Massilia Violacea Sp. Nov., Isolated from Riverbank Soil Salvador Embarcadero-Jime´Nez,1 A´ Lvaro Peix,2,3 Jose´ Mariano Igual,2,3 Flor N

International Journal of Systematic and Evolutionary Microbiology (2016), 66, 707–711 DOI 10.1099/ijsem.0.000776

Massilia violacea sp. nov., isolated from riverbank soil Salvador Embarcadero-Jime´nez,1 A´ lvaro Peix,2,3 Jose´ Mariano Igual,2,3 Flor N. Rivera-Ordun˜a1 and En Tao Wang1

Correspondence 1Departamento de Microbiologıá, Escuela Nacional de Ciencias Biolo´gicas, Instituto Politećnico En Tao Wang Nacional, Mexico City, Mexico [email protected] 2Instituto de Recursos Naturales y Agrobiologıá de Salamanca, Consejo Superior de Investigaciones Cientı´ficas (IRNASA-CSIC), Salamanca, Spain 3Unidad Asociada Grupo de Interaccioń Planta-Microorganismo, Universidad de Salamanca-IRNASA (CSIC), Salamanca, Spain

A bacterial strain designated CAVIOT was isolated during the course of a study of culturable bacteria in a riverbank soil sample from Tlaxcala, Mexico. The strain was subjected to a polyphasic taxonomic characterization. Strain CAVIOT was aerobic, Gram-stain-negative, non-spore-forming and rod-shaped. Colonies grown on R2A agar at 28 8C were pale violet, mucoid, rounded, smooth and glossy. The strain was motile and catalase- and oxidase-positive, and maximum growth temperature was 35 8C. Strain CAVIOT was classiﬁed within the genus Massilia as its 16S rRNA gene sequence was closely related to those of Massilia umbonata LP01T (97.5 % similarity), Massilia dura 16T (97.2 %) and Massilia plicata 76T (97.1 %). The predominant respiratory quinone was Q8. The major fatty acids were summed feature 3

(C16 : 1v7c/C16 : 1v6c), C16 : 0 and summed feature 8 (C18 : 1v7c/C18 : 1v6c). The predominant polar lipids were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and an unknown phospholipid. The DNA G+C content was 65.0 mol% (Tm). DNA–DNA hybridization results showed values below 25 % with respect to the type strains of the closest related species. Therefore, strain CAVIOT can be differentiated from previously described species of the genus Massilia and represents a novel species, for which the name Massilia violacea sp. nov. is proposed. The type strain is CAVIOT (5CECT 8897T5LMG 28941T).

The genus Massilia belongs to family Oxalobacteraceae, from glacial ice (Shen et al., 2015) and Massilia umbonata class Betaproteobacteria. It was first described by La Scola from soil samples (Rodrı´guez-Dıáz et al., 2014). et al. (1998) with the type species Massilia timonae, isolated In the present study, strain CAVIOT was isolated from a from blood of an immunocompromised patient with a cer- soil sample collected from the riverbank of Zahuapan ebellar injury. Later, species of the genus Naxibacter were River at Las Cascadas de Atlihuetzia, Tlaxcala, Mexico, transferred to Massilia and the description of this genus during an investigation of the diversity of culturable bac- was emended by Ka¨mpfer et al. (2011). At the time of writ- teria associated with soil from the riverbank. The Zahuapan ing, 27 species with validity published names have been River is an important source of water for agriculture in described, including Massilia oculi isolated from clinical south–central Mexico. Serial dilutions of sampled soil specimens (Ka¨mpfer et al., 2012), Massilia aerilata from were inoculated on caseine-peptone-starch agar (per litre: air samples (Weon et al., 2008), Massilia aurea from casein, 0.5 g; bacto-peptone, 0.5 g; starch, 1.0 g; glycerol, water (Gallego et al., 2006), Massilia eurypsychrophila 1g; K2HPO4, 0.2 g; MgSO4 . 0.7H2O, 0.05 g; FeCl3, 0.004 g; agar, 15 g; pH 8.0), and incubated at 28 8C for 48 h. A bacterial colony producing a violet pigment was selected and subcultured on R2A agar (Reasoner & Abbreviations: ML, maximum-likelihood; NJ, neighbour-joining. Geldreich, 1985) to obtain pure cultures. Cells were stained The GenBank/EMBL/DDBJ accession number for the 16S rRNA according to the classical Gram procedure. Growth of gene sequence of CAVIOT is KT003718. the strain was also observed using nutrient agar (Becton One supplementary table and three supplementary figures are available Dickinson), Lysogeny broth (Becton Dickinson), tripticase with the online Supplementary Material. soy agar (Becton Dickinson), MacConkey agar (Merck)

