International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1051–1056
DOI 10.1099/ijs.0.058404-0
Mariniluteicoccus flavus gen. nov., sp. nov., a new member of the family Propionibacteriaceae, isolated from a deep-sea sediment Dao-Feng Zhang,1,23 Hong-Fei Wang,13 Zi-Jun Xiong,2 Xin-Peng Tian,3 Lan Liu,2 Xiao-Mei Zhang,2 Zhao Jiang,2 Si Zhang3 and Wen-Jun Li1,2 1
Correspondence
Key Laboratory of Biogeography and Bioresource in Arid Land, CAS, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, U¨ru¨mqi 830011, PR China
Si Zhang [email protected]
2
Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education and Laboratory for Conservation and Utilization of Bio-Resources, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, PR China
Wen-Jun Li [email protected]
3
Key Laboratory of Tropical Marine Bio-resources and Ecology, CAS; RNAM Center for Marine Microbiology, CAS; Guangdong Key Laboratory of Marine Materia Medica; and South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, PR China
A Gram-staining-positive, aerobic, non-motile, irregular coccus, designated strain YIM M13146T, was isolated from a sediment sample collected from the South China Sea at a depth of 2439 m, and its taxonomic position was determined by a polyphasic approach. Optimal growth of the strain was observed at 30 6C (range 5–40 6C), pH 7.0 (pH 6.0–9.0) and 0–1 % NaCl (0–6 %, w/v) on/in tryptic soy agar/broth. Strain YIM M13146T had the major cellular fatty acid anteiso-C15 : 0, the predominant respiratory menaquinone MK-9(H4), peptidoglycan type A3c (LL-DAP–Gly) containing alanine, glycine, glutamic acid and LL-diaminopimelic acid (LL-DAP) and the polar lipids phosphatidylcholine, diphosphatidylglycerol, one unknown phospholipid and several glycolipids. The G+C content of the DNA was 67.2 mol%. Phenotypic and chemotaxonomic characteristics together with 16S rRNA gene sequence analyses showed that strain YIM M13146T was distinct from its close phylogenetic relatives in the genera Propioniferax and Granulicoccus of the family Propionibacteriaceae. Hence, a new genus and species, Mariniluteicoccus flavus gen. nov., sp. nov., is proposed. The type strain of Mariniluteicoccus flavus is YIM M13146T (5DSM 25892T5CCTCC AB 2012055T).
The ocean covers more than 70 % of the Earth’s surface, and extreme environments at depths greater than 200 m (the deep-sea) account for about 64 % of the Earth’s surface (Bull, 2011). The South China Sea is Chinese largest tropical marginal sea, with a complex and diverse submarine sediment environment, and is rich in biological resources, but the utilization of microbial resources in the deep-sea sediment environment is still in its infancy. Up to now, researchers taking part in this project have isolated, identified and published many novel actinobacterial taxa, such as Sciscionella marina (Tian et al., 2009a), Streptomyces nanshensis (Tian et al., 2009b), Marinactinospora 3These authors contributed equally to this work. Abbreviation: DAP, diaminopimelic acid. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain YIM M13146T is KF564278. Three supplementary figures are available with the online version of this paper.
058404 G 2014 IUMS
Printed in Great Britain
thermotolerans (Tian et al., 2009c), Janibacter alkaliphilus (Li et al., 2012a), Nocardiopsis coralliicola (Li et al., 2012b), Rhodococcus nanhaiensis (Li et al., 2012c), Streptomyces nanhaiensis (Tian et al., 2012a), Streptomyces oceani (Tian et al., 2012b), Streptomyces glycovorans, Streptomyces xishensis and Streptomyces abyssalis (Xu et al., 2012), Pseudonocardia antitumoralis (Tian et al., 2013), Mycobacterium sediminis (Zhang et al., 2013a) and Streptomonospora nanhaiensis (Zhang et al., 2013b). There are clearly still many unknown, uncultured micro-organisms in the South China Sea. In this study, we report an actinobacterium of a new genus of the family Propionibacteriaceae isolated from the South China Sea. The family Propionibacteriaceae was described by Delwiche (1957) and its description has been emended by Stackebrandt et al. (1997) and Zhi et al. (2009) on the basis of 16S rRNA gene sequence analyses. Currently, there are 17 known genera in the family, according to the List of Prokaryotic Names with Standing in Nomenclature (http:// www.bacterio.net/index.html). Strain YIM M13146T was 1051
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isolated from a sediment sample collected from the South China Sea (18u 1.6549 N 112u 30.2039 E) at a depth of 2439 m, by the serial dilution technique using HP agar medium [5 g fucose, 1 g proline, 1 g (NH4)2SO4, 2 g CaCl2, 1 g K2HPO4, B vitamin mixture (0.5 mg each thiamine hydrochloride, riboflavin, niacin, pyridoxine, calcium pantothenate, inositol and p-aminobenzoic acid and 0.25 mg biotin), 35 g NaCl, 12 g agar, 1000 ml distilled water; pH 7.2] at an incubation temperature of 28 uC for 1 month. The strain was cultivated and maintained on tryptic soy agar (TSA) and stored as an aqueous glycerol suspension (20 %, v/v) at 280 uC. Gram staining was carried out by using the standard Gram reaction and was confirmed by using the KOH lysis test (Cerny, 1978). Cell motility was tested by the hanging-drop technique (Skerman, 1967) using phase-contrast microscopy (Olympus). Microscopic observation of strain YIM M13146T grown on TSA for 7 days at 28 uC was made by light microscopy (BH 2; Olympus). Cells negatively stained with 2 % uranyl acetate were used for the detection of the presence of flagella using a JEM-2100 transmission electron microscope (Vreeland et al. 1980). Growth was tested at 5, 10, 15, 20, 28, 30, 35, 40, 45 and 50 uC on TSA by incubating the cultures for 14 days. The ability of the strain to grow at pH 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 [using the buffer system described by Xu et al. (2005)] and 0, 1, 2, 3, 4, 6, 7, 8, 9 and 10 % (w/v) NaCl (pH 7) was examined at 28 uC for 14 days with tryptic soy broth (TSB) as the basal medium. Anaerobic cultivation was performed on TSA using a Whitley A35 anaerobic workstation (Don Whitley Scientific). Carbon source utilization was tested using the Biolog GEN III MicroPlate according to the manufacturer’s instructions. Catalase activity was detected by the production of bubbles after the addition of a drop of 3 % (v/v) H2O2. Other physiological and biochemical tests were performed by using the API ZYM and API 20E strips (bioMe´rieux) according to the manufacturer’s instructions. Biomass for analyses of cellular fatty acids was obtained from cultures grown on TSA (Difco) for 3 days, and that for the other tests was grown for 7 days. Cellular fatty acid analysis was performed by using the Microbial Identification System (Sherlock version 6.1; MIDI database TSBA6). The amino acid composition in peptidoglycan hydrolysates was analysed by TLC (Hasegawa et al. 1983) and HPLC (Tang et al. 2009). Menaquinones were extracted by using the methods of Collins et al. (1977) and separated by HPLC (Tamaoka et al., 1983). Polar lipids were extracted and analysed by two-dimensional TLC according to Embley & Wait (1994). The genomic DNA G+C content was determined by using the HPLC method of Mesbah et al. (1989).
