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1 Departamento de Microbiología y Genética, Edificio Departamental, Lab. 209, Campus Miguel de Unamuno, Universidad de Salamanca, 37007 Salamanca, Spain
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany
Correspondence
Martha E. Trujillo
mett{at}usal.es
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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An extended phylogenetic tree and the cellular fatty acid profile of strain NAR01T are available as supplementary data in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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described has increased significantly in the last year (Kroppenstedt et al., 2005; Thawai et al., 2005a, b; Trujillo et al., 2005) and the genus currently holds 27 species with validly published names. While most strains have been isolated from soil collected in diverse geographical regions, members of the genus Micromonospora also appear to be closely associated with plant roots (Coombs & Franco, 2003; Valdés et al., 2005; M. E. Trujillo, unpublished observations). In the present paper, we report on the isolation of a novel Micromonospora strain, NAR01T, from surface-sterilized, nitrogen-fixing root nodules obtained from the plant Coriaria myrtifolia, which was collected in Salamanca, Spain.
The root nodules used for the isolation of strain NAR01T were washed several times with sterile distilled water and were then surface sterilized in HgCl2 (2.5 % w/v) for 2 min. The nodules were rinsed several times with sterile distilled water and then crushed using a sterile glass rod. The homogenized plant tissue was inoculated onto yeast extract/mannitol agar (Vincent, 1970) and the plates were incubated at 28 °C for 10 days. The isolation plates were then examined under a stereoscopic microscope: several small orange colonies were readily observed. Strain NAR01T was selected because of its capacity to degrade xylan, as shown by the ability of the micro-organism to grow on XED medium (Rivas et al., 2003), which contained this carbohydrate as the sole carbon source.
The cultural characteristics of strain NAR01T were studied on several media, namely yeast extract-malt extract agar (ISP 2), Bennett's agar (Jones, 1949), oatmeal agar (ISP 3), SA1 agar (Trujillo et al., 2005) and yeast extract/mannitol agar. Abundant growth was observed on Bennett's, ISP 2 and SA1 agars, while moderate growth was obtained on ISP 3 and yeast extract/mannitol agar media. Colonies were an intense orange colour, folded and raised, turning darker after 3 weeks; neither aerial hyphae nor diffusible pigments were produced. Differences in substrate-mycelium colour found between NAR01T and Micromonospora species are presented in Table 1.
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for Micromonospora endolithica. Gram staining (Doetsch, 1981) was performed using 3-day-old cultures: strain NAR01T was Gram-positive.
Extraction of genomic DNA, PCR amplification of the 16S rRNA gene and sequencing of the purified PCR products were performed as described previously (Rivas et al., 2003). The sequence of strain NAR01T was manually aligned and compared with other sequences obtained from GenBank/EMBL. Phylogenetic distances were calculated with the Kimura two-parameter model, and the tree topologies were inferred using the least-squares (De Soete, 1983), maximum-parsimony (Fitch, 1971) and neighbour-joining methods (Saitou & Nei, 1987). Bootstrap replications (1000) were performed using the MEGA program as described by Kumar et al. (2001).
An almost-complete 16S rRNA gene sequence (1451 nt) was obtained for NAR01T and compared with those deposited in the public databases. The highest levels of similarity were with M. endolithica DSM 44398T (98.94 %) and Micromonospora chersina DSM 44151T (98.4 %). The results of the phylogenetic analysis using various tree-making algorithms were very similar (data not shown). A subset of the closest phylogenetic relatives of strain NAR01T based on the neighbour-joining method are presented in Fig. 1. A phylogenetic tree that includes all Micromonospora species with validly published names is available as Supplementary Fig. S1 in IJSEM Online.
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; Rhuland et al., 1955). Whole-cell sugars were analysed according to Staneck & Roberts (1974). Menaquinones were extracted and purified using the method of Minnikin et al. (1984) and then analysed by HPLC (model 1100; Hewlett Packard). Polar lipids were extracted and identified by two-dimensional TLC (Minnikin et al., 1984). Cellular fatty acid methyl esters were prepared from cells grown for 24 h on trypticase soy agar (Schröder et al., 1997).
The chemotaxonomic markers found were in agreement with the results of phylogenetic analyses, confirming the classification of strain NAR01T within the genus Micromonospora, and also revealing differences at the species level. The novel isolate contained meso-diaminopimelic acid, while the whole-cell sugars detected were arabinose, glucose, mannose, ribose and xylose. Specifically, the presence of glucose and mannose and the absence of galactose and rhamnose distinguished the novel isolate from M. endolithica DSM 44398T. Other differences in sugar composition between NAR01T and its closest phylogenetic neighbours are given in Table 1
. Large amounts of menaquinone MK-10(H4) (>60 %) and smaller amounts of MK-9(H4) and MK-10(H6) were detected. The polar lipid composition included phosphatidylglycerol, phosphatidylinositol and phosphatidylethanolamine, a profile that corresponds to Lechevalier's phospholipid pattern II (Lechevalier et al., 1977). Some unidentified glycolipids were also detected. Significant amounts of iso-15 : 0 (24 %), iso-16 : 0 (17 %), 17 : 0 (10 %) and 10-methyl-17 : 0 (16 %) were detected in the cellular fatty acid profile of NAR01T. The detailed fatty acid composition of strain NAR01T is presented in Supplementary Table S1 (available in IJSEM Online). The G+C content, determined using the thermal melting method (Mandel & Marmur, 1968), was found to be 70.2 mol%.
