Mucilaginibacter carri sp. nov., isolated from a car air conditioning system

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1 International Journal of Systematic and Evolutionary Microbiology (2016), 66, DOI /ijsem Mucilaginibacter carri sp. nov., isolated from a car air conditioning system Dong-Uk Kim, 1 Hyosun Lee, 1 Hyun Kim, 1 Song-Gun Kim, 2,3 So Yoon Park 4 and Jong-Ok Ka 1 Correspondence Jong-Ok Ka joka@snu.ac.kr 1 Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul , Republic of Korea 2 Resource Center/Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology, Daejeon , Republic of Korea 3 University of Science and Technology, Yuseong-gu, Daejeon , Republic of Korea 4 Research & Development Division, Hyundai Motor Group, Uiwang, , Republic of Korea A Gram-stain-negative, pink bacterial strain, designated PR0008K T, was isolated from an automobile evaporator core in Korea. The cells were obligately aerobic and rod-shaped. The strain grew at C (optimum, 20 8C), at ph 5 8 (optimum, 7), and in the presence of % (w/v) NaCl. Phylogenetically, the strain was closely related to members of the genus Mucilaginibacter ( % 16S rrna sequence similarities) and showed a high sequence similarity with Mucilaginibacter litoreus BR-18 T, Mucilaginibacter lutimaris BR-3 T and Mucilaginibacter soli R9-65 T (97.0 %, 96.9 % and 96.9 % 16S rrna sequence similarity, respectively). It contained summed feature 3 (C 16 : 1 v7c and/or C 16 : 1 v6c), C 16 : 0, iso-c 17 : 0 3-OH and C 16 : 0 as the predominant fatty acids and MK-7 as the major menaquinone. The polar lipids were phosphatidylethanolamine, one unknown aminophospholipid, two unknown aminolipids and two unknown polar lipids. The DNA G+C content of this strain was 47.4 mol%. Based on the phenotypic, genotypic and chemotaxonomic data, strain PR0008K T represents a novel species in the genus Mucilaginibacter, for which the name Mucilaginibacter carri sp. nov. (5KACC T 5NBRC T ) is proposed. The genus Mucilaginibacter is a member of the family Sphingobacteriaceae and was first proposed by Pankratov et al. (2007) and later amended by Urai et al. (2008) and Baik et al. (2010) on the basis of the classification of the family Sphingobacteriaceae. The genus possesses oxidase activity, can hydrolyse aesculin, pectin, starch and xylan, and the predominant quinone is MK-7. The major fatty acids are straight- and branched-chain saturated fatty acids. At the time of writing, the genus Mucilaginibacter contains 38 species with validly published names. These species have been isolated from various environments such as Arctic tundra, rhizosphere, soil, tidal flat sediment, marine sand, water and wetland (Baik et al., 2010; Jeon et al., 2009; Jiang et al., 2012; Joung et al., 2014; Joung et al., 2015; Kang et al., 2011; Kim et al., 2010; Kim et al., 2012a; Lee et al., 2013; Yoon et al., 2012). In this study, we describe a novel strain of a novel species of Abbreviations: AL, aminolipid; AP, aminophospholipid; L, lipid; PE, phosphatidylethanolamine. The GenBank/EMBL/DDBJ accession number for the 16S rrna gene sequence of strain PR0008K T is JX the genus Mucilaginibacter, designated PR0008K T, isolated from a car air conditioner that operated in Korea. The aim of this study was to determine the exact taxonomic position of this novel strain using a polyphasic approach. Strain PR0008K T was isolated from a biofilm growing on the surface of an automobile evaporator. The evaporator cores were disassembled from one sports utility vehicle and four sedans, which had been operating for more than one year in Korea. Aluminium heat exchanger fins were harvested from evaporator cores using sterilized long-nose pliers. A mass of 10 g of harvested aluminium fins were blended with 195 ml PBS using an automatic blender (max400, Hanil). Samples of 100 ml suspended PBS were plated on R2A agar using the standard dilution plating technique. After plating, plates were incubated at 28 8C for 7 days and a pink bacterial strain, PR0008K T, was isolated. The 16S rrna gene sequence was determined as described previously (Kim & Ka, 2014). Almost full-length similarity was determined using the EzTaxon server (Kim et al., 2012b) and sequences from PR0008K T and related taxa (retrieved from the NCBI database) were aligned with G 2016 IUMS Printed in Great Britain

2 Mucilaginibacter carri sp. nov. SINA (v1.2.11) according to the SILVA seed alignment [ Pruesse et al. (2012)]. The method of Jukes & Cantor (1969) was used to calculate evolutionary distances. Phylogenetic trees were reconstructed by neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony methods (Fitch, 1971) using the MEGA6 software program (Tamura et al. 2013). In each case, bootstrap values were calculated based on 1000 replications (Felsenstein, 1985). Bacterial growth at 4, 10, 15, 20, 25, 28, 37, 40 and 45 8C was assessed after 5 days of incubation in R2A broth. The ph range for growth was determined after 5 days of incubation at 28 8C in R2A broth adjusted to ph 5 10 at intervals of 1.0 ph unit. Tolerance to NaCl was tested in R2A broth supplemented with 0, 0.5, 1, 1.5 and 2.0 % NaCl (w/v) after 5 days of incubation. Growth of strain PR0008K T was tested on R2A agar (BD), Luria-Bertani agar (BD), nutrient agar (BD), trypticase soy agar (BD) and MacConkey agar (BD) at 28 8C for 5 days. Growth under anaerobic conditions was determined with R2A medium in a Bactron anaerobic/environmental chamber (Sheldon Manufacturing) and an anaerobic atmosphere generation bag (Thermo) and also with fluid thioglycollate medium (BD). The Gram reaction of strain PR0008K T was determined using a Colour Gram 2 kit (biomérieux). Cellular morphology was observed under an inverted light microscope (Zeiss; AXIO), with cells grown for 2 days at 28 8C on R2A agar. For negative staining, a 2 day-old culture grown on R2A (BD) at 28 8C was allowed to attach onto the carbon grid for 1 min and stained with 2 % (w/v) phosphotungstic acid for 1 min. The grids were inspected on a TEM (JEOL; JEM1010) at an operating voltage of 80 kv. For pigments analysis, cells grown on R2A agar for 2 days at 28 8C were scraped from the R2A agar surface and the KOH test was performed according to Bernardet et al. (2002). Oxidase and catalase activities were tested using oxidase reagent and ID colour catalase reagent, respectively (biomérieux). Degradation of casein, starch, DNA, chitin and cellulose was determined using the methods of Smibert & Krieg (1994) after 7 days of incubation. Other physiological and biochemical characteristics were examined with API 20E, API 20NE, API ZYM and API 50CH systems (biomérieux), according to the manufacturer s instruction. Cells of strain PR0008K T grown on R2A agar medium at 28 8C for 2 days were used for API tests. Fatty acids were extracted, methylated and separated by gas chromatography (model 6890; Hewlett Packard) according to the protocol of the Sherlock Microbial Identification System after strain PR0008K T and related species of the genus Mucilaginibacter were grown on R2A agar medium for 3 days at 25 8C in our laboratory. Fatty acid methyl esters were identified and quantified using the TSBA 6 database (version 6.10) of the Sherlock Microbial Identification System (MIDI). Fatty acid analysis was performed in triplicate. For isoprenoid quinone analysis, 100 mg of freeze-dried cells were treated with chloroform/methanol (2 : 1, v/v) overnight. Preparative TLC was performed using Kieselgel 60 F254 plates (20620 cm, 0.5 mm thick; Merck) with petroleum ether/diethyl ether (9 : 1, v/v) as the solvent. The resulting band was marked under short-wavelength UV light, scraped from the plate and redissolved in acetone. Finally, the menaquinone profile was analysed using reversed-phase HPLC (LC20AD system; Shimadzu) with an ODS-2 C18 column ( mm, Phenomenex) and a UV detector at 270 nm. Polar lipids were extracted and separated using twodimensional TLC according to the method of Komagata & Suzuki (1987). To identify the specific moieties of lipids, the following spraying methods were applied: 0.5 % (w/v) alpha-naphthol in methanol and water (1 : 1, v/v) followed by spraying with 95 % (v/v) sulfuric acid for glycolipids, 0.25 % (w/v) ninhydrin in acetone for amino lipids, molybdenum blue reagent (Sigma) for phospholipids (Counsell & Murray, 1986) and 5 % (w/v) molybdophosphoric acid hydrate (Merck) in ethanol for total lipids. Determination of the DNA G+C content (mol%) was examined by real-time PCR analysis (Gonzalez & Saiz- Jimenez, 2002). Strain PR0008K T was deposited in the Korea Agricultural Culture Collection (KACC) of South Korea and the NITE Biological Resource Center (NBRC) of Japan. Sequence analyses of the 16S rrna gene showed that strain PR0008K T has 16S rrna sequence similarities of % with members of the genus Mucilaginibacter. Only strain Mucilaginibacter litoreus BR-18 T (97.0 %), showed more than 97 % 16S rrna sequence similarity with the isolate. The strain showed 16S rrna sequence similarities of nearly 97 % with Mucilaginibacter lutimaris BR-3 T (96.9 %) and Mucilaginibacter soli R9-65 T (96.9 %). Therefore, these three strains of species of the genus Mucilaginibacter were used as reference strains in this study. The neighbour-joining tree showed that strain PR0008K T was grouped with members of the genus Mucilaginibacter (Fig. 1). Cells of strain PR0008K T were Gram-stain-negative, obligately aerobic, rod-shaped and mm in size. Colonies were circular with entire edges, convex, transparent, shiny and pinkish on R2A agar plates. The strain grew on Luria-Bertani agar (BD), trypticase soy agar (BD), nutrient agar (BD) and R2A agar (BD), but not on MacConkey agar (BD). The temperature range for growth was C, with optimum growth at 20 8C. The ph range for growth was ph 5 8, with optimum growth at ph 7. The NaCl tolerance range was % (w/v). Flexirubin-type pigments were not produced. The strain was positive for catalase and oxidase. Casein and starch were hydrolysed in contrast to chitin, cellulose and DNA. The results, showing the morphological, physiological and biochemical characteristics that differentiate strain PR0008K T from closely related species of the genus Mucilaginibacter are listed in Table

3 D.-U. Kim and others Mucilaginibacter sabulilitoris SMS-12 T (JQ739458) Mucilaginibacter lappiensis ANJLI2 T (DQ234446) Mucilaginibacter pineti M47C3B T (KF483876) Mucilaginibacter dorajii DR-f4 T (GU139697) Mucilaginibacter flavus HME6839 T (HQ449707) Mucilaginibacter soyangensis HME6664 T (HM590829) Mucilaginibacter ximonensis XM-003 T (EU729366) Mucilaginibacter calamicampi WR-R1Y T (KC537738) Mucilaginibacter auburnensis JM-1070 T (KF892548) Mucilaginibacter composti TR6-03 T (AB267719) Mucilaginibacter myungsuensis HMD1056 T (GQ144415) Mucilaginibacter boryungensis BDR-9 T (HM061614) Mucilaginibacter gynuensis YC7003 T (KC247157) Mucilaginibacter mallensis MP1X4 T (FN400859) Mucilaginibacter frigoritolerans FT22 T (FN400860) Mucilaginibacter gracilis TPT18 T (AM490403) 100 Mucilaginibacter paludis DSM T (AEIH ) Mucilaginibacter polysacchareus DRP28 T (HM748604) Mucilaginibacter angelicae GG-w14 T (HM627214) Mucilaginibacter oryzae B9 T (EU109722) Mucilaginibacter gossypii Gh-67 T (EU672804) Mucilaginibacter gossypiicola Gh-48 T (EU672805) Mucilaginibacter kameinonensis SCK T (AB330392) Mucilaginibacter lutimaris BR-3 T (HQ455786) Mucilaginibacter rigui WPCB133 T (EU747841) Mucilaginibacter litoreus BR-18 T (JF999998) Mucilaginibacter carri PR0008K T (JX089330) Mucilaginibacter soli R9-65 T (JF701183) Mucilaginibacter defluvii A5 T (JQ955677) Mucilaginibacter herbaticus DR-9 T (JN695632) Mucilaginibacter koreensis TF8 T (JQ966281) Mucilaginibacter daejeonensis Jip 10 T (AB267717) Mucilaginibacter jinjuensis YC7004 T (JQ765855) 98 Mucilaginibacter polytrichastri RG4-7 T (KC560020) Fig. 1. Neighbour-joining phylogenetic tree reconstructed from comparative analysis of 16S rrna gene sequences showing the relationship between strain PR0008K T and members of the genus Mucilaginibacter. Bootstrap values (expressed as percentages of 1000 replications) greater than 50 % are shown at branching points. The dots indicate that the corresponding branches were also recovered in the maximum-parsimony tree. Bar, nt substitutions per position. Cellular fatty acid profiles of strain PR0008K T, M. litoreus KCTC T, M. lutimaris KCTC T and M. soli KACC T are shown in Table 2. All species contained summed feature 3 (C 16 : 1 v7c and/or C 16 : 1 v6c), iso-c 15 : 0, and iso-c 17 : 0 3-OH as the major cellular fatty acids. The predominant fatty acids of strain PR0008K T were similar to those of members of genus Mucilaginibacter. The predominant menaquinone of strain PR0008K T was MK-7, which was typical of members of the genus Mucilaginibacter. The polar lipids of strain PR0008K T were phosphatidylethanolamine (PE), one unknown aminophospholipid (AP), two unknown aminolipids (AL1-2) and five unknown polar lipids (L1 2) (Fig. 2). PE was the predominant polar lipid of strain PR0008K T and other species of the genus Mucilaginibacter (Jiang et al., 2012; Kim et al., 2012a; Yoon et al., 2012). The DNA G+C content of strain PR0008K T was 47.4 mol%, which lies within the ranges for all other species of the genus Mucilaginibacter with validly published names International Journal of Systematic and Evolutionary Microbiology 66

