU979537)

T 0.91 0.98 Propionibacterium propionicum DSM 43307 (AJ003058) ‘Propionibacterium acidipropionici ’ NCFB 570 (AJ704570) Tessaracoccus lubricantis KSS-17SeT (FM178840) 0.94 Tessaracoccus bendigoensis Ben 106T (AF038504) 0.99 T 0.58 Tessaracoccus flavescens SST-39 (AM393882) Brooklawnia cerclae BL-34T (DQ196625) 0.85 1 Propionimicrobium lymphophilum DSM 4903T (AJ003056) Aestuariimicrobium kwangyangense R27T (DQ830982) Friedmanniella antarctica DSM 11053T (Z78206) 1 Friedmanniella capsulata Ben 108T (AF084529) T 1 Propionicicella superfundia BL-10 (DQ176646) 0.92 1 Propionicimonas paludicola Wd T (AB078858) T 0.76 Microlunatus phosphovorus DSM 10555 (Z78207) T 0.91 0.91 Microlunatus panaciterrae Gsoil 954 (AB271051) Microlunatus soli CC-012602 T (FJ807672) Auraticoccus monumenti MON 2.2T (FN552748)

Fig. 2. Maximum-likelihood tree based on nearly full-length 16S rRNA gene sequences from representative type species and strains of the family Propionibacteriaceae with the two novel strains of this study. The tree was built by FastTree 2.1.5 using Auraticoccus monumenti as an outgroup (Alonso-Vega et al., 2011). Bootstrap values >0.58 are indicated at nodes and were based on 1000 replicates (Price et al., 2010). A similar topology was found by the nighbour-joining method using the Tamura– Nei model (Tamura et al., 2004, 2013) in Geneious 8.0.5, except the internal branching within the clade of Luteococcus- Aestuariimicrobium was different. There were a total of 1351 positions in the final dataset. Bar 0.05 substitutions per position.

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Table 1. Distinguishing features of related genera within the family Propionibacteriaceae

Genera: 1, Raineyella gen. nov. (data from this study); 2, Propioniferax (Yokota et al., 1994; Yokota, 2015); 3, Granulicoccus (Maszenan et al., 2007); 4, Luteococcus (Collins et al., 2000); 5, Propionibacterium (Patrick & McDowell, 2015); 6, Tessaracoccus (Seviour & Maszenan, 2015); 7, Brooklawnia (da Costa et al., 2015); 8, Propionimicrobium (Stackebrandt, 2015); 9, Aestuariimicrobium (Seshadri, 2015). +, Positive; À, neagtive; ±, variable across strains/species; ND, no data available; FAn, facultative anaerobe; An, anaerobe. All genera include Gram-positive species produc- ing propionic acids and do not form endospores.

Feature 1 2 3 4 5 6 7 8 9

Cell morphology Rods* Rods* Cocci Rods* Rods* Cocci Rods* Rods* Rods*/ cocci

Relation to O2 FAn FAn FAn FAn FAn FAn FAn An Aerobe Catalase activity ± + + + + + + ± + NaCl requirement (%, w/v) 0.25–0.5 (0–7) 0–7.5 0.01 ND ND ND 0–3 ND 0–3 Optimum temperature for 26 (3–41) 37 (10–40) 30 (15–37) 37 35 (30–37) 25 (20–37) 37 36–37 30 (4–40)  growth (range) ( C) Growth substrates Sugars + + + + + + + + + Proteo-products + + + À ± + + À + Amino acids + À + À + + + À + Urease ± + + ÀÀÀ ND ND ± DNA G+C content (mol%) 70.2 59–63 69 66–68 60–70 74 68 56 69

*All rods of the family Propionibacteriaceae are pleomorphic.

dominated the hits. The genome encoded five CRISPR and well on D-proline but not on L-proline (Pikuta & Hoover, 14 transposases, suggesting that phages and horizontal gene 2014). transfers were an important component of the isolate’s past. Fatty acid profiles of the novel isolates are also similar to those Greater than thirty potentially catabolic dehydrogenases of the two phylogenetically closest species Granulicoccus phe- were indicative of a heterotrophic metabolism (Schriek nolivorans (Maszenan et al., 2007) and Propioniferax innocua et al., 2007). The genome also encoded two catalases, which (Pitcher & Collins, 1991; Yokota et al., 1994), both of which confirmed the catalase-positive test. possess large amounts of anteiso-pentadecanoic acid (anteiso Notwithstanding the different geographic locations and C15 : 0, ~50 %), and pentadecanoic acid (C15 : 0). In contrast to ecosystems of both isolates, their genotypic and phenotypic Granulicoccus phenolivorans, the novel isolate LZ-22T does characteristics are consistent with proposed affiliation to not contain iso-pentadecanoic dimethyl ester (iso-C15 : 0 the same species in spite of small variations. For instance, DMA). T LZ-22 grows best on lactose, which is not utilized by strain T T In conclusion, we summarize that strains LZ-22 and 10J 10J. Likewise, strain LZ-22 is catalase-positive while strain represent a novel species of a new genus within the family 10J is catalase-negative. A unique metabolic feature of strain T Propionibacteriaceae with proposed name Raineyella antarc- LZ-22 was the ability to grow on some D-amino acids (D- tica gen. nov., sp. nov. met, D-leu, D-his and D-val). While both L- and D- enan- tiomers of methionine and leucine supported growth, only the D- chirality enantiomers but not L- enantiomers of histi- Description of Raineyella gen. nov. dine and valine were utilized. At the same time, amino acids ¢ alanine, serine and proline were metabolized by strain LZ- Raineyella (Rai.ney.el la. N.L. fem. n. Raineyella the name is T dedicated to the British microbiologist Frederick Andrew 22 in traditional L- enantiomeric form, and the D- enan- Rainey, President of Bergey’s International Society for Micro- tiomers were neglected. A variety of genes associated with bial Systematics (2012–2014), a member of the Bergey’s Man- racemase and isomerases activities were found in the T ual Trust, and who has significantly contributed to bacterial genome sequence of strain LZ-22 . The ability to metabo- phylogeny and . lize alternate chiral enantiomers of amino acids and sugars by extremophilic anaerobes has been previously studied by Clusters within the Family Propionibacteriaceae. Faculta- our group as a part of the Shadow Biosphere project (Pikuta tively anaerobic and catalase-variable. Pleomorphic rods et al., 2006a; Pikuta & Hoover, 2010), and this is not the that stain Gram-positive. Non spore-forming. The major first report of preferable anaerobic growth with D-amino fatty acid is anteiso-pentadecanoic acid (anteiso-C15 : 0). In acids along with absolute disregard of their L-enantiomers. the quinone system, MK-9(H4) is predominant and minor T For example, Carnobacterium pleistocenium FTR1 grew amounts of MK-8(H4) are present. Chemoorganotrophs, Downloaded from www.microbiologyresearch.org by 5534 International Journal of Systematic and Evolutionary Microbiology 66 IP: 179.27.78.74 On: Mon, 06 Aug 2018 13:58:54 Raineyella antarctica gen. nov., sp. nov. psychrotolerant or mesophilic, athalassic with neutral pH Acknowledgements range for growth. We express gratitude to Col. James Pritzker and The Tawani Founda- The type species is Raineyella antarctica. tion, which provided a primary funding for the 2008 Tawani Interna- tional Schirmacher Oasis/Lake Untersee, Antarctica Expeditions, and we also thank National Aeronautics and Space Administration (NASA) and the National Science Foundation (NSF). We express special grati- tude to Dr Valerie Lukin of the Arctic and Antarctic Research Insti- Description of Raineyella antarctica tute/Russian Antarctic Expedition (AARI/RAE) of the Russian sp. nov. Federation, Mirella Kruger of Antarctic Logistics Centre International ¢ (ALCI), the team from the United States, Russia and Austria for sci- Raineyella antarctica (ant.arc ti.ca. L. fem. adj. antarctica of entific and engineering assistance, equipment and logistic support the continent of Antarctica). during expeditions. We also thank the Russian support team at Novolazarevsky Station, Antarctica for their kind hospitality and Shows the following characteristics in addition to those listed assistance. We thank Prof. Larry Shimkets (Bio Science, UGA) for his in the genus description. Cells are motile by gliding or not, help with photography of our new isolate, Ying Xiao, MD (LSU) for with sizes 0.6–1.0Â3.5–10 µm. Cells have large inclusions of assistance with TEM photomicrographs, and Dr Sara Cline (Athens volutin or polyphosphate granules. Psychrotolerant meso- State University) for assistance with UV spectrophotometry. We also  philes. Growth occurs within temperature range of 3–41 C acknowledge the generous assistance of Nikos C. Kyrpides, Tanja  with an optimum at 24–28 C, no growth occurs at 0 or 45 Woyke, Nicole Shapiro, and the other members of the JGI microbial   genome sequencing team. The work conducted by the US Depart- C. Survives pasteurization at 70 C for 30 min. Salinity range ment of Energy Joint Genome Institute, a DOE Office of Science is 0–7 % (w/v) NaCl with optimum at 0.25 %, and pH range User Facility, is supported by the Office of Science of the US Depart- is 4–9 with optimum at pH 7.8. Chemoorganoheterotroph ment of Energy under Contract No. DE-AC02-05CH11231. with fermentative metabolism. Anaerobic growth occurs on: yeast extract, peptone, chitin, D-glucose, sucrose, D-fructose, D-ribose, maltose, D-galactose, cellobiose, lactose, inulin, References starch, trehalose, L-cysteine, L-cystine, L-alanine, L-proline, Alonso-Vega, P., Carro, L., Martínez-Molina, E. & Trujillo, M. E. 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