Downloaded from www.microbiologyresearch.org by 000776 G 2015 IUMS Printed in Great Britain 707 IP: 54.70.40.11 On: Tue, 06 Nov 2018 20:58:47 S. Embarcadero-Jime´nez and others

Massilia lutea 101T (AY966001) 87 Massilia albidiflava 45T (AY965999) 69 Massilia umbonata LP01T (HM053474) 0.05 Massilia dura 16T (AY965998) Massilia violacea CAVIOT (KT003718)

54 Massilia plicata 76T (AY966000) Massilia lurida D5T (HQ839786) 53 Massilia flava Y9T (HM777013) Massilia namucuonensis 333-1-0411T (JF99985) Massilia eurypsychrophila B528-3T (KJ361508) Massilia niabensis 5420S-26T (EU808006) Massilia aurea AP13T (AM231588) T 53 Massilia brevitalea byr23-80 (EF546777) 99 Massilia jejuensis 5317J-18T (FJ9699486) Massilia oculi CCUG 43427AT (FR773700) Massilia timonae UR/MT95T (U544770) Massilia alkalitolerans YIM 31775T (AY679161) 97 T Massilia varians CC 7478 (AM774587) Massilia haematophila CCM 7480T (AM774589) 58 Massilia suwonensis 5414S-25T (FJ969487) TS3T (HM130516) 50 Massilia tieshanensis Massilia niastensis 5516S-1T (EU808005) 71 Massilia aerilata 5516S-11T (EF688526) 54 KACC 17471T (KC574383) 65 Massilia kyonggiensis 86 'Massilia arvi' THG-RS2O (KJ810584) 58 Massilia norwichensis NS9T (HG798294) Massilia yuzhufengensis Y1243-1T (JQ409016) Massilia consociata CCUG 58010T (FN814307) Burkholderia cenocepacia CCM 4899T (AF148556)

Fig. 1. ML phylogenetic tree based on 16S rRNA gene sequences of strain CAVIOT and species of the genus Massilia. Numbers at nodes indicate bootstrap percentages (based on 1000 resamplings) $50 %. The nodes marked with ﬁlled circles were also obtained with the NJ algorithm (see Fig. S1). Bar, 0.05 substitutions per nucleotide.

