International Journal of Systematic and Evolutionary Microbiology (2014), 64, 2775–2780
DOI 10.1099/ijs.0.057638-0
Halopolyspora alba gen. nov., sp. nov., isolated from sediment Hangxian Lai,13 Xiaomin Wei,13 Yingying Jiang,1 Xiu Chen,2 Qinyuan Li,2 Yi Jiang,2 Chenglin Jiang2 and Leionid Gillerman3 Correspondence Hangxian Lai [email protected] Yi Jiang [email protected]
1
College of Natural Resources and Environment, Northwest A&F University, Yangling, Shaanxi, 712100, PR China
2
Yunnan Institute of Microbiology and Life Sciences Lab Center, Yunnan University, Kunming, 650091, PR China
3
J. Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede Boker, 84990, Israel
A novel halophilic, filamentous actinomycete, designated strain AFM 10251T, was isolated from a sediment sample collected from the Dead Sea, Israel. The isolate grew with 10–35 % multi-salts, and did not grow without NaCl or MgCl2. The isolate formed a white aerial mycelium, and long chains of arthrospores with more than 10 spores per chain. The spores were spherical or oval with warty surfaces, and sterile mycelium was present between individual spores. The isolate contained meso-diaminopimelic acid and a small proportion of LL-diaminopimelic acid as cell-wall diamino acids, and galactose and arabinose as whole-cell sugars. The major menaquinone was MK-9(H4). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and three unknown phospholipids. Major fatty acids were iso-C16 : 0, iso-C17 : 0, iso-C15 : 0 and anteiso-C17 : 0. The DNA G+C content of strain AFM 10251T was 66.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain AFM 10251T and the genus Actinopolyspora formed a distinct lineage. Analysis of the secondary structures of variable areas of the 16S rRNA gene showed that strain AFM 10251T was different from all recognized species of the genus Actinopolyspora and members of the family Pseudonocardiaceae. Analysis of the signature nucleotides of the 16S rRNA gene showed that strain AFM 10251T and Actinopolyspora halophila formed a single group, but with base pair differences at positions 127 : 234 and 183 : 194. On the basis of analysis of chemical and molecular characteristics, strain AFM 10251T is considered to represent a novel species of a new genus in the family Actinopolysporaceae, for which the name Halopolyspora alba gen. nov., sp. nov. is proposed. The type strain of Halopolyspora alba is AFM 10251T (5DSM 45976T5CGMCC 4.7114T).
The genus Actinopolyspora, of the family Nocardiaceae, was proposed by Gochnauer et al. (1975), with the description of Actinopolyspora halophila as a halophilic actinomycete. The genus was subsequently placed in the family Pseudonocardiaceae of the suborder Pseudonocardineae (Stackebrandt et al., 1997; Xu et al., 2007). A new suborder Actinopolysporineae and the family Actinopolysporaceae were proposed by Zhi et al. (2009). Goodfellow & Trujillo (2012) then proposed the order ‘Actinopolysporales’ including only one 3These authors contributed equally to this work. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain AFM 10251T is KF022042. Three supplementary figures are available with the online version of this paper.
057638 G 2014 IUMS
Printed in Great Britain
family and one genus, Actinopolyspora. At the time of writing, the genus Actinopolyspora comprises eight recognized species: Actinopolyspora alba (Tang et al., 2011), Actinopolyspora algeriensis (Meklat et al., 2012), Actinopolyspora erythraea (Tang et al., 2011), Actinopolyspora halophila (Gochnauer et al., 1975), Actinopolyspora iraqiensis (Ruan et al., 1994), Actinopolyspora lacussalsi (Guan et al., 2013), Actinopolyspora mortivallis (Yoshida et al., 1991) and Actinopolyspora xinjiangensis (Guan et al., 2010). During the course of studies on halophilic actinomycete communities in saline lakes, several novel strains were obtained. A new genus belonging to the family Actinopolysporaceae (Zhi et al., 2009; Goodfellow & Trujillo, 2012) is proposed to accommodate one of these halophilic actinomycetes based on the results of the present polyphasic taxonomic study. 2775
H. Lai and others
multi-salts. The pH (3.0–12.0) for growth was determined as described by Xu et al. (2005) in peptone water medium supplemented with 10 % (w/v) multi-salts. Tolerance to multi-salts, NaCl and MgCl2 at different concentrations (0–35 %, w/v) was tested on CMKA medium. Gram staining was carried out by using the standard Gram reaction (Murray et al., 1994). Utilization of carbon and nitrogen sources was examined by using the medium of Smibert & Krieg (1994) supplemented with various substrates and 15 % multi-salts. Catalase activity was determined by assessing bubble production with 3 % (v/v) H2O2, according to the methods used by Smibert & Krieg (1994). Anaerobic growth was assessed in liquid medium at 40 uC. Other physiological tests were carried out as described by Gordon et al. (1974). Fig. 1. Scanning electron micrograph of spore chains of strain AFM 10251T grown on PDA medium for 30 days at 28 6C. Bar, 3 mm.
Strain AFM 10251T was isolated from a sediment sample collected from the Dead Sea, Israel, by using CMKA medium. This CMKA medium contained (per litre) 0.5 g Casein acids hydrolysate, 1.5 g mannitol, 1 g KNO3, 2 g (NH4)2SO4, 0.5 g K2HPO4, 0.5 g CaCO3, 20 g agar and 20 % (w/v) multi-salts. The multi-salts comprised 49 % (w/ w) MgCl2, 32 % (w/w) NaCl, 14 % (w/w) CaCl2 and 5 % (w/w) KCl. The organism was grown and maintained on modified potato dextrose agar (PDA) medium plates containing (per litre) 200 g potato (boiled and filtered), 2.5 g glucose, 18 g agar and 20 % (w/v) multi-salts. Cell morphology was examined by light microscopy (B5; Motic) and scanning electron microscopy (S-4800; Hitachi) after 15–30 days of incubation on modified PDA medium with 20 % (w/v) multi-salts. Cultural characteristics were observed on Czapek’s agar (Waksman, 1967), nutrient agar, PDA and ISP media 2–5 (Shirling & Gottlieb, 1966) supplemented with 20 % (w/v) multi-salts at 28 uC. The colours of substrate and aerial mycelia and soluble pigments were determined with colour chips from the ISCC-NBS colour charts (Kelly, 1964). Growth was tested over a range of temperatures (10–55 uC) as described by Xu et al. (2005) in peptone water medium supplemented with 15 % (w/v)
Strain AFM 10251T was Gram-stain-positive and grew aerobically. Aerial mycelium was white, well developed, branched and produced long chains of arthrospores with more than 10 spores per chain (Fig. 1). The spore chains were straight or flexuous. Sterile mycelium was present between individual spores. The spores were spherical or oval (1.2 mm in diameter) with warty surfaces. The spores were non-motile. Sporangia were not observed. Fragments of substrate mycelia were observed. Cultural characteristics and physiological and biochemical features are detailed in Table 1 and in the description of the species below. Growth was good on PDA, nutrient agar, ISP 4 and ISP 5, moderate on ISP 3, and weak on Czapek’s agar and ISP 2. The aerial mycelium was white on all the test media except nutrient agar and ISP 2. The substrate mycelium was light yellow on PDA, nutrient agar and ISP 2, and pale yellow on ISP 4. No soluble pigment was produced on any of the test media. Temperature and pH ranges for growth of strain AFM 10251T were 20–50 uC and pH 5.0–10.0, with optimal growth at 45 uC and pH 7.0. The multi-salts, NaCl and MgCl2 concentration ranges for growth were 10–35, 10–35 and 5–30 %, respectively, with optimal growth occurring at 20, 20–25 and 15 %, respectively. Other physiological and biochemical characteristics of strain AFM 10251T are given in the species description. Biomass of strain AFM 10251T was cultured on modified PDA medium at 28 uC for 14 days. Menaquinones were
Table 1. Cultural characteristics of strain AFM 10251T Medium
Growth
Colour of: Aerial mycelium
PDA Czapek’s agar Nutrient agar Yeast/malt extract agar (ISP 2) Oatmeal agar (ISP 3) Inorganic salts/starch agar (ISP 4) Glycerol/asparagine agar (ISP 5)
2776
Good Poor Good Poor Moderate Good Good
White White Yellowish white Colourless White White white
Substrate mycelium
Soluble pigment
Light yellow Colourless Light yellow Light yellow Colourless Pale yellow Colourless
None None None None None None None
International Journal of Systematic and Evolutionary Microbiology 64
Halopolyspora alba gen. nov., sp. nov.
