International Journal of Systematic and Evolutionary Microbiology (2014), 64, 2223–2228
DOI 10.1099/ijs.0.057737-0
Mucilaginibacter pineti sp. nov., isolated from Pinus pinaster wood from a mixed grove of pines trees Gabriel Paiva,1 Pedro Abreu,1 Diogo Neves Proenc¸a,1 Susana Santos,1 Maria Fernanda Nobre2 and Paula V. Morais1,3 Correspondence
1
Paula V. Morais
2
[email protected]
IMAR-CMA, University of Coimbra, 3004-517 Coimbra, Portugal CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal
3
Department of Life Sciences, FCTUC, University of Coimbra, 3004-517 Coimbra, Portugal
Bacterial strain M47C3BT was isolated from the endophytic microbial community of a Pinus pinaster tree branch from a mixed grove of pines. Phylogenetic analysis of 16S rRNA gene sequences showed that this organism represented one distinct branch within the family Sphingobacteriaceae, most closely related to the genus Mucilaginibacter. Strain M47C3BT formed a distinct lineage, closely related to Mucilaginibacter dorajii KACC 14556T, with which it shared 97.2 % 16S rRNA gene sequence similarity. The other members of the genus Mucilaginibacter included in the same clade were Mucilaginibacter lappiensis ATCC BAA-1855T sharing 97.0 % similarity and Mucilaginibacter composti TR6-03T that had a lower similarity (95.7 %). The novel strain was Gram-staining-negative, formed rod-shaped cells, grew optimally at 26 6C and at pH 7, and was able to grow with up to 0.3 % (w/v) NaCl. The respiratory quinone was menaquinone 7 (MK-7) and the major fatty acids of the strain were summed feature 3 (C16 : 1v7c/iso-C15 : 0 2-OH), iso-C15 : 0 and iso-C17 : 0 3-OH, representing 73.5 % of the total fatty acids. The major components of the polar lipid profile of strain M47C3BT consisted of phosphatidylethanolamine, three unidentified aminophospholipids, one unidentified aminolipid and three unidentified polar lipids. The G+C content of the DNA was 40.6 mol%. On the basis of the phylogenetic analysis and physiological and biochemical characteristics we propose the name Mucilaginibacter pineti sp. nov. for the novel species represented by strain M47C3BT (5CIP 110632T5LMG 28160T).
The genus Mucilaginibacter of the family Sphingobacteriaceae, phylum Bacteroidetes, was first described by Pankratov et al. (2007) to group Gram-staining-negative, non-spore-forming and non-motile rods producing large amounts of extracellular polymeric substances. The genus was proposed to include two novel species, Mucilaginibacter paludis and Mucilaginibacter gracilis. At the time of writing, the genus Mucilaginibacter comprises 22 species with validly published names: M. paludis 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), M. frigoritolerans, M. lappiensis and M. mallensis (Ma¨nnisto¨ et al., 2010), M. boryungensis (Kang et al., 2011), M. composti (Cui et al., 2011), M. dorajii (Kim et al., 2010), M. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain M47C3BT is KF483876. Two supplementary figures are available with the online version of this paper.
057737 G 2014 IUMS
Printed in Great Britain
myungsuensis (Joung & Joh, 2011), M. angelicae (Kim et al., 2012a), M. litoreus (Yoon et al., 2012), M. lutimaris (Kim et al., 2012b), M. polysacchareus (Han et al., 2012), M. soli (Jiang et al., 2012) and M. jinjuensis (Khan et al., 2013). Members of the genus Mucilaginibacter have been isolated from very different environments including peat bogs, soils, dried rice straw, wetland freshwater (An et al., 2009; Baik et al., 2010; Madhaiyan et al., 2010; Pankratov et al., 2007; Urai et al., 2008) and also cold environments (Ma¨nnisto¨ et al., 2010). Pinus pinaster trees from a mixed grove of pines in Malhada, Oliveira do Hospital, Portugal, were sampled and the bark and sapwood of each cross-section were removed under sterile conditions for the study of the diversity of the endophytic community. Strain M47C3BT, forming mucoid, light-pink colonies, was isolated from dilutions of wood chips plated on R2A agar (Difco) incubated at 25 uC, for three days. The strain was maintained in the same medium supplemented with 15 % (v/v) glycerol at 280 uC after subculture and purification. 2223
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Cell morphology and motility were examined by phasecontrast microscopy (Leitz Diaplan, UK) after growth on R2A agar at 26 uC for 48 h. Growth on several bacteriological media was tested using R2A agar, tryptic soy agar (TSA; Difco), nutrient agar (NA; Difco) and R2 liquid (Difco) incubated at 26 uC for 5 days. The temperature range (4, 15, 20, 22, 26, 30, 37, 40, 42, 45 uC) and optimum temperature for growth were examined on R2A agar and in R2 liquid incubated for up to 5 days. Salt tolerance was tested in R2A agar and R2 liquid supplemented with NaCl at the final concentrations of 0 to 3 % (w/v), in 0.5 % increments (agar) and 0.1 % increments (liquid), at 26 uC, incubated for up to 5 days. The pH range for growth was examined at 26 uC in the same medium by using 50 mM MES (pH 3, 4, 5, 6, 7), HEPES (pH 6, 7, 8), TAPS (pH 8, 9) and CAPSO (pH 10) over a pH range from pH 5.0 to 10.0, with intervals of 0.5 pH units. The Gram-staining reaction and the presence of cytochrome oxidase and catalase were determined after 24 h of incubation on R2A agar as described by Smibert & Krieg (1994). The ability to hydrolyse agar aesculin, casein, xylan, gelatin, arbutin,
