2010-Mucilaginibacter myungsuensis
Mucilaginibacter myungsuensis sp.nov.,isolated from a mesotrophic artificial lake
Yochan Joung and Kiseong Joh
Correspondence Kiseong Joh ksjoh@hufs.ac.kr Department of Bioscience and Biotechnology,Hankuk University of Foreign Studies,Wangsan, Yongin,Gyeonggi449-791,Republic of Korea
A non-motile,pale-yellow bacterium,designated strain HMD1056T,was isolated from an artificial lake located within the campus of Hankuk University of Foreign Studies,Yongin,Korea.The major fatty acids were summed feature3(iso-C15:02-OH and/or C16:1v7c;49.1%)and iso-C15:0(22.4%). The major respiratory quinone was MK-7.The DNA G+C content was46.9mol%.A phylogenetic tree based on16S rRNA gene sequences showed that strain HMD1056T formed a lineage within the genus Mucilaginibacter and was closely related to the type strains of Mucilaginibacter ximonensis (95.4%sequence similarity),Mucilaginibacter kameinonensis(94.5%)and Mucilaginibacter paludis (93.4%).On the basis of the evidence presented in this study,strain HMD1056T represents a novel species of the genus Mucilaginibacter,for which the name Mucilaginibacter myungsuensis sp.nov.is proposed.The type strain is HMD1056T(5KCTC22746T5CECT7550T).
The genus Mucilaginibacter was proposed by Pankratov et al. (2007)and members of the genus are characterized by the ability to produce large amounts of extracellular polymeric substances and to hydrolyse pectin,xylan,pullulan and starch.Strains of Mucilaginibacter may also contain iso-C15:0as the major fatty acid and other hydroxy fatty acids as major components.At the time of writing,the genus Mucilaginibacter includes12species with validly published names:Mucilaginibacter paludis(the type species)and M. gracilis(Pankratov et al.,2007),M.kameinonensis(Urai et al.,2008),M.daejeonensis(An et al.,2009),M.ximonensis (Luo et al.,2009),M.oryzae(Jeon et al.,2009),M.rigui (Baik et al.,2010),M.gossypii and M.gossypiicola (Madhaiyan et al.,2010)and M.frigoritolerans,https://www.wendangku.net/doc/b18964590.html,ppiensis and M.mallensis(Ma¨nnisto¨et al.,2010).Mucilaginibacter species have been isolated from rice straw,various soils, acidic Sphagnum peat bog,freshwater and lichen.
In the course of a study on the microbial diversity of a mesotrophic artificial lake within the campus of Hankuk University of Foreign Studies in Yongin,Republic of Korea, pale-yellow colonies were isolated on R2A agar at30u C using the standard dilution plating technique.Isolate HMD1056T was maintained on R2A agar at30u C and the culture was suspended in water containing20% glycerol(w/v)for storage at280u C.
