International Journal of Systematic and Evolutionary Microbiology (2013), 63, 3375–3378 DOI 10.1099/ijs.0.047159-0

Mycetocola zhadangensis sp. nov., isolated from snow

Liang Shen,1 Yongqin Liu,1 Tandong Yao,1 Shichang Kang,1 Yanan Wang,2 Nianzhi Jiao,3 Hongcan Liu,4 Yuguang Zhou,4 Baiqing Xu1 and Xiaobo Liu1

Correspondence 1Key Laboratory of Tibetan Environmental Changes and Land Surface Processes, Institute of Yongqin Liu Tibetan Plateau Research, Chinese Academy of Sciences, Beijing, 100085, PR China [email protected] 2Key Laboratory of Microbial Engineering, The Institute of Biology, Henan Academy of Sciences, Zhengzhou, 450008, PR China 3State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, 361005, PR China 4Institute of Microbiology, China General Microbiological Culture Collection Center, Chinese Academy of Sciences, Beijing, 100101, PR China

A Gram-stain-positive, aerobic, short rod-shaped bacterium, strain ZD1-4T, was isolated from the Zhadang Glacier snow pit. The 16S rRNA gene sequence of the isolate showed highest similarity (98.8 %) to that of Mycetocola manganoxydans MB1-14T. The major fatty acids of strain ZD1-4T

were anteiso-C15 : 0,C16 : 0,C18 : 0 and anteiso-C17 : 0. It possessed diphosphatidylglycerol as one of the major polar lipids, and MK-10 and MK-11 as the predominant isoprenoid quinones. The T DNA G+C content of strain ZD1-4 was 63.8±0.2 mol% (Tm). A number of phenotypic characteristics distinguished this bacterium from the type strains of other of the genus Mycetocola. Moreover, the novel isolate showed only approximately 50 % DNA–DNA relatedness with M. manganoxydans MB1-14T. According to these genotypic and phenotypic data, it is evident that strain ZD1-4T represents a novel species of the genus Mycetocola, for which the name Mycetocola zhadangensis sp. nov. is proposed. The type strain is ZD1-4T (5KACC 16570T5CGMCC 1.12042T).

At the time of writing, the genus Mycetocola includes five Genomic DNA was extracted according to Marmur (1961). species with validly published names, Mycetocola lacteus, The 16S rRNA genes for all purified were amplified M. saprophilus, M. tolaasinivorans, M. reblochoni and M. with universal primers 27F (59-AGAGTTTGATCCTGGCT- manganoxydans (Tsukamoto et al., 2001; Bora et al., 2008; CAG-39) and 1492R (59-CGGTTACCTTGTTACGACTT-39) Luo et al., 2012). Members of the genus are Gram-positive, (Embley, 1991). The PCR products were further purified and aerobic, non-sporulating, short rod-shaped bacteria, which sequenced at Boai Yonghua (Beijing). The 16S rRNA gene contain anteiso-C15 : 0 and anteiso-C17 : 0 as typical fatty sequences were compared with available sequences via the acids, MK-10 as the predominant isoprenoid quinone and EzTaxon-e server (http://eztaxon-e.ezbiocloud.net) (Kim et al., lysine as the diagnostic cell-wall diamino acid. 2012) and GenBank to determine the approximate phylogen- etic affiliation for the novel isolates. Phylogenetic trees were Snow samples were collected from Zhadang Glacier on the reconstructed using the neighbour-joining and maximum- Qinghai–Tibetan Plateau of China. Thawed water from snow parsimony methods with MEGA 5.05 software (Tamura et al., samples was used as the initial isolation source. After 2011) (Fig. 1). A representative yellow strain, ZD1-4T,was incubation at 24 uC for about 4 weeks, 50 bacterial colonies phylogenetically related most closely to M. manganoxydans were seen on R2A agar plates (Reasoner & Geldreich, 1985). MB1-14T (98.8 % 16S rRNA gene sequence similarity) and was They were restreaked several times and confirmed for purity by well-distinguished from the type strains of other species of the checking the sequencing map and microscopic examination. genus Mycetocola.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene Phenotypic and genotypic analyses were conducted to sequence of Mycetocola zhadangensis ZD1-4T is JQ409017. further determine the taxonomic position of the novel Two supplementary figures are available with the online version of this isolate. Cell morphology was examined by transmission paper. electron microscopy (JEM-1230; JEOL) (Fig. S1 available in