and TY agar (per litre: tryptone, 5 g; yeast extract, 3 g; CaCl2, (59-TACGGYTACCTTGTTACGACTT-39) (Lane, 1991). 0.6 g; agar, 15 g; pH 7.2), but it grew poorly, forming punc- Amplification was performed in a volume of 25 mlwith tate and non-pigmented colonies in the tested media. 50 ng of template DNA, 10 pmol of each primer and 1 unit of DreamTaq DNA Polymerase (Thermo Scientific), using a Cellular morphology was observed in detail by trans- Veriti 96-well thermal cycler (Applied Biosystems). The fol- mission electron microscopy (JEOL JEM-1010) after lowing PCR conditions were used: an initial denaturation negative staining with phosphotungstic acid. Cells were step at 94 8C for 5 min; 25 cycles of 1 min at 94 8C, 1 min rod-shaped (2.0–2.6 mm in length and 0.5–0.7 mm in diam- at 57 8C and 1 min at 72 8C; and finally an extension step at eter) and motile by polar and lateral flagella (Fig. S1, avail- 72 8C for 5 min. The PCR product was electrophoresed and able in the online Supplementary Material). stained with ethidium bromide. The amplicon was purified For 16S rRNA gene sequencing and comparison analysis, using a GeneJET PCR Purification kit (Thermo Scientific), DNA extraction was performed according to the protocol according to the manufacturer’s instructions. Sequencing of Hoffman & Winston (1987). The almost full- was conducted using a BigDye ABI PRISM Terminator length 16S rRNA gene was amplified using primers Cycle Sequencing kit in an ABI PRISM 3730XL Sequencer 27F (59-AGAGTTTGATCMTGGCTCAG-39) and 1492R by the Macrogen Sequencing service. Comparison of the 16S