elastin, starch, DNA, chitin, and Tweens 20, 40, 60 and 80 at a concentration of 1.0 % (w/v or v/v) in R2A agar, after incubation at 26 uC for up to 5 days, was determined as described by Tindall et al. (2007). Other physiological properties and enzyme activities were determined using the API ZYM and API 20NE test strips (bioMe´rieux) at 26 uC according to the manufacturer’s instructions. Singlecarbon-source assimilation was determined using API 50 CH test strips (bioMe´rieux) after incubation at 26 uC for up to 7 days, as described by Morais et al. (2004). API 50 CH strips were also used for evaluation of acid production from single carbon sources after incubation at 26 uC for up to 5 days, according to the manufacturer’s instructions. The ability of the strain to oxidize different carbon sources was assessed using Biolog GN2 MicroPlates, incubated at 26 uC. The results were recorded daily for up to 7 days using a MicroPlate reader (Sunrise Xread Plus version V 4.30; Tecan). The test for flexirubin-like pigments was performed by soaking cells grown on R2A agar at 26 uC for 2 days with 20 % (w/v) KOH (Fautz & Reichenbach, 1980). Congo red adsorption was tested by using R2A-Congo red agar (25 mg
Table 1. Differential characteristics of strain M47C3BT compared with the type strains of Mucilaginibacter genus Strains: 1, M47C3BT (data from this study); 2, M. dorajii KACC 14556 T; 3, M. lappiensis ATCC BAA-1856T. All strains are positive for (API ZYM) alkaline phosphatase, leucine arylamidase, Valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase and are negative for lipase (C14), a-chymotrypsine. All strains cannot assimilate (API 20NE and API 50 CH) D-Mannitol. Data for reference strains are from Kim et al. (2010) and Ma¨nnisto¨ et al. (2010). +, positive; w+, weakly positive; –, negative; ND, not determined. Characteristic Colony colour Catalase/oxidase Cell length (mm) Temperature range (uC) Max. NaCl (%, w/v) tolerance pH range Acid production from: D-Arabinose D-Galactose D-Glucose N-Acetylglucosamine Amygdalin D-Cellobiose D-Maltose D-Lactose D-Trehalose Assimilation of: L-Arabinose N-acetylglucosamine Maltose D-Glucose D-Mannose Enzyme activities: Esterase C4 Esterase lipase C8 DNA G+C content (mol%)
2224
1
2
3
Light Pink +/+ 1–3 15–30 0.3 5.5–8.0
Light Yellow +/+ 1.1–1.8 4–30 1 5.0–8.0
Light Pink +/+ 1–3 0–31 1.5 4.5–8.0
– – 2 2 2 2 2 2 2
+ + + w+ w+ + + + +
+ + + w+ + + + + +
– – – – –
+ + + + w+
+ + + + w+
– – 40.64
w+ w+ 42.6
w+ + 43.5
International Journal of Systematic and Evolutionary Microbiology 64
Mucilaginibacter pineti sp. nov. from pine wood
Congo red l21) incubated at 26 uC for 2 days (Freeman et al., 1989). Antibiotic-sensitivity tests were performed for strain M47C3BT by using discs (Oxoid) containing the following: lincomycin (15 mg), ampicillin (10 mg), amoxicillin+clavulanic acid (30 mg), gentamicin (30 mg), polymyxin B (300 U), chloramphenicol (100 mg), erythromycin (15 mg), vancomycin (30 mg), streptomycin (50 mg), rifampicin (30 mg), tetracycline (30 mg) or kanamycin (30 mg). Cells for polar lipids and lipoquinone analysis were grown on R2A agar at 26 uC for 48 h and then harvested and lyophilized. Polar lipids were extracted and two-dimensional TLC was performed on silica gel G plates (10610 cm, 0.25 mm thickness; Merck) using chloroform/methanol/water (65 : 25 : 4, by vol.) in the first direction and chloroform/acetic acid/methanol/water (80 : 15 : 12 : 4, by vol.) in the second direction (da Costa et al., 2006). The polar lipids were identified using differential staining with molybdophosphoric acid (total polar lipids), ninhydrin (aminolipids) and anaphthol/sulfuric acid (glycolipids). Phosphatidylethanolamine was used as a standard. Lipoquinones were extracted from freeze-dried cells, purified by TLC and separated by HPLC (da Costa et al., 2011a). Cells for fatty acids analysis were grown on R2A agar in sealed plastic plates for 48 h (Morais et al., 2004). The fatty acid methyl esters (FAMEs) were obtained from fresh wet biomass and were separated, identified and quantified using the standard MIS Library Generation Software (Sherlock Microbial ID System, 2TSBA 6 database, version 6.0; MIDI) as described previously (da Costa et al., 2011b). The DNA G+C content of the genome was determined by HPLC as described by Mesbah et al. (1989). The 16S rRNA gene was amplified by PCR and sequenced as described by Morais