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92 Mycetocola lacteus CM-10T (AB012648) 100 0.005 Mycetocola saprophilus CM-01T (AB012647) 54 Mycetocola tolaasinivorans CM-05T (AB012646) 74 Mycetocola reblochoni R-20377T (DQ062097)

ZD1-4T (JQ409017) 61 Mycetocola zhadangensis

93 Mycetocola manganoxydans MB1-14T (GU217690)

Microbacterium sediminicola YM10-847T (AB286021)

Labedella gwakjiensis KSW2-17T (DQ533552)

Cryobacterium luteum Hh15T (HQ845193)

Cryobacterium roopkundense RuGl7T (EF467640)

87 Cryobacterium arcticum SK-1T (GQ406814)

69 Cryobacterium psychrotolerans 0549T (DQ515963)

Frondihabitans peucedani RS-15T (FM998017)

99 Subtercola frigoramans K265T (AF224723)

87 Agreia pratensis P 229/10T (AJ310412)

Fig. 1. Neighbour-joining phylogenetic tree for strain ZD1-4T, based on 16S rRNA gene sequence analysis. Filled circles indicate that the corresponding branches were also recovered in the maximum-parsimony tree. Bootstrap values based on 1000 replications are listed as percentages; only values .50 % are shown. Bar, 0.005 substitutions per nucleotide position.

IJSEM Online). Phenotypic characteristics such as Gram Kandler (1972). Cell-wall hydrolysates of strain ZD1-4T staining, catalase activity, and hydrolysis of casein, contained lysine as the diagnostic diamino acid. hypoxanthine, Tween 80 and starch were performed using For isoprenoid quinone and polar lipid analyses, cells were the methods described by Smibert & Krieg (1994). Growth harvested after 48 h of growth at 28 uC. Isoprenoid at 0, 5, 10, 15, 20, 25, 30, 35 and 40 uC was measured in quinones were analysed as described by Hiraishi et al. R2A broth. The pH range (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, (1998) performed on a Waters Acquity Ultra Performance 7.5, 8.0, 8.5, 9.0, 9.5, 10.0) for growth was also determined LC (UPLC)-Q-TOF-MS spectrometer in electrospraying using R2A broth. The pH of the medium was adjusted with ionization mode (Romano et al., 2006). The isoprenoid citrate phosphate buffer or Tris/HCl buffer (Breznak & quinones of strain ZD1-4T were MK-9 (19.3 %), MK-10 Costilow, 1994). Other physiological and biochemical (63.0 %) and MK-11 (17.3 %). Polar lipids were extracted properties were determined with API 20NE and API and analysed by two-dimensional TLC (Altenburger et al., ZYM strips according to the manufacturer’s instructions 1996; Tindall et al., 1990). The novel isolate contained (bioMe´rieux). A number of differences between strain diphosphatidylglycerol and several unknown phospholi- T ZD1-4 and other recognized type strains of the genus pids (Fig. S2). The absence of phosphatidylglycerol Mycetocola are shown in Table 1. distinguished strain ZD1-4T from its closest relative, M. T + Strain ZD1-4T was cultivated on R2A agar at 28 uC for 48 h manganoxydans MB1-14 . The genomic DNA G C and cells were harvested. Whole-cell fatty acid methyl esters content was estimated from the midpoint value (Tm)of were extracted and prepared using the standard protocol of the thermal denaturation profile (Mandel et al., 1970). The + T ± the Microbial Identification System (MIDI, version 6.0). As DNA G C content of strain ZD1-4 was 63.8 0.2 mol% ± shown in Table 2, the fatty acid profile of strain ZD1-4T (mean SD of n55 determinations). was consistent with that of the genus Mycetocola. However, DNA–DNA hybridization experiments between strain T T T strain ZD1-4 has a relatively higher content of C16 : 0, ZD1-4 and M. manganoxydans MB1-14 were performed C18 : 0 and C18 : 1 than those of other members of the genus. using methods described previously (De Ley et al., 1970; The possession of unsaturated fatty acid C18 : 1 may be Huss et al., 1983; Jahnke, 1992). The mean (±SD) DNA attributed to its adaptation to the cold environment reassociation rate between strain ZD1-4T and M. (Corien, 2008). Cell-wall preparations were prepared manganoxydans MB1-14T was 50±3.5 % (data were taken according to the method described by Schleifer & from three independent experiments). This indicated that

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Table 1. Differential characteristics of strain ZD1-4T and phylogenetically related species of the genus Mycetocola

Strains: 1, ZD1-4T;2,M. manganoxydans MB1-14T;3,M. reblochoni R-20377T;4,M. saprophilus NRRL B-24119T;5,M. tolaasinivorans NRRL B- T T 24120 ;6,M. lacteus NRRL B-24121 . All data are from this study except where indicated. +, Positive; 2, negative; (+), weakly positive; ND, not determined.