4 Mucilaginibacter carri sp. nov. Table 1. Differential phenotypic characteristics of strain PR0008K T and related species of the genus Mucilaginibacter Strains: 1, PR0008K T ;2,M. lutimaris KCTC T ;3,M. soli KACC T ;4,M. litoreus KCTC T. All data were obtained from this study. All strains were positive in tests for acetoin production, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-as-biphosphohydrolase, a-glucosidase, and N-acetyl-b-glucosaminidase. All strains were negative in tests for b-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, lipase (C14), trypsin, a-chymotrypsin, citrate utilization, H 2 S production, urease, tryptophan deaminase, indole production, fermentation/oxidation of D-mannitol, inositol, D-sorbitol, L-rhamnose, NO 2 production, assimilation of D-mannitol, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. All strains were weakly positive for the utilization of D-galactose. All strains were negative for the utilization of glycerol, erythritol, L-xylose, D-adonitol, methyl b-d-xylopyranoside, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, glycogen, xylitol, D-fucose, D- and L-arabitol, potassium gluconate and potassium 2-ketogluconate. +, Positive; (+), weakly positive; 2, negative. Characteristic Hydrolysis of: Gelatin Aesculin (+) Fermentation/oxidation of: D-Glucose D-Sucrose D-Melibiose (+) + (+) 2 Amygdalin 2 + (+) 2 L-Arabinose (+) Assimilation of: D-Glucose (+) + (+) 2 L-Arabinose (+) D-Mannose (+) + (+) 2 N-Acetyl-D-glucosamine D-Maltose (+) Enzyme activities: Cystine arylamidase a-galactosidase b-galactosidase b-glucuronidase b-glucosidase a-mannosidase (+) a-fucosidase + (+) (+) 2 Utilization of: D-Arabinose (+) L-Arabinose (+) 2 (+) 2 D-Ribose 2 (+) (+) 2 D-Xylose 2 (+) (+) 2 D-Glucose (+) D-Fructose (+) D-Mannose (+) L-Sorbose 2 2 (+) (+) Methyl a-d-mannopyranoside (+) + (+) 2 Methyl a-d-glucopyranoside (+) N-Acetylglucosamine 2 + (+) 2 Table 1. cont. Characteristic Amygdalin Arbutin (+) Aesculin Salicin (+) D-Cellobiose D-Maltose D-Lactose D-Melibiose (+) + (+) 2 D-Saccharose (Sucrose) (+) + + (+) D-Trehalose (+) + (+) 2 Inulin 2 (+) 2 2 D-Melezitose 2 (+) 2 2 D-Raffinose 2 + (+) 2 Amidon (Starch) (+) (+) (+) 2 Gentiobiose (+) D-Turanose (+) + (+) 2 D-Lyxose 2 (+) (+) 2 D-Tagatose 2 (+) 2 2 L-Fucose 2 (+) (+) 2 Potassium 5-ketogluconate (+) Table 2. Cellular fatty acid profiles of PR0008K T and related species of the genus Mucilaginibacter Strains: 1, PR0008K T ;2,M. lutimaris KCTC T ;3,M. soli KACC T ;4,M. litoreus KCTC T. All data were obtained from this study. All strains were collected after growth on R2A agar at 25 8C for 3 days. Values are percentages of total fatty acids, and only fatty acids representing more than 1 % for at least one of the strains are shown., Not detected; TR, trace amounts (,1 %). Fatty acid iso-c 15 : C 16 : 1 v5c C 16 : iso-c 15 : 0 3-OH iso-c 17 : C 16 : 0 3-OH 1.9 TR 1.7 iso-c 17 : 0 3-OH *Summed features *Summed features represent groups of two or three fatty acids that cannot be separated using the MIDI system. Summed feature 3 comprises C 16 : 1 v7c and/or C 16 : 1 v6c; summed feature 4 comprises iso-c 17 : 1 I and/or anteiso B; summed feature 9 comprises iso-c 17 : 1 v9c and/or C 16 : 0 10-methyl

