Mariniluteicoccus endophyticus sp. nov., an endophytic actinobacterium isolated from root of Ocimum basilicum

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1 International Journal of Systematic and Evolutionary Microbiology (2016), 66, DOI /ijsem Mariniluteicoccus endophyticus sp. nov., an endophytic actinobacterium isolated from root of Ocimum basilicum Bing-Bing Liu, 1,2 3 Wei Chen, 3 3 Xiao Chu, 2 Ying Yang, 3 Nimaichand Salam, 1 Wei-Yao Hu, 3 Rui Gao, 3 Yan-Qing Duan 3 and Wen-Jun Li 1,2 Correspondence Yan-Qing Duan dyanqing@aliyun.com Wen-Jun Li liwenjun3@mail.sysu.edu.cn 1 State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou , PR China 2 Yunnan Institute of Microbiology, Yunnan University, Kunming, , PR China 3 China Tobacco Yunnan Industrial Co. Ltd, Kunming, , PR China Two strains of Gram-stain-positive, aerobic, non-motile, irregular coccus-shaped bacteria, designated YIM 2617 T and YIM , were isolated from the root of Ocimum basilicum. Phylogenetic analysis on the basis of 16S rrna gene sequence comparison revealed that the two strains were closely related to Mariniluteicoccus flavus YIM M13146 T (98.1 and 98.0 % similarities, respectively) and formed a robust clade with M. flavus in the neighbour-joining tree. Optimum growth of the strains was observed at C, at ph and in the presence of 0 2 % (w/v) NaCl. The chemotaxonomic profiles of the strains comprised anteiso-c 15 : 0 as the major cellular fatty acid, MK-9(H 4 ) as the predominant menaquinone, alanine, glycine, glutamic acid and LL-diaminopimelic acid as peptidoglycan components, and phosphatidylcholine and diphosphatidylglycerol as the major polar lipids. The G+C contents of the genomic DNA of strains YIM 2617 T and YIM were 66.4 and 66.6 mol%, respectively. DNA DNA hybridizations of the two strains with M. flavus YIM M13146 T gave mean relatedness values of and %, respectively, while the relatedness value between them was %. On the basis of the data recorded from the present study, strains YIM 2617 T and YIM represent a novel species of the genus Mariniluteicoccus, for which the name Mariniluteicoccus endophyticus sp. nov. is proposed. The type strain is YIM 2617 T (5KCTC T 5DSM T 5JCM T ). The genus Mariniluteicoccus was first reported by Zhang et al. (2014) for a novel actinobacterium isolated from marine sediment collected at a depth of 2439 m of the South China Sea. The genus is affiliated to the family Propionibacteriaceae and comprised, at the time of writing, a single species, Mariniluteicoccus flavus (Zhang et al., 2014). The major chemotaxonomic characteristics of this genus are the presence of anteiso-c 15 : 0 as the major cellular fatty acid, MK-9(H 4 ) as the predominant respiratory menaquinone, and alanine, glycine, glutamic acid and LL-diaminopimelic acid in the peptidoglycan. The major polar lipids 3These authors contributed equally to this work. The GenBank/EMBL/DDBJ accession numbers for the 16S rrna gene sequences of strains YIM 2617 T and YIM are KP and KP793092, respectively. One supplementary table and three supplementary figures are available with the online Supplementary Material. comprised phosphatidylcholine, diphosphatidylglycerol, unidentified polar lipids and several unidentified glycolipids. Endophytes are bacteria that reside within the living tissues of plants without substantially harming them (Misaghi & Donndelinger, 1990). Associations between plants and micro-organisms are very complex. For instance, rhizobacteria show beneficial effects on plant growth via the provision of essential elements, inhibition of colonization by pathogenic micro-organisms or by helping the plant to overcome stress responses to environmental insults (Hallmann et al., 1997). Strains YIM 2617 T and YIM were isolated from a root sample of Ocimum basilicum collected from Impression Tobacco farm in Shilin County, Yunnan Province, south-west China ( E N). The root samples were washed with double distilled water to remove adhering soil particles, and surface sterilized as described by Li et al. (2008). Subsequently, these samples G 2016 IUMS Printed in Great Britain

2 Mariniluteicoccus endophyticus sp. nov. were ground in liquid nitrogen, shaken in saline solution (0.9 %, w/v, NaCl) and centrifuged at 73 g to remove the plant tissues. Aliquots (120 ml) of the supernatant were added to Biolog EcoPlates and incubated at 30 8C for 2 weeks. Chambers containing arginine as substrate changed in colour to blue, indicating the utilization of arginine as a carbon source. The aliquot from this chamber was then diluted to concentration and spread onto plant agar 11 (PA11) medium which contained the following (g l 21 ): soluble starch (5), arginine (2), KNO 3 (1), K 2 HPO 4.3H 2 O (0.5), MgSO 4.7H 2 O (0.5), CaCO 3 (1), NaCl (5) and trace salts solution (1 ml). The ph of the medium was adjusted to 7.0. The trace salts solution was made up of the following salts (g l 21 ): FeSO 4.7H 2 O (0.02), MnCl 2.2H 2 O (0.01), ZnSO 4.7H 2 O (0.01) and CuSO 4.5H 2 O (0.01). Inoculated plates were then incubated at 30 8C for 3 weeks. Two yellow-coloured colonies, designated strains YIM 2617 T and YIM , were selected from among other colonies for further study. These strains were stored as 20 % (v/v) glycerol suspensions at 280 8C. The phenotypic features of the isolates were determined on trypticase soy agar (TSA; Difco), unless otherwise stated. Gram staining was carried out by the non-staining method using 3 % KOH (Buck, 1982). Cell motility was established by the development of turbidity in a test tube containing semi-solid medium (Leifson, 1960). Morphological characteristics were characterized using light microscopy (BH-2; Olympus) and scanning electron microscopy (Quanta 200; FEI). The temperature range for growth was examined in TSA incubated at 4, 10, 15, 20, 28, 30, 32, 37, 42, 45 and 50 8C. Tolerance to NaCl was determined after 5 days of growth on TSA medium supplemented with 0 7 % (w/v) NaCl (at intervals of 0.5 %). The initial ph range for growth (ph ) was examined at 30 8C in tryptose soy broth (TSB; Difco) by using the following buffer systems: ph , 0.1 M citric acid/0.1 M sodium citrate; ph , 0.1 M KH 2 PO 4 /0.1 M NaOH; ph , 0.1 M NaHCO 3 /0.1 M Na 2 CO 3. Hydrolysis of starch, casein, gelatin and Tweens 20, 40, 60 and 80 was tested according to the methods of Smibert & Krieg (1994). Anaerobic growth was tested on TSA medium at 30 8C for 21 days using the Whitley A35 anaerobic workstation (Don Whitley Scientific). Oxidase and catalase activities were determined using oxidase reagent (biomérieux) and based on bubble production in 5 % (v/v) H 2 O 2, respectively. Carbon and nitrogen source utilization experiments were carried out as described by Gordon et al. (1974). Antibiotic susceptibility was examined using antibiotic discs on TSA medium (Groth et al., 2004). Other physiological and enzyme activities were determined with the API 20NE, API 50CH and API ZYM strips (biomérieux) according to the manufacturer s instructions. The tests were performed simultaneously for the two strains along with M. flavus YIM T grown under similar experimental conditions. The amino acid compositions in peptidoglycan hydrolysates were analysed by TLC (Hasegawa et al., 1983) and HPLC (Tang et al., 2009). For cellular fatty acid analysis, strains YIM 2617 T and YIM were grown on TSA for 3 days. The resulting biomass was harvested, methylated and analysed by using the Microbial Identification System (Sherlock Version 6.1; MIDI database: TSBA6). Menaquinones were extracted following 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 as described by Embley & Wait (a) (b) Fig. 1. Electron microscopy of cells of strains YIM 2617 T (a) and YIM (b). The strains were grown on TSA medium for 5 days at 28 8C. Bar, 5 mm