et al. (2004), and aligned against representative reference sequences of the most closely related members of the genus Mucilaginibacter obtained from the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al., 2012c) and then aligned by SINA (v1.2.9) using the SILVA SEED as reference alignment (http://www.arb-silva.de/aligner/; Pruesse et al., 2012). Evolutionary distances were calculated (Jukes & Cantor, 1969), and phylogenetic dendrograms were reconstructed using the neighbour-joining (Saitou & Nei, 1987) and maximum-likelihood (Olsen et al., 1994) algorithms included in the ARB software package (Ludwig et al., 2004). Tree topologies were evaluated by performing bootstrap analysis (Felsenstein, 1985) of 1000 datasets by using the ARB software package. T
Strain M47C3B was Gram-staining-negative, non-sporeforming and appeared as rod-shaped cells (0.09–0.1 mm in width and 1–3 mm in length). After 48 h of incubation on R2A agar, colonies were circular, smooth, glistening and light pink. Biochemical and physiological characteristics of strain M47C3BT are summarized in Table 1 and in the species description. Strain M47C3BT had an optimum temperature for grow at 26 uC and was not able to grow at temperatures below 15 uC, in contrast to its closest relatives, M. dorajii and M. lappiensis (Table 1). Strain M47C3BT was able to grow from pH 5.5 up to pH 8.0 with optimal growth at pH 7, and http://ijs.sgmjournals.org
was positive for catalase and weakly positive for oxidase. Strain M47C3BT could grow in R2A agar and R2 liquid containing up to 0.3 % (w/v) NaCl, while its closest relatives grew with 1 to 1.5 % (w/v) NaCl. Colonies on R2A-Congo red agar were non-pigmented and flexirubin-type pigments were not formed. Strain M47C3BT was able to assimilate very few carbon sources in comparison with M. dorajii and M. lappiensis (Table 1). The strain was able to hydrolyse casein and starch, and was weakly urease-positive. The major polar lipids were phosphatidylethanolamine, three unidentified aminophospholipids, one unidentified aminolipid and three unidentified polar lipids. No glycolipids were detected (Fig. S1, available in the online Supplementary Material). The major fatty acids of strain M47C3BT were summed feature 3 (C16 : 1v7c/iso-C15 : 0 2-OH), iso-C15 : 0 and iso-C17 : 0 Table 2. Fatty acid composition of strain M47C3BT and the type strains of closely related members of the genus Mucilaginibacter Strains: 1, M47C3BT (data from this study); 2, M. dorajii KACC 14556T (Kim et al., 2010); 3, M. lappiensis ATCC BAA-1855T (Ma¨nnisto¨ et al., 2010). Values are percentages of total fatty acids. TR, Traces (,0.5 %); –, not detected. Fatty acid iso-C13 : 0 3-OH C14 : 0 C14 : 0 2-OH C15 : 0 C15 : 0 2-OH C15 : 0 3-OH iso-C15 : 0 iso-C15 : 0 3-OH C15 : 1v6c anteiso-C15 : 0 C16 : 0 C16 : 0 3-OH iso-C16 : 0 iso-C16 : 0 3-OH C16 : 1v5c C17 : 0 C17 : 0 3-OH iso-C17 : 0 iso-C17 : 0 3-OH C17 : 1v8c iso-C17 : 1v9c C18 : 0 C18 : 1v7c Summed feature 2* Summed feature 3* Summed feature 9*
1
2
3
– 0.7 0.3 0.8 – – 17.7 2.2 0.2 0.3 7.7 3.3 – – 6.5 – – 0.5 10.2 – – – – 0.2 45.5 2.2
0.9 1.2 – 6.5 1.2
TR
TR
22.6 1.3 1.2 0.8 6.5 2.3 1.2 0.6 4.7 0.6 0.6 TR
6.5 0.9 1.7 3.4 1.6 – 30.9 –
1.1 – 3.4 1.1 – 28.9 1.4 2.9 – 6.4 – – – 2.9 – – 0.8 7.8 TR
2.4 – – – 39.9 –
*Summed features combine groups of two or more fatty acids that cannot be separated by GLC with the MIDI system. Summed feature 2 contains C12 : 0 aldehyde, C14 : 0 3-OH and/or iso-C16 : 1. Summed feature 3 contains C16 : 1v7c and/or iso-C15 : 0 2-OH. Summed feature 9 contains iso-C17 : 1v9c and/or C16 : 0 10 methyl. 2225
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3-OH, which accounted for 73.5 % of the total fatty acids (Table 2). Menaquinone 7 (MK-7) was the major respiratory quinone. The almost complete 16S rRNA gene sequence of strain M47C3BT (1496 bp) was aligned with those of representative members of all species of the genus Mucilaginibacter of the family Sphingobacteriaceae, showing this organism to be a representative of a novel species belonging to the genus Mucilaginibacter. According to the phylogenetic tree reconstructed by using the neighbourjoining method, the closest phylogenetic relatives were M. dorajii KACC 14556T (97.2 %) and M. lappiensis ATCC BAA-1855T (97.0 %) (Fig. 1). The tree topology was confirmed by using a maximum-likelihood analysis (Fig. S2). The G+C content of the DNA, as determined by HPLC (Mesbah et al., 1989), was 40.6 mol%. The phenotypic characterization, enzymic activities, biochemical and physiological characteristics (Table 1), fatty acid profile (Table 2) and phylogenetic evidence indicate that strain M47C3BT represents a novel species of the genus Mucilaginibacter, for which we propose the name Mucilaginibacter pineti sp. nov. Description of Mucilaginibacter pineti sp. nov. Mucilaginibacter pineti (pi.ne9ti. L. gen. n. pineti of a pine wood, pine grove).