Kim et al., 2012). Multiple alignments with sequences of the most closely related bacteria retrieved from the GenBank/EMBL/DDBJ database were carried out using the CLUSTAL_X 1.8 program (Thompson et al., 1997). Phylogenetic trees were reconstructed by using the neighbourjoining (Saitou & Nei, 1987), maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) treemaking algorithms by using the software package MEGA version 5.05 (Tamura et al., 2011). The stability of relationships was assessed by performing bootstrap analyses with 1000 resamplings (Felsenstein, 1985). Strain YIM M13146T grew well on TSA and International Streptomyces Project (ISP) medium 2. Round, yellow colonies with wet, smooth surfaces were formed stably on TSA and ISP 2 within the first week, and the surfaces became dry and wrinkled during the second week. No diffusible pigment was observed in either medium. Investigations of 7day-old cultures of strain YIM M13146T revealed that cells of the strain were Gram-stain-positive, irregular cocci, 0.5– 1.0 mm in diameter, and non-motile, with no flagella, and occurred singly or in pairs or triads (Fig. 1). The isolate could grow at pH 6.0–9.0, 0–6 % (w/v) NaCl and 5–40 uC; optimum growth occurred at 30 uC, 0–1 % (w/v) NaCl and pH 7.0. Detailed physiological characteristics are shown in Table 1 and the species description. The profile of cellular fatty acids (.1 %) of strain YIM M13146T showed the presence of anteiso-C13 : 0 (3.26 %), iso-C14 : 0 (2.11 %), C14 : 0 (1.16 %), iso-C15 : 0 (5.08 %), anteiso-C15 : 0 (63.1 %), C14 : 0 2-OH (1.4 %), iso-C16 : 1 H (2.91 %), iso-C16 : 0 (3.72 %), C16 : 0 (2.7 %), anteisoC17 : 1v9c (2.42 %), anteiso-C17 : 0 (2.51 %), summed feature 3 (C16 : 1v7c and/or C16 : 1v6c; 2.92 %) and summed feature 9 (iso-C17 : 1v9c and/or 10-methyl C16 : 0; 1.37 %). The polar lipids of strain YIM M13146T were phosphatidylcholine, diphosphatidylglycerol, an unknown phosphoglycolipid and several glycolipids, with phosphatidylglycerol and two unknown polar lipids as minor components (Fig. S1, available in the online Supplementary Material). TLC analysis indicated that strain YIM M13146T possessed
Extraction of genomic DNA, PCR amplification and sequencing of the 16S rRNA gene were carried out as described by Li et al. (2007). The 16S rRNA gene sequence obtained in this study was compared with sequences from EzBioCloud using BLAST (http://eztaxon-e.ezbiocloud.net/;
Fig. 1. Transmission electron micrograph of cells of strain YIM M13146T grown on TSA for 7 days at 28 6C. Bar, 1 mm.
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International Journal of Systematic and Evolutionary Microbiology 64
Mariniluteicoccus flavus gen. nov., sp. nov.
Table 1. Phenotypic and biochemical characteristics of strain YIM M13146T and its closest phylogenetic neighbours Strains: 1, YIM M13146T; 2, Granulicoccus phenolivorans PG 02T (data from Maszenan et al., 2007); 3, Propioniferax innocua ATCC 49929T (Yokota et al., 1994). ND, No data available. All strains showed positive reactions for gelatin hydrolysis and negative reactions for production of H2S and indole and showed peptidoglycan type A3c (LL-DAP–Gly) and optimum growth at pH 7. Character O2 requirement Cell morphology Cell size (mm) Isolation source Growth temperature (uC) Optimum Range pH range for growth Presence of: Oxidase Catalase Urease Major menaquinone(s) Nitrate reduction Major polar lipids* Major fatty acid(s) (.10 %)D DNA G+C content (mol%)
1
2
3
Aerobe Cocci, single and in pairs or triads 0.5–1.0 Marine sediment
Facultative anaerobe Cocci, single and in pairs 0.3–1.4 Phenol-degrading granules
Facultative anaerobe Pleomorphic rods 0.5–1.2 Human epidermal surface
30 5–40 6.0–9.0
30 15–37 5.0–8.5
37 10–40
2 2 2 MK-9(H4) + DPG, PC, PGL, GL ai-C15 : 0 67.2
2 + + MK-9(H4), MK-8(H4) 2 PG, DPG, GL i-C15 : 0, i-C15 : 0 DMA 69
+ + + MK-9(H4) + PE, PG, GL ai-C15 : 0, i-C15 : 0 62
ND
*DPG, Disphosphatidylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; GL, unknown glycolipid(s); PGL, unknown phosphoglycolipid. Dai, Anteiso-branched; i, iso-branched; i-C15 : 0 DMA, 1,1-dimethoxy isopentadecane.