Almost-complete sequences of the16S rRNA gene were obtained for strain HMD1056T,as described previously (Cho&Giovannoni,2003).Identification of phylogenetic neighbours and calculation of pairwise16S rRNA gene sequence similarity were performed by using the EzTaxon server(https://www.wendangku.net/doc/b18964590.html,/;Chun et al.,2007). Phylogenetic and molecular evolutionary analyses were conducted by using MEGA version4(Tamura et al.,2007). Phylogenetic trees were inferred using the maximum-likelihood(Felsenstein,1981),maximum-parsimony (Fitch,1971)and neighbour-joining(Saitou&Nei,1987) algorithms.The robustness of the topologies for the maximum-likelihood and neighbour-joining trees was evaluated by means of bootstrap analysis(Felsenstein, 1985)based on1000resamplings of the sequences.All phylogenetic trees generated in this study indicated that strain HMD1056T belonged to the genus Mucilaginibacter (Fig.1).Strain HMD1056T formed a coherent clade with M.ximonensis XM-003T(95.4%)within the phylogeneti-cally well-resolved Mucilaginibacter clade.This phyloge-netic inference,coupled with16S rRNA gene sequence similarities of,97%(Wayne et al.,1987)between strain HMD1056T and strains of other Mucilaginibacter species, suggested that the strain should be assigned to the genus Mucilaginibacter as a representative of a novel species. M.paludis KACC13450T,M.gracilis KACC13451T,M. kameinonensis KCTC22227T and M.ximonensis KCTC 22437T were used as reference strains for phenotypic analysis and determination of cellular fatty acid profiles. Cell morphology was examined by light microscopy.Gram staining was determined using the bioMe′rieux Gram stain kit according to the manufacturer’s instructions.Anaerobic growth was tested on R2A agar at25u C by using a GasPak EZ anaerobic container system(BD)according to the manufacturer’s instructions.Cellular pigments were extracted with acetone/methanol(1:1,v/v)and their absorption spectra were determined using a scanning UV/visible spectrophotometer(UV6101A;Shimadzu).The
The GenBank/EMBL/DDBJ accession number for the16S rRNA gene
sequence of strain HMD1056T is GQ144415.
International Journal of Systematic and Evolutionary Microbiology(2011),61,1506–1510DOI10.1099/ijs.0.022798-0 1506022798G2011IUMS Printed in Great Britain
presence of flexirubin-type pigments was investigated by using the bathochromatic shift test with a KOH solution (Bernardet et al.,2005;Fautz &Reichenbach,1980).Catalase and oxidase tests were performed according to standard methods (MacFaddin,1980).The temperature range and optimum for growth were assessed on MA at 4,10–30u C (at 5u C intervals),37and 42u C over 72h.The pH range for growth was determined in R2A broth at 20u C.The medium was adjusted to pH 5.0–10.0(at intervals of 1.0pH unit)using 0.1mM sodium acetate buffer (pH 5.0–7.0),0.1mM phosphate buffer (pH 7.0–8.0)and 0.1mM sodium carbonate buffer (pH 9.0–10.0).Growth in the presence of 0.5%and 1.0–5.0%(at intervals of 1.0%)NaCl (w/v)was also tested in R2A broth.Hydrolysis of casein [3%skimmed milk (Difco),v/v ],CM-cellulose [1.0%CM-cellulose (Sigma),w/v ],crystalline cellulose (Whatman No.1filter paper,1%w/v)and starch (1.0%,w/v)was tested using R2A agar as the basal medium.MacConkey agar (Difco)and DNase test agar (Difco)were used to test for growth and production of DNase,respectively.Other biochemical tests and carbon source oxidation tests were performed using API 20NE,
API 50CH and API ZYM strips (bioMe
′rieux)and Biolog GN2MicroPlates,according to the manufacturers’instruc-tions.The DNA G +C content was determined by using HPLC (Mesbah &Whitman,1989).For fatty acid methyl ester analysis,strain HMD1056T and the four reference strains were grown on MA at 25u C for 3days and fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser,1990).Isoprenoid quinones were isolated according to Minnikin et al.(1984)and analysed by HPLC as described by Collins (1985).Morphological,cultural,physiological and biochemical characteristics of strain HMD1056T are listed in Table 1and in the species description.Strain HMD1056T exhibited a number of phenotypic similarities to species of the genus Mucilaginibacter ,including growth temperature range,pale-yellow pigmentation and obligately aerobic growth.However,several characteristics of HMD1056T clearly differentiated this strain from the type strains of Mucilaginibacter species (Table 1).
