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Other Gram-Positive Bacteria

Sporosarcina antarctica sp. nov., a psychrophilic bacterium isolated from the Antarctic

Yong Yu, Yu-Hua Xin, Hong-Can Liu, Bo Chen, Jun Sheng, Zhen-Ming Chi, Pei-Jin Zhou, De-Chao Zhang

  • 1SOA Key Laboratory for Polar Science, Polar Research Institute of China, Shanghai 200136, PR China
  • 2China General Microbiological Culture Collection Center and State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China
  • 3UNESCO Chinese Center of Marine Biotechnology, Ocean University of China, Qingdao 266003, PR China
  • Correspondence
    De-Chao Zhang
    zhangdechao{at}yahoo.cn

    • International Journal of Systematic and Evolutionary Microbiology 2008; 58(9):2114–2117 · https://doi.org/10.1099/ijs.0.65838-0

    View at publisher PubMed

    Abstract

    A Gram-positive, psychrophilic, rod-shaped bacterium, designated strain N-05T, was isolated from soil samples collected off King George Island, west Antarctica (6 ° 13′ 31″ S 5 ° 57′ 08″ W). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain N-05T was related to members of the genus Sporosarcina and had highest 16S rRNA gene sequence similarity with the type strain of Sporosarcina macmurdoensis (98.0 %). The temperature range for growth of strain N-05T was 0–23 °C, with optimum growth occurring at 17–18 °C and approximately pH 6.0–8.0. Strain N-05T had MK-7 as the major menaquinone and anteiso-C15 : 0 and C16 : 1ω7c alcohol as major fatty acids. The genomic DNA G+C content was 39.2 mol%. On the basis of phenotypic characteristics, phylogenetic analysis and DNA–DNA relatedness data, strain N-05T is considered to represent a novel species of the genus Sporosarcina, for which the name Sporosarcina antarctica is proposed. The type strain is N-05T (=CGMCC 1.6503T=JCM 14646T).

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain N-05T is EF154512.

    • A table detailing the cellular fatty acid content of strain N-05T in comparison with other recognized Sporosarcina species is available as supplementary material with the online version of this paper.

    The genus Sporosarcina, which belongs to the family Bacillaceae, was proposed by Kluyver & van Niel (1936) to accommodate bacteria that have spherical or oval-shaped cells and contain MK-7 as the major menaquinone. At the time of writing, the genus Sporosarcina comprises nine recognized species, namely Sporosarcina ureae (Claus et al., 1983; Claus & Fahmy, 1986), S. globispora (Larkin & Stokes, 1967; Yoon et al., 2001), S. psychrophila (Nakamura, 1984; Yoon et al., 2001), S. pasteurii (Chester, 1898; Yoon et al., 2001), S. aquimarina (Yoon et al., 2001), S. macmurdoensis (Reddy et al., 2003), S. koreensis and S. soli (Kwon et al., 2007), and S. saromensis (An et al., 2007). Here we report on the characterization of a novel psychrophilic bacterium of the genus Sporosarcina isolated from soil samples collected off King George Island, west Antarctica (6 ° 13′ 31″ S 5 ° 57′ 08″ W).

    In the laboratory, frozen soil collected off King George Island was suspended in an equal volume of liquid PYG medium (0.5 % bacto peptone, 0.02 % yeast extract, 0.5 % glucose, 0.3 % beef extract, 0.05 % NaCl and 0.15 % MgSO4 . 7H2O, w/v, pH adjusted to 7.0; Zhang et al., 2007) supplemented with cycloheximide, nystatin and nalidixic acid (all at 25 μg ml−1) and shaken at 10 °C for 4 days at 150 r.p.m. The culture was further diluted (1 : 10) and spread onto PYG agar plates and incubated at 4 °C for 2 weeks. Strain N-05T was obtained in pure culture after three successive transfers to fresh PYG agar and stored at −80 °C in 30 % (v/v) glycerol. S. macmurdoensis DSM 15428T was obtained from the DSMZ and routinely grown on ABM agar (0.5 % peptone, 0.2 % yeast extract and 1.5 % agar, w/v, pH 7.2) at 18 °C and used as a reference strain.

    DNA was extracted and purified as described by Sambrook et al. (1989). The gene encoding 16S rRNA was amplified by PCR with two universal primers (Zhang et al., 2007). Sequencing reactions were carried out by using an ABI BigDye 3.1 sequencing kit (Applied Biosystems) and an automated DNA sequencer (model ABI3730; Applied Biosystems). The nearly complete 16S rRNA gene sequence (1446 bp) of strain N-05T was submitted to GenBank and EMBL to search for similar sequences by using the blast algorithm. A phylogenetic tree was constructed by using Kimura's two-parameter and pairwise-deletion model analysis implemented in the program mega version 3.0 (Kumar et al., 2004). Resultant tree topologies were evaluated by bootstrap analysis based on 1000 replicates. Phylogenetic analysis (Fig. 1⇓) showed that strain N-05T was grouped with members of the genus Sporosarcina, and formed a distinct cluster with S. macmurdoensis DSM 15428T (98.0 % 16S rRNA gene sequence similarity) supported by a high bootstrap value (100 %) in the neighbour-joining tree. Levels of 16S rRNA gene sequence similarity between strain N-05T and other strains used in the phylogenetic analysis were less than 95.6 %. A similar topology was found in the tree generated with the maximum-parsimony algorithm (data not shown).

    Figure image not available in archive
    Fig. 1.

    Phylogenetic relationships between strain N-05T, recognized members of the genus Sporosarcina and other related species based on 16S rRNA gene sequences. The tree was constructed by using the neighbour-joining method. Numbers at nodes represent levels of bootstrap support (%) based on a neighbour-joining analysis of 1000 resampled datasets. GenBank accession numbers are given in parentheses. Bar, 1 % sequence divergence.

    Cell morphology of strain N-05T was examined with an Axioplan 2 microscope. Colony morphology was observed following incubation on PYG at 17 °C. Growth temperature was determined with a TN3F temperature-gradient incubator (Advantec). The pH range for growth was determined by using PYG broth at various pH values adjusted with HCl or NaOH (both at 1 mol l−1). Anaerobic growth was investigated in PYG medium in an anaerobic test tube filled with nitrogen gas. General physiological tests were performed by using conventional methods (Dong & Cai, 2001). Acid production from carbohydrates was determined as described by Leifson (1963). API strips (API 20 E, API 20 NE, API ZYM; bioMérieux) were used to determine the physiological and biochemical characteristics of strain N-05T according to the manufacturer's instructions. The API strips were incubated for 4 days at 17 °C. All of the tests were performed in duplicate. Morphological, physiological and biochemical characteristics of strain N-05T are given in the species description below or are shown in Table 1⇓.

    Table 1.

    Differential phenotypic characteristics between strain N-05T and recognized Sporosarcina species

    Strains: 1, N-05T (S. antarctica sp. nov.); 2, S. macmurdoensis CMS 21wT (data from Reddy et al., 2003); 3, S. aquimarina SW28T (Yoon et al., 2001); 4, S. globispora DSM 4T (Nakamura, 1984; Rüger, 1983); 5, S. psychrophila IAM 12468T (Nakamura, 1984); 6, S. pasteurii NCIMB 8841T (Yoon et al., 2001); 7, S. ureae DSM 2281T (Claus & Fahmy, 1986); 8, S. koreensis F73T (Kwon et al., 2007); 9, S. soli I80T (Kwon et al., 2007); 10, S. saromensis HG645T (An et al., 2007). +, Positive; −, negative; w, weak; na, no data available.

    Respiratory quinones were extracted and purified according to Collins (1985) and were analysed by HPLC (Wu et al., 1989) by using menaquinone 7 (MK-7) from S. macmurdoensis DSM 15428T as a reference. Cell-wall peptidoglycan was prepared and analysed in accordance with the methods of Komagata & Suzuki (1987) and Zhang et al. (2007). For analysis of fatty acid methyl esters, cell mass of strain N-05T was harvested from PYG plates after incubation at 17 °C for 4 days. The fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI (Microbial Identification) system (Sasser, 1990). Strain N-05T contained MK-7 as the major menaquinone. The peptidoglycan of the isolate contained a cell-wall type of l-Lys–d-Glu (variation A4α). The predominant cellular fatty acids of strain N-05T were anteiso-C15 : 0 (39.75 % of the total), C16 : 1ω7c alcohol (18.89 %), iso-C17 : 1 I and/or anteiso-C17 : 1 B (11.91 %), iso-C14 : 0 (7.55 %), iso-C16 : 0 (7.01 %), anteiso-C17 : 0 (4.23 %), C16 : 1ω11c (3.67 %), iso-C15 : 0 (1.36 %), iso-C16 : 1 (1.30 %) and C16 : 0 (1.19 %). The fatty acid profile of strain N-05T was similar to those of recognized Sporosarcina species (Yoon et al., 2001), although strain N-05T contained a greater proportion of unsaturated fatty acids (C16 : 1ω7c alcohol, iso-C17 : 1 I and/or anteiso-C17 : 1 B), which contribute to the maintenance of membrane fluidity at low temperature. The fatty acid profiles of strain N-05T and other recognized Sporosarcina species are detailed in Supplementary Table S1 available in IJSEM Online.