isolated according to Collins (1994) and were analysed by HPLC (Kroppenstedt, 1982). Polar lipids were analysed according to the methods of Minnikin et al. (1984). Wholecell sugars and cell-wall amino acids were detected by using precolumn derivatization with 1-phenyl-3-methyl-5-pyrazolone by HPLC (Agilent 1100) (Tang et al., 2009). Biomass for fatty acid analysis was obtained after growth on tripticase soy agar at 28 uC for 10 days. Extraction and analysis of fatty acids were performed as described by Sasser (1990) by using the Microbial Identification System (MIDI) (Sherlock version 6.1; MIDI database TSBA6). Cell-wall diamino acids of strain AFM 10251T were meso-diaminopimelic acid and a small proportion of LLdiaminopimelic acid. Whole-cell sugars were galactose and arabinose. The major menaquinone system was MK-9(H4) (93 %). The polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and three unknown phospholipids (PL1–3) (Fig. S1, available in the online Supplementary Material). Major fatty acids were iso-C16 : 0 (20.47 %), iso-C17 : 0 (20.49 %), iso-C15 : 0 (16.92 %), anteiso-C17 : 0 (14.50 %), summed feature 9 (iso-C17 : 1v9c and/or 10-methyl C16 : 0, 7.38 %) and C17 : 1v8c (6.34 %). For 16S rRNA gene sequence analysis, isolation of chromosomal DNA, PCR amplification and direct sequencing of the purified products were carried out as described by Li et al. (2007). The 16S rRNA gene sequence of strain AFM 10251T was aligned manually with reference strains retrieved from DDBJ/EMBL/GenBank via the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al., 2012). Multiple alignments with sequences of its most closely related species were carried out by using the program CLUSTAL X (Thompson et al., 1997). Phylogenetic trees were reconstructed using MEGA version 5.0 (Tamura et al., 2011) with the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) algorithms. The topologies of the phylogenetic trees were evaluated by using the bootstrap resampling method of Felsenstein (1985) with 1000 replicates. The G+C content of the DNA was determined according to the method of Marmur (1961) and was determined by reversed-phase HPLC of nucleosides according to Mesbah et al. (1989). Fig. 2 shows the phylogenetic tree of strain AFM 10251T and members of the genus Actinopolyspora, together with other representatives of the family Pseudonocardiaceae (Zhi et al., 2009; Goodfellow &Trujillo, 2012) based on 16S rRNA gene sequences. Strain AFM 10251T formed a robust clade with members of the genus Actinopolyspora with bootstrap support of 95 %, while levels of 16S rRNA gene sequence similarity between strain AFM 10251T and the type strains of species of the genus Actinopolyspora were below 92 %. The G+C content of the DNA of strain AFM 10251T was 66.7 mol%. Actinopolyspora iraqiensis was described by Ruan et al. (1994), but Tang et al. (2011) reported that this species was http://ijs.sgmjournals.org
a heterotypic synonym of Saccharomonospora halophila (Al-Zarban et al., 2002). The result of phylogenetic analysis in the present study showed that Actinopolyspora iraqiensis A.S. 4.1193T was placed in the cluster of the genus Saccharomonospora but is not a heterotypic synonym of Saccharomonospora halophila. To examine the secondary structures of nine variable areas (V1–V9) of the 16S rRNA gene we used the procedures described by Bouthinon & Soldano (1999) and Akutsu (2000). The 16S rRNA gene sequences of strain AFM 10251T and the type strains of five recognized species of the genus Actinopolyspora and related genera of the family Pseudonocardiaceae were cut by using the program CLUSTAL X and the secondary structures were evaluated and viewed via the programs RNA structure 3.7 (De Rijk & De Wachter, 1997) and RnaViz 2.0 (De Rijk et al., 2003). The results are shown in Figs S2 and S3. Actinokineospora diospyrosa and Amycolatopsis japonica, and Saccharopolyspora halophila and Saccharomonospora viridis were similar in patterns of secondary structure and in cycle and stem numbers, respectively (Fig. S2). Strain AFM 10251T had two cycles and two stems, while Actinopolyspora halophila had four cycles and four stems. The latter two also differed in base size and sequence. The secondary structure of variable region V3 of strain AFM 10251T was different from all species of the genus Actinopolyspora and genus of the family Pseudonocardiaceae. The secondary structure of region V4 showed that the left stem of six species was the same in base composition and size, except 1 bp of Saccharomonospora viridis; patterns of strain AFM 10251T, Actinopolyspora halophila, Actinokineospora diospyrosa and Amycolatopsis japonica were the same, and had three cycles and three stems, but differed in base composition and size; Saccharopolyspora halophila had four cycles and four stems, while Saccharomonospora viridis had five cycles and five stems. The signature nucleotides of the 16S rRNA gene sequence of strain AFM 10251T and five related genera were analysed using the methods described by Stackebrandt et al. (1997) (Table 2). The results showed that the species of four genera of the family Pseudonocardiaceae, Actinokineospora diospyrosa, Amycolatopsis japonica, Saccharopolyspora halophila and Saccharomonospora viridis, formed one group, but with base pair differences at positions 183 : 194 and 1001 : 1039; strain AFM 10251T and Actinopolyspora halophila formed another group, but with base pair differences at positions 127 : 234 and 183 : 194. Based on analysis of chemical and molecular characteristics, strain AFM 10251T is shown to be a member of the family Actinopolysporaceae. However, it differs from the genus Actinopolyspora. Therefore, strain AFM 10251T is considered to represent a novel species of a new genus in the family Actinopolysporaceae, for which the name Halopolyspora alba gen. nov., sp. nov. is proposed. 2777