LL-diaminopimelic
acid (LL-DAP) in the cell wall. Further HPLC analysis of the peptidoglycan showed the presence of alanine, glycine and glutamic acid (2 : 1 : 1). These results confirmed that the peptidoglycan type of the isolate was A3c (LL-DAP–Gly). The predominant menaquinone of strain YIM M13146T was MK-9(H4) and the DNA G+C content was 67.2 mol%. An almost-complete 16S rRNA gene sequence was obtained for isolate YIM M13146T (1536 bp). The sequence showed 90.0–95.2 % similarity to members of family Propionibacteriaceae. Comparative 16S rRNA gene sequence analysis showed that the strain was located stably in the family Propionibacteriaceae together with the genera Granulicoccus (95.2 % similarity to the type strain of the type species; 63/ 1315 mismatches) (Maszenan et al., 2007) and Propioniferax (94.1 % similarity to the type strain of the type species; 86/ 1451 mismatches) (Yokota et al., 1994) in all three treemaking methods (Figs 2, S2 and S3). The results of 16S rRNA gene sequence comparisons demonstrated that strain YIM M13146T was most closely related to the genera Granulicoccus and Propioniferax of the family Propionibacteriaceae. Strain YIM M13146T, which was located phylogenetically in the family Propionibacteriaceae, exhibited obvious differences from its closely related neighbours Granulicoccus phenolivorans PG 02T and Propioniferax innocua ATCC 49929T. The predominant menaquinone [MK-9(H4)] and http://ijs.sgmjournals.org
peptidoglycan type [A3c (LL-DAP–Gly)] were consistent with the properties of Granulicoccus and Propioniferax, while strain YIM M13146T possessed phosphatidylcholine and an unknown phosphoglycolipid as major polar lipids, whereas the genera Granulicoccus and Propioniferax lack both of them. The isolate contained anteiso-C15 : 0 as the major fatty acid, while those of Granulicoccus phenolivorans PG 02T were iso-C15 : 0 and iso-C15 : 0 DMA (1,1-dimethoxy isopentadecane) and those of Propioniferax innocua ATCC 49929T were anteiso-C15 : 0 and iso-C15 : 0. Additionally, the cell morphology of strain YIM M13146T was also visibly different from that of Granulicoccus phenolivorans PG 02T and Propioniferax innocua ATCC 49929T. The distinctions between the novel isolate and its two most closely related phylogenetic neighbours are summarized in Table 1. On the basis of phenotypic, chemotaxonomic and phylogenetic analysis, strain YIM M13146T is considered to represent a novel species of a new genus in the family Propionibacteriaceae, for which the name Mariniluteicoccus flavus gen. nov., sp. nov. is proposed. Description of Mariniluteicoccus gen. nov. Mariniluteicoccus [Ma.ri9ni.lu.te9i.coc9cus. L. adj. marinus of the sea; L. adj. luteus yellow; N.L. masc. n. coccus (from Gr. masc. n. kokkos a grain, a seed) a coccus; N.L. masc. n. Mariniluteicoccus yellow coccus living in the sea]. 1053
D.-F. Zhang and others
98*
Friedmanniella okinawensis FB1T (AB445455) Friedmanniella flava W6T (HQ839787)
98*
0.01
Friedmanniella sagamiharensis FB2T (AB445456) 99* 72*
Friedmanniella lucida FA2T (AB445454) Microlunatus panaciterrae Gsoil 954T (AB271051)
50*
Auraticoccus monumenti MON 2.2T (FN552748) Naumannella halotolerans WS 4616T (FR832425) Propionicicella superfundia BL 10T (DQ176646)
81* 80* 56*
Propionicimonas paludicola DSM 15597T (FR733712) Micropruina glycogenica Lg2T (AB012607) Propioniferax innocua ATCC 49929T (AF227165)
68
Mariniluteicoccus flavus YIM M13146T (KF564278)
93* 86*
Granulicoccus phenolivorans PG 02T (AY566575) Aestuariimicrobium kwangyangense R27T (DQ830982)
100* 75
53
Luteococcus peritonei CCUG 38120T (AJ132334) Luteococcus japonicus DSM 10546T (Z78208) Tessaracoccus lubricantis KSS 17SeT (FM178840)
72*
Brooklawnia cerclae BL 34T (DQ196625)
100
Propioniciclava tarda WR06T (AB298731) Propionibacterium freudenreichii ATCC 6207T (X53217)
74* 99*
Arachnia propionica DSM 43307T (AJ003058) Nocardioides daejeonensis MJ31T (JF937066)
76
Kribbella flavida DSM 17836T (CP001736) Glycomyces harbinensis ATCC 43155T (D85483) Nocardiopsis dassonvillei DSM 43111T (CP002040)
Fig. 2. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences of strain YIM M13146T and members of the family Propionibacteriaceae. Bootstrap values (expressed as percentages of 1000 replications) ¢50 % are shown at branch points. Asterisks denote nodes that were also recovered using the maximum-likelihood and maximum-parsimony methods. Bar, 0.01 substitutions per nucleotide position.