Downloaded from www.microbiologyresearch.org by 708 International Journal of Systematic and Evolutionary Microbiology 66 IP: 54.70.40.11 On: Tue, 06 Nov 2018 20:58:47 Massilia violacea sp. nov. rRNA gene sequence of strain CAVIOT against those of type Table 1. Cellular fatty acid profiles of strain CAVIOT and its strains of bacterial species in the EzTaxon-e database (http:// closest related strains www.ezbiocloud.net/eztaxon) (Kim et al.,2012)revealedan Strains: 1, CAVIOT;2,M. umbonata DSM 26121T;3,M. dura DSM affiliation of the novel strain with the genus Massilia. The clo- T T sest relative was M. umbonata LP01T (DSM 26121T)at97.5% 17513 ;4,M. plicata DSM 17505 . All data were obtained in this pairwise similarity (34 nt differences), followed by Massilia study, under the same conditions from cells grown on R2A agar for 8 2 , dura 16T (DSM 17513T) (97.2 % similarity, 39 nt differences) 24 h at 28 C. , Not detected or 1%. T T and Massilia plicata 76 (DSM 17505 )(97.1%similarity, Fatty acid 1 2 3 4 41 nt differences). The remaining Massilia species showed similarity values lower than 96 %. The 16S rRNA gene iso-C10 : 0 –––– T sequence of strain CAVIO was aligned with those from all C10 : 0 –––– recognized species of the genus Massilia by using CLUSTAL X C10 : 0 3-OH 3.6 4.3 4.7 5.5 v2.1 (Larkin et al., 2007). The MEGA 6.06 package (Tamura C12 : 0 3.6 6.3 4.3 3.5 et al., 2013) was used to obtain phylogenetic trees by applying C12 : 0 2-OH 2.1 – – – the neighbour-joining (NJ; Saitou & Nei, 1987) and maxi- C12 : 0 3-OH – – – – mum-likelihood (ML; Felsenstein, 1981) methods. The substi- C14 : 0 – 3.1 1.7 1.2 tution models employed were Kimura’s two-parameter for NJ C14 : 0 2-OH – 2.5 2.5 2.7 and TN93+I + G for ML. Bootstrap analysis was based on C16 : 0 32.3 23.9 28.9 32.0 v 1000 resamplings (Felsenstein, 1985). According to the ML iso-C17 : 1 5c 1.5 1.3 2.5 – tree (Fig. 1), CAVIOT clustered in an independent branch C17 : 0 cyclo 2.7 – – – formed by Massilia lutea, Massilia albidiflava, M. umbonata, iso-C17 : 0 3-OH 1.1 – – – M. dura, M. plicata and Massilia lurida. This relationship was C18 : 0 – – 2.2 – Summed feature 3 42.9 44.7 44.9 46.8 congruent with the tree obtained by NJ analysis (Fig. S2). * Summed feature 8D 7.7 7.5 5.3 4.9 DNA–DNA hybridization was carried according to the v v method of Ezaki et al. (1989), following the recommen- *C16 : 1 7c/C16 : 1 6c. T D v v dations of Willems et al. (2001). CAVIO was hybridized C18 : 1 7c/C18 : 1 6c. with the type strains of the three most closely related Massilia species showing more than 97 % 16S rRNA gene sequence similarity. Mean hybridization values were T j25 % in all cases (Table S1). Therefore, strain CAVIOT Tindall (1990a, b). Strain CAVIO contained Q8 as the represents a different species of Massilia taking the rec- principal quinone. The polar lipids were phosphatidylgly- ommendation of a threshold value of 70 % DNA–DNA