DOI: 10.15468/5h43b5
title: Roseovarius ramblicola sp. nov., a moderately halophilic bacterium isolated from saline soil in Spain

description: This dataset contains the digitized treatments in Plazi based on the original journal article Castro, David J., Cerezo, Isabel, Sampedro, Inmaculada, Martínez-Checa, Fernando (2018): Roseovarius ramblicola sp. nov., a moderately halophilic bacterium isolated from saline soil in Spain. International Journal of Systematic and Evolutionary Microbiology 68 (6): 1851-1856, DOI: 10.1099/ijsem.0.002744, URL: http://dx.doi.org/10.1099/ijsem.0.002744AbstractStrain D15 T was isolated from a soil sample taken from Rambla Salada (Murcia), south-eastern Spain, by using the dilutionto-extinction method. The strain, a Gram-stain-negative aerobic bacteria, is non-motile, ovoid- or rod-shaped, catalase- and oxidase-positive, and grows at NaCl concentrations within the range 0.5 – 10 % (w/v) [optimum 3 % (w/v)], at 5 – 30 Ǫ C (optimum 28 Ǫ C) and at pH 6 – 9 (optimum pH 7.0). The 16S rRNA gene sequence indicates that it belongs to the genus Roseovarius in the class Alphaproteobacteria. Its closest relatives are Roseovarius tolerans EL-172 T and Roseovarius azorensis SSW 084 T, to which the strain shows 16S rRNA gene-sequence similarity values of 96.1 and 95.3 %, respectively. The DNA G+C content is 63 mol%. The major fatty acids (&gt;5 % of the total fatty acids) of strain D15 T are C 18: 1 Ɯ 7 c, C16: 0 and C12: 0. The only detected isoprenoid quinone of strain D15 T is ubiquinone 10 (Q-10). The polar lipid profile contains phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, aminolipid and three polar lipids. Based on the phylogenetic, genotypic, phenotypic and chemotaxonomic data, the strain represents a novel species of the genus Roseovarius, for which the name Roseovarius ramblicola sp. nov. is proposed. Strain D15 T (= CECT 9424= LMG 30322) is the type strain.The genus Roseovarius belongs to the family Rhodobacteraceae within the class Alphaproteobacteria and it was established by Labrenz et al. [1]. At the time of writing, the genus comprises 20 species.The genus Roseovarius accommodates Gram-stain-negative, aerobic, ovoid- or rod-shaped members characterized as being bacteriochlorophyll a (BChla)-positive, oxidase- and catalase-positive, and requiring Na + for growth. Most of the bacteria of this genus have been isolated from marine environments including seawater or tidal flats [2 – 6].In this study we describe strain D15 T, a moderately halophilic bacterium isolated from saline soil in Rambla Salada (Murcia).Strain D15 T was isolated from a sample of soil taken from Rambla Salada (Murcia) south-eastern Spain, 38 Ǫ 07′ 27.1″ N 1 Ǫ 07′ 01.4″ W, a hypersaline rambla (a steep-sided river bed, normally dry but subject to flash flooding). The culture medium used for the isolation of the bacteria was S3, a lownutrient medium [7] supplemented with 3 % (w/v) sea-salt solution [8] and the cultivation method used was the dilution-to-extinction approach.In this approach serial dilutions from 1 g soil were prepared, previously sonicated for 30 s, in 10 ml supplemented S3 medium. The number of micro-organisms in each dilution was determined using a Petroff Hausser counting chamber using methylene blue as contrast. A 48-well microtiter plate, containing 490 µl supplemented S3 medium, was inoculated with 10 µl dilution containing 100 bacteria per millilitre (approximately 1 bacterium per well). The extinction cultures were then incubated at 25 Ǫ C for 30 days. The growth wells were then re-isolated in Reasoner’ s 2A agar (R 2A) medium plates [9]. The pure strain was maintained and grown routinely in R 2A [9] at 30 Ǫ C with 3 % (w/v) sea-salt solution [8] as well as in marine agar (MA: 2216, Difco).The advantage of this method is the improvement of strain recovery, especially from species that are apparently uncultivable or from slow-growing species [10 – 13].Optimum growth conditions were evaluated by growing the strain in R 2A medium at 0.5, 1, 3, 5, 7.5, 10, 15, 20, 25 and 30 % (w/v) NaCl concentrations. The effect of different temperatures (4, 15, 20, 25, 30, 32, 35, 40 and 45 Ǫ C) and different pH values (4, 5, 6, 7, 8, 9, 10 and 11) were assessed in R 2A medium at 3 % w/v NaCl.Gram staining was performed according to the method of [14]. Catalase activity was determined by bubble production in 3 % (v/v) H 2 O 2 solution. Oxidase activity was examined using 1 % (v/v) tetramethyl-p-phenylenediamine [15]. Growth under anaerobic conditions was determined in an anaerobic jar using AnaeroGen (Oxoid) and an anaerobic indicator (Oxoid) on R 2A medium supplemented with 3 % (w/v) sea-salt solution [8]. Phenotypic