Cells are Gram-staining-positive, aerobic, non-motile, irregular cocci. The genus is a member of the family Propionibacteriaceae. The major cellular fatty acid is anteiso-C15 : 0, the predominant menaquinone is MK-9(H4), the peptidoglycan type is A3c (LL-DAP–Gly) containing alanine, glycine, glutamic acid and LL-diaminopimelic acid in a molar ratio of 2 : 1 : 1 : 2 and the polar lipids are phosphatidylcholine, diphosphatidylglycerol, an unknown phosphoglycolipid and several unknown glycolipids. The G+C content of the DNA of the type strain of the type species is 67.2 mol%. The type species is Mariniluteicoccus flavus. 1054
Description of Mariniluteicoccus flavus sp. nov. Mariniluteicoccus flavus (fla9vus. L. masc. adj. flavus yellow, reflecting the colour of colonies of the type strain). The characteristics are the same as those described for the genus with the following additions. Cells are 0.5–1.0 mm in diameter. Optimal growth occurs at 30 uC (range 5–40 uC), pH 7.0 (range pH 6.0–9.0) and 0–1 % NaCl (range 0–6 %, w/v) on/in TSA/TSB. Production of catalase is negative. Positive reactions from the API ZYM system are observed for alkaline phosphatase, esterase (C4), esterase lipase (C8), International Journal of Systematic and Evolutionary Microbiology 64
Mariniluteicoccus flavus gen. nov., sp. nov.
leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, bgalactosidase, a-glucosidase and b-glucosidase; negative reactions for lipase (C14), trypsin, a-chymotrypsin, agalactosidase, b-glucuronidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. Positive reactions from the API 20E system for b-galactosidase, the Voges–Proskauer test, gelatin hydrolysis and nitrate reductase, while negative reactions are observed for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, H2S production, tryptophan deaminase, urease, indole production, oxidase and fermentation under anaerobic conditions. Positive reactions from the GEN III system for utilization of dextrin, maltose, cellobiose, gentiobiose, sucrose, turanose, methyl b-D-glucoside, D-salicin, Nacetyl-D-glucosamine, N-acetyl-b-D-mannosamine, N-acetyl neuraminic acid, a-D-glucose, D-mannose, D-fructose, Dgalactose, D- and L-fucose, L-rhamnose, inosine, D-arabitol, glycerol, D-glucose 6-phosphate, D-fructose 6-phosphate, Dserine, glycyl L-proline, L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, L-histidine, L-pyroglutamic acid, L-serine, D-galacturonic acid, L-galactonic acid lactone, D-gluconic acid, D-glucuronic acid, glucuronamide, quinic acid, Dsaccharic acid, D-lactic acid methyl ester, L-lactic acid, aketoglutaric acid, acetoacetic acid and acetic acid and tolerance of pH 6, 1 and 4 % NaCl, 1 % sodium lactate, troleandomycin, nalidixic acid and lithium chloride. Negative reactions from the GEN III system for utilization of trehalose, stachyose, raffinose, lactose, melibiose, Nacetyl-D-galactosamine, 3-methyl glucose, D-sorbitol, Dmannitol, myo-inositol, D-aspartic acid, gelatin, pectin, mucic acid, p-hydroxyphenylacetic acid, methyl pyruvate, citric acid, D- and L-malic acid, bromosuccinic acid, Tween 40, c-aminobutyric acid, a-hydroxybutyric acid, b-hydroxyDL-butyric acid, a-ketobutyric acid, propionic acid and formic acid and tolerance of pH 5, 8 % NaCl, fusidic acid, Dserine, rifamycin SV, minocycline, lincomycin, guanidine hydrochloride, niaproof 4, vancomycin, tetrazolium violet, tetrazolium blue, potassium tellurite, aztreonam, sodium butyrate and sodium bromate. T
T
The type strain YIM M13146 (5DSM 25892 5CCTCC AB 2012055T) was isolated from a sediment sample collected from the South China Sea (18u 1.6549 N 112u 30.2039 E) at a depth of 2439 m.
Acknowledgements The authors are grateful to Professor George M. Garrity from Michigan State University for the Latin construction of the new taxon name. This research was supported by the National Basic Research Program of China (no. 2010CB833801) and the National Natural Science Foundation of China (nos 30900002 and 312700040). W.-J. L. was also supported by ‘Hundred Talents Program’ of the Chinese Academy of Sciences.
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Zhang, D. F., Chen, X., Zhang, X. M., Zhi, X. Y., Yao, J. C., Jiang, Y., Xiong, Z. & Li, W. J. (2013a). Mycobacterium sediminis sp. nov. and
Tian, X. P., Tang, S. K., Dong, J. D., Zhang, Y. Q., Xu, L. H., Zhang, S. & Li, W. J. (2009c). Marinactinospora thermotolerans gen. nov., sp. nov.,
Mycobacterium arabiense sp. nov., two rapidly growing members of the genus Mycobacterium. Int J Syst Evol Microbiol 63, 4081–4086.
a marine actinomycete isolated from a sediment in the northern South China Sea. Int J Syst Evol Microbiol 59, 948–952.