H. Lai and others
Actinopolyspora alba YIM 90480T (GQ480940) Actinopolyspora lacussalsi TRM 40139T (JX485633) 0.02 Actinopolyspora erythraea YIM 90600T (GQ480939) 100 Actinopolyspora xinjiangensis TRM 40136T (GU479394) 94 99 Actinopolyspora algeriensis H19T (HQ918195) 95 Actinopolyspora halophila ATCC 27976T (X54287) 99 Actinopolyspora mortivallis DSM 44261T (DQ883812) Halopolyspora alba gen. nov., sp. nov. AFM10251T (KF022042) Actinokineospora diospyrosa NRRL B-24047T (AF114797) 70 Saccharothrix syringae NRRL B-16468T (AF114812) 87 100 Saccharothrix coeruleofusca NRRL B-16115T (AF114805) 92 Actinokineospora enzanensis IFO 16517T (AB058395) Actinoalloteichus nanshanensis NEAU 119T (GQ926935) 58 Saccharomonospora cyanea DSM 44106T (Z38018) 86 Saccharomonospora glauca SB-37 (Z38006) 51 Saccharomonospora azurea SB-01 (Z38022) 48 Saccharomonospora viridis DSM 43017T (CP001683) Actinopolyspora iraqiensis AS 4.1193T (EF372522) 92 100 100 Saccharomonospora halophila DSM 44411T (AJ278497) 66 Saccharomonospora saliphila YIM 90502T (DQ367416) Amycolatopsis minnesotensis 32U-2T (DQ076482) 99 Amycolatopsis japonica DSM 44213T (AJ508236) 99 Amycolatopsis halophila YIM 93223T (FJ606836) 58 Saccharopolyspora rectivirgula DSM 43747T (JN010255) 98 Saccharopolyspora hordei DSM 44065T (FN179275) 96 Saccharopolyspora hirsuta ATCC 27875T (U93341) 91 Saccharopolyspora spinosa DSM 44228T (AF002818) 43 Saccharopolyspora rosea IMMIB L-1070T (AM992060) 78 Saccharopolyspora halophila YIM 90500T (DQ923129) Saccharopolyspora gloriosae YIM 60513T (EU005371) 59 32 Saccharopolyspora cebuensis SPE 10-1T (EF030715) 57 Saccharopolyspora erythraea NRRL 2338T (AM420293) 100 Saccharopolyspora taberi DSM 43856T (AF002819) Glycomyces harbinensis IFO 14487T (D85483) 51 100
Fig. 2. Neighbour-joining tree based on 16S rRNA gene sequences showing the phylogenetic relationship between strain AFM 10251T and closely related species of the family Actinopolysporaceae. Bootstrap values, expressed as percentages of 1000 replications, are given at branch points. Accession numbers are given in parentheses. Bar, 1 substitution per 50 nt.
Description of Halopolyspora gen. nov.
Description of Halopolyspora alba sp. nov.
Halopolyspora (Ha.lo.po.ly.spo9ra. Gr. n. hals salt; Gr. adj. polus many; Gr. fem. n. spora seed and in biology a spore; N.L. fem. n. Halopolyspora saline many-spored bacteria).
Halopolyspora alba (al9ba. L. fem. adj. alba white).
Aerobic, and Gram-stain-positive. Aerial mycelium is well developed, branched and forms long chains of arthrospores. Spores are non-motile, and sclerotia or sporangia are not observed. Fragments of substrate mycelia are observed. Optimal growth occurs with 20 % (w/v) multi-salts. Cell wall contains meso-diaminopimelic acid and a small proportion of LL-diaminopimelic acid. Whole-cell hydrolysates contain arabinose and galactose. Cells contain diphosphatidylglycerol and phosphatidylcholine as diagnostic phospholipids. Main branched-chain fatty acids are iso-C16 : 0, iso-C17 : 0, isoC15 : 0 and anteiso-C17 : 0. The predominant menaquinone is MK-9(H4). The type species is Halopolyspora alba sp. nov.
Has the following characteristics in addition to those given for the genus. Arthrospores have more than 10 spores per chain. Spore chains are straight or flexuous. Sterile mycelium is present between spores. Spores are spherical or oval (1.2 mm in diameter) with warty surfaces. Forms a light yellow to pale yellow substrate mycelium and no soluble pigment is produced. Optimal growth occurs at 45 uC and pH 7. Utilizes D-sorbitol, lactose, inositol, trehalose, dextrin, sucrose, D-ribose, D-fructose, D-xylose, L-rhamnose, salicin, cellobiose, L-sorbose, raffinose, D-mannitol, D-mannose, D-galactitol, D-glucose, starch, L-proline, L-aspartic acid, glycine, L-arginine, L-cysteine, L-tyrosine, L-methionine, Lornithine, L-phenylalanine, L-serine, L-alanine, L-histidine, ammonium citrate, diammonium phosphate, potassium nitrate, ammonium acetate, ammonium nitrate and sodium acetate as sole carbon or nitrogen sources, but not Darabinose, maltose, D-galactose, L-glutamic acid, ammonium
2778
International Journal of Systematic and Evolutionary Microbiology 64
Halopolyspora alba gen. nov., sp. nov.
Table 2. Signature nucleotides of the 16S rRNA gene of strain AFM 10251T and the type strains of its closest genera in the families Actinopolysporaceae and Pseudonocardiaceae Strains: 1, AFM 251T; 2, Actinopolyspora halophila ATCC 27976T; 3, Actinokineospora diospyrosa NRRL B-24047T; 4, Amycolatopsis japonica DSM 44213T; 5, Saccharopolyspora halophila YIM 90500T; 6, Saccharomonospora viridis DSM 43017T. Escherichia coli positions 127 : 234 183 : 194 502 : 543 603 : 635 610 747 952 : 1229 986 : 1219 987 : 1218 1001 : 1039 1308 : 1329
1
2
3
4
5
6
G:C U:G G:C C:G A U U:A U:A G:C C: G C:G
A:U C:G G:C C:G A U U:A U:A G:C C:G C:G
G:C U:G A:U C:G A A U:A U:A G:C U: G C:G
G:C U:U A:U C:G A A U:A U:A G:C C:G C:G
G:C G:C A:U C:G A A U:A U:A G:C G:G C:G
G:C U:2 A:U C:G A A U:A U:A G:C C:G C:G
dihydrogen citrate or ammonium molybdate. Positive for catalase, nitrate reduction, indole production, and hydrolysis of Tween 20, Tween 80, starch and urea. Negative for milk peptonization and coagulation, methyl red and Voges– Proskauer tests, H2S production, and hydrolysis of casein, gelatin, cellulose and hippurate. The polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and three unknown phospholipids. The type strain, AFM 10251T (5DSM 45976T5CGMCC 4.7114T), was isolated from a sediment sample collected from the Dead Sea, Israel. The DNA G+C content of the type strain is 66.7 mol% (HPLC).
