Massilia violacea sp . nov . , isolated from riverbank soil

Correspondence En Tao Wang entaowang@yahoo.com.mx Departamento de Microbiologı́a, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City, Mexico Instituto de Recursos Naturales y Agrobiologı́a de Salamanca, Consejo Superior de Investigaciones Cientı́ficas (IRNASA-CSIC), Salamanca, Spain Unidad Asociada Grupo de Interacción Planta-Microorganismo, Universidad de Salamanca-IRNASA (CSIC), Salamanca, Spain

The genus Massilia belongs to family Oxalobacteraceae, class Betaproteobacteria.It was first described by La Scola et al. (1998) with the type species Massilia timonae, isolated from blood of an immunocompromised patient with a cerebellar injury.Later, species of the genus Naxibacter were transferred to Massilia and the description of this genus was emended by Ka ¨mpfer et al. (2011).At the time of writing, 27 species with validity published names have been described, including Massilia oculi isolated from clinical specimens (Ka ¨mpfer et al., 2012), Massilia aerilata from air samples (Weon et al., 2008), Massilia aurea from water (Gallego et al., 2006), Massilia eurypsychrophila from glacial ice (Shen et al., 2015) and Massilia umbonata from soil samples (Rodrı ´guez-Dı ´az et al., 2014).
In the present study, strain CAVIO T was isolated from a soil sample collected from the riverbank of Zahuapan River at Las Cascadas de Atlihuetzia, Tlaxcala, Mexico, during an investigation of the diversity of culturable bacteria associated with soil from the riverbank.The Zahuapan River is an important source of water for agriculture in south-central Mexico.Serial dilutions of sampled soil were inoculated on caseine-peptone-starch agar (per litre: casein, 0.5 g; bacto-peptone, 0.5 g; starch, 1.0 g; glycerol, 1 g; K 2 HPO 4 , 0.2 g; MgSO 4 .0.7H 2 O, 0.05 g; FeCl 3 , 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 & Geldreich, 1985) to obtain pure cultures.Cells were stained according to the classical Gram procedure.Growth of the strain was also observed using nutrient agar (Becton Dickinson), Lysogeny broth (Becton Dickinson), tripticase soy agar (Becton Dickinson), MacConkey agar (Merck) Abbreviations: ML, maximum-likelihood; NJ, neighbour-joining.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of CAVIO T is KT003718.and TY agar (per litre: tryptone, 5 g; yeast extract, 3 g; CaCl 2 , 0.6 g; agar, 15 g; pH 7.2), but it grew poorly, forming punctate and non-pigmented colonies in the tested media.Cellular morphology was observed in detail by transmission electron microscopy (JEOL JEM-1010) after negative staining with phosphotungstic acid.Cells were rod-shaped (2.0-2.6 mm in length and 0.5-0.7 mm in diameter) and motile by polar and lateral flagella (Fig. S1, available in the online Supplementary Material).
For 16S rRNA gene sequencing and comparison analysis, DNA extraction was performed according to the protocol of Hoffman & Winston (1987).The almost fulllength 16S rRNA gene was amplified using primers 27F (59-AGAGTTTGATCMTGGCTCAG-39) and 1492R (59-TACGGYTACCTTGTTACGACTT-39) (Lane, 1991).Amplification was performed in a volume of 25 ml with 50 ng of template DNA, 10 pmol of each primer and 1 unit of DreamTaq DNA Polymerase (Thermo Scientific), using a Veriti 96-well thermal cycler (Applied Biosystems).The following PCR conditions were used: an initial denaturation step at 94 8C for 5 min; 25 cycles of 1 min at 94 8C, 1 min at 57 8C and 1 min at 72 8C; and finally an extension step at 72 8C for 5 min.The PCR product was electrophoresed and stained with ethidium bromide.The amplicon was purified using a GeneJET PCR Purification kit (Thermo Scientific), according to the manufacturer's instructions.Sequencing was conducted using a BigDye ABI PRISM Terminator Cycle Sequencing kit in an ABI PRISM 3730XL Sequencer by the Macrogen Sequencing service.Comparison of the 16S rRNA gene sequence of strain CAVIO T against those of type strains of bacterial species in the EzTaxon-e database (http:// www.ezbiocloud.net/eztaxon)(Kim et al., 2012) revealed an affiliation of the novel strain with the genus Massilia.The closest relative was M. umbonata LP01 T (DSM 26121 T ) at 97.5 % pairwise similarity (34 nt differences), followed by Massilia dura 16 T (DSM 17513 T ) (97.2 % similarity, 39 nt differences) and Massilia plicata 76 T (DSM 17505 T ) (97.1 % similarity, 41 nt differences).The remaining Massilia species showed similarity values lower than 96 %.The 16S rRNA gene sequence of strain CAVIO T was aligned with those from all recognized species of the genus Massilia by using CLUSTAL X v2.1 (Larkin et al., 2007).The MEGA 6.06 package (Tamura et al., 2013) was used to obtain phylogenetic trees by applying the neighbour-joining (NJ; Saitou & Nei, 1987) and maximum-likelihood (ML; Felsenstein, 1981) methods.The substitution models employed were Kimura's two-parameter for NJ and TN93+I + G for ML.Bootstrap analysis was based on 1000 resamplings (Felsenstein, 1985).According to the ML tree (Fig. 1), CAVIO T clustered in an independent branch formed by Massilia lutea, Massilia albidiflava, M. umbonata, M. dura, M. plicata and Massilia lurida.This relationship was congruent with the tree obtained by NJ analysis (Fig. S2).