Zhang, D. F., Pan, H. Q., He, J., Zhang, X. M., Zhang, Y. G., Klenk, H.-P., Hu, J. C. & Li, W. J. (2013b). Description of Streptomonospora
Tian, X.-P., Long, L.-J., Wang, F.-Z., Xu, Y., Li, J., Zhang, J., Zhang, C.-S., Zhang, S. & Li, W.-J. (2012a). Streptomyces nanhaiensis sp. nov.,
sediminis sp. nov. and Streptomonospora nanhaiensis sp. nov., and reclassification of Nocardiopsis arabia Hozzein & Goodfellow 2008 as Streptomonospora arabica comb. nov. and emended description of the genus Streptomonospora. Int J Syst Evol Microbiol 63, 4447–4455.
a marine streptomycete isolated from a deep sea sediment. Int J Syst Evol Microbiol 62, 864–868. Tian, X. P., Xu, Y., Zhang, J., Li, J., Chen, Z., Kim, C. J., Li, W. J., Zhang, C. S. & Zhang, S. (2012b). Streptomyces oceani sp. nov., a new obligate
Zhi, X. Y., Li, W. J. & Stackebrandt, E. (2009). An update of the
marine actinomycete isolated from a deep-sea sample of seep authigenic carbonate nodule in South China Sea. Antonie van Leeuwenhoek 102, 335–343.
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.
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DOI 10.1099/ijs.0.058404-0
Mariniluteicoccus flavus gen. nov., sp. nov., a new member of the family Propionibacteriaceae, isolated from a deep-sea sediment Dao-Feng Zhang,1,23 Hong-Fei Wang,13 Zi-Jun Xiong,2 Xin-Peng Tian,3 Lan Liu,2 Xiao-Mei Zhang,2 Zhao Jiang,2 Si Zhang3 and Wen-Jun Li1,2 1
Correspondence
Key Laboratory of Biogeography and Bioresource in Arid Land, CAS, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, U¨ru¨mqi 830011, PR China
Si Zhang [email protected]
2
Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education and Laboratory for Conservation and Utilization of Bio-Resources, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, PR China
Wen-Jun Li [email protected]
3
Key Laboratory of Tropical Marine Bio-resources and Ecology, CAS; RNAM Center for Marine Microbiology, CAS; Guangdong Key Laboratory of Marine Materia Medica; and South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, PR China
A Gram-staining-positive, aerobic, non-motile, irregular coccus, designated strain YIM M13146T, was isolated from a sediment sample collected from the South China Sea at a depth of 2439 m, and its taxonomic position was determined by a polyphasic approach. Optimal growth of the strain was observed at 30 6C (range 5–40 6C), pH 7.0 (pH 6.0–9.0) and 0–1 % NaCl (0–6 %, w/v) on/in tryptic soy agar/broth. Strain YIM M13146T had the major cellular fatty acid anteiso-C15 : 0, the predominant respiratory menaquinone MK-9(H4), peptidoglycan type A3c (LL-DAP–Gly) containing alanine, glycine, glutamic acid and LL-diaminopimelic acid (LL-DAP) and the polar lipids phosphatidylcholine, diphosphatidylglycerol, one unknown phospholipid and several glycolipids. The G+C content of the DNA was 67.2 mol%. Phenotypic and chemotaxonomic characteristics together with 16S rRNA gene sequence analyses showed that strain YIM M13146T was distinct from its close phylogenetic relatives in the genera Propioniferax and Granulicoccus of the family Propionibacteriaceae. Hence, a new genus and species, Mariniluteicoccus flavus gen. nov., sp. nov., is proposed. The type strain of Mariniluteicoccus flavus is YIM M13146T (5DSM 25892T5CCTCC AB 2012055T).
The ocean covers more than 70 % of the Earth’s surface, and extreme environments at depths greater than 200 m (the deep-sea) account for about 64 % of the Earth’s surface (Bull, 2011). The South China Sea is Chinese largest tropical marginal sea, with a complex and diverse submarine sediment environment, and is rich in biological resources, but the utilization of microbial resources in the deep-sea sediment environment is still in its infancy. Up to now, researchers taking part in this project have isolated, identified and published many novel actinobacterial taxa, such as Sciscionella marina (Tian et al., 2009a), Streptomyces nanshensis (Tian et al., 2009b), Marinactinospora 3These authors contributed equally to this work. Abbreviation: DAP, diaminopimelic acid. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain YIM M13146T is KF564278. Three supplementary figures are available with the online version of this paper.