Fitch, W. M. (1971). Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20, 406–416. Gochnauer, M. B., Leppard, G. G., Komaratat, P., Kates, M., Novitsky, T. & Kushner, D. J. (1975). Isolation and characterization of
Actinopolyspora halophila, gen. et sp. nov., an extremely halophilic actinomycete. Can J Microbiol 21, 1500–1511. Goodfellow, M. & Trujillo, M. E. (2012) Order II. Actinopolysporales ord. nov. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, Vol. 5, pp. 162–163. Edited by M. Goodfellow, P. Ka¨mpfer, H. J. Busse, M. E. Trujillo, K.-i. Suzuki, W. Ludwig & W. B. Whitman. New York: Springer. Gordon, R. E., Barnett, D. A., Handerhan, J. E. & Pang, C. H.-N. (1974). Nocardia coeliaca, Nocardia autotrophica, and the nocardin
strain. Int J Syst Bacteriol 24, 54–63. Guan, T. W., Liu, Y., Zhao, K., Xia, Z. F., Zhang, X. P. & Zhang, L. L. (2010). Actinopolyspora xinjiangensis sp. nov., a novel exteremely
References
halophilic actinomycete isolated from a salt lake in Xinjiang, China. Antonie van Leeuwenhoek 98, 447–453.
Akutsu, T. (2000). Dynamic programming algorithms for RNA
Guan, T. W., Wei, B., Zhang, Y., Xia, Z. F., Che, Z. M., Chen, X. G. & Zhang, L. L. (2013). Actinopolyspora lacussalsi sp. nov., an extremely
secondary structure prediction with pseudoknots. Discrete Appl Math 104, 45–62. Al-Zarban, S. S., Al-Musallam, A. A., Abbas, I., Stackebrandt, E. & Kroppenstedt, R. M. (2002). Saccharomonospora halophila sp. nov., a
halophilic actinomycete isolated from a salt lake. Int J Syst Evol Microbiol 63, 3009–3013. Kelly, K. L. (1964). Inter-Society Color Council – National Bureau of
novel halophilic actinomycete isolated from marsh soil in Kuwait. Int J Syst Evol Microbiol 52, 555–558.
Standards Color Name Charts Illustrated with Centroid Colors. Washington, DC: US Government Printing Office.
Bouthinon, D. & Soldano, H. (1999). A new method to predict the consensus secondary structure of a set of unaligned RNA sequences. Bioinformatics 15, 785–798.
Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e: a
Collins, M. D. (1994). Isoprenoid quinones. In Chemical Methods in
Prokaryotic Systematics, pp. 265–309. Edited by M. Goodfellow & A. G. O’Donnell. Chichester: Wiley. De Rijk, P. & De Wachter, R. (1997). RnaViz, a program for the
visualisation of RNA secondary structure. Nucleic Acids Res 25, 4679– 4684. De Rijk, P., Wuyts, J. & De Wachter, R. (2003). RnaViz 2: an improved
representation of RNA secondary structure. Bioinformatics 19, 299–300. Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a
maximum likelihood approach. J Mol Evol 17, 368–376. Felsenstein, J. (1985). Confidence limits on phylogenies: an approach
using the bootstrap. Evolution 39, 783–791. http://ijs.sgmjournals.org
prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62, 716–721. Kroppenstedt, R. M. (1982). Separation of bacterial menaquinones by
HPLC using reverse phase (RP18) and a silver loaded ion exchanger as stationary phases. J Liq Chromatogr 5, 2359–2367. Li, W. J., Xu, P., Schumann, P., Zhang, Y. Q., Pukall, R., Xu, L. H., Stackebrandt, E. & Jiang, C. L. (2007). Georgenia ruanii sp. nov., a
novel actinobacterium isolated from forest soil in Yunnan (China), and emended description of the genus Georgenia. Int J Syst Evol Microbiol 57, 1424–1428. Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218. Meklat, A., Bouras, N., Zitouni, A., Mathieu, F., Lebrihi, A., Schumann, P., Spro¨er, C., Klenk, H. P. & Sabaou, N. (2012).
2779
H. Lai and others Actinopolyspora algeriensis sp. nov., a novel halophilic actinomycete isolated from a Saharan soil. Extremophiles 16, 771–776.
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise
Tang, S. K., Wang, Y., Chen, Y., Lou, K., Cao, L. L., Xu, L. H. & Li, W. J. (2009). Zhihengliuella alba sp. nov., and emended description of the
measurement of the G+C content of deoxyribonucleic acid by highperformance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
genus Zhihengliuella. Int J Syst Evol Microbiol 59, 2025–2032.
Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated
Tang, S. K., Wang, Y., Klenk, H. P., Shi, R., Lou, K., Zhang, Y. J., Chen, C., Ruan, J. S. & Li, W. J. (2011). Actinopolyspora alba sp. nov.
procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.
and Actinopolyspora erythraea sp. nov., isolated from a salt field, and reclassification of Actinopolyspora iraqiensis Ruan et al. 1994 as a heterotypic synonym of Saccharomonospora halophila. Int J Syst Evol Microbiol 61, 1693–1698.
Murray, R. G. E., Doetsch, R. N. & Robinow, C. F. (1994). Determinative
and cytological light microscopy. In Methods for General and Molecular Bacteriology, pp. 22–41. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology. Ruan, J. S., Al-Tai, A. M., Zhou, Z. H. & Qu, L. H. (1994). Actinopolyspora
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible
strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
iraqiensis sp. nov., a new halophilic actinomycete isolated from soil. Int J Syst Bacteriol 44, 759–763.
Waksman, S. A. (1967). The Actinomycetes. A Summary of Current
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new
Xu, P., Li, W. J., Tang, S. K., Zhang, Y. Q., Chen, G. Z., Chen, H. H., Xu, L. H. & Jiang, C. L. (2005). Naxibacter alkalitolerans gen. nov., sp. nov.,
method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425. Sasser, M. (1990). Identification of bacteria by gas chromatography
Knowledge. New York: Ronald Press.
of cellular fatty acids. USFCC Newsl 20, 16.
a novel member of the family ‘Oxalobacteraceae’ isolated from China. Int J Syst Evol Microbiol 55, 1149–1153.
Shirling, E. B. & Gottlieb, D. (1966). Methods for characterization of
Xu, L. H., Li, W. J., Liu, Z. H. & Jiang, C. L. (2007). Actinomecete
Streptomyces species. Int J Syst Bacteriol 16, 313–340.
Systematics—Principle, Methods and Practice. Beijing: Academic Press.
Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization.
In Methods for General and Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.
Yoshida, M., Matsubara, J. S., Kudo, T. & Horikoshi, K. (1991).
Stackebrandt, E., Rainey, F. A. & Ward-Rainey, N. L. (1997). Proposal
Zhi, X. Y., Li, W. J. & Stackebrandt, E. (2009). An update of the
for a new hierarchic classification system, Actinobacteria classis nov. Int J Syst Bacteriol 47, 479–491.
Actinopolyspora mortivallis sp. nov., a moderately halophilic actinomycete. Int J Syst Bacteriol 41, 15–20.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using
structure and 16S rRNA gene sequence-based definition of higher ranks of the class Actinobacteria, with the proposal of two new suborders and four new families and emended descriptions of the existing higher taxa. Int J Syst Evol Microbiol 59, 589–608.