3 B.-B. Liu and others (1994). The DNA G+C content was measured using the HPLC method of Mesbah et al. (1989). Taxonomic relationships were examined further by DNA DNA hybridization according to the fluorometric micro-well method (Ezaki et al., 1989; Christensen et al., 2000). Genomic DNA extraction, PCR amplification and sequencing of the 16S rrna gene were carried out according to the method of Li et al. (2007). The identification of phylogenetic neighbours and calculation of sequence similarities were achieved via the EzTaxon-e server (Kim et al., 2012). Multiple alignments were further performed with related taxa obtained from public databases using the CLUSTAL X software package (Thompson et al., 1997). Evolutionary distances were calculated using Kimura s two-parameter model (Kimura, 1983). Phylogenetic trees were reconstructed by the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Fitch, 1971) and maximumlikelihood (Felsenstein, 1981) methods using the software package MEGA version 5.0 (Tamura et al., 2011). The topologies of the phylogenetic trees were evaluated by the bootstrap analysis of Felsenstein (1985) with 1000 replicates. Cells of strains YIM 2617 T and YIM were Gramstain-positive and had irregular coccus shapes with size ranging between 0.7 and 1.1 mm (Fig. 1). Growth was observed at C (optimum C), with % (w/v) NaCl (optimum 0 2 % NaCl) and at ph (optimum ph ). The strains were positive for catalase, oxidase and nitrate reduction tests. The strains were susceptible to the following antibiotics (mg per disc, unless otherwise indicated): amikacin (30), cefuroxime sodium (30), chloramphenicol (30), ciprofloxacin (5), erythromycin (15), norfloxacin (10), novobiocin (30), penicillin (10 IU), piperacillin (100), tetracycline (30) and vancomycin (30) but resistant to ethylhydrocupreine (5), gentamicin (10), oxacillin (1), polymyxin B (300 IU) and sulfamethoxazode/trimethoprim (23.75/1.25). Detailed physiological characteristics are given in the species description. Phenotypic properties useful in distinguishing the two strains from M. flavus YIM M13146 T are listed in Table 1. Analysis of the almost-complete 16S rrna gene sequence indicated that strains YIM 2617 T and YIM belong to the genus Mariniluteicoccus. Comparison of the sequences with related 16S rrna gene sequences retrieved from the GenBank/EMBL/DDBJ databases clearly demonstrated that the two strains showed closest sequence similarity to M. flavus YIM M13146 T (98.1 and 98.0 %, respectively). However, the level of 16S rrna gene sequence similarity between strains YIM 2617 T and YIM was 99.1 %. Phylogenetic analysis showed that strains YIM 2617 T and YIM were grouped with the only recognized member of the genus Mariniluteicoccus and together formed a distinct clade with M. flavus YIM M13146 T in all three phylogenetic trees (Fig. 2 and Figs S1 and S2, available in the online Supplementary Material). Table 1. Differential phenotypic and biochemical characteristics between strains YIM 2617 T and YIM with their closest phylogenetic neighbour Strains: 1, M. flavus YIM M13146 T ; 2, YIM 2617 T ; 3, YIM All strains were grown on TSA medium for 5 days. +, Positive; 2, negative; W, weakly positive. Characteristic Cell morphology (size, mm) Isolation