characterization, including biochemical characters, sugar fermentation and enzymatic tests, were carried out by using API 20NE, API 50 CH and API ZYM strips (bioMerieux) following the manufacturer’ s instructions. Sensitivity to antimicrobial compounds was assayed according to the conventional Kirby–Bauer method [16].Transmission electron micrographs of strain D15 T are available as supplementary material (Fig. S1, available in the online version of this article). The results obtained are detailed in the species description.The cells of strain D15 T were non motile, ovoid- or rodshaped, Gram-stain-negative, catalase- and oxidase-positive, and strictly aerobic. They could grow at 0.5–10 % (w/v) NaCl, optimally at 3 % (w/v) NaCl. The temperature range for growth was 5–30 Ǫ C, with the optimum being 28 Ǫ C. The pH range for growth was 6–9 with optimum growth at pH 7.0. Other characteristics of strain D15 T are given in the species description and those that differ with the type strains in the genus Roseovarius are shown in Table 1.The DNA G+C content of strain D15 T was estimated from the midpoint value (Tm) of their DNA [17]. Tm was determined by the graphic method [18] and the DNA G+C content was calculated by using the equation described by [19]. The G+C content of reference DNA of Escherichia coli NCTC 9001 T was 50.9 mol% [20]. The DNA G+C content was 63 mol%, a similar value to members of the genus Roseovarius [5, 21] (Table 1).Phylogenetic analyses based on the 16S rRNA gene were performed as described elsewhere [22]. In this case, the PCR product was cloned into the pGEM-T cloning vector (Promega) according to the manufacturer’ s recommendations, and transformed into Escherichia coli DH5-a. The identification of phylogenetic neighbours was initially carried out by the BLASTN [23] program against the GenBank/EMBL/ DDBJ database containing type strains with validly published prokaryotic names and representatives of uncultured phylotypes. The 16S rRNA sequence of strain D15 T was also implemented into the current release of the LTP ARB database ‘All-species Living Tree Project’ [24]. We also carried out the identification of phylogenetic neighbours and a calculation of pairwise 16S rRNA gene sequence similarity by using the EzTaxon server (www.ezbiocloud.net; [25]). Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 7 [26]. Clustering was determined using the neighbour-joining, maximum-parsimony and maximum-likelihood algorithms and the evolutionary distances were computed using the Jukes–Cantor method [27]. The analysis involved 29 nucleotide sequences and the stability of the clusters was ascertained by performing a bootstrap analysis (1000 replications).Characteristic 1 2 3 Colony pigmentation White Non-pigmented Red Growth range: Temperature 5–30 15–40 8.5–33.5 pH 6.0–9.0 7.0–9.0 6.2–9.0 NaCl (% w/v) 0.5–10 0.5–7.0 1.0–8.0 Bacteriochlorophyll a + -- + Enzymatic activities in API ZYM: Nitrate reductase- - + -- Valine arylamidase- - -- -- Cystine arylamidase- - -- -- Trypsin- - -- -- Acid phosphatase- - -- + Alcaline phosphatase- - -- -- Naphthol-AS-BI-phosphohydrolase- - + -- Esterase C4 -- + -- Esterase lipase C8 -- + -- Acid production in API 50CH from: D- Galactose- - -- -- D- Fructose- - -- -- Potassium 5-ketogluconate- - + -- Polar lipids PC, PE, PG, AL, L PC, PE, PG, DPG, L, AL PC, PE, DPG, PL DNA G+C content (mol%) 63 61.9 62.2–63.8Phylogenetic analyses based on these three methods indicated that strain D15 T was included in the cluster which make up the Roseovarius species, resulting in highly similar tree topologies. Fig. 1 shows the tree containing the phylogroup in which our new strain is included according to the maximum-likelihood algorithm.Fig. 1. Maximum-likelihood phylogenetic tree based on nearly complete 16S rRNA gene sequences showing the relationships between strain D15 T, type species of the genus Roseovarius and the closest related species in the family Rhodobacteraceae. Filled circles indicate nodes that were also recovered in the maximum-parsimony and neighbour-joining trees based on the same sequences. Numbers at nodes are levels of bootstrap support (percentages) based on analyses of 1000 re-sampled datasets; only values above 50 % are shown. Bar, 0.05 nt changes per position. The GenBank/EMBL/DDBJ accession number of each sequence is shown in parentheses.Table 2. Fatty-acid profile of the strain D15T and the type strains of the genus Roseovarius 1, strain D15T; 2, R. azorensis DSM 100674T, 3, R. tolerans EL-172T. All data from this study, except that for strain 3, which was taken from Labrenz et al. [1]. Cells were grown on MA at 30 Ǫ C for 48 h. The major fatty acids (&gt;5 % of the total fatty acids) of strain