058404 G 2014 IUMS
Printed in Great Britain
thermotolerans (Tian et al., 2009c), Janibacter alkaliphilus (Li et al., 2012a), Nocardiopsis coralliicola (Li et al., 2012b), Rhodococcus nanhaiensis (Li et al., 2012c), Streptomyces nanhaiensis (Tian et al., 2012a), Streptomyces oceani (Tian et al., 2012b), Streptomyces glycovorans, Streptomyces xishensis and Streptomyces abyssalis (Xu et al., 2012), Pseudonocardia antitumoralis (Tian et al., 2013), Mycobacterium sediminis (Zhang et al., 2013a) and Streptomonospora nanhaiensis (Zhang et al., 2013b). There are clearly still many unknown, uncultured micro-organisms in the South China Sea. In this study, we report an actinobacterium of a new genus of the family Propionibacteriaceae isolated from the South China Sea. The family Propionibacteriaceae was described by Delwiche (1957) and its description has been emended by Stackebrandt et al. (1997) and Zhi et al. (2009) on the basis of 16S rRNA gene sequence analyses. Currently, there are 17 known genera in the family, according to the List of Prokaryotic Names with Standing in Nomenclature (http:// www.bacterio.net/index.html). Strain YIM M13146T was 1051
D.-F. Zhang and others
isolated from a sediment sample collected from the South China Sea (18u 1.6549 N 112u 30.2039 E) at a depth of 2439 m, by the serial dilution technique using HP agar medium [5 g fucose, 1 g proline, 1 g (NH4)2SO4, 2 g CaCl2, 1 g K2HPO4, B vitamin mixture (0.5 mg each thiamine hydrochloride, riboflavin, niacin, pyridoxine, calcium pantothenate, inositol and p-aminobenzoic acid and 0.25 mg biotin), 35 g NaCl, 12 g agar, 1000 ml distilled water; pH 7.2] at an incubation temperature of 28 uC for 1 month. The strain was cultivated and maintained on tryptic soy agar (TSA) and stored as an aqueous glycerol suspension (20 %, v/v) at 280 uC. Gram staining was carried out by using the standard Gram reaction and was confirmed by using the KOH lysis test (Cerny, 1978). Cell motility was tested by the hanging-drop technique (Skerman, 1967) using phase-contrast microscopy (Olympus). Microscopic observation of strain YIM M13146T grown on TSA for 7 days at 28 uC was made by light microscopy (BH 2; Olympus). Cells negatively stained with 2 % uranyl acetate were used for the detection of the presence of flagella using a JEM-2100 transmission electron microscope (Vreeland et al. 1980). Growth was tested at 5, 10, 15, 20, 28, 30, 35, 40, 45 and 50 uC on TSA by incubating the cultures for 14 days. The ability of the strain to grow at pH 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 [using the buffer system described by Xu et al. (2005)] and 0, 1, 2, 3, 4, 6, 7, 8, 9 and 10 % (w/v) NaCl (pH 7) was examined at 28 uC for 14 days with tryptic soy broth (TSB) as the basal medium. Anaerobic cultivation was performed on TSA using a Whitley A35 anaerobic workstation (Don Whitley Scientific). Carbon source utilization was tested using the Biolog GEN III MicroPlate according to the manufacturer’s instructions. Catalase activity was detected by the production of bubbles after the addition of a drop of 3 % (v/v) H2O2. Other physiological and biochemical tests were performed by using the API ZYM and API 20E strips (bioMe´rieux) according to the manufacturer’s instructions. Biomass for analyses of cellular fatty acids was obtained from cultures grown on TSA (Difco) for 3 days, and that for the other tests was grown for 7 days. Cellular fatty acid analysis was performed by using the Microbial Identification System (Sherlock version 6.1; MIDI database TSBA6). The amino acid composition in peptidoglycan hydrolysates was analysed by TLC (Hasegawa et al. 1983) and HPLC (Tang et al. 2009). Menaquinones were extracted by using the methods of Collins et al. (1977) and separated by HPLC (Tamaoka et al., 1983). Polar lipids were extracted and analysed by two-dimensional TLC according to Embley & Wait (1994). The genomic DNA G+C content was determined by using the HPLC method of Mesbah et al. (1989).
Kim et al., 2012). Multiple alignments with sequences of the most closely related bacteria retrieved from the GenBank/EMBL/DDBJ database were carried out using the CLUSTAL_X 1.8 program (Thompson et al., 1997). Phylogenetic trees were reconstructed by using the neighbourjoining (Saitou & Nei, 1987), maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) treemaking algorithms by using the software package MEGA version 5.05 (Tamura et al., 2011). The stability of relationships was assessed by performing bootstrap analyses with 1000 resamplings (Felsenstein, 1985). Strain YIM M13146T grew well on TSA and International Streptomyces Project (ISP) medium 2. Round, yellow colonies with wet, smooth surfaces were formed stably on TSA and ISP 2 within the first week, and the surfaces became dry and wrinkled during the second week. No diffusible pigment was observed in either medium. Investigations of 7day-old cultures of strain YIM M13146T revealed that cells of the strain were Gram-stain-positive, irregular cocci, 0.5– 1.0 mm in diameter, and non-motile, with no flagella, and occurred singly or in pairs or triads (Fig. 1). The isolate could grow at pH 6.0–9.0, 0–6 % (w/v) NaCl and 5–40 uC; optimum growth occurred at 30 uC, 0–1 % (w/v) NaCl and pH 7.0. Detailed physiological characteristics are shown in Table 1 and the species description. The profile of cellular fatty acids (.1 %) of strain YIM M13146T showed the presence of anteiso-C13 : 0 (3.26 %), iso-C14 : 0 (2.11 %), C14 : 0 (1.16 %), iso-C15 : 0 (5.08 %), anteiso-C15 : 0 (63.1 %), C14 : 0 2-OH (1.4 %), iso-C16 : 1 H (2.91 %), iso-C16 : 0 (3.72 %), C16 : 0 (2.7 %), anteisoC17 : 1v9c (2.42 %), anteiso-C17 : 0 (2.51 %), summed feature 3 (C16 : 1v7c and/or C16 : 1v6c; 2.92 %) and summed feature 9 (iso-C17 : 1v9c and/or 10-methyl C16 : 0; 1.37 %). The polar lipids of strain YIM M13146T were phosphatidylcholine, diphosphatidylglycerol, an unknown phosphoglycolipid and several glycolipids, with phosphatidylglycerol and two unknown polar lipids as minor components (Fig. S1, available in the online Supplementary Material). TLC analysis indicated that strain YIM M13146T possessed
Extraction of genomic DNA, PCR amplification and sequencing of the 16S rRNA gene were carried out as described by Li et al. (2007). The 16S rRNA gene sequence obtained in this study was compared with sequences from EzBioCloud using BLAST (http://eztaxon-e.ezbiocloud.net/;
Fig. 1. Transmission electron micrograph of cells of strain YIM M13146T grown on TSA for 7 days at 28 6C. Bar, 1 mm.
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International Journal of Systematic and Evolutionary Microbiology 64
Mariniluteicoccus flavus gen. nov., sp. nov.