source Cocci, single/ pairs/triads ( ) Marine sediment Cocci ( ) Root of Ocimum basilicum Cocci ( ) Root of Ocimum basilicum Growth temperature (8C) Optimum Range Optimum ph for growth NaCl for growth (%, w/v) Optimum Range Hydrolysis of: Tweens 20, 40, Tween 80 2 W W Gelatin Oxidase Catalase API ZYM results Trypsin a-chymotrypsin b-galactosidase Utilization of: Adipate Arginine Aspartic acid D-Galactose L-Rhamnose Raffinose Trehalose DNA G+C content (mol%) Strains YIM 2617 T and YIM had identical chemotaxonomic characteristics, which were in accordance with those of M. flavus YIM M13146 T. The TLC patterns indicated that strains YIM 2617 T and YIM contained LL-diaminopimelic acid as the major diagnostic diamino acid. Further analysis by HPLC showed the presence of alanine, glycine and glutamic acid in the peptidoglycan hydrolysates. The major cellular fatty acids (.1 %) of strains YIM 2617 T and YIM were anteiso-c 15 : 0, iso- C 15 : 0, anteiso-c 13 : 0, iso-c 14 : 0, iso-c 16 : 0, C 16 : 0 and summed feature 4 (iso-c 17 : 1 I and/or anteiso-c 17 : 1 B) (Table S1). The fatty acid profiles of the two strains were in agreement with that of M. flavus YIM M13146 T. The predominant menaquinone of the two strains 1308 International Journal of Systematic and Evolutionary Microbiology 66

4 Mariniluteicoccus endophyticus sp. nov * 83* 83* 89* * 86* 99* 98* 100 * 87 Propionicimonas paludicola DSM T (FR733712) Micropruina glycogenica JCM T (AB012607) Propionicicella superfundia DSM T (DQ176646) Naumannella halotolerans WS4616 T (FR832425) Auraticoccus monumenti MON 2.2 T (FN552748) Friedmanniella antarctica DSM T (Z78206) Microlunatus panaciterrae DSM T (AB271051) Propioniferax innocua ATCC T (AF227165) Granulicoccus phenolivorans JCM T (AY566575) Mariniluteicoccus flavus YIM M13146 T (KF564278) Mariniluteicoccus endophyticus YIM 2617 T (KP793091) Mariniluteicoccus endophyticus YIM (KP793092) Luteococcus japonicus DSM T (Z78208) Aestuariimicrobium kwangyangense JCM T (DQ830982) Tessaracoccus lubricantis DSM T (FM178840) Propionimicrobium lymphophilum DSM 4903 T (AJ003056) Brooklawnia cerclae JCM T (DQ ) Propionibacterium freudenreichii ATCC 6207 T (X53217) Propioniciclava tarda DSM T (AB298731) Glycomyces harbinensis ATCC T (D85483) Nocardiopsis dassonvillei DSM T (CP002040) Fig. 2. Neighbour-joining phylogenetic tree based on nearly complete 16S rrna gene sequences of strains YIM 2617 T and YIM and some related members of the family Propionibacteriaceae. Percentage bootstrap values.50 % (based on 1000 resamplings) are given at branch points. GenBank accession numbers of the type strains of each species are given in parentheses. Bar, 0.01 substitutions per nucleotide position. Asterisks indicate branches that were also recovered in the maximum-parsimony and minimum-evolution trees (see Figs S1 and S2). was MK-9(H 4 ). Polar lipids of strains YIM 2617 T and YIM comprised phosphatidylcholine, diphosphatidylglycerol, one unidentified phospholipid, seven unidentified glycolipids and four unidentified polar lipids (Fig. S3). The genomic DNA G+C contents were 66.4 and 66.6 mol%, respectively. Mean levels of DNA DNA relatedness between YIM 2617 T and YIM and M. flavus YIM M13146 T were and %, respectively, which are significantly lower than the threshold value of 70 % for species delineation (Stackebrandt & Goebel, 1994). However, the DNA DNA relatedness between YIM 2617 T and YIM was %, indicating that the two strains should be assigned to the same species. Based on the results from the phenotypic (Tables 1 and S1), chemotaxonomic and phylogenetic analysis, strains YIM 2617 T and YIM are considered to represent a novel species of the genus