An actinomycete strain was recovered from a pond where radon is known to be dissolved. A polyphasic study was undertaken to identify the new isolate. The 16S rRNA gene sequence of strain WA201T showed closest similarity to the type strains of Micromonospora carbonacea (98?5 %) and Micromonospora matsumotoense (98?1 %). The chemotaxonomic results confirmed the taxonomic position of the isolate in the genus Micromonospora. DNA-DNA relatedness values supported the classification of this isolate as a novel species. A number of physiological and biochemical tests were able to distinguish strain WA201 T from its closest phylogenetic neighbours. Therefore, it is proposed that isolate WA201 T (=DSM 44830 T =LMG 22229 T ) be considered the type strain representing a novel species, Micromonospora mirobrigensis sp. nov. (Ørskov, 1923) are reported to inhabit soil, water, marine environments and sediments (Lüdemann & Brodsky, 1963;Kawamoto, 1989). Micromonospora endolithica, isolated from an extreme Antarctic sandstone environment, has been described recently (Hirsch et al., 2004), showing that this genus is distributed in very different environments. Kasai et al. (2000) redefined and reduced the genus to 14 species based on DNA-DNA relatedness, 16S rRNA and gyrB phylogenetic data. We report the classification of an organism representing a novel Micromonospora species.
Representatives of the genus Micromonospora
Isolate WA201T was recovered from a water sample of a pond, located on a former uranium mine (Ciudad Rodrigo, Spain) where radon is known to be dissolved in water (Lozano et al., 2002). The isolation procedure using soil extract agar pH 6?5 at 28 u C was described previously by Trujillo et al. (2004). Long-term maintenance of strain WA201T was accomplished by storage in glycerol suspension (20 %, w/v) at 280 uC. The isolate was cultured on Bennett's agar, glucose-yeast extract agar and nutrient agar to check for growth rate. Growth was slow on these media and therefore a basal medium (SA1) was designed to improve it. SA1 contained glucose, 10 g; yeast extract (Difco), 3 g; tryptone (Difco), 5 g; tryptose (Difco), 2 g; starch (Fluka), 2 g; CaCO 3 , 100 mg; CoCl 2 , traces; ferric citrate, traces; Bacto-agar (Difco), 18 g; and distilled water, 1 l. The pure culture was then routinely grown on SA1 agar.Extraction of genomic DNA, PCR amplification of the 16S rRNA gene and sequencing of the purified PCR products were described previously (Rivas et al., 2003). The sequence of isolate WA201T was manually aligned and compared with representative sequences of members of the order Actinomycetales obtained from GenBank/EMBL. Phylogenetic distances were calculated with the Kimura twoparameter model and tree topologies were inferred using the least-squares (De Soete, 1983), maximum-parsimony (Fitch, 1972) and neighbour-joining methods (Saitou & Nei, 1987). One thousand bootstrap replications were performed using the MEGA program as described by Kumar et al. (2001).Morphological features were studied on glucose-yeast extract ag...