2780
International Journal of Systematic and Evolutionary Microbiology 64
DOI 10.1099/ijs.0.057638-0
Halopolyspora alba gen. nov., sp. nov., isolated from sediment Hangxian Lai,13 Xiaomin Wei,13 Yingying Jiang,1 Xiu Chen,2 Qinyuan Li,2 Yi Jiang,2 Chenglin Jiang2 and Leionid Gillerman3 Correspondence Hangxian Lai [email protected] Yi Jiang [email protected]
1
College of Natural Resources and Environment, Northwest A&F University, Yangling, Shaanxi, 712100, PR China
2
Yunnan Institute of Microbiology and Life Sciences Lab Center, Yunnan University, Kunming, 650091, PR China
3
J. Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede Boker, 84990, Israel
A novel halophilic, filamentous actinomycete, designated strain AFM 10251T, was isolated from a sediment sample collected from the Dead Sea, Israel. The isolate grew with 10–35 % multi-salts, and did not grow without NaCl or MgCl2. The isolate formed a white aerial mycelium, and long chains of arthrospores with more than 10 spores per chain. The spores were spherical or oval with warty surfaces, and sterile mycelium was present between individual spores. The isolate contained meso-diaminopimelic acid and a small proportion of LL-diaminopimelic acid as cell-wall diamino acids, and galactose and arabinose as whole-cell sugars. The major menaquinone was MK-9(H4). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and three unknown phospholipids. Major fatty acids were iso-C16 : 0, iso-C17 : 0, iso-C15 : 0 and anteiso-C17 : 0. The DNA G+C content of strain AFM 10251T was 66.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain AFM 10251T and the genus Actinopolyspora formed a distinct lineage. Analysis of the secondary structures of variable areas of the 16S rRNA gene showed that strain AFM 10251T was different from all recognized species of the genus Actinopolyspora and members of the family Pseudonocardiaceae. Analysis of the signature nucleotides of the 16S rRNA gene showed that strain AFM 10251T and Actinopolyspora halophila formed a single group, but with base pair differences at positions 127 : 234 and 183 : 194. On the basis of analysis of chemical and molecular characteristics, strain AFM 10251T is considered to represent a novel species of a new genus in the family Actinopolysporaceae, for which the name Halopolyspora alba gen. nov., sp. nov. is proposed. The type strain of Halopolyspora alba is AFM 10251T (5DSM 45976T5CGMCC 4.7114T).
The genus Actinopolyspora, of the family Nocardiaceae, was proposed by Gochnauer et al. (1975), with the description of Actinopolyspora halophila as a halophilic actinomycete. The genus was subsequently placed in the family Pseudonocardiaceae of the suborder Pseudonocardineae (Stackebrandt et al., 1997; Xu et al., 2007). A new suborder Actinopolysporineae and the family Actinopolysporaceae were proposed by Zhi et al. (2009). Goodfellow & Trujillo (2012) then proposed the order ‘Actinopolysporales’ including only one 3These authors contributed equally to this work. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain AFM 10251T is KF022042. Three supplementary figures are available with the online version of this paper.
057638 G 2014 IUMS
Printed in Great Britain
family and one genus, Actinopolyspora. At the time of writing, the genus Actinopolyspora comprises eight recognized species: Actinopolyspora alba (Tang et al., 2011), Actinopolyspora algeriensis (Meklat et al., 2012), Actinopolyspora erythraea (Tang et al., 2011), Actinopolyspora halophila (Gochnauer et al., 1975), Actinopolyspora iraqiensis (Ruan et al., 1994), Actinopolyspora lacussalsi (Guan et al., 2013), Actinopolyspora mortivallis (Yoshida et al., 1991) and Actinopolyspora xinjiangensis (Guan et al., 2010). During the course of studies on halophilic actinomycete communities in saline lakes, several novel strains were obtained. A new genus belonging to the family Actinopolysporaceae (Zhi et al., 2009; Goodfellow & Trujillo, 2012) is proposed to accommodate one of these halophilic actinomycetes based on the results of the present polyphasic taxonomic study. 2775
H. Lai and others
multi-salts. The pH (3.0–12.0) for growth was determined as described by Xu et al. (2005) in peptone water medium supplemented with 10 % (w/v) multi-salts. Tolerance to multi-salts, NaCl and MgCl2 at different concentrations (0–35 %, w/v) was tested on CMKA medium. Gram staining was carried out by using the standard Gram reaction (Murray et al., 1994). Utilization of carbon and nitrogen sources was examined by using the medium of Smibert & Krieg (1994) supplemented with various substrates and 15 % multi-salts. Catalase activity was determined by assessing bubble production with 3 % (v/v) H2O2, according to the methods used by Smibert & Krieg (1994). Anaerobic growth was assessed in liquid medium at 40 uC. Other physiological tests were carried out as described by Gordon et al. (1974). Fig. 1. Scanning electron micrograph of spore chains of strain AFM 10251T grown on PDA medium for 30 days at 28 6C. Bar, 3 mm.
Strain AFM 10251T was isolated from a sediment sample collected from the Dead Sea, Israel, by using CMKA medium. This CMKA medium contained (per litre) 0.5 g Casein acids hydrolysate, 1.5 g mannitol, 1 g KNO3, 2 g (NH4)2SO4, 0.5 g K2HPO4, 0.5 g CaCO3, 20 g agar and 20 % (w/v) multi-salts. The multi-salts comprised 49 % (w/ w) MgCl2, 32 % (w/w) NaCl, 14 % (w/w) CaCl2 and 5 % (w/w) KCl. The organism was grown and maintained on modified potato dextrose agar (PDA) medium plates containing (per litre) 200 g potato (boiled and filtered), 2.5 g glucose, 18 g agar and 20 % (w/v) multi-salts. Cell morphology was examined by light microscopy (B5; Motic) and scanning electron microscopy (S-4800; Hitachi) after 15–30 days of incubation on modified PDA medium with 20 % (w/v) multi-salts. Cultural characteristics were observed on Czapek’s agar (Waksman, 1967), nutrient agar, PDA and ISP media 2–5 (Shirling & Gottlieb, 1966) supplemented with 20 % (w/v) multi-salts at 28 uC. The colours of substrate and aerial mycelia and soluble pigments were determined with colour chips from the ISCC-NBS colour charts (Kelly, 1964). Growth was tested over a range of temperatures (10–55 uC) as described by Xu et al. (2005) in peptone water medium supplemented with 15 % (w/v)
Strain AFM 10251T was Gram-stain-positive and grew aerobically. Aerial mycelium was white, well developed, branched and produced long chains of arthrospores with more than 10 spores per chain (Fig. 1). The spore chains were straight or flexuous. Sterile mycelium was present between individual spores. The spores were spherical or oval (1.2 mm in diameter) with warty surfaces. The spores were non-motile. Sporangia were not observed. Fragments of substrate mycelia were observed. Cultural characteristics and physiological and biochemical features are detailed in Table 1 and in the description of the species below. Growth was good on PDA, nutrient agar, ISP 4 and ISP 5, moderate on ISP 3, and weak on Czapek’s agar and ISP 2. The aerial mycelium was white on all the test media except nutrient agar and ISP 2. The substrate mycelium was light yellow on PDA, nutrient agar and ISP 2, and pale yellow on ISP 4. No soluble pigment was produced on any of the test media. Temperature and pH ranges for growth of strain AFM 10251T were 20–50 uC and pH 5.0–10.0, with optimal growth at 45 uC and pH 7.0. The multi-salts, NaCl and MgCl2 concentration ranges for growth were 10–35, 10–35 and 5–30 %, respectively, with optimal growth occurring at 20, 20–25 and 15 %, respectively. Other physiological and biochemical characteristics of strain AFM 10251T are given in the species description. Biomass of strain AFM 10251T was cultured on modified PDA medium at 28 uC for 14 days. Menaquinones were
Table 1. Cultural characteristics of strain AFM 10251T Medium
Growth
Colour of: Aerial mycelium
PDA Czapek’s agar Nutrient agar Yeast/malt extract agar (ISP 2) Oatmeal agar (ISP 3) Inorganic salts/starch agar (ISP 4) Glycerol/asparagine agar (ISP 5)
2776
Good Poor Good Poor Moderate Good Good
White White Yellowish white Colourless White White white
Substrate mycelium
Soluble pigment
Light yellow Colourless Light yellow Light yellow Colourless Pale yellow Colourless
None None None None None None None
International Journal of Systematic and Evolutionary Microbiology 64
Halopolyspora alba gen. nov., sp. nov.