Table 1. Phenotypic and biochemical characteristics of strain YIM M13146T and its closest phylogenetic neighbours Strains: 1, YIM M13146T; 2, Granulicoccus phenolivorans PG 02T (data from Maszenan et al., 2007); 3, Propioniferax innocua ATCC 49929T (Yokota et al., 1994). ND, No data available. All strains showed positive reactions for gelatin hydrolysis and negative reactions for production of H2S and indole and showed peptidoglycan type A3c (LL-DAP–Gly) and optimum growth at pH 7. Character O2 requirement Cell morphology Cell size (mm) Isolation source Growth temperature (uC) Optimum Range pH range for growth Presence of: Oxidase Catalase Urease Major menaquinone(s) Nitrate reduction Major polar lipids* Major fatty acid(s) (.10 %)D DNA G+C content (mol%)
1
2
3
Aerobe Cocci, single and in pairs or triads 0.5–1.0 Marine sediment
Facultative anaerobe Cocci, single and in pairs 0.3–1.4 Phenol-degrading granules
Facultative anaerobe Pleomorphic rods 0.5–1.2 Human epidermal surface
30 5–40 6.0–9.0
30 15–37 5.0–8.5
37 10–40
2 2 2 MK-9(H4) + DPG, PC, PGL, GL ai-C15 : 0 67.2
2 + + MK-9(H4), MK-8(H4) 2 PG, DPG, GL i-C15 : 0, i-C15 : 0 DMA 69
+ + + MK-9(H4) + PE, PG, GL ai-C15 : 0, i-C15 : 0 62
ND
*DPG, Disphosphatidylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; GL, unknown glycolipid(s); PGL, unknown phosphoglycolipid. Dai, Anteiso-branched; i, iso-branched; i-C15 : 0 DMA, 1,1-dimethoxy isopentadecane.
LL-diaminopimelic
acid (LL-DAP) in the cell wall. Further HPLC analysis of the peptidoglycan showed the presence of alanine, glycine and glutamic acid (2 : 1 : 1). These results confirmed that the peptidoglycan type of the isolate was A3c (LL-DAP–Gly). The predominant menaquinone of strain YIM M13146T was MK-9(H4) and the DNA G+C content was 67.2 mol%. An almost-complete 16S rRNA gene sequence was obtained for isolate YIM M13146T (1536 bp). The sequence showed 90.0–95.2 % similarity to members of family Propionibacteriaceae. Comparative 16S rRNA gene sequence analysis showed that the strain was located stably in the family Propionibacteriaceae together with the genera Granulicoccus (95.2 % similarity to the type strain of the type species; 63/ 1315 mismatches) (Maszenan et al., 2007) and Propioniferax (94.1 % similarity to the type strain of the type species; 86/ 1451 mismatches) (Yokota et al., 1994) in all three treemaking methods (Figs 2, S2 and S3). The results of 16S rRNA gene sequence comparisons demonstrated that strain YIM M13146T was most closely related to the genera Granulicoccus and Propioniferax of the family Propionibacteriaceae. Strain YIM M13146T, which was located phylogenetically in the family Propionibacteriaceae, exhibited obvious differences from its closely related neighbours Granulicoccus phenolivorans PG 02T and Propioniferax innocua ATCC 49929T. The predominant menaquinone [MK-9(H4)] and http://ijs.sgmjournals.org
peptidoglycan type [A3c (LL-DAP–Gly)] were consistent with the properties of Granulicoccus and Propioniferax, while strain YIM M13146T possessed phosphatidylcholine and an unknown phosphoglycolipid as major polar lipids, whereas the genera Granulicoccus and Propioniferax lack both of them. The isolate contained anteiso-C15 : 0 as the major fatty acid, while those of Granulicoccus phenolivorans PG 02T were iso-C15 : 0 and iso-C15 : 0 DMA (1,1-dimethoxy isopentadecane) and those of Propioniferax innocua ATCC 49929T were anteiso-C15 : 0 and iso-C15 : 0. Additionally, the cell morphology of strain YIM M13146T was also visibly different from that of Granulicoccus phenolivorans PG 02T and Propioniferax innocua ATCC 49929T. The distinctions between the novel isolate and its two most closely related phylogenetic neighbours are summarized in Table 1. On the basis of phenotypic, chemotaxonomic and phylogenetic analysis, strain YIM M13146T is considered to represent a novel species of a new genus in the family Propionibacteriaceae, for which the name Mariniluteicoccus flavus gen. nov., sp. nov. is proposed. Description of Mariniluteicoccus gen. nov. Mariniluteicoccus [Ma.ri9ni.lu.te9i.coc9cus. L. adj. marinus of the sea; L. adj. luteus yellow; N.L. masc. n. coccus (from Gr. masc. n. kokkos a grain, a seed) a coccus; N.L. masc. n. Mariniluteicoccus yellow coccus living in the sea]. 1053
D.-F. Zhang and others
98*
Friedmanniella okinawensis FB1T (AB445455) Friedmanniella flava W6T (HQ839787)
98*
0.01
Friedmanniella sagamiharensis FB2T (AB445456) 99* 72*
Friedmanniella lucida FA2T (AB445454) Microlunatus panaciterrae Gsoil 954T (AB271051)
50*
Auraticoccus monumenti MON 2.2T (FN552748) Naumannella halotolerans WS 4616T (FR832425) Propionicicella superfundia BL 10T (DQ176646)
81* 80* 56*
Propionicimonas paludicola DSM 15597T (FR733712) Micropruina glycogenica Lg2T (AB012607) Propioniferax innocua ATCC 49929T (AF227165)
68
Mariniluteicoccus flavus YIM M13146T (KF564278)
93* 86*
Granulicoccus phenolivorans PG 02T (AY566575) Aestuariimicrobium kwangyangense R27T (DQ830982)
100* 75
53
Luteococcus peritonei CCUG 38120T (AJ132334) Luteococcus japonicus DSM 10546T (Z78208) Tessaracoccus lubricantis KSS 17SeT (FM178840)
72*
Brooklawnia cerclae BL 34T (DQ196625)
100
Propioniciclava tarda WR06T (AB298731) Propionibacterium freudenreichii ATCC 6207T (X53217)
74* 99*
Arachnia propionica DSM 43307T (AJ003058) Nocardioides daejeonensis MJ31T (JF937066)
76
Kribbella flavida DSM 17836T (CP001736) Glycomyces harbinensis ATCC 43155T (D85483) Nocardiopsis dassonvillei DSM 43111T (CP002040)
Fig. 2. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences of strain YIM M13146T and members of the family Propionibacteriaceae. Bootstrap values (expressed as percentages of 1000 replications) ¢50 % are shown at branch points. Asterisks denote nodes that were also recovered using the maximum-likelihood and maximum-parsimony methods. Bar, 0.01 substitutions per nucleotide position.