Cells on R2A agar are small, smooth, glistening and light pink. Cells are Gram-staining-negative, rod-shaped (0.09– 0.1 mm in width and 1–3 mm in length) after 48 h of incubation on R2A agar and non-spore-forming. Grows on NA and on R2A agar but not on TSA. Growth occurs between 15 and 30 uC (optimal at 26 uC). Growth occurs at pH 5.5–8.0 (optimal at pH 7). Growth occurs with up to 0.3 % (w/v) NaCl. Catalase- and oxidase-positive. Colonies are not coloured on R2A-Congo red agar and flexirubintype pigments are not formed. Resistant to ampicillin (10 mg), gentamicin (30 mg), polymyxin B (300 U) and kanamycin (30 mg), and susceptible to rifampicin (30 mg), streptomycin (50 mg), vancomycin (30 mg) and tetracycline (30 mg). Positive for hydrolysis of aesculin, starch and DNA (weakly positive). Negative for hydrolysis of xylan, elastin, arbutin, gelatin, Tween 20, Tween 40, Tween 60 and Tween 80. Negative for nitrate reduction, indole production and glucose fermentation. Positive for alkaline phosphatase, leucine arylamidase, valine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and afucosidase. Weakly positive for cystine arylamidase. Negative for esterase (C4), esterase lipase (C8), lipase (C14), achymotrypsin and b-glucuronidase (API ZYM). Positive for
Mucilaginibacter gossypiicola Gh-48T (EU672805) Mucilaginibacter gossypii Gh-67T (EU672804) Mucilaginibacter kameinonensis SCKT (AB330392) Mucilaginibacter oryzae B9T (EU109722) Mucilaginibacter angelicae GG-w14T (HM627214) Mucilaginibacter polysacchareus DRP28T (HM748604) Mucilaginibacter pineti M47C3BT (KF483876) Mucilaginibacter dorajii DR-f4T (GU139697) Mucilaginibacter lappiensis ANJLI2T (DQ234446) Mucilaginibacter composti TR6-03T (AB267719) Mucilaginibacter rigui WPCB133T (EU747841) 99 Mucilaginibacter lutimaris BR-3T (HQ455786) Mucilaginibacter soli R9-65T (JF701183) Mucilaginibacter litoreus BR-18T (JF999998) Mucilaginibacter gracilis TPT18T (AM490403) 97 Mucilaginibacter paludis TPT56T (AM490402) Mucilaginibacter frigoritolerans FT22T (FN400860) Mucilaginibacter gynuensis YC7003T (KC247157) Mucilaginibacter mallensis MP1X4T (FN400859) Mucilaginibacter myungsuensis HMD1056T (GQ144415) Mucilaginibacter ximonensis XM-003T (EU729366) Mucilaginibacter boryungensis BDR-9T (HM061614) Mucilaginibacter daejeonensis Jip 10T (AB267717) Mucilaginibacter jinjuensis YC7004T (JQ765855) Parasegetibacter luojiensis RHYL-37T (EU877263) Segetibacter koreensis Gsoil 664T (AB267478) Sediminibacterium salmoneum NJ-44T (EF407879) 72
0.10
72
86 71
Fig. 1. Phylogenetic dendrogram based on a comparison of the 16S rRNA gene sequences of M47C3BT strain and the closest phylogenetic relatives. The tree was created using the neighbour-joining method. Numbers indicate percentages of bootstrap sampling, derived from 1000 replications; values below 70 % are not shown. Filled circles indicate node branches conserved when the tree was reconstructed using the maximum-parsimony algorithm. The isolate characterized in this study is indicated in bold type. Bar, one inferred nucleotide substitution per 10 nt. 2226
International Journal of Systematic and Evolutionary Microbiology 64
Mucilaginibacter pineti sp. nov. from pine wood
aesculin and p-nitrophenyl-b-D-galactopyranoside (API 20 NE). Assimilates the carbon sources aesculin ferric citrate (API 20 NE and API50 CH), p-nitrophenyl-b-D-galactopyranoside (PNPG) (API 20 NE), and methyl a-D-mannopyranoside, arbutin, potassium 2-ketogluconate and potassium 5-ketogluconate (API 50 CH). The other organic substrates included in API 50 CH and API 20 NE are not utilized. Acid is produced from (API 50 CH) D-ribose, D-xylose, D-galactose, D-glucose, D-mannitol, D-sorbitol and aesculin ferric citrate. Acid is produced weakly from methyl bD-xylopyranoside, D-mannose, melibiose, sucrose, gentiobiose and potassium 5-ketogluconate. Tests using the Biolog GN2 system show dextrin is oxidized, a-cyclodextrin, glycogen, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, cellobiose, D-fructose, L-fucose, D-galactose, gentiobiose, a-D-glucose, a-lactose, lactulose, maltose, D-mannose, melibiose, methyl b-D-glucoside, D-psicose, raffinose, sucrose, trehalose, turanose, D-gluconic acid, D-glucosaminic acid, a-ketobutyric acid, L-alaninamide, L-alanine, L-alanyl glycine, L-glutamic acid, L-proline, L-serine and L-threonine are weakly oxidized, and the remaining substrates are not oxidized. The major components of the polar lipid profile of strain M47C3BT consisted of phosphatidylethanolamine, three unidentified aminophospholipids, one unidentified aminolipid and three unidentified polar lipids. No glycolipids are detected. The major fatty acids are summed feature 3 (C16 : 1v7c/iso-C15 : 0 2-OH), iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 0 and C16 : 1v5c. The major respiratory lipoquinone is menaquinone 7 (MK-7). The type strain, M47C3BT (5CIP 110632T5LMG 28160T) was isolated from the wood of a Pinus pinaster in Malhada, Coimbra, Portugal. The DNA G+C content of the type strain is 40.6 %.
Acknowledgements We are indebted to Professor J. P. Euze´by (E´cole National Ve´te´rinaire, Toulouse, France) for the etymology of the new organisms’ name. This research was financially supported by Program of National Action for Pine Wood Nematode control: ‘The disease of Pine Wood Nematode’, by Instituto de Financiamento da Agricultura e Pescas (IFAP) and Autoridade Florestal Nacional (AFN) and by the Fundac¸a˜o para a Cieˆncia e Tecnologia (FCT), Portugal, under the PTDC/AGR-CFL/115373/2009 project. G. P. was supported by FCT, Portugal, fellowship DRH36/11/448. D. N. P. was supported by FCT, Portugal, graduate fellowship SFRH/BD/61311/2009.