isolated according to Collins (1994) and were analysed by HPLC (Kroppenstedt, 1982). Polar lipids were analysed according to the methods of Minnikin et al. (1984). Wholecell sugars and cell-wall amino acids were detected by using precolumn derivatization with 1-phenyl-3-methyl-5-pyrazolone by HPLC (Agilent 1100) (Tang et al., 2009). Biomass for fatty acid analysis was obtained after growth on tripticase soy agar at 28 uC for 10 days. Extraction and analysis of fatty acids were performed as described by Sasser (1990) by using the Microbial Identification System (MIDI) (Sherlock version 6.1; MIDI database TSBA6). Cell-wall diamino acids of strain AFM 10251T were meso-diaminopimelic acid and a small proportion of LLdiaminopimelic acid. Whole-cell sugars were galactose and arabinose. The major menaquinone system was MK-9(H4) (93 %). The polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and three unknown phospholipids (PL1–3) (Fig. S1, available in the online Supplementary Material). Major fatty acids were iso-C16 : 0 (20.47 %), iso-C17 : 0 (20.49 %), iso-C15 : 0 (16.92 %), anteiso-C17 : 0 (14.50 %), summed feature 9 (iso-C17 : 1v9c and/or 10-methyl C16 : 0, 7.38 %) and C17 : 1v8c (6.34 %). For 16S rRNA gene sequence analysis, isolation of chromosomal DNA, PCR amplification and direct sequencing of the purified products were carried out as described by Li et al. (2007). The 16S rRNA gene sequence of strain AFM 10251T was aligned manually with reference strains retrieved from DDBJ/EMBL/GenBank via the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al., 2012). Multiple alignments with sequences of its most closely related species were carried out by using the program CLUSTAL X (Thompson et al., 1997). Phylogenetic trees were reconstructed using MEGA version 5.0 (Tamura et al., 2011) with the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) algorithms. The topologies of the phylogenetic trees were evaluated by using the bootstrap resampling method of Felsenstein (1985) with 1000 replicates. The G+C content of the DNA was determined according to the method of Marmur (1961) and was determined by reversed-phase HPLC of nucleosides according to Mesbah et al. (1989). Fig. 2 shows the phylogenetic tree of strain AFM 10251T and members of the genus Actinopolyspora, together with other representatives of the family Pseudonocardiaceae (Zhi et al., 2009; Goodfellow &Trujillo, 2012) based on 16S rRNA gene sequences. Strain AFM 10251T formed a robust clade with members of the genus Actinopolyspora with bootstrap support of 95 %, while levels of 16S rRNA gene sequence similarity between strain AFM 10251T and the type strains of species of the genus Actinopolyspora were below 92 %. The G+C content of the DNA of strain AFM 10251T was 66.7 mol%. Actinopolyspora iraqiensis was described by Ruan et al. (1994), but Tang et al. (2011) reported that this species was http://ijs.sgmjournals.org
a heterotypic synonym of Saccharomonospora halophila (Al-Zarban et al., 2002). The result of phylogenetic analysis in the present study showed that Actinopolyspora iraqiensis A.S. 4.1193T was placed in the cluster of the genus Saccharomonospora but is not a heterotypic synonym of Saccharomonospora halophila. To examine the secondary structures of nine variable areas (V1–V9) of the 16S rRNA gene we used the procedures described by Bouthinon & Soldano (1999) and Akutsu (2000). The 16S rRNA gene sequences of strain AFM 10251T and the type strains of five recognized species of the genus Actinopolyspora and related genera of the family Pseudonocardiaceae were cut by using the program CLUSTAL X and the secondary structures were evaluated and viewed via the programs RNA structure 3.7 (De Rijk & De Wachter, 1997) and RnaViz 2.0 (De Rijk et al., 2003). The results are shown in Figs S2 and S3. Actinokineospora diospyrosa and Amycolatopsis japonica, and Saccharopolyspora halophila and Saccharomonospora viridis were similar in patterns of secondary structure and in cycle and stem numbers, respectively (Fig. S2). Strain AFM 10251T had two cycles and two stems, while Actinopolyspora halophila had four cycles and four stems. The latter two also differed in base size and sequence. The secondary structure of variable region V3 of strain AFM 10251T was different from all species of the genus Actinopolyspora and genus of the family Pseudonocardiaceae. The secondary structure of region V4 showed that the left stem of six species was the same in base composition and size, except 1 bp of Saccharomonospora viridis; patterns of strain AFM 10251T, Actinopolyspora halophila, Actinokineospora diospyrosa and Amycolatopsis japonica were the same, and had three cycles and three stems, but differed in base composition and size; Saccharopolyspora halophila had four cycles and four stems, while Saccharomonospora viridis had five cycles and five stems. The signature nucleotides of the 16S rRNA gene sequence of strain AFM 10251T and five related genera were analysed using the methods described by Stackebrandt et al. (1997) (Table 2). The results showed that the species of four genera of the family Pseudonocardiaceae, Actinokineospora diospyrosa, Amycolatopsis japonica, Saccharopolyspora halophila and Saccharomonospora viridis, formed one group, but with base pair differences at positions 183 : 194 and 1001 : 1039; strain AFM 10251T and Actinopolyspora halophila formed another group, but with base pair differences at positions 127 : 234 and 183 : 194. Based on analysis of chemical and molecular characteristics, strain AFM 10251T is shown to be a member of the family Actinopolysporaceae. However, it differs from the genus Actinopolyspora. Therefore, strain AFM 10251T is considered to represent a novel species of a new genus in the family Actinopolysporaceae, for which the name Halopolyspora alba gen. nov., sp. nov. is proposed. 2777