cerol, phosphatidylethanolamine, diphosphatidylglycerol relatedness (Wayne et al., 1987). and an unknown phospholipid (Fig. S3). Comparing with the closest species M. flava, M. lurida and M. umbonata, For base composition analysis, DNA was prepared according we observed that strain CAVIOT shared the common to Chun & Goodfellow (1995). The G+C content of the DNA polar lipids found in species of the genus Massilia. Only was determined using the thermal denaturation method M. flava had an unknown aminolipid (Wang et al., (Mandel & Marmur, 1968). The G+C content of strain 2012). For further characterization of strain CAVIOT,a CAVIOT was 65.0 mol%. This value is within the range series of phenotypic and physiological tests were done. obtained for Massilia species (Ka¨mpfer et al., 2011). Growth at several temperatures (4, 10, 28, 35 and 37 8C) and at different NaCl concentrations (0.3, 0.5, 1.0, 1.5, The cellular fatty acids were analysed by using the 2.0 and 2.5 %, w/v) was investigated on R2A agar. Microbial Identification System (MIDI, Microbial ID) To inoculate these media, a suspension (0.5 McFarland Sherlock 6.1 and the library RTSBA 6 version 6.21 nephelometer standard) was prepared and drops were (Sasser, 1990). Strain CAVIOT and the closest related placed on the agar plates. The assay was monitored for species (the same as used for DNA–DNA hybridization) 5 days. pH tolerance range (5.0, 5.8, 6.5, 7.2, 8.0, 8.3, 8.5, were grown on R2A agar plates for 24 h at 28 8C. The 8.8, 9.2 and 9.6) was observed in R2A broth for 5 days at major fatty acids of CAVIOT were C (32.3 %), 16 : 0 28 8C. Hydrolysis of the following substrates was tested C v7c/C v6c in summed feature 3 (42.9 %) and 16 : 1 16 : 1 using R2A agar: starch (1.5 %, w/v), CM-cellulose (0.1 %) C v7c/C v6c in summed feature 8 (7.7 %). As can 18 : 1 18 : 1 and Tween 80 (1 and 0.01 %, w/v, CaCl ). The motility be seen, CAVIOT showed a similar fatty acid profile to 2 of bacterial cells was observed in semisolid R2A with other species of the genus Massilia (Table 1). 0.3 % agar. Oxidase and catalase were tested after 24 h of For analyses of respiratory quinones and polar lipids, strain incubation at 28 8C. Additionally, API 20NE medium (bio- CAVIOT was cultured for 24 h in tripticase soy broth Me´rieux) and Biolog Gen III Microplates (Biolog) were (Becton Dikinson) at 28 8C to obtain biomass. These ana- used following the manufacturers’ instructions. The results lyses were carried out by the Identification Service at the from these systems were recorded after 48 h of incubation Deutsche Sammlung von Mikroorganismen und Zellkultu- at 28 8C. Differential phenotypic characteristics between ren from freeze-dried cells using the protocols described by strain CAVIOT and its closest relatives are reported

Downloaded from www.microbiologyresearch.org by http://ijs.microbiologyresearch.org 709 IP: 54.70.40.11 On: Tue, 06 Nov 2018 20:58:47 S. Embarcadero-Jime´nez and others