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International Journal of Systematic and Evolutionary Microbiology 64
DOI 10.1099/ijs.0.057737-0
Mucilaginibacter pineti sp. nov., isolated from Pinus pinaster wood from a mixed grove of pines trees Gabriel Paiva,1 Pedro Abreu,1 Diogo Neves Proenc¸a,1 Susana Santos,1 Maria Fernanda Nobre2 and Paula V. Morais1,3 Correspondence
1
Paula V. Morais
2
[email protected]
IMAR-CMA, University of Coimbra, 3004-517 Coimbra, Portugal CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal
3
Department of Life Sciences, FCTUC, University of Coimbra, 3004-517 Coimbra, Portugal
Bacterial strain M47C3BT was isolated from the endophytic microbial community of a Pinus pinaster tree branch from a mixed grove of pines. Phylogenetic analysis of 16S rRNA gene sequences showed that this organism represented one distinct branch within the family Sphingobacteriaceae, most closely related to the genus Mucilaginibacter. Strain M47C3BT formed a distinct lineage, closely related to Mucilaginibacter dorajii KACC 14556T, with which it shared 97.2 % 16S rRNA gene sequence similarity. The other members of the genus Mucilaginibacter included in the same clade were Mucilaginibacter lappiensis ATCC BAA-1855T sharing 97.0 % similarity and Mucilaginibacter composti TR6-03T that had a lower similarity (95.7 %). The novel strain was Gram-staining-negative, formed rod-shaped cells, grew optimally at 26 6C and at pH 7, and was able to grow with up to 0.3 % (w/v) NaCl. The respiratory quinone was menaquinone 7 (MK-7) and the major fatty acids of the strain were summed feature 3 (C16 : 1v7c/iso-C15 : 0 2-OH), iso-C15 : 0 and iso-C17 : 0 3-OH, representing 73.5 % of the total fatty acids. The major components of the polar lipid profile of strain M47C3BT consisted of phosphatidylethanolamine, three unidentified aminophospholipids, one unidentified aminolipid and three unidentified polar lipids. The G+C content of the DNA was 40.6 mol%. On the basis of the phylogenetic analysis and physiological and biochemical characteristics we propose the name Mucilaginibacter pineti sp. nov. for the novel species represented by strain M47C3BT (5CIP 110632T5LMG 28160T).
The genus Mucilaginibacter of the family Sphingobacteriaceae, phylum Bacteroidetes, was first described by Pankratov et al. (2007) to group Gram-staining-negative, non-spore-forming and non-motile rods producing large amounts of extracellular polymeric substances. The genus was proposed to include two novel species, Mucilaginibacter paludis and Mucilaginibacter gracilis. At the time of writing, the genus Mucilaginibacter comprises 22 species with validly published names: M. paludis 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), M. frigoritolerans, M. lappiensis and M. mallensis (Ma¨nnisto¨ et al., 2010), M. boryungensis (Kang et al., 2011), M. composti (Cui et al., 2011), M. dorajii (Kim et al., 2010), M. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain M47C3BT is KF483876. Two supplementary figures are available with the online version of this paper.
057737 G 2014 IUMS
Printed in Great Britain
myungsuensis (Joung & Joh, 2011), M. angelicae (Kim et al., 2012a), M. litoreus (Yoon et al., 2012), M. lutimaris (Kim et al., 2012b), M. polysacchareus (Han et al., 2012), M. soli (Jiang et al., 2012) and M. jinjuensis (Khan et al., 2013). Members of the genus Mucilaginibacter have been isolated from very different environments including peat bogs, soils, dried rice straw, wetland freshwater (An et al., 2009; Baik et al., 2010; Madhaiyan et al., 2010; Pankratov et al., 2007; Urai et al., 2008) and also cold environments (Ma¨nnisto¨ et al., 2010). Pinus pinaster trees from a mixed grove of pines in Malhada, Oliveira do Hospital, Portugal, were sampled and the bark and sapwood of each cross-section were removed under sterile conditions for the study of the diversity of the endophytic community. Strain M47C3BT, forming mucoid, light-pink colonies, was isolated from dilutions of wood chips plated on R2A agar (Difco) incubated at 25 uC, for three days. The strain was maintained in the same medium supplemented with 15 % (v/v) glycerol at 280 uC after subculture and purification. 2223
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Cell morphology and motility were examined by phasecontrast microscopy (Leitz Diaplan, UK) after growth on R2A agar at 26 uC for 48 h. Growth on several bacteriological media was tested using R2A agar, tryptic soy agar (TSA; Difco), nutrient agar (NA; Difco) and R2 liquid (Difco) incubated at 26 uC for 5 days. The temperature range (4, 15, 20, 22, 26, 30, 37, 40, 42, 45 uC) and optimum temperature for growth were examined on R2A agar and in R2 liquid incubated for up to 5 days. Salt tolerance was tested in R2A agar and R2 liquid supplemented with NaCl at the final concentrations of 0 to 3 % (w/v), in 0.5 % increments (agar) and 0.1 % increments (liquid), at 26 uC, incubated for up to 5 days. The pH range for growth was examined at 26 uC in the same medium by using 50 mM MES (pH 3, 4, 5, 6, 7), HEPES (pH 6, 7, 8), TAPS (pH 8, 9) and CAPSO (pH 10) over a pH range from pH 5.0 to 10.0, with intervals of 0.5 pH units. The Gram-staining reaction and the presence of cytochrome oxidase and catalase were determined after 24 h of incubation on R2A agar as described by Smibert & Krieg (1994). The ability to hydrolyse agar aesculin, casein, xylan, gelatin, arbutin,
