Micromonospora pisi sp. nov., isolated from root nodules of Pisum sativum

The genus Micromonospora (Ørskov, 1923) is the type genus of the family Micromonosporaceae. At the time of writing, this family comprised 22 genera, including the recently described taxon Polymorphospora (Tamura et al., 2006). Phylogenetically, the genus Polymorphospora shows a close relationship to the genus Micromonospora (Tamura et al., 2006), and also shares various chemotaxonomic markers including cell-wall chemotype II, phospholipid type II and peptidoglycan type A1γ. The main differences between Micromonospora and Polymorphospora species are found in the cell-wall sugar composition and fatty acid type. Additionally, Polymorphospora rubra is reported to produce short spore chains and polymorphic spores (Tamura et al., 2006).

Many species in the genus Micromonospora have been described in the past few years and, at the time of writing, the genus comprised 37 recognized species, including species isolated from root nodules of Coriaria sp. and Lupinus angustifolius (Trujillo et al., 2006b, 2007). Here we report the isolation and taxonomic characterization of a novel strain, GUI 15^T, from surface-sterilized, nitrogen-fixing root nodules obtained from a plant of Pisum sativum collected in Zamora, Spain.

Root nodules used for the isolation of strain GUI 15^T were processed as described by Trujillo et al. (2006b), by using yeast extract-mannitol agar (Vincent, 1970) and incubation at 28 °C for 15 days. Isolation plates were examined routinely under a stereoscopic microscope until bacterial growth was observed. Selected orange colonies were transferred to SA1 agar (Trujillo et al., 2005) to obtain abundant biomass for long-term maintenance as glycerol suspensions (20 %, v/v) at −80 °C. The strain investigated was maintained routinely on SA1 agar. Strain GUI 15^T was selected because of its capacity to degrade xylan, and the colour of its substrate mycelium, which was beige to light yellow on ISP 2 agar (Shirling & Gottlieb, 1966).

Cultural characteristics of strain GUI 15^T were studied on several media, namely Bennett's (Jones, 1949), oatmeal (ISP 3; Shirling & Gottlieb, 1966), SA1 (Trujillo et al., 2005), yeast-extract malt-extract (ISP 2) and yeast extract-mannitol agars. The novel strain showed good growth on all media except oatmeal agar, on which growth was poor and slow; neither aerial hyphae nor diffusible pigments were produced on these test media. The colour of the substrate mycelium was generally pale yellow and colonies were folded and raised, turning brown after 3 weeks. Detailed results of the cultural characteristics of strain GUI 15^T on the various test media are given in Supplementary Table S1 in IJSEM Online. Cell morphology and motility were observed by using phase-contrast microscopy (Leica; CTR MIC) of 7-day-old cultures grown on ISP 2 agar. Spore production was examined on 2-week-old cultures on ISP 2 agar by using scanning electron microscopy (DSM 940; Zeiss). Strain GUI 15^T produced extensively branching and non-fragmenting hyphae (0.3–0.5 μm in diameter). No motility was observed. Mainly single, smooth, spherical, non-motile spores (0.6–0.8 μm in diameter) were produced, although in some cases pairs of spores were observed (Fig. 1⇓). This spore morphology corresponds to that described for members of the genus Micromonospora but not to that for Polymorphospora, which is reported to produce spore chains (Tamura et al., 2006). Gram and acid-fast stains (Doetsch, 1981) were used with 4-day-old cultures. Strain GUI 15^T was Gram-positive and non-acid-fast.

DNA extraction for PCR amplification of the 16S rRNA and gyrase B subunit (gyrB) genes was performed as described previously (Trujillo et al., 2007). Amplification and sequencing conditions for the 16S rRNA gene followed the methods described by Rivas et al. (2003). The 16S rRNA gene sequence of strain GUI 15^T was aligned manually and compared with other sequences obtained from the GenBank/EMBL/DDBJ databases. Phylogenetic distances were calculated with the Kimura two-parameter model (Kimura, 1980) and tree topologies were inferred by using the maximum-parsimony (Fitch, 1971) and neighbour-joining methods (Saitou & Nei, 1987). Bootstrap replications (1000) were performed by using the mega4 program (Tamura et al., 2007).