Mariniluteicoccus, for which the name Mariniluteicoccus endophyticus sp. nov. is proposed. Description of Mariniluteicoccus endophyticus sp. nov. Mariniluteicoccus endophyticus (en.do.phy9ti.cus. Gr. pref. endo within; Gr. n. phyton plant; L. masc. suff. -icus adjectival suffix used with the sense of belonging to; N.L. masc. adj. endophyticus within plant, endophytic, pertaining to the isolation from plant tissues). Gram-stain-negative, non-motile, irregular cocci ( mm in diameter). Strictly anaerobic. Grows at ph (optimum, ) and at uc (optimum, uc). Grows in the presence of % (w/v) NaCl (optimum, 0 2 %). Positive for oxidase and catalase activities but negative for H 2 S and indole production tests. Nitrate is reduced to nitrite. Hydrolyses Tweens 20, 40, 60 and 80 but not starch, gelatin or casein. Utilizes adipate, L-alanine, L-arginine, L-aspartate, cellobiose, D-fructose, gluconate, D-glucose, glycerol, maltose, malic acid, D-mannose, raffinose, L-rhamnose, proline, starch, sucrose and trehalose as the sole carbon or nitrogen source, but not D-arabinose, N-acetylglucosamine, capric acid, citric acid, lactose, lysine, D-mannitol, phenylacetic acid, sorbitol, sorbose, xylitol or xylose. Variable utilization of D-galactose as carbon source. In the API ZYM system, positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase, naphthol-as-bi-phosphohydrolase, a-glucosidase and b-glucosidase, but negative for lipase (C14), a-galactosidase, b-galactosidase, b-glucuronidase, N-acetyl-b-glucosaminidase, a-mannosidase and b-fucosidase. In the API 50 CHB system, acid is produced from arbutin, aesculin, cellobiose, D-fructose, gentiobiose, D-glucose, glycerol, glycogen, maltose,

5 B.-B. Liu and others D-mannose, methyl a-d-glucopyranoside salicin, starch, sucrose, trehalose and turanose, but not from N-acetylglucosamine adonitol, amygdalin, DL-arabinose, DL-arabitol, dulcitol, D-galactose, erythritol, DL-fucose, gluconate, inositol, inulin, 2-ketogluconate, 5-ketogluconate, lactose, D-lyxose, D-mannitol, methyl a-d-mannopyranoside, melibiose, methyl b-d-xylopyranoside, raffinose, L-rhamnose, D-ribose, sorbitol, L-sorbose, D-tagatose, xylitol or DLxylose. The cell peptidoglycan layers contain LL-diaminopimelic acid, alanine, glycine and glutamic acid. The predominant menaquinone is MK-9(H 4 ) and the major fatty acid is anteiso-c 15 : 0. The polar lipid profile consists of phosphatidylcholine, diphosphatidylglycerol, one phospholipid, four unidentified polar lipids and seven unidentified glycolipids. The type strain, YIM 2617 T (5KCTC T 5DSM T 5JCM T ), was isolated from the root of Ocimum basilicum, collected from Shilin city, south-west China. The DNA G+C content of the type strain is 66.6 mol%. YIM is a second strain of the species. Acknowledgements This research was supported by projects of China Tobacco Yunnan Industrial Co. Ltd (Nos. 2014YL01 and 2015CP01). W-J. L. was also supported by a Guangdong Province Higher Vocational Colleges & Schools Pearl River Scholar Funded Scheme (2014). References Buck, J. D. (1982). Nonstaining (KOH) method for determination of gram reactions of marine bacteria. Appl Environ Microbiol 44, Christensen, H., Angen, O., Mutters, R., Olsen, J. E. & Bisgaard, M. (2000). DNA-DNA hybridization determined in micro-wells using covalent attachment of DNA. Int J Syst Evol Microbiol 50, Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E. (1977). Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100, Embley, T. M. & Wait, R. (1994). Structural lipids of eubacteria. In Chemical Methods in Prokaryotic Systematics, pp Edited by M. Goodfellow & A. G. O. Donnell. 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