Table 2. Differential phenotypic characteristics between strain CAVIOT and its closest related species

Taxa: 1, strain CAVIOT;2,M. umbonata DSM 26121T;3,M. dura DSM 17513T;4,M. plicata DSM 17505T;5,M. lutea 101T (Zhang et al., 2006); 6, M. albidiﬂava 45T (Zhang et al., 2006); 7, M. lurida D5T (Luo et al., 2013); 8, M. ﬂava (Wang et al., 2012). Data were obtained in this study unless indicated otherwise. +, Positive, 2, negative; W, weakly positive; ND, no data available.

Characteristic 1 2 3 4 5* 6* 7* 8*

Colony colour Violet Yellow Pale white Yellow to pale brown Yellow Pale white to yellow Pale yellow Yellow Growth at 37 8C 2 ++ + + + + + Oxidase + 2 + 2 ++ ++ Aesculin 2 + 2 + ND ND + ND D-Glucose + W ++++++ L-Arabinose 2 + 2 + 22 ++ D-Mannose 2 ++ + ND ND + W Starch 2 + 2 +++ 2 + D-Mannitol 2 + 222222 N-Acetyl-D-glucosamine 2 W 22++ +2 Potassium gluconate 2 W + WNDND 2 ND Malic acid + W 2 + ND ND + ND

*Data obtained from the literature. in Table 2. Taken together, the phenotypic, chemotaxo- acid is positive. Negative results are obtained for assimila- nomic and genotypic data support the classification of tion of gentiobiose, sucrose, turanose, stachyose, raffinose, T strain CAVIO as a novel species within the genus Massilia, a-lactose, melibiose, methyl b-D-glucoside, D-salicin, N-acetyl- for which the name Massilia violacea sp. nov. is proposed. D-glucosamine, N-acetyl-b-D-mannosamine, N-acetyl-neura- minic acid, D-mannose, D-fructose, D-galactose, 3-methyl Description of Massilia violacea sp. nov. glucose, D-fucose, L-fucose, L-rhamnose, D-serine, D-sorbitol, D-mannitol, D-arabitol, myo-inositol, glycerol, gelatin, 9 Massilia violacea (vi.o.la ce.a. L. fem. adj. violacea of violet, L-alanine, L-arginine, L-pyroglutamic acid, L-serine, pectin, referring to the colony colour). D-galacturonic acid, L-galactonic acid lactone, D-gluconic Cells are Gram-stain-negative, aerobic, non-spore-forming, acid, D-glucuronic acid, mucic acid, quinic acid, D-saccharic rod-shaped, 2.0–2.6 mm in length and 0.5–0.7 mm in diam- acid, p-hydroxyphenylacetic acid, D-lactic acid methyl c a eter, and motile by polar and lateral flagella. Colonies are ester, D-malic acid, -aminobutyric acid, -hydroxybutyric a violet, circular, convex, mucoid and usually 2.0–5.0 mm acid, -ketobutyric acid, acetoacetic acid and formic acid. diameter on R2A agar medium after 48 h of incubation Assimilation of inosine, glucuronamide, citric acid, propio- at 28 uC. Growth occurs on R2A agar medium at 10– nic acid and acetic acid is weak. Resistant to nalidixic acid, 35 uC, at pH 5.8–9.2 and with 0–1% (w/v) NaCl, with rifamycin and aztreonam, but sensitive to fusidic acid, tro- 28 uC, pH 8.0 and 0% NaCl for optimum growth. The leandomycin, minocycline, lincomycin and vancomycin. main respiratory quinone is Q8. The major fatty acids are Unable to grow in the presence of guanidine HCl, Niaproof 4, lithium chloride, potassium tellurite, sodium bromate C16:0,C16:1v7c/C16:1v6c (summed feature 3) and and sodium butyrate. C18:1v7c/C18:1v6c (summed feature 8). The main polar lipids are phosphatidylglycerol, phosphatidylethanolamine, Thetypestrain,CAVIOT (5CECT 8897T5LMG 28941T), diphosphatidylglycerol and an unknown phospholipid. was isolated from riverbank soil in Tlaxcala, Mexico. The Oxidase and catalase reactions are positive; interestingly, DNA G+C content of the type strain is 65.0 mol%. catalase activity is weak in comparison with that of the closest type strains. Tween 80 is hydrolysed but not starch or Acknowledgements CM-cellulose. According to the API 20NE system, assimilation of D-glucose and L-malic acid is positive, while This research was funded by grants from the Instituto Politećnico Nacio- assimilation of D-mannose, D-mannitol and potassium glu- nal (IPN) (SIP 20140124 and 20150597). S. E. J. received a doctoral fel- conate is negative. Hydrolysis of aesculin is negative. lowship provided by CONACyT in the period 2012–2015 (number In Biolog Gen III plates, assimilation of dextrin, maltose, 230841) and an international mobility fellowship (290936). Special thanks to the Escuela Nacional de Ciencias Biolo´gicas of IPN and Insti- trehalose, cellobiose, N-acetyl-D-galactosamine, D-glucose tuto de Recursos Naturales y AgroBiologıá de Salamanca (IRNASA) of 6-phosphate, D-fructose 6-phosphate, D-aspartic acid, CSIC for support and facilities to carry out this work. We thank Dr glycyl L-proline, L-aspartic acid, L-glutamic acid, L- Martha Helena Ramı´rez-Bahena from IRNASA-CSIC for support and histidine, methyl pyruvate, L-lactic acid, a-ketoglutaric acid, helpful discussions. Special thanks to Dr E´dgar O´ liver Lo´pez Villegas bromosuccinic acid, Tween 40 and b-hydroxy-DL-butyric from ENCB-IPN for his advice with electron microscopy.