PCR amplification of the gyrB gene of strains GUI 15^T, Micromonospora pattaloongensis TJ2-2^T and P. rubra TT 97-42^T was performed by using the REDExtract-N-Amp Plant PCR kit (Sigma). Each PCR mixture (20 μl final volume) contained 10 μl REDExtract-N-Amp PCR Readymix, 0.4 μl of each primer (20 μM), 2 μl extraction/dilution mixture (50 : 50 v/v) and 1 μl template DNA. For the design of gyrB primers, sequences of the following Micromonospora species previously determined by Kasai et al. (2000) were used: M. aurantiaca (NBRC 16125^T), M. carbonacea (NBRC 14108^T), M. chalcea (NBRC 13503^T), M. chersina (NBRC 15963^T), M. coerulea (NBRC 13504^T), M. echinospora (NBRC 13149^T), M. halophytica (NBRC 14112^T), M. nigra (NBRC 16103^T), M. inositola (ATCC 21773^T), M. olivasterospora (NBRC 14304^T), M. purpureochromogenes (NBRC 13324^T) and M. rosaria (NBRC 13697^T). These sequences were aligned via the clustal_x program and were searched for primers similar to those described by Richert et al. (2007) but more specific for the genus Micromonospora. Two overlapping PCR products were amplified to obtain a final composite sequence of 1110 nt. In the first amplification, a 500-bp fragment was obtained by using primers GYF1 (5′-TCCGGYGGYCTGCACGGCGT-3′; position 19–38) and GYR1B (5′-CGGAAGCCCTCYTCGTGSGT-3′; position 548–567). The amplification was performed with an initial denaturation at 95 °C for 9 min, followed by 35 cycles with denaturation at 95 °C for 1 min, annealing at 62 °C for 1 min and extension at 72 °C for 2 min, followed by a step at 72 °C for 7 min. The second fragment of 900 bp was amplified by using primers GYF3 (5′-ACSGTCGACTTCGACTTCCA-3′, position 220–239) and GYR3B (5′-CAGCACSAYCTTGTGGTA-3′, position 1210–1226). The amplification was performed with an initial denaturation at 95 °C for 9 min, followed by 35 cycles with denaturation at 95 °C for 1 min, annealing at 54 °C for 1 min and extension at 72 °C for 2 min, followed by a step at 72 °C for 7 min. Sequencing was carried out by using primers GYF1, GYF3, GYF4 (5′-ACCCACGAGGAGGGCTTCCG-3′, position 548–567) GYR1B and GYR3B. The primer positions correspond to the nucleotides in the gyrB gene sequence of Microbacterium imperiale 33884 (AB074922.1). Phylogenetic analysis was performed as described above.

An almost-complete 16S rRNA gene sequence (1452 nt) was obtained for strain GUI 15^T and was compared with those deposited in the public databases. The highest levels of 16S rRNA gene sequence similarity were with Micromonospora pattaloongensis JCM 12833^T (98.7 %) and P. rubra TT 97-42^T (98.5 %). Additionally, identification of its closest phylogenetic neighbours and calculation of pairwise 16S rRNA gene sequence similarities were performed via the EzTaxon server (; Chun et al., 2007), which yielded similar results.

The results of the phylogenetic analysis with various tree-making algorithms were very similar (data not shown). Fig. 2⇓ shows the relationship between strain GUI 15^T and its closest phylogenetic neighbours based on 16S rRNA gene sequences. The selected strains formed two subclades, one of which comprised P. rubra TT 97-42^T, Micromonospora pattaloongensis TJ2-2^T, Micromonospora olivasterospora DSM 43868^T and strain GUI 15^T. This clustering was supported by bootstrap values which ranged from 62 to 77 %. A tree containing all recognized species of the genus Micromonospora at the time of writing and other representatives of the family Micromonosporaceae is provided as Supplementary Fig. S1 (in IJSEM Online). This tree showed that the grouping of the above four strains was identical. From these results, the taxonomic affiliation of strain GUI 15^T at the genus level could not be determined.

To clarify the affiliation of strain GUI 15^T to its closest phylogenetic neighbours, the sequences of the gyrB gene for strain GUI 15^T, P. rubra TT 97-42^T and Micromonospora pattaloongensis TJ2-2^T were obtained and compared. Levels of gyrB gene sequence similarity between strain GUI 15^T and Micromonospora pattaloongensis TJ2-2^T and P. rubra TT 97-42^T were 89.6 and 90.5 %, respectively, whereas P. rubra TT 97-42^T and M. pattaloongensis TJ2-2^T shared 88.1 % gyrB gene sequence similarity. Levels of gyrB gene sequence similarity between strain GUI 15^T and other available Micromonospora species ranged from 90.3 to 93.1 %. The phylogenetic analysis shown in Fig. 3⇓ included all available sequences for the type strains of Micromonospora species and other members of the family Micromonosporaceae. All Micromonospora strains were recovered in one cluster including strain GