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 species. 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 Propionibacteriaceae (~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. Downloaded from www.microbiologyresearch.org by 001552 ã 2016 IUMS Printed in Great Britain 5529 IP: 164.73.160.156 On: Mon, 18 Dec 2017 20:07:00 E. V. Pikuta and others 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 bacteria 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.