Phenotypic characteristics were studied using several standard methods. Catalase and oxidase activities were determined as described previously (Trujillo et al., 2006). The hydrolysis of arbutin, aesculin, casein, gelatin, Tweens 20 and 80, starch, tyrosine, urea and xylan was investigated as described by Trujillo et al. (2005), using SA1 agar as a basal medium. Tests for the utilization of various substrates as sole carbon and energy sources were performed according to Williams et al. (1983). The temperature range for growth and the NaCl tolerance were recorded using ISP 2 agar as the basal medium. SA1 agar supplemented with appropriate buffer systems was used for pH-tolerance studies. Enzyme activities for strains NAR01T, M. endolithica DSM 44398T, M. chersina DSM 44151T and Micromonospora echinospora DSM 43816T were performed using API ZYM and API Coryne kits (bioMérieux) according to the manufacturer's instructions.
The novel isolate, NAR01T, presented a phenotypic profile that clearly distinguished it from its closest phylogenetic neighbours (Table 1). An important difference found between NAR01T and its closest neighbour, M. endolithica DSM 44398T, was the capacity of the latter organism to grow in 7 % NaCl (NAR01T tolerated concentrations of only 1 %). Xylan degradation also differentiated NAR01T from M. endolithica DSM 44398T, M. chersina DSM 44151T and Micromonospora inositola DSM 43819T. Furthermore, NAR01T was negative for tyrosine hydrolysis whereas M. endolithica DSM 44398T and Micromonospora rosaria DSM 803T were positive. Additional phenotypic data are given in the species description.
Finally, DNA–DNA hybridizations between NAR01T, M. endolithica DSM 44398T and M. chersina DSM 44151T were carried out to confirm the species status of the novel isolate. DNA was isolated and purified on hydroxyapatite according to the method of Cashion et al. (1977). Hybridization measurements were obtained according to the spectrophotometric method of De Ley et al. (1970), with the modification of Huß et al. (1983). DNA–DNA reassociation values of 11.5 and 25 % were obtained between NAR01T and M. endolithica DSM 44398T and M. chersina DSM 44151T, respectively. These percentages fall well below the 70 % value recommended for defining genomic species (Wayne et al., 1987). Furthermore, they support the idea that Micromonospora strains represent individual genospecies when 16S rRNA gene sequence values are below 99 % (Kroppenstedt et al., 2005).
Strain NAR01T can be clearly differentiated from currently described Micromonospora species on the basis of the genotypic and phenotypic data. Thus, isolate NAR01T represents the type strain of a novel species, for which we propose the name Micromonospora coriariae sp. nov.
Description of Micromonospora coriariae sp. nov.
Micromonospora coriariae (co.ri.a'ri.ae. N.L. gen. n. coriariae of Coriaria, pertaining to the isolation of the type strain from root nodules of Coriaria myrtifolia).
Gram-positive, chemo-organotrophic, aerobic actinobacterium. Colonies are intensely orange, raised and folded. Well-developed substrate mycelium carrying single spores mostly on the tips of the hyphae. Aerial mycelium is absent. Diffusible pigments are not produced. pH range for growth is 7–9; does not grow below pH 6.5. Optimum growth temperature is 28 °C. Oxidase- and catalase-positive. Arbutin, aesculin, casein, gelatin, starch and Tween 80 are degraded, but Tween 20, tyrosine and urea are not. The following are used as carbon sources: alanine, arabinose, arginine, cellobiose, fructose, gluconate, glucose, histidine, mannose, melibiose, pyruvate, raffinose, rhamnose, salicin, serine, starch, trehalose and xylose. The following substrates are not used as carbon sources: ascorbic acid, meso-erythritol, galactose, glutaric acid, lysine, melezitose, proline, propionic acid, quinic acid, sorbitol, sorbose, sucrose, tyrosine, valine and xylitol. Positive enzymic reactions are obtained for the following: acid phosphatase, alkaline phosphatase, cystine arylamidase, esterase (C8), leucine arylamidase, 
-galactosidase, 
-galactosidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, pyrazinamidase and valine arylamidase. Negative reactions are obtained for the following enzymes: -chymotrypsin, -fucosidase, -glucuronidase, -mannoside, naphthol-AS-BI-phosphohydrolase and pyrrolidonyl arylamidase. Other physiological and biochemical characteristics are shown in Table 1. Whole-cell sugars present are glucose, xylose, arabinose, mannose and ribose. The major menaquinone is MK-10(H4).
The type strain, NAR01T (=DSM 44875T=LMG 23557T), was isolated from root nodules of Coriaria myrtifolia.
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ACKNOWLEDGEMENTS |
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