Characteristic 1 2 3 4 5 6

Isolation source Snow Desert soil* CheeseD Fungus fruiting bodiesd Fungus fruiting bodiesd Fungus fruiting bodiesd Temperature range (uC) 0–40 20–35* 20–30D .4–33d .4–33d .4–33d Nitrate reduction 22* 2* +* 2* +* Assimilation of: Glucose 2 ++D +++ Arabinose 2 ++D 222 Mannose 2 ++D +++ Mannitol 2 ++D +++ Maltose 22 +* + 2 + Malic acid 22 +* + 22 Production of (API ZYM): Alkaline phosphatase 2 + 2* 222 Esterase lipase (C4) ++ 2* + (+) + Esterase lipase (C8) ++ +* +++ Valine arylamidase + (+) 2* 222 Cystine arylamidase + (+) 2* 222 Acid phosphatase ++ 2* +++ a-Glucosidase ++ +* +++ b-Glucosidase 2 ++* +++

*Data from Luo et al. (2012). DData from Bora et al. (2008). dData from Tsukamoto et al. (2001).

Table 2. Chemotaxonomic markers of strain ZD1-4T and type strain ZD1-4T was not a member of M. manganoxydans.It strains of related species of the genus Mycetocola was shown that the 16S rRNA gene sequence similarities between strain ZD1-4T and the type strains of other species Strains: 1, ZD1-4T;2,M. manganoxydans MB1-14T (data from Luo of the genus Mycetocola were less than 97 %. This suggested et al., 2012); 3, M. reblochoni R-20377T (Luo et al., 2012); 4, M. that strain ZD1-4T could not be affiliated to any recognized saprophilus NRRL B-24119T (Luo et al., 2012); 5, M. tolaasinivorans species of the genus Mycetocola. NRRL B-24120T (Luo et al., 2012); 6, M. lacteus NRRL B-24121T (Luo et al., 2012). Values are percentages of the total. 2, Not detected or Based on the genotypic and phenotypic data presented, the T ,1%; ND, not determined. novel psychrotolerant bacterium ZD1-4 represents a novel species of the genus Mycetocola, for which the name Component 1 2 3 4 5 6 Mycetocola zhadangensis sp. nov. is proposed. Fatty acids C12 : 0 2.1 22222 Description of Mycetocola zhadangensis sp. nov. iso-C14 : 0 22221.3 2 Mycetocola zhadangensis (zha.dang.en9sis. N.L. masc. adj. C14 : 0 4.6 22222 zhadangensis of or belonging to Zhadang Glacier, Tibetan anteiso-C15 : 1 3.7 2.2 2.9 222 iso-C15 : 0 1.7 2.4 1.0 222 Plateau, China, where the type strain was isolated). anteiso-C15 : 0 28.9 46.9 30.0 53.7 64.4 55.0 Cells are aerobic, Gram-stain-positive and rod-shaped. iso-C16 : 0 7.4 4.6 11.0 13.0 14.9 12.8 Yellow, round, smooth, convex and opaque colonies are C 21.2 8.7 9.0 4.5 1.9 2.6 16 : 0 produced on R2A agar plates after incubation at 28 uC for anteiso-C 9.1 12.4 20.2 23.0 15.4 26.5 17 : 0 u u C v9c 3.1 22222 2–3 days. Grows at 0–40 C (optimally at 25 C) on R2A 18 : 1 agar and at pH 5–8 (optimally at pH 7). Negative for C18 : 0 17.5 6.3 6.3 222 Menaquinones nitrate reduction. In API ZYM tests, positive for produc- MK-9 19.3 8.3 20.7 ND ND 33.0 tion of esterase (C4), esterase lipase (C8), leucine MK-10 63.0 70.9 68.4 ND ND 47.2 arylamidase, valine arylamidase, cystine arylamidase, acid MK-11 17.3 19.8 5.4 ND ND 7.2 phosphatase, naphthol-AS-BI-phosphohydrolase, a-gluco- sidase and N-acetyl-b-glucosaminidase, but negative for

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