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 1IMAR-CMA, University of Coimbra, 3004-517 Coimbra, Portugal Paula V. Morais 2CNC-Center for Neuroscience and Cell Biology, University of Coimbra, [email protected] 3004-517 Coimbra, Portugal 3Department 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 Sphingobac- myungsuensis (Joung & Joh, 2011), M. angelicae (Kim teriaceae, phylum Bacteroidetes, was first described by et al., 2012a), M. litoreus (Yoon et al., 2012), M. lutimaris Pankratov et al. (2007) to group Gram-staining-negative, (Kim et al., 2012b), M. polysacchareus (Han et al., 2012), M. non-spore-forming and non-motile rods producing large soli (Jiang et al., 2012) and M. jinjuensis (Khan et al., 2013). amounts of extracellular polymeric substances. The genus Members of the genus Mucilaginibacter have been isolated was proposed to include two novel species, Mucilaginibacter from very different environments including peat bogs, soils, paludis and Mucilaginibacter gracilis. At the time of writing, dried rice straw, wetland freshwater (An et al., 2009; Baik the genus Mucilaginibacter comprises 22 species with validly et al., 2010; Madhaiyan et al., 2010; Pankratov et al., 2007; published names: M. paludis and M. gracilis (Pankratov Urai et al., 2008) and also cold environments (Ma¨nnisto¨ et al., 2007), M. kameinonensis (Urai et al., 2008), M. daejeo- et al., 2010). nensis (An et al., 2009), M. ximonensis (Luo et al., 2009), M. oryzae (Jeon et al., 2009), M. rigui (Baik et al., 2010), Pinus pinaster trees from a mixed grove of pines in Malhada, M. gossypii and M. gossypiicola (Madhaiyan et al., 2010), Oliveira do Hospital, Portugal, were sampled and the bark M. frigoritolerans, M. lappiensis and M. mallensis (Ma¨nnisto¨ and sapwood of each cross-section were removed under sterile conditions for the study of the diversity of the et al., 2010), M. boryungensis (Kang et al., 2011), M. composti T (Cui et al., 2011), M. dorajii (Kim et al., 2010), M. endophytic community. Strain M47C3B , forming mucoid, light-pink colonies, was isolated from dilutions of wood The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene chips plated on R2A agar (Difco) incubated at 25 uC, for sequence of strain M47C3BT is KF483876. three days. The strain was maintained in the same medium Two supplementary figures are available with the online version of this supplemented with 15 % (v/v) glycerol at 280 uC after paper. subculture and purification. 057737 G 2014 IUMS Printed in Great Britain 2223 G. Paiva and others Cell morphology and motility were examined by phase- elastin, starch, DNA, chitin, and Tweens 20, 40, 60 and 80 contrast microscopy (Leitz Diaplan, UK) after growth on at a concentration of 1.0 % (w/v or v/v) in R2A agar, after R2A agar at 26 uC for 48 h. Growth on several bacterio- incubation at 26 uC for up to 5 days, was determined as logical media was tested using R2A agar, tryptic soy agar described by Tindall et al. (2007). Other physiological (TSA; Difco), nutrient agar (NA; Difco) and R2 liquid properties and enzyme activities were determined using the (Difco) incubated at 26 uC for 5 days. The temperature API ZYM and API 20NE test strips (bioMe´rieux) at 26 uC range (4, 15, 20, 22, 26, 30, 37, 40, 42, 45 uC) and optimum according to the manufacturer’s instructions. Single- temperature for growth were examined on R2A agar and in carbon-source assimilation was determined using API 50 R2 liquid incubated for up to 5 days. Salt tolerance was CH test strips (bioMe´rieux) after incubation at 26 uC for tested in R2A agar and R2 liquid supplemented with NaCl up to 7 days, as described by Morais et al. (2004). API 50 at the final concentrations of 0 to 3 % (w/v), in 0.5 % CH strips were also used for evaluation of acid production increments (agar) and 0.1 % increments (liquid), at 26 uC, from single carbon sources after incubation at 26 uC for up incubated for up to 5 days. The pH range for growth was to 5 days, according to the manufacturer’s instructions. The examined at 26 uC in the same medium by using 50 mM ability of the strain to oxidize different carbon sources was MES (pH 3, 4, 5, 6, 7), HEPES (pH 6, 7, 8), TAPS (pH 8, assessed using Biolog GN2 MicroPlates, incubated at 26 uC. 9) and CAPSO (pH 10) over a pH range from pH 5.0 to The results were recorded daily for up to 7 days using a 10.0, with intervals of 0.5 pH units. The Gram-staining MicroPlate reader (Sunrise Xread Plus version V 4.30; reaction and the presence of cytochrome oxidase and Tecan). The test for flexirubin-like pigments was performed catalase were determined after 24 h of incubation on R2A by soaking cells grown on R2A agar at 26 uC for 2 days with agar as described by Smibert & Krieg (1994). The ability to 20 % (w/v) KOH (Fautz & Reichenbach, 1980). Congo red hydrolyse agar aesculin, casein, xylan, gelatin, arbutin, 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 1 2 3 Colony colour Light Pink Light Yellow Light Pink Catalase/oxidase +/++/++/+ Cell length (mm) 1–3 1.1–1.8 1–3 Temperature range (uC) 15–30 4–30 0–31 Max. NaCl (%, w/v) tolerance 0.3 1 1.5 pH range 5.5–8.0 5.0–8.0 4.5–8.0 Acid production from: D-Arabinose – ++ D-Galactose – ++ D-Glucose 2 ++ N-Acetylglucosamine 2 w+ w+ Amygdalin 2 w++ D-Cellobiose 2 ++ D-Maltose 2 ++ D-Lactose 2 ++ D-Trehalose 2 ++ Assimilation of: L-Arabinose – ++ N-acetylglucosamine – ++ Maltose – ++ D-Glucose – ++ D-Mannose – w+ w+ Enzyme activities: Esterase C4 – w+ w+ Esterase lipase C8 – w++ DNA G+C content (mol%) 40.64 42.6 43.5 2224 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 was positive for catalase and weakly positive for oxidase. et al., 1989). Antibiotic-sensitivity tests were performed for Strain M47C3BT could grow in R2A agar and R2 liquid strain M47C3BT by using discs (Oxoid) containing the containing up to 0.3 % (w/v) NaCl, while its closest relatives following: lincomycin (15 mg), ampicillin (10 mg), amox- grew with 1 to 1.5 % (w/v) NaCl. Colonies on R2A-Congo red icillin+clavulanic acid (30 mg), gentamicin (30 mg), poly- agar were non-pigmented and flexirubin-type pigments were myxin B (300 U), chloramphenicol (100 mg), erythromycin not formed. Strain M47C3BT was able to assimilate very few (15 mg), vancomycin (30 mg), streptomycin (50 mg), rifam- carbon sources in comparison with M. dorajii and M. picin (30 mg), tetracycline (30 mg) or kanamycin (30 mg). lappiensis (Table 1). The strain was able to hydrolyse casein and starch, and was weakly urease-positive. The major polar Cells for polar lipids and lipoquinone analysis were grown lipids were phosphatidylethanolamine, three unidentified on R2A agar at 26 uC for 48 h and then harvested and aminophospholipids, one unidentified aminolipid and three lyophilized.