D15T are highlighted in bold. --, Not detected. Data represent percentages of total fatty acids.The most closely related species were Roseovarius tolerans EL-172 T and Roseovarius azorensis SSW 084 T to which strain D15 T showed 16S rRNA gene sequence similarity values of 96.1 and 95.3 %, respectively.Fatty acid 1 2 3 C12:0 2-OH 0.9 0.3 2.4 C12:0 3-OH- - 6.0 -- C12:1 3-OH 3.6 0.2 3.6 C12:0 7.1 2.9 -- C16:0 11.9 13.4 6.2 C18:0 1.8 0.8 0.8 C16:1 -- -- 0.8 C18:2 -- -- s C16:0 2-OH 2.5 2.3 -- C18:1 Ɯ 9 c 0.7 2.4 -- C18:1 Ɯ 7 c 11-methyl 2.3 5.9 -- Cyclo-C19: 0 Ɯ 8 c 0.9 4.0 -- Summed features:* 3 0.8 0.8 -- 8 61.5 56.9 70.2 *Summed feature 3: C16: 1 Ɯ 7 c /C16: 1 Ɯ 6 c, summed feature 8: C18: 1 Ɯ 7 c.The fatty acids of strain D15 T and R. azorensis DSM 100674 T were analysed at the Spanish Type Culture Collection. Cells were grown on MA for 48 h incubation at 30 Ǫ C (stationary growth phase). The whole-cell composition of the fatty acids was determined by gas chromatography using the MIDI Microbial Identification System [28]. The fatty-acid profile was obtained with an Agilent 6850 gas chromatograph using the TSBA6 145 database [29]. Analysis of the polar lipids and respiratory quinones of strain D15 T was carried out by the Identification Service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany. Polar lipids are extracted from 100 mg freeze dried cell material using a chloroform: methanol: 0.3 % aqueous NaCl mixture 1: 2: 0.8 (v/v/v) [30]. The extraction solvent was stirred overnight and the cell debris pelleted by centrifugation. Polar lipids were recovered into the chloroform phase by adjusting the chloroform: methanol:0.3 % aqueous NaCl mixture to a ratio of 1: 1: 0.9 (v/v/v).Polar lipids were separated by two-dimensional silica gel thin-layer chromatography (Macherey–Nagel Art. No. 818 135). The first direction was developed in chloroform– methanol–water (65: 25: 4, v/v/v), and the second in chloroform–methanol–acetic acid–water (80: 12: 15: 4, v/v/v/v). Total lipid material was detected using molybdatophosphoric acid and specific functional groups detected using spray reagents specific for defined functional groups [31]. The two-stage method described by [32, 33] was used to first extract the respiratory lipoquinones followed by the polar lipids.The major fatty acids (&gt;5 % of the total fatty acids) of strain D15 T were C 18: 1 Ɯ 7 c (61.5 %), C 16: 0 (11.9 %) and C 12: 0 (7.1 %) (Table 2). The fatty-acid profile of strain D15 T was similar to the profile described for R. tolerans EL-172 T [1] and R. azorensis DSM 100674 T, as shown in Table 2. The only isoprenoid quinone detected was ubiquinone Q 10 in accordance with the lipoquinone present in the described species of the genus Roseovarius. The polar lipid profile of strain D15 T was composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, aminolipid and three unidentified polar lipids (Fig. S3). This pattern was different from the reference strains previously described [1, 5, 21]. For example, it had no diphosphatidylglycerol compared with R. tolerans EL-172 T and R. azorensis DSM 100674 T, but it did have phosphatidylcholine, phosphatidylethanolamine as well as the two type strains of genus Roseovarius considered in this study.To analyse the presence of bacteriochlorophyll a, puf genes (photosynthetic reaction centre genes pufL and pufM) were amplified by PCR using puf Lf (5′-CTKTTCGACT TCTGGGTSGG-3′) and pufM r (5′-CATSGTCCAGCGCCAGAA-3′) as the pair of primers, following the protocol described by [34]. The PCR product of about 1.5 kb 168 (Fig. S2) was cloned into the pGEM-T cloning vector (Promega) according to the manufacturer’ s recommendations, transformed into Escherichia coli DH5-a and sequenced with T 7 and SP6 primers. The pufL and pufM gene sequences of strain D15 T showed 89 and 87 % similarity to the sequences of pufL and pufM genes of R. tolerans EL-172 T (DDBJ/EMBL/GenBank accession number DQ915720) and R. mucosus DFL-24 T (DDBJ/EMBL/GenBank accession number CP020474) respectively, and 81 % similarity to the sequences of pufL and pufM genes of R. denitrificans Och 114 T (DDBJ/EMBL/GenBank accession number DQ915721) and R. halotolerans Och 210 T (DDBJ/ EMBL/GenBank accession number DQ915719).Fig. S2 shows the PCR products of the pufLM genes amplified from total DNA of strain D15 T and the absence of these genes in another type strain of genus Roseovarius like Roseovarius pacificus LMG 24575 T.Accordingly, on the basis of differences in phenotypic and chemotaxonomic characteristics, and genetic distinctiveness, strain D15 T should be recognized as representing a novel species of the genus Roseovarius, for which we propose the name Roseovarius ramblicola.