DNA-DNA hybridization was carried according to the method of Ezaki et al. (1989), following the recommendations of Willems et al. (2001).CAVIO T was hybridized 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 j25 % in all cases (Table S1).Therefore, strain CAVIO T represents a different species of Massilia taking the recommendation of a threshold value of 70 % DNA-DNA relatedness (Wayne et al., 1987).
For base composition analysis, DNA was prepared according to Chun & Goodfellow (1995).The G+C content of the DNA was determined using the thermal denaturation method (Mandel & Marmur, 1968).The G+C content of strain CAVIO T was 65.0 mol%.This value is within the range obtained for Massilia species (Ka ¨mpfer et al., 2011).
The cellular fatty acids were analysed by using the Microbial Identification System (MIDI, Microbial ID) Sherlock 6.1 and the library RTSBA 6 version 6.21 (Sasser, 1990).Strain CAVIO T and the closest related species (the same as used for DNA-DNA hybridization) were grown on R2A agar plates for 24 h at 28 8C.The major fatty acids of CAVIO T were C 16 : 0 (32.3 %), C 16 : 1 v7c/C 16 : 1 v6c in summed feature 3 (42.9%) and C 18 : 1 v7c/C 18 : 1 v6c in summed feature 8 (7.7 %).As can be seen, CAVIO T showed a similar fatty acid profile to other species of the genus Massilia (Table 1).
For analyses of respiratory quinones and polar lipids, strain CAVIO T was cultured for 24 h in tripticase soy broth (Becton Dikinson) at 28 8C to obtain biomass.These analyses were carried out by the Identification Service at the Deutsche Sammlung von Mikroorganismen und Zellkulturen from freeze-dried cells using the protocols described by Tindall (1990a, b).Strain CAVIO T contained Q8 as the principal quinone.The polar lipids were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and an unknown phospholipid (Fig. S3).Comparing with the closest species M. flava, M. lurida and M. umbonata, we observed that strain CAVIO T shared the common polar lipids found in species of the genus Massilia.Only M. flava had an unknown aminolipid (Wang et al., 2012).For further characterization of strain CAVIO T , a series of phenotypic and physiological tests were done.Growth at several temperatures (4, 10, 28, 35 and 37 8C) and at different NaCl concentrations (0.3, 0.5, 1.0, 1.5, 2.0 and 2.5 %, w/v) was investigated on R2A agar.To inoculate these media, a suspension (0.5 McFarland nephelometer standard) was prepared and drops were placed on the agar plates.The assay was monitored for 5 days.pH tolerance range (5.0, 5.8, 6.5, 7.2, 8.0, 8.3, 8.5, 8.8, 9.2 and 9.6) was observed in R2A broth for 5 days at 28 8C.Hydrolysis of the following substrates was tested using R2A agar: starch (1.5 %, w/v), CM-cellulose (0.1 %) and Tween 80 (1 and 0.01 %, w/v, CaCl 2 ).The motility of bacterial cells was observed in semisolid R2A with 0.3 % agar.Oxidase and catalase were tested after 24 h of incubation at 28 8C.Additionally, API 20NE medium (bio-Me ´rieux) and Biolog Gen III Microplates (Biolog) were used following the manufacturers' instructions.The results from these systems were recorded after 48 h of incubation at 28 8C.Differential phenotypic characteristics between strain CAVIO T and its closest relatives are reported in Table 2. Taken together, the phenotypic, chemotaxonomic and genotypic data support the classification of strain CAVIO T as a novel species within the genus Massilia, for which the name Massilia violacea sp.nov. is proposed.
Description of Massilia violacea sp.nov.
Oxidase and catalase reactions are positive; interestingly, catalase activity is weak in comparison with that of the closest type strains.Tween 80 is hydrolysed but not starch or CM-cellulose.According to the API 20NE system, assimilation of D-glucose and L-malic acid is positive, while assimilation of D-mannose, D-mannitol and potassium gluconate is negative.Hydrolysis of aesculin is negative.
The type strain, CAVIO T (5CECT 8897 T 5LMG 28941 T ), was isolated from riverbank soil in Tlaxcala, Mexico.The DNA G+C content of the type strain is 65.0 mol%.
Fig.1.ML phylogenetic tree based on 16S rRNA gene sequences of strain CAVIO T 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.

Table 1 .
Cellular fatty acid profiles of strain CAVIO T and its closest related strains Strains: 1, CAVIO T ; 2, M. umbonata DSM 26121 T ; 3, M. dura DSM 17513 T ; 4, M. plicata DSM 17505 T .All data were obtained in this study, under the same conditions from cells grown on R2A agar for 24 h at 28 8C.2, Not detected or ,1 %.
ND*Data obtained from the literature.