Cells are Gram-staining-positive, aerobic, non-motile, irregular cocci. The genus is a member of the family Propionibacteriaceae. The major cellular fatty acid is anteiso-C15 : 0, the predominant menaquinone is MK-9(H4), the peptidoglycan type is A3c (LL-DAP–Gly) containing alanine, glycine, glutamic acid and LL-diaminopimelic acid in a molar ratio of 2 : 1 : 1 : 2 and the polar lipids are phosphatidylcholine, diphosphatidylglycerol, an unknown phosphoglycolipid and several unknown glycolipids. The G+C content of the DNA of the type strain of the type species is 67.2 mol%. The type species is Mariniluteicoccus flavus. 1054
Description of Mariniluteicoccus flavus sp. nov. Mariniluteicoccus flavus (fla9vus. L. masc. adj. flavus yellow, reflecting the colour of colonies of the type strain). The characteristics are the same as those described for the genus with the following additions. Cells are 0.5–1.0 mm in diameter. Optimal growth occurs at 30 uC (range 5–40 uC), pH 7.0 (range pH 6.0–9.0) and 0–1 % NaCl (range 0–6 %, w/v) on/in TSA/TSB. Production of catalase is negative. Positive reactions from the API ZYM system are observed for alkaline phosphatase, esterase (C4), esterase lipase (C8), International Journal of Systematic and Evolutionary Microbiology 64
Mariniluteicoccus flavus gen. nov., sp. nov.
leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, bgalactosidase, a-glucosidase and b-glucosidase; negative reactions for lipase (C14), trypsin, a-chymotrypsin, agalactosidase, b-glucuronidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. Positive reactions from the API 20E system for b-galactosidase, the Voges–Proskauer test, gelatin hydrolysis and nitrate reductase, while negative reactions are observed for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, H2S production, tryptophan deaminase, urease, indole production, oxidase and fermentation under anaerobic conditions. Positive reactions from the GEN III system for utilization of dextrin, maltose, cellobiose, gentiobiose, sucrose, turanose, methyl b-D-glucoside, D-salicin, Nacetyl-D-glucosamine, N-acetyl-b-D-mannosamine, N-acetyl neuraminic acid, a-D-glucose, D-mannose, D-fructose, Dgalactose, D- and L-fucose, L-rhamnose, inosine, D-arabitol, glycerol, D-glucose 6-phosphate, D-fructose 6-phosphate, Dserine, glycyl L-proline, L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, L-histidine, L-pyroglutamic acid, L-serine, D-galacturonic acid, L-galactonic acid lactone, D-gluconic acid, D-glucuronic acid, glucuronamide, quinic acid, Dsaccharic acid, D-lactic acid methyl ester, L-lactic acid, aketoglutaric acid, acetoacetic acid and acetic acid and tolerance of pH 6, 1 and 4 % NaCl, 1 % sodium lactate, troleandomycin, nalidixic acid and lithium chloride. Negative reactions from the GEN III system for utilization of trehalose, stachyose, raffinose, lactose, melibiose, Nacetyl-D-galactosamine, 3-methyl glucose, D-sorbitol, Dmannitol, myo-inositol, D-aspartic acid, gelatin, pectin, mucic acid, p-hydroxyphenylacetic acid, methyl pyruvate, citric acid, D- and L-malic acid, bromosuccinic acid, Tween 40, c-aminobutyric acid, a-hydroxybutyric acid, b-hydroxyDL-butyric acid, a-ketobutyric acid, propionic acid and formic acid and tolerance of pH 5, 8 % NaCl, fusidic acid, Dserine, rifamycin SV, minocycline, lincomycin, guanidine hydrochloride, niaproof 4, vancomycin, tetrazolium violet, tetrazolium blue, potassium tellurite, aztreonam, sodium butyrate and sodium bromate. T
T
The type strain YIM M13146 (5DSM 25892 5CCTCC AB 2012055T) was isolated from a sediment sample collected from the South China Sea (18u 1.6549 N 112u 30.2039 E) at a depth of 2439 m.
Acknowledgements The authors are grateful to Professor George M. Garrity from Michigan State University for the Latin construction of the new taxon name. This research was supported by the National Basic Research Program of China (no. 2010CB833801) and the National Natural Science Foundation of China (nos 30900002 and 312700040). W.-J. L. was also supported by ‘Hundred Talents Program’ of the Chinese Academy of Sciences.
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