Downloaded from www.microbiologyresearch.org by 710 International Journal of Systematic and Evolutionary Microbiology 66 IP: 54.70.40.11 On: Tue, 06 Nov 2018 20:58:47 Massilia violacea sp. nov.

References Mandel, M. & Marmur, J. (1968). Use of ultraviolet absorbance temperature profile for determining the guanine plus cytosine Chun, J. & Goodfellow, M. (1995). A phylogenetic analysis of the content of DNA. Methods Enzymol 12B 195–206. genus Nocardia with 16S rRNA gene sequences. Int J Syst Bacteriol Reasoner, D. J. & Geldreich, E. E. (1985). A new medium for the 45 , 240–245. enumeration and subculture of bacteria from potable water. Appl Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluoromeric Environ Microbiol 49, 1–7. deoxyribonucleic acid-deoxyribonucleic acid acid hybridization in Rodrı´guez-Dıáz, M., Cerrone, F., Sa´ nchez-Peinado, M., SantaCruz- microdilution wells as an alternative to membrane filter Calvo, L., Pozo, C. & Lo´ pez, J. G. (2014). Massilia umbonata hybridization in which radioisotopes are used to determine genetic sp. nov., able to accumulate poly-(-hydroxybutyrate, isolated from a relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229. sewage sludge compost-soil microcosm. Int J Syst Evol Microbiol 64, Felsenstein, J. (1981). Evolutionary trees from DNA sequences: 131–137. a maximum likelihood approach. J Mol Evol 17, 368–376. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new Felsenstein, J. (1985). Confidence limits on phylogenies: an approach method for reconstructing phylogenetic trees. Mol Biol Evol 4 (406), using the bootstrap. Evolution 39, 783–791. 425. Gallego, V., Sa´ nchez-Porro, C., Garcıá, M. T. & Ventosa, A. (2006). Sasser, M. (1990). Identification of bacteria by gas chromatography of Massilia aurea sp. nov., isolated from drinking water. Int J Syst Evol cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Inc. Microbiol 56, 2449–2453. Shen, L., Liu, Y., Gu, Z., Xu, B., Wang, N., Jiao, N., Liu, H. & Zhou, Y. Hoffman, C. S. & Winston, F. (1987). A ten-minute DNA preparation (2015). Massilia eurypsychrophila sp. nov. a facultative psychrophilic from yeast efficiently releases autonomous plasmids for bacteria isolated from ice core. Int J Syst Evol Microbiol 65, 2124–2129. transformation of Escherichia coli. Gene 57, 267–272. Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013). Ka¨ mpfer, P., Lodders, N., Martin, K. & Falsen, E. (2011). Revision of MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol the genus Massilia La Scola et al. 2000, with an emended description Evol 30, 2725–2729. of the genus and inclusion of all species of the genus Naxibacter as Tindall, B. J. (1990a). A comparative study of the lipid composition new combinations, and proposal of Massilia consociata sp. nov. Int of Halobacterium saccharovorum from various sources. Syst Appl J Syst Evol Microbiol 61, 1528–1533. Microbiol 13, 128–130. Ka¨ mpfer, P., Lodders, N., Martin, K. & Falsen, E. (2012). Massilia oculi Tindall, B. J. (1990b). Lipid composition of Halobacterium sp. nov., isolated from a human clinical specimen. Int J Syst Evol lacusprofundi. FEMS Microbiol Lett 66, 199–202. Microbiol 62, 364–369. Wang, J., Zhang, J., Pang, H., Zhang, Y., Li, Y. & Fan, J. (2012). Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Massilia flava sp. nov., isolated from soil. Int J Syst Evol Microbiol Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e: 62, 580–585. a prokaryotic 16S rRNA gene sequence database with phylotypes that Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, represent uncultured species. Int J Syst Evol Microbiol 62, 716–721. O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & La Scola, B., Birtles, R. J., Mallet, M. N. & Raoult, D. (1998). Massilia other authors (1987). International Committee on Systematic timonaegen. nov., sp. nov., isolated from blood of an Bacteriology. Report of the ad hoc committee on reconciliation of immunocompromised patient with cerebellar lesions. J Clin approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464. 36 Microbiol , 2847–2852. Weon, H. Y., Kim, B. Y., Son, J. A., Jang, H. B., Hong, S. K., Go, S. J. & Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid Kwon, S. W. (2008). Massilia aerilata sp. nov., isolated from an air Techniques in Bacterial Systematics, pp. 115–175. Edited by sample. Int J Syst Evol Microbiol 58, 1422–1425. E. Stackebrandt & M. Goodfellow. Chichester: Wiley. Willems, A., Doignon-Bourcier, F., Goris, J., Coopman, R., de Lajudie, P., Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, De Vos, P. & Gillis, M. (2001). DNA-DNA hybridization study of P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A. & other Bradyrhizobium strains. Int J Syst Evol Microbiol 51, 1315–1322. authors (2007). CLUSTAL W and CLUSTAL_X version 2.0. Zhang, Y. Q., Li, W. J., Zhang, K. Y., Tian, X. P., Jiang, Y., Xu, L. H., Jiang, 23 Bioinformatics , 2947–2948. C. L. & Lai, R. (2006). Massilia dura sp. nov., Massilia albidiflava Luo, X., Xie, Q., Wang, J., Pang, H., Fan, J. & Zhang, J. (2013). Massilia sp. nov., Massilia plicata sp. nov. and Massilia lutea sp. nov., isolated lurida sp. nov., isolated from soil. Int J Syst Evol Microbiol 63, 2118–2123. from soils in China. Int J Syst Evol Microbiol 56,459–463.

Downloaded from www.microbiologyresearch.org by http://ijs.microbiologyresearch.org 711 IP: 54.70.40.11 On: Tue, 06 Nov 2018 20:58:47