The fatty acid profile of strain HMD1056T contained summed feature 3(iso-C 15:02-OH and/or C 16:1v 7c ;49.1%)and iso-C 15:0(22.4%)as major components.The complete fatty acid profile of strain HMD1056T is given in Table 2.The major fatty acid profile was similar to those of other Mucilaginibacter species,although there were differences in the relative proportions of some fatty acids (Table 2).The respiratory quinones were MK-7(major component)and MK-6(minor),as reported for other members of genus Mucilaginibacter .The DNA G +C content of strain HMD1056T was 46.9mol%,a value within the range reported for members of genus Mucilaginibacter .The phenotypic data confirm the phylo-genetic affiliation of strain HMD1056T to the genus Mucilaginibacter .Therefore,strain HMD1056T should be classified in the genus Mucilaginibacter as a member of novel species,for which the name Mucilaginibacter myungsuensis sp.nov.is proposed.
Description of Mucilaginibacter myungsuensis sp.nov.
Mucilaginibacter myungsuensis (myung.su.en 9sis.N.L.masc.adj.myungsuensis pertaining to Myungsu,the name of the mesotrophic freshwater lake from which the type strain was isolated).
Cells are Gram-negative,non-motile,aerobic rods,0.7–0.8m m in diameter and 1.4–1.5m m long.Colonies on R2A are pale yellow,mucoid,convex and round and approxi-mately 5mm in diameter after 2days at 25u C.Good growth occurs on blood agar,TSA and R2A agar.No growth occurs on DNA test agar or MacConkey agar.Growth occurs in the presence of 0–1.0%(w/v)NaCl (optimum 0.5%),at pH 5–9(optimum pH 7)and at 10–30u C (optimum 30u C).Oxidase and catalase activities and aesculin hydrolysis activity are present,but arginine dihydrolase,lysine decarboxylase,ornithine decarboxylase,tryptophan deaminase and L -phenylalanine deaminase activities are absent.Casein and starch are hydrolysed,but gelatin and tyrosine are not.Citrate is not utilized.Flexirubin-type pigments are produced.In the API ZYM gallery,alkaline phosphatase,esterase (C4),esterase lipase (C8),leucine arylamidase,valine arylamidase,
cystine
Fig. 1.Neighbour-joining phylogenetic tree based on nearly complete 16S rRNA gene sequences showing the relationships between strain HMD1056T and strains of the genus Mucilaginibacter.Percentages at nodes are levels of bootstrap support (.50%)based on neighbour-joining analyses of 1000resampled datasets.Filled and open circles respectively indicate notes recovered by all three treeing methods or by two treeing methods.Sphingo-bacterium spiritivorum ATCC 33861T was used as an outgroup.Bar,0.01substitutions per nucleotide position.
Mucilaginibacter myungsuensis sp.nov.
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Table1.Differential characteristics of strain HMD1056T and type strains of closely related Mucilaginibacter species
Strains:1,HMD1056T;2,M.paludis KACC13450T;3,M.gracilis KACC13451T;4,M.kameinonensis KCTC22227T;5,M.ximonensis KCTC22437T (data in columns1–5from this study unless indicated);6,M.gossypii Gh-67T;7,M.gossypiicola Gh-48T(data in columns6and7from Madhaiyan et al.,2010);8,M.frigoritolerans FT22T;9,https://www.wendangku.net/doc/b18964590.html,ppiensis ANJL12T;10,M.mallensis MP1X4T(data in columns8–10from Ma¨nnisto¨et al.,2010). +,Positive;W,weakly positive;2,negative;NA,no data available.
