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 Microbiologı´a, Escuela Nacional de Ciencias Biolo´gicas, Instituto Polite´cnico En Tao Wang Nacional, Mexico City, Mexico [email protected] 2Instituto de Recursos Naturales y Agrobiologı´a de Salamanca, Consejo Superior de Investigaciones Cientı´ficas (IRNASA-CSIC), Salamanca, Spain 3Unidad Asociada Grupo de Interaccio´n 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-Dı´az 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.