5 D.-U. Kim and others AP AL1 L1 AL2 Fig. 2. Two-dimensional TLC showing the total polar lipid profile of strain PR0008K T. PE, phosphatidylethanolamine; AP, unknown aminophospholipid; AL1 2, unknown aminolipid; L1 2, unknown polar lipid (that did not stain with any of the specific spray reagents applied to detect a phosphate or an amino moiety). In conclusion, strain PR0008K T has the main characteristics of species of the genus Mucilaginibacter. Strain PR0008K T is Gram-stain-negative and produces non-diffusible yellow pigments, which are not of the flexirubin-type. In addition, phylogenetic analysis (based on 16S rrna sequences), major fatty acid composition, the predominant menaquinone and the range of G+C contents support strain PR0008K T being a member of the genus Mucilaginibacter. However, the strain is distinguishable from other species of the genus Mucilaginibacter due to a combination of physiological and biochemical features. Therefore, on the basis of the polyphasic data presented here, we suggest that strain PR0008K T represents a novel species of the genus Mucilaginibacter, for which we propose the name Mucilaginibacter carri sp. nov. L2 Description of Mucilaginibacter carri sp. nov. Mucilaginibacter carri (car ri. L. gen. n. carri of a vehicle). Cells are Gram-stain-negative, obligately aerobic, rodshaped, mm in size after cultivation for 3 days at 28 uc on R2A agar medium. Colonies on R2A agar medium are circular with entire edges, convex, transparent, shiny and pinkish. Non-diffusible yellow pigments, which are not of the flexirubin-type are produced. Aerobic growth occurs on Luria-Bertani agar, trypticase soy agar, nutrient agar and R2A agar, but not on MacConkey agar. Growth occurs aerobically at uc (optimum,20uc) PE and at ph 5 8 (optimum, 7). Cells grow in the absence of NaCl, but can tolerate up to 1.5 % (w/v) NaCl. Catalasepositive and oxidase-positive. Casein, starch, aesculin and gelatin are hydrolysed, but chitin, cellulose, DNA and arginine are not hydrolysed. Nitrate is not reduced to nitrite. Acetoin is produced, but indole and H 2 Sarenotproduced. The strain produces alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-as- BI-phosphohydrolase, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-bglucosaminidase, a-mannosidase and a-fucosidase. The strain does not produce lipase (C14), trypsin, a-chymotrypsin, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urease or tryptophan deaminase. The following substrates are not assimilated: D-mannitol, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. Positive for fermentation/oxidation of D-glucose, sucrose, melibiose (weakly) and L-arabinose (weakly). Negative for the fermentation/oxidation of D-mannitol, inositol, D-sorbitol, L-rhamnose and amygdalin. Positive for the utilization of D-arabinose (weakly), L-arabinose (weakly), D-galactose (weakly), D-glucose, D-mannose, methyl a-d-mannopyranoside (weakly), methyl a-d-glucopyranoside (weakly), arbutin (weakly), aesculin, salicin (weakly), D-cellobiose, maltose, lactose, melibiose (weakly), sucrose (weakly), trehalose (weakly), starch (weakly), gentiobiose (weakly) and turanose (weakly). Negative for the utilization of glycerol, erythritol, D-ribose, D-xylose, L-xylose, D-adonitol, methyl b-d-xylopyranoside, D-fructose, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, N-acetylglucosamine, amygdalin, inulin, melezitose, raffinose, glycogen, xylitol, D-lyxose, D-tagatose, D- and L-fucose, D- andl-arabitol, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketogluconate. The major cellular fatty acids are summed feature 3 (C 16 : 1 v7c and/or C 16 : 1 v6c), iso-c 15 : 0 and iso-c 17 : 0 3-OH. The major menaquinone is MK-7. The polar lipids are phosphatidylethanolamine, an unknown aminophospholipid, two unknown aminolipids and two unknown polar lipids. The type strain, PR0008K T (5KACC T 5NBRC T ), was isolated from an automobile evaporator core in Korea. The DNA G+C content of the type strain is 47.4 mol%. Acknowledgements This work was supported by a grant from the Regional SubGenBank Support Program of Rural Development Administration, Republic of Korea. References Baik, K. S., Park, S. C., Kim, E. M., Lim, C. H. & Seong, C. N. (2010). Mucilaginibacter rigui sp. nov., isolated from wetland freshwater, and emended description of the genus Mucilaginibacter. Int J Syst Evol Microbiol 60, Bernardet, J. F., Nakagawa, Y., Holmes, B. & Subcommittee on the taxonomy of Flavobacterium and Cytophaga-like bacteria of the 1758 International Journal of Systematic and Evolutionary Microbiology 66

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