elastin, starch, DNA, chitin, and Tweens 20, 40, 60 and 80 at a concentration of 1.0 % (w/v or v/v) in R2A agar, after incubation at 26 uC for up to 5 days, was determined as described by Tindall et al. (2007). Other physiological properties and enzyme activities were determined using the API ZYM and API 20NE test strips (bioMe´rieux) at 26 uC according to the manufacturer’s instructions. Singlecarbon-source assimilation was determined using API 50 CH test strips (bioMe´rieux) after incubation at 26 uC for up to 7 days, as described by Morais et al. (2004). API 50 CH strips were also used for evaluation of acid production from single carbon sources after incubation at 26 uC for up to 5 days, according to the manufacturer’s instructions. The ability of the strain to oxidize different carbon sources was assessed using Biolog GN2 MicroPlates, incubated at 26 uC. The results were recorded daily for up to 7 days using a MicroPlate reader (Sunrise Xread Plus version V 4.30; Tecan). The test for flexirubin-like pigments was performed by soaking cells grown on R2A agar at 26 uC for 2 days with 20 % (w/v) KOH (Fautz & Reichenbach, 1980). Congo red adsorption was tested by using R2A-Congo red agar (25 mg
Table 1. Differential characteristics of strain M47C3BT compared with the type strains of Mucilaginibacter genus Strains: 1, M47C3BT (data from this study); 2, M. dorajii KACC 14556 T; 3, M. lappiensis ATCC BAA-1856T. All strains are positive for (API ZYM) alkaline phosphatase, leucine arylamidase, Valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase and are negative for lipase (C14), a-chymotrypsine. All strains cannot assimilate (API 20NE and API 50 CH) D-Mannitol. Data for reference strains are from Kim et al. (2010) and Ma¨nnisto¨ et al. (2010). +, positive; w+, weakly positive; –, negative; ND, not determined. Characteristic Colony colour Catalase/oxidase Cell length (mm) Temperature range (uC) Max. NaCl (%, w/v) tolerance pH range Acid production from: D-Arabinose D-Galactose D-Glucose N-Acetylglucosamine Amygdalin D-Cellobiose D-Maltose D-Lactose D-Trehalose Assimilation of: L-Arabinose N-acetylglucosamine Maltose D-Glucose D-Mannose Enzyme activities: Esterase C4 Esterase lipase C8 DNA G+C content (mol%)
2224
1
2
3
Light Pink +/+ 1–3 15–30 0.3 5.5–8.0
Light Yellow +/+ 1.1–1.8 4–30 1 5.0–8.0
Light Pink +/+ 1–3 0–31 1.5 4.5–8.0
– – 2 2 2 2 2 2 2
+ + + w+ w+ + + + +
+ + + w+ + + + + +
– – – – –
+ + + + w+
+ + + + w+
– – 40.64
w+ w+ 42.6
w+ + 43.5
International Journal of Systematic and Evolutionary Microbiology 64
Mucilaginibacter pineti sp. nov. from pine wood
Congo red l21) incubated at 26 uC for 2 days (Freeman et al., 1989). Antibiotic-sensitivity tests were performed for strain M47C3BT by using discs (Oxoid) containing the following: lincomycin (15 mg), ampicillin (10 mg), amoxicillin+clavulanic acid (30 mg), gentamicin (30 mg), polymyxin B (300 U), chloramphenicol (100 mg), erythromycin (15 mg), vancomycin (30 mg), streptomycin (50 mg), rifampicin (30 mg), tetracycline (30 mg) or kanamycin (30 mg). Cells for polar lipids and lipoquinone analysis were grown on R2A agar at 26 uC for 48 h and then harvested and lyophilized. Polar lipids were extracted and two-dimensional TLC was performed on silica gel G plates (10610 cm, 0.25 mm thickness; Merck) using chloroform/methanol/water (65 : 25 : 4, by vol.) in the first direction and chloroform/acetic acid/methanol/water (80 : 15 : 12 : 4, by vol.) in the second direction (da Costa et al., 2006). The polar lipids were identified using differential staining with molybdophosphoric acid (total polar lipids), ninhydrin (aminolipids) and anaphthol/sulfuric acid (glycolipids). Phosphatidylethanolamine was used as a standard. Lipoquinones were extracted from freeze-dried cells, purified by TLC and separated by HPLC (da Costa et al., 2011a). Cells for fatty acids analysis were grown on R2A agar in sealed plastic plates for 48 h (Morais et al., 2004). The fatty acid methyl esters (FAMEs) were obtained from fresh wet biomass and were separated, identified and quantified using the standard MIS Library Generation Software (Sherlock Microbial ID System, 2TSBA 6 database, version 6.0; MIDI) as described previously (da Costa et al., 2011b). The DNA G+C content of the genome was determined by HPLC as described by Mesbah et al. (1989). The 16S rRNA gene was amplified by PCR and