H. Lai and others
Actinopolyspora alba YIM 90480T (GQ480940) Actinopolyspora lacussalsi TRM 40139T (JX485633) 0.02 Actinopolyspora erythraea YIM 90600T (GQ480939) 100 Actinopolyspora xinjiangensis TRM 40136T (GU479394) 94 99 Actinopolyspora algeriensis H19T (HQ918195) 95 Actinopolyspora halophila ATCC 27976T (X54287) 99 Actinopolyspora mortivallis DSM 44261T (DQ883812) Halopolyspora alba gen. nov., sp. nov. AFM10251T (KF022042) Actinokineospora diospyrosa NRRL B-24047T (AF114797) 70 Saccharothrix syringae NRRL B-16468T (AF114812) 87 100 Saccharothrix coeruleofusca NRRL B-16115T (AF114805) 92 Actinokineospora enzanensis IFO 16517T (AB058395) Actinoalloteichus nanshanensis NEAU 119T (GQ926935) 58 Saccharomonospora cyanea DSM 44106T (Z38018) 86 Saccharomonospora glauca SB-37 (Z38006) 51 Saccharomonospora azurea SB-01 (Z38022) 48 Saccharomonospora viridis DSM 43017T (CP001683) Actinopolyspora iraqiensis AS 4.1193T (EF372522) 92 100 100 Saccharomonospora halophila DSM 44411T (AJ278497) 66 Saccharomonospora saliphila YIM 90502T (DQ367416) Amycolatopsis minnesotensis 32U-2T (DQ076482) 99 Amycolatopsis japonica DSM 44213T (AJ508236) 99 Amycolatopsis halophila YIM 93223T (FJ606836) 58 Saccharopolyspora rectivirgula DSM 43747T (JN010255) 98 Saccharopolyspora hordei DSM 44065T (FN179275) 96 Saccharopolyspora hirsuta ATCC 27875T (U93341) 91 Saccharopolyspora spinosa DSM 44228T (AF002818) 43 Saccharopolyspora rosea IMMIB L-1070T (AM992060) 78 Saccharopolyspora halophila YIM 90500T (DQ923129) Saccharopolyspora gloriosae YIM 60513T (EU005371) 59 32 Saccharopolyspora cebuensis SPE 10-1T (EF030715) 57 Saccharopolyspora erythraea NRRL 2338T (AM420293) 100 Saccharopolyspora taberi DSM 43856T (AF002819) Glycomyces harbinensis IFO 14487T (D85483) 51 100
Fig. 2. Neighbour-joining tree based on 16S rRNA gene sequences showing the phylogenetic relationship between strain AFM 10251T and closely related species of the family Actinopolysporaceae. Bootstrap values, expressed as percentages of 1000 replications, are given at branch points. Accession numbers are given in parentheses. Bar, 1 substitution per 50 nt.
Description of Halopolyspora gen. nov.
Description of Halopolyspora alba sp. nov.
Halopolyspora (Ha.lo.po.ly.spo9ra. Gr. n. hals salt; Gr. adj. polus many; Gr. fem. n. spora seed and in biology a spore; N.L. fem. n. Halopolyspora saline many-spored bacteria).
Halopolyspora alba (al9ba. L. fem. adj. alba white).
Aerobic, and Gram-stain-positive. Aerial mycelium is well developed, branched and forms long chains of arthrospores. Spores are non-motile, and sclerotia or sporangia are not observed. Fragments of substrate mycelia are observed. Optimal growth occurs with 20 % (w/v) multi-salts. Cell wall contains meso-diaminopimelic acid and a small proportion of LL-diaminopimelic acid. Whole-cell hydrolysates contain arabinose and galactose. Cells contain diphosphatidylglycerol and phosphatidylcholine as diagnostic phospholipids. Main branched-chain fatty acids are iso-C16 : 0, iso-C17 : 0, isoC15 : 0 and anteiso-C17 : 0. The predominant menaquinone is MK-9(H4). The type species is Halopolyspora alba sp. nov.
Has the following characteristics in addition to those given for the genus. Arthrospores have more than 10 spores per chain. Spore chains are straight or flexuous. Sterile mycelium is present between spores. Spores are spherical or oval (1.2 mm in diameter) with warty surfaces. Forms a light yellow to pale yellow substrate mycelium and no soluble pigment is produced. Optimal growth occurs at 45 uC and pH 7. Utilizes D-sorbitol, lactose, inositol, trehalose, dextrin, sucrose, D-ribose, D-fructose, D-xylose, L-rhamnose, salicin, cellobiose, L-sorbose, raffinose, D-mannitol, D-mannose, D-galactitol, D-glucose, starch, L-proline, L-aspartic acid, glycine, L-arginine, L-cysteine, L-tyrosine, L-methionine, Lornithine, L-phenylalanine, L-serine, L-alanine, L-histidine, ammonium citrate, diammonium phosphate, potassium nitrate, ammonium acetate, ammonium nitrate and sodium acetate as sole carbon or nitrogen sources, but not Darabinose, maltose, D-galactose, L-glutamic acid, ammonium
2778
International Journal of Systematic and Evolutionary Microbiology 64
Halopolyspora alba gen. nov., sp. nov.
Table 2. Signature nucleotides of the 16S rRNA gene of strain AFM 10251T and the type strains of its closest genera in the families Actinopolysporaceae and Pseudonocardiaceae Strains: 1, AFM 251T; 2, Actinopolyspora halophila ATCC 27976T; 3, Actinokineospora diospyrosa NRRL B-24047T; 4, Amycolatopsis japonica DSM 44213T; 5, Saccharopolyspora halophila YIM 90500T; 6, Saccharomonospora viridis DSM 43017T. Escherichia coli positions 127 : 234 183 : 194 502 : 543 603 : 635 610 747 952 : 1229 986 : 1219 987 : 1218 1001 : 1039 1308 : 1329
1
2
3
4
5
6
G:C U:G G:C C:G A U U:A U:A G:C C: G C:G
A:U C:G G:C C:G A U U:A U:A G:C C:G C:G
G:C U:G A:U C:G A A U:A U:A G:C U: G C:G
G:C U:U A:U C:G A A U:A U:A G:C C:G C:G
G:C G:C A:U C:G A A U:A U:A G:C G:G C:G
G:C U:2 A:U C:G A A U:A U:A G:C C:G C:G
dihydrogen citrate or ammonium molybdate. Positive for catalase, nitrate reduction, indole production, and hydrolysis of Tween 20, Tween 80, starch and urea. Negative for milk peptonization and coagulation, methyl red and Voges– Proskauer tests, H2S production, and hydrolysis of casein, gelatin, cellulose and hippurate. The polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and three unknown phospholipids. The type strain, AFM 10251T (5DSM 45976T5CGMCC 4.7114T), was isolated from a sediment sample collected from the Dead Sea, Israel. The DNA G+C content of the type strain is 66.7 mol% (HPLC).