Characteristic12345678910
Isolation source Water Sphagnum
peat bog a*Sphagnum
peat bog a
Soil b Soil c Soil Soil Tundra
soil
Lichen Tundra
soil
DNA G+C content
(mol%)
46.946.1a42.4a43.7b43.4c46.744.244.043.546.0
Major menaquinone(s)7,67,6a7,6a7b7c777,67,67,6 Growth temperature(u C)10–3010–3010–3010–3010–3020–3020–300–330–3123to33 Flexirubin-type pigments+22+2NA NA+2+
API20NE results
Glucose fermentation222++22222 Nitrate reduction22222++22+ Urease22222+2222 Gelatinase22222222+2
API ZYM results
Esterase(C4)++++2NA NA222 Cystine arylamidase+22+2NA NA2+2 Trypsin+22+2NA NA2+2 b-Glucuronidase22+++NA NA222 b-Galactosidase2++++NA NA+++ a-Glucosidase+++++NA NA+++ b-Glucosidase22+++NA NA++2
Acid production from:
D-Arabinose++2+2222+2 L-Arabinose2++2222+2+ D-Xylose+++22++222 D-Glucose++++2+++++ D-Fructose++++222W2+ D-Mannose++++++++W+ L-Rhamnose22222+2222 Melibiose++++++W+++ Trehalose++D++++++++ Melezitose+22+2+++2+ Utilization of(Biolog
GN2):
a-Cyclodextrin22+22+++2+ Dextrin2+++2++++2 Glycogen2++22++222 N-Acetyl-D-
galactosamine
22222++2+2
N-Acetyl-D-
glucosamine
22222+++++
Adonitol22222++222 Cellobiose++22++++++ D-Fructose+++22+2+W+ L-Fucose2++22++2+W D-Galactose2++22++W++ Gentiobiose++2+++++++ Lactose2+222+++++ Lactulose2222+++++2 Maltose++++2+++++ D-Mannose2+++++++++ Melibiose2+++++++++
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Mucilaginibacter myungsuensis sp.nov. Table1.cont.
Characteristic12345678910 Methyl b-D-glucoside22++++++++ Raffinose2++++++2+2 Trehalose2++2++++++ Turanose2++2++++++ L-Alanine222++++2+2 L-Alanyl glycine2+2+2+++++ L-Asparagine22222+22+2 L-Glutamic acid+2+++2++++ L-Proline++2++2+++W L-Serine2++222++22 L-Threonine2+2++2++2W
2,3-Butanediol22222++222 Glycerol2+2+2+++++
2+222+++22 DL-a-Glycerol
phosphate
2++2+22+W2 a-D-Glucose
1-phosphate
*Data taken from:a,Pankratov et al.(2007);b,Urai et al.(2008);c,Luo et al.(2009).
D Reported as negative by Luo et al.(2009).
Table2.Cellular fatty acid compositions of strain HMD1056T and type strains of closely related Mucilaginibacter species Strains:1,HMD1056T;2,M.paludis KACC13450T;3,M.gracilis KACC13451T;4,M.kameinonensis KCTC22227T;5,M.ximonensis KCTC22437T (data in columns1–5from this study);6,M.gossypii Gh-67T;7,M.gossypiicola Gh-48T(data in columns6and7from Madhaiyan et al.,2010);8, M.frigoritolerans FT22T;9,https://www.wendangku.net/doc/b18964590.html,ppiensis ANJL12T;10,M.mallensis MP1X4T(data in columns8–10from Ma¨nnisto¨et al.,2010).All strains were grown for2days at25u C on R2A.Values are percentages of total fatty acids.Only fatty acids that account for more than1%for one or more strains are indicated.tr,Trace;ND,not detected/not reported.