sequenced as described by Morais et al. (2004), and aligned against representative reference sequences of the most closely related members of the genus Mucilaginibacter obtained from the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al., 2012c) and then aligned by SINA (v1.2.9) using the SILVA SEED as reference alignment (http://www.arb-silva.de/aligner/; Pruesse et al., 2012). Evolutionary distances were calculated (Jukes & Cantor, 1969), and phylogenetic dendrograms were reconstructed using the neighbour-joining (Saitou & Nei, 1987) and maximum-likelihood (Olsen et al., 1994) algorithms included in the ARB software package (Ludwig et al., 2004). Tree topologies were evaluated by performing bootstrap analysis (Felsenstein, 1985) of 1000 datasets by using the ARB software package. T
Strain M47C3B was Gram-staining-negative, non-sporeforming and appeared as rod-shaped cells (0.09–0.1 mm in width and 1–3 mm in length). After 48 h of incubation on R2A agar, colonies were circular, smooth, glistening and light pink. Biochemical and physiological characteristics of strain M47C3BT are summarized in Table 1 and in the species description. Strain M47C3BT had an optimum temperature for grow at 26 uC and was not able to grow at temperatures below 15 uC, in contrast to its closest relatives, M. dorajii and M. lappiensis (Table 1). Strain M47C3BT was able to grow from pH 5.5 up to pH 8.0 with optimal growth at pH 7, and http://ijs.sgmjournals.org
was positive for catalase and weakly positive for oxidase. Strain M47C3BT could grow in R2A agar and R2 liquid containing up to 0.3 % (w/v) NaCl, while its closest relatives grew with 1 to 1.5 % (w/v) NaCl. Colonies on R2A-Congo red agar were non-pigmented and flexirubin-type pigments were not formed. Strain M47C3BT was able to assimilate very few carbon sources in comparison with M. dorajii and M. lappiensis (Table 1). The strain was able to hydrolyse casein and starch, and was weakly urease-positive. The major polar lipids were phosphatidylethanolamine, three unidentified aminophospholipids, one unidentified aminolipid and three unidentified polar lipids. No glycolipids were detected (Fig. S1, available in the online Supplementary Material). The major fatty acids of strain M47C3BT were summed feature 3 (C16 : 1v7c/iso-C15 : 0 2-OH), iso-C15 : 0 and iso-C17 : 0 Table 2. Fatty acid composition of strain M47C3BT and the type strains of closely related members of the genus Mucilaginibacter Strains: 1, M47C3BT (data from this study); 2, M. dorajii KACC 14556T (Kim et al., 2010); 3, M. lappiensis ATCC BAA-1855T (Ma¨nnisto¨ et al., 2010). Values are percentages of total fatty acids. TR, Traces (,0.5 %); –, not detected. Fatty acid iso-C13 : 0 3-OH C14 : 0 C14 : 0 2-OH C15 : 0 C15 : 0 2-OH C15 : 0 3-OH iso-C15 : 0 iso-C15 : 0 3-OH C15 : 1v6c anteiso-C15 : 0 C16 : 0 C16 : 0 3-OH iso-C16 : 0 iso-C16 : 0 3-OH C16 : 1v5c C17 : 0 C17 : 0 3-OH iso-C17 : 0 iso-C17 : 0 3-OH C17 : 1v8c iso-C17 : 1v9c C18 : 0 C18 : 1v7c Summed feature 2* Summed feature 3* Summed feature 9*
1
2
3
– 0.7 0.3 0.8 – – 17.7 2.2 0.2 0.3 7.7 3.3 – – 6.5 – – 0.5 10.2 – – – – 0.2 45.5 2.2
0.9 1.2 – 6.5 1.2
TR
TR
22.6 1.3 1.2 0.8 6.5 2.3 1.2 0.6 4.7 0.6 0.6 TR
6.5 0.9 1.7 3.4 1.6 – 30.9 –
1.1 – 3.4 1.1 – 28.9 1.4 2.9 – 6.4 – – – 2.9 – – 0.8 7.8 TR
2.4 – – – 39.9 –
*Summed features combine groups of two or more fatty acids that cannot be separated by GLC with the MIDI system. Summed feature 2 contains C12 : 0 aldehyde, C14 : 0 3-OH and/or iso-C16 : 1. Summed feature 3 contains C16 : 1v7c and/or iso-C15 : 0 2-OH. Summed feature 9 contains iso-C17 : 1v9c and/or C16 : 0 10 methyl. 2225
G. Paiva and others
3-OH, which accounted for 73.5 % of the total fatty acids (Table 2). Menaquinone 7 (MK-7) was the major respiratory quinone. The almost complete 16S rRNA gene sequence of strain M47C3BT (1496 bp) was aligned with those of representative members of all species of the genus Mucilaginibacter of the family Sphingobacteriaceae, showing this organism to be a representative of a novel species belonging to the genus Mucilaginibacter. According to the phylogenetic tree reconstructed by using the neighbourjoining method, the closest phylogenetic relatives were M. dorajii KACC 14556T (97.2 %) and M. lappiensis ATCC BAA-1855T (97.0 %) (Fig. 1). The tree topology was confirmed by using a maximum-likelihood analysis (Fig. S2). The G+C content of the DNA, as determined by HPLC (Mesbah et al., 1989), was 40.6 mol%. The phenotypic characterization, enzymic activities, biochemical and physiological characteristics (Table 1), fatty acid profile (Table 2) and phylogenetic evidence indicate that strain M47C3BT represents a novel species of the genus Mucilaginibacter, for which we propose the name Mucilaginibacter pineti sp. nov. Description of Mucilaginibacter pineti sp. nov. Mucilaginibacter pineti (pi.ne9ti. L. gen. n. pineti of a pine wood, pine grove).