Fitch, W. M. (1971). Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20, 406–416. Gochnauer, M. B., Leppard, G. G., Komaratat, P., Kates, M., Novitsky, T. & Kushner, D. J. (1975). Isolation and characterization of
Actinopolyspora halophila, gen. et sp. nov., an extremely halophilic actinomycete. Can J Microbiol 21, 1500–1511. Goodfellow, M. & Trujillo, M. E. (2012) Order II. Actinopolysporales ord. nov. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, Vol. 5, pp. 162–163. Edited by M. Goodfellow, P. Ka¨mpfer, H. J. Busse, M. E. Trujillo, K.-i. Suzuki, W. Ludwig & W. B. Whitman. New York: Springer. Gordon, R. E., Barnett, D. A., Handerhan, J. E. & Pang, C. H.-N. (1974). Nocardia coeliaca, Nocardia autotrophica, and the nocardin
strain. Int J Syst Bacteriol 24, 54–63. Guan, T. W., Liu, Y., Zhao, K., Xia, Z. F., Zhang, X. P. & Zhang, L. L. (2010). Actinopolyspora xinjiangensis sp. nov., a novel exteremely
References
halophilic actinomycete isolated from a salt lake in Xinjiang, China. Antonie van Leeuwenhoek 98, 447–453.
Akutsu, T. (2000). Dynamic programming algorithms for RNA
Guan, T. W., Wei, B., Zhang, Y., Xia, Z. F., Che, Z. M., Chen, X. G. & Zhang, L. L. (2013). Actinopolyspora lacussalsi sp. nov., an extremely
secondary structure prediction with pseudoknots. Discrete Appl Math 104, 45–62. Al-Zarban, S. S., Al-Musallam, A. A., Abbas, I., Stackebrandt, E. & Kroppenstedt, R. M. (2002). Saccharomonospora halophila sp. nov., a
halophilic actinomycete isolated from a salt lake. Int J Syst Evol Microbiol 63, 3009–3013. Kelly, K. L. (1964). Inter-Society Color Council – National Bureau of
novel halophilic actinomycete isolated from marsh soil in Kuwait. Int J Syst Evol Microbiol 52, 555–558.
Standards Color Name Charts Illustrated with Centroid Colors. Washington, DC: US Government Printing Office.
Bouthinon, D. & Soldano, H. (1999). A new method to predict the consensus secondary structure of a set of unaligned RNA sequences. Bioinformatics 15, 785–798.
Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e: a
Collins, M. D. (1994). Isoprenoid quinones. In Chemical Methods in
Prokaryotic Systematics, pp. 265–309. Edited by M. Goodfellow & A. G. O’Donnell. Chichester: Wiley. De Rijk, P. & De Wachter, R. (1997). RnaViz, a program for the
visualisation of RNA secondary structure. Nucleic Acids Res 25, 4679– 4684. De Rijk, P., Wuyts, J. & De Wachter, R. (2003). RnaViz 2: an improved
representation of RNA secondary structure. Bioinformatics 19, 299–300. Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a
maximum likelihood approach. J Mol Evol 17, 368–376. Felsenstein, J. (1985). Confidence limits on phylogenies: an approach
using the bootstrap. Evolution 39, 783–791. http://ijs.sgmjournals.org
prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62, 716–721. Kroppenstedt, R. M. (1982). Separation of bacterial menaquinones by
HPLC using reverse phase (RP18) and a silver loaded ion exchanger as stationary phases. J Liq Chromatogr 5, 2359–2367. Li, W. J., Xu, P., Schumann, P., Zhang, Y. Q., Pukall, R., Xu, L. H., Stackebrandt, E. & Jiang, C. L. (2007). Georgenia ruanii sp. nov., a
novel actinobacterium isolated from forest soil in Yunnan (China), and emended description of the genus Georgenia. Int J Syst Evol Microbiol 57, 1424–1428. Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218. Meklat, A., Bouras, N., Zitouni, A., Mathieu, F., Lebrihi, A., Schumann, P., Spro¨er, C., Klenk, H. P. & Sabaou, N. (2012).
2779
H. Lai and others Actinopolyspora algeriensis sp. nov., a novel halophilic actinomycete isolated from a Saharan soil. Extremophiles 16, 771–776.
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise
Tang, S. K., Wang, Y., Chen, Y., Lou, K., Cao, L. L., Xu, L. H. & Li, W. J. (2009). Zhihengliuella alba sp. nov., and emended description of the
measurement of the G+C content of deoxyribonucleic acid by highperformance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
genus Zhihengliuella. Int J Syst Evol Microbiol 59, 2025–2032.
Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated
Tang, S. K., Wang, Y., Klenk, H. P., Shi, R., Lou, K., Zhang, Y. J., Chen, C., Ruan, J. S. & Li, W. J. (2011). Actinopolyspora alba sp. nov.
procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.
and Actinopolyspora erythraea sp. nov., isolated from a salt field, and reclassification of Actinopolyspora iraqiensis Ruan et al. 1994 as a heterotypic synonym of Saccharomonospora halophila. Int J Syst Evol Microbiol 61, 1693–1698.
Murray, R. G. E., Doetsch, R. N. & Robinow, C. F. (1994). Determinative
and cytological light microscopy. In Methods for General and Molecular Bacteriology, pp. 22–41. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology. Ruan, J. S., Al-Tai, A. M., Zhou, Z. H. & Qu, L. H. (1994). Actinopolyspora
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible
strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
iraqiensis sp. nov., a new halophilic actinomycete isolated from soil. Int J Syst Bacteriol 44, 759–763.
Waksman, S. A. (1967). The Actinomycetes. A Summary of Current
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new
Xu, P., Li, W. J., Tang, S. K., Zhang, Y. Q., Chen, G. Z., Chen, H. H., Xu, L. H. & Jiang, C. L. (2005). Naxibacter alkalitolerans gen. nov., sp. nov.,
method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425. Sasser, M. (1990). Identification of bacteria by gas chromatography
Knowledge. New York: Ronald Press.
of cellular fatty acids. USFCC Newsl 20, 16.
a novel member of the family ‘Oxalobacteraceae’ isolated from China. Int J Syst Evol Microbiol 55, 1149–1153.
Shirling, E. B. & Gottlieb, D. (1966). Methods for characterization of
Xu, L. H., Li, W. J., Liu, Z. H. & Jiang, C. L. (2007). Actinomecete
Streptomyces species. Int J Syst Bacteriol 16, 313–340.
Systematics—Principle, Methods and Practice. Beijing: Academic Press.
Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization.
In Methods for General and Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.
Yoshida, M., Matsubara, J. S., Kudo, T. & Horikoshi, K. (1991).
Stackebrandt, E., Rainey, F. A. & Ward-Rainey, N. L. (1997). Proposal
Zhi, X. Y., Li, W. J. & Stackebrandt, E. (2009). An update of the
for a new hierarchic classification system, Actinobacteria classis nov. Int J Syst Bacteriol 47, 479–491.
Actinopolyspora mortivallis sp. nov., a moderately halophilic actinomycete. Int J Syst Bacteriol 41, 15–20.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using
structure and 16S rRNA gene sequence-based definition of higher ranks of the class Actinobacteria, with the proposal of two new suborders and four new families and emended descriptions of the existing higher taxa. Int J Syst Evol Microbiol 59, 589–608.
2780
International Journal of Systematic and Evolutionary Microbiology 64