Fatty acid12345678910
C14:0 2.6 1.6 1.1 1.7tr 1.2 1.7tr tr tr
iso-C15:024.423.220.622.933.225.820.021.918.926.0 anteiso-C15:0tr15.8ND12.5tr ND ND 1.6ND tr
C15:1v6c 1.1 1.8tr 1.1 3.1tr tr ND ND ND
C15:1v5c ND tr ND ND ND 2.8 1.3 1.1ND tr
C15:0ND ND ND ND ND ND ND 1.2tr 1.3
C16:1v5c 3.78.8 5.0 6.8 3.7 2.8 1.3 2.7 6.8 4.0
C16:0 4.5 3.77.9 5.8 2.315.017.0 3.1 3.7 4.8
iso-C15:03-OH 2.9 1.7 2.0 2.6 3.7 1.9 4.5 2.9 2.2 2.9
C15:02-OH ND 1.2tr 1.4tr ND tr tr ND tr
C17:1v8c ND 1.0tr tr tr tr tr ND ND ND
C16:03-OH 1.4tr 3.3 1.4 1.2 5.1 4.6tr 3.2tr
C18:0tr tr tr tr ND 1.6ND ND ND tr
iso-C17:03-OH 5.5 3.0 4.9 6.7 6.0 6.5 6.116.414.312.5
C17:02-OH ND tr ND ND ND ND ND0.9tr 1.4
iso-C17:1ND ND ND ND ND ND ND 4.1 2.79.5 Summed feature3*49.130.549.334.237.733.039.638.745.032.9 Summed feature9* 1.6tr 1.4ND 4.6tr tr ND ND ND
*Summed features represent groups of two or three fatty acids that could not be separated using the MIDI system.Summed feature3comprised iso-C15:02-OH and/or C16:1v7c and summed feature9comprised iso-C17:1v9c and/or10-methyl C16:0.
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arylamidase,trypsin,acid phosphatase,naphthol-AS-BI-
phosphohydrolase,a-galactosidase,a-glucosidase,N-
acetyl-b-glucosaminidase and a-fucosidase activities are
present,but lipase(C14),a-chymotrypsin,b-galactosidase, b-glucuronidase,b-glucosidase and a-mannosidase activ-ities are absent.The following compounds are utilized as
sole carbon sources in Biolog GN2microplates:cellobiose, D-fructose,gentiobiose,a-D-glucose,maltose,sucrose,L-glutamic acid and L-proline.The following carbon sources are not utilized:a-cyclodextrin,dextrin,glycogen,Tweens
40and80,N-acetyl-D-galactosamine,N-acetyl-D-glucosa-
mine,adonitol,L-arabinose,D-arabitol,i-erythritol,L-
fucose,D-galactose,myo-inositol,lactose,lactulose, D-mannitol,D-mannose,melibiose,methyl b-D-glucoside, D-psicose,raffinose,L-rhamnose,D-sorbitol,trehalose, turanose,xylitol,pyruvic acid methyl ester,succinic acid monomethyl ester,acetic acid,cis-aconitic acid,citric acid, formic acid,D-galactonic acid lactone,D-galacturonic acid, D-gluconic acid,D-glucosaminic acid,D-glucuronic acid, a-,b-and c-hydroxybutyric acids,p-hydroxyphenylacetic acid,itaconic acid,a-ketobutyric acid,a-ketoglutaric acid, a-ketovaleric acid,DL-lactic acid,malonic acid,propionic acid,quinic acid,D-saccharic acid,sebacic acid,succinic acid,bromosuccinic acid,succinamic acid,glucuronamide, L-alaninamide,D-and L-alanine,L-alanyl glycine,L-asparagine,L-aspartic acid,glycyl L-aspartic acid,glycyl L-glutamic acid,L-histidine,hydroxy-L-proline,L-leucine, L-ornithine,L-phenylalanine,L-pyroglutamic acid,D-and L-serine,L-threonine,DL-carnitine,c-aminobutyric acid, urocanic acid,inosine,uridine,thymidine,phenylethyla-mine,putrescine,2-aminoethanol,2,3-butanediol,glycerol, DL-a-glycerol phosphate,a-D-glucose1-phosphate and D-glucose6-phosphate.The major fatty acids are summed feature3(iso-C15:02-OH and/or C16:1v7c)and iso-C15:0. The DNA G+C content of the type strain is46.9mol%. The type strain,HMD1056T(5KCTC22746T5CECT 7550T),was isolated from a mesotrophic freshwater lake in Wangsan,Yongin,Gyeonggi,Republic of Korea.
Acknowledgements
This research was supported by the project on survey and excavation of Korean indigenous species of the National Institute of Biological Resources(NIBR)under the Ministry of Environment,Korea.
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