Cells on R2A agar are small, smooth, glistening and light pink. Cells are Gram-staining-negative, rod-shaped (0.09– 0.1 mm in width and 1–3 mm in length) after 48 h of incubation on R2A agar and non-spore-forming. Grows on NA and on R2A agar but not on TSA. Growth occurs between 15 and 30 uC (optimal at 26 uC). Growth occurs at pH 5.5–8.0 (optimal at pH 7). Growth occurs with up to 0.3 % (w/v) NaCl. Catalase- and oxidase-positive. Colonies are not coloured on R2A-Congo red agar and flexirubintype pigments are not formed. Resistant to ampicillin (10 mg), gentamicin (30 mg), polymyxin B (300 U) and kanamycin (30 mg), and susceptible to rifampicin (30 mg), streptomycin (50 mg), vancomycin (30 mg) and tetracycline (30 mg). Positive for hydrolysis of aesculin, starch and DNA (weakly positive). Negative for hydrolysis of xylan, elastin, arbutin, gelatin, Tween 20, Tween 40, Tween 60 and Tween 80. Negative for nitrate reduction, indole production and glucose fermentation. Positive for alkaline phosphatase, leucine arylamidase, valine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and afucosidase. Weakly positive for cystine arylamidase. Negative for esterase (C4), esterase lipase (C8), lipase (C14), achymotrypsin and b-glucuronidase (API ZYM). Positive for
Mucilaginibacter gossypiicola Gh-48T (EU672805) Mucilaginibacter gossypii Gh-67T (EU672804) Mucilaginibacter kameinonensis SCKT (AB330392) Mucilaginibacter oryzae B9T (EU109722) Mucilaginibacter angelicae GG-w14T (HM627214) Mucilaginibacter polysacchareus DRP28T (HM748604) Mucilaginibacter pineti M47C3BT (KF483876) Mucilaginibacter dorajii DR-f4T (GU139697) Mucilaginibacter lappiensis ANJLI2T (DQ234446) Mucilaginibacter composti TR6-03T (AB267719) Mucilaginibacter rigui WPCB133T (EU747841) 99 Mucilaginibacter lutimaris BR-3T (HQ455786) Mucilaginibacter soli R9-65T (JF701183) Mucilaginibacter litoreus BR-18T (JF999998) Mucilaginibacter gracilis TPT18T (AM490403) 97 Mucilaginibacter paludis TPT56T (AM490402) Mucilaginibacter frigoritolerans FT22T (FN400860) Mucilaginibacter gynuensis YC7003T (KC247157) Mucilaginibacter mallensis MP1X4T (FN400859) Mucilaginibacter myungsuensis HMD1056T (GQ144415) Mucilaginibacter ximonensis XM-003T (EU729366) Mucilaginibacter boryungensis BDR-9T (HM061614) Mucilaginibacter daejeonensis Jip 10T (AB267717) Mucilaginibacter jinjuensis YC7004T (JQ765855) Parasegetibacter luojiensis RHYL-37T (EU877263) Segetibacter koreensis Gsoil 664T (AB267478) Sediminibacterium salmoneum NJ-44T (EF407879) 72
0.10
72
86 71
Fig. 1. Phylogenetic dendrogram based on a comparison of the 16S rRNA gene sequences of M47C3BT strain and the closest phylogenetic relatives. The tree was created using the neighbour-joining method. Numbers indicate percentages of bootstrap sampling, derived from 1000 replications; values below 70 % are not shown. Filled circles indicate node branches conserved when the tree was reconstructed using the maximum-parsimony algorithm. The isolate characterized in this study is indicated in bold type. Bar, one inferred nucleotide substitution per 10 nt. 2226
International Journal of Systematic and Evolutionary Microbiology 64
Mucilaginibacter pineti sp. nov. from pine wood
aesculin and p-nitrophenyl-b-D-galactopyranoside (API 20 NE). Assimilates the carbon sources aesculin ferric citrate (API 20 NE and API50 CH), p-nitrophenyl-b-D-galactopyranoside (PNPG) (API 20 NE), and methyl a-D-mannopyranoside, arbutin, potassium 2-ketogluconate and potassium 5-ketogluconate (API 50 CH). The other organic substrates included in API 50 CH and API 20 NE are not utilized. Acid is produced from (API 50 CH) D-ribose, D-xylose, D-galactose, D-glucose, D-mannitol, D-sorbitol and aesculin ferric citrate. Acid is produced weakly from methyl bD-xylopyranoside, D-mannose, melibiose, sucrose, gentiobiose and potassium 5-ketogluconate. Tests using the Biolog GN2 system show dextrin is oxidized, a-cyclodextrin, glycogen, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, cellobiose, D-fructose, L-fucose, D-galactose, gentiobiose, a-D-glucose, a-lactose, lactulose, maltose, D-mannose, melibiose, methyl b-D-glucoside, D-psicose, raffinose, sucrose, trehalose, turanose, D-gluconic acid, D-glucosaminic acid, a-ketobutyric acid, L-alaninamide, L-alanine, L-alanyl glycine, L-glutamic acid, L-proline, L-serine and L-threonine are weakly oxidized, and the remaining substrates are not oxidized. The major components of the polar lipid profile of strain M47C3BT consisted of phosphatidylethanolamine, three unidentified aminophospholipids, one unidentified aminolipid and three unidentified polar lipids. No glycolipids are detected. The major fatty acids are summed feature 3 (C16 : 1v7c/iso-C15 : 0 2-OH), iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 0 and C16 : 1v5c. The major respiratory lipoquinone is menaquinone 7 (MK-7). The type strain, M47C3BT (5CIP 110632T5LMG 28160T) was isolated from the wood of a Pinus pinaster in Malhada, Coimbra, Portugal. The DNA G+C content of the type strain is 40.6 %.
Acknowledgements We are indebted to Professor J. P. Euze´by (E´cole National Ve´te´rinaire, Toulouse, France) for the etymology of the new organisms’ name. This research was financially supported by Program of National Action for Pine Wood Nematode control: ‘The disease of Pine Wood Nematode’, by Instituto de Financiamento da Agricultura e Pescas (IFAP) and Autoridade Florestal Nacional (AFN) and by the Fundac¸a˜o para a Cieˆncia e Tecnologia (FCT), Portugal, under the PTDC/AGR-CFL/115373/2009 project. G. P. was supported by FCT, Portugal, fellowship DRH36/11/448. D. N. P. was supported by FCT, Portugal, graduate fellowship SFRH/BD/61311/2009.
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