    The DNA G+C content was determined by using the thermal denaturation method with Escherichia coli K-12 as reference, and DNA–DNA hybridization experiments were performed using the liquid renaturation method (De Ley et al., 1970) as modified by Huß et al. (1983); both experiments were carried out by using a DU800 spectrophotometer (Beckman) with a thermal controller. The DNA G+C content of strain N-05T was 39.2 mol%. The level of DNA–DNA relatedness between strain N-05T and S. macmurdoensis DSM 15428T was as low as 41.7 %, much lower than the threshold (70 %) recommended for species demarcation (Wayne et al., 1987), thus verifying that strain N-05T represents a species distinct from S. macmurdoensis. 16S rRNA gene sequence similarities of less than 97.0 % were found between strain N-05T and the type strains of other recognized species of the genus Sporosarcina. It has been suggested that in bacterial strains showing less than 97 % 16S rRNA gene sequence similarity, levels of DNA–DNA hybridization are less than 70 % (Stackebrandt & Goebel, 1994). Therefore, strain N-05T is genotypically distinct from all recognized species of the genus Sporosarcina.

    Based on the phenotypic, phylogenetic and genomic evidence presented, strain N-05T is considered to represent a novel species of the genus Sporosarcina, for which the name Sporosarcina antarctica sp. nov. is proposed.

    Description of Sporosarcina antarctica sp. nov.

    Sporosarcina antarctica (ant.arc′ti.ca. L. fem. adj. antarctica southern, of the Antarctic, the geographical origin of the type strain).

    Cells are Gram-positive, psychrophilic, non-motile rods, 0.3–0.6 μm by 1.1–2.6 μm in size. Colonies are light yellow, smooth, circular and convex with entire margins. Produces catalase and cytochrome oxidase. Growth occurs under anaerobic conditions on PYG medium. Growth occurs at 0–23 °C and pH 5.0–10.0, with optimum growth at 17–18 °C and approximately pH 6.0–8.0. Growth occurs in the presence of 0–9 % (w/v) NaCl. Nitrate is not reduced and hydrogen sulfide is not produced. Negative for indole production and the Voges–Proskauer recation. Negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, leucine arylamidase, valine arylamidase, cystine arylamidase, urease, gelatinase, caseinase, trypsin, amylase, lecithinase, acid phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, α-fucosidase and α-chymotrypsin. Positive for naphthol-AS-BI-phosphohydrolase and alkaline phosphatase. d-Glucose and α-ketobutyric acid can be utilized as sole carbon sources, but not sucrose, lactose, galactose, trehalose, cellobiose, l-rhamnose, melibiose, raffinose, d-sorbitol, inositol, maltose, glucose, glycerol, fructose, melezitose, l-arabinose, d-mannose, mannitol, N-acetylglucosamine, malate, succinate, capric acid, adipic acid or phenylacetic acid. Acid is not produced from the following carbohydrates: d-glucose, fructose, sucrose, lactose, galactose, trehalose, cellobiose, l-rhamnose, melibiose, raffinose, d-sorbitol, inositol, maltose, glycerol, melezitose, l-arabinose, d-mannose or mannitol. MK-7 is the major menaquinone. The cell wall contains peptidoglycans of the l-Lys–d-Glu type (variation A4α). Predominant cellular fatty acids are anteiso-C15 : 0 (39.75 % of the total), C16 : 1ω7c alcohol (18.89 %), iso-C17 : 1 I and/or anteiso-C17 : 1 B (11.91 %), iso-C14 : 0 (7.55 %), iso-C16 : 0 (7.01 %), anteiso-C17 : 0 (4.23 %) and C16 : 1ω11c (3.67 %). The DNA G+C content of the type strain is 39.2 mol%.

    The type strain, N-05T (=CGMCC 1.6503T=JCM 14646T), was isolated from soil samples collected off King George Island, west Antarctica (6 ° 13′ 31″ S 5 ° 57′ 08″ W).

    Acknowledgments

    This work was supported by the National Natural Science Foundation of China (30500001), the National Basic Research Program of China (2004CB719601) and the National Infrastructure of Natural Resources for Science and Technology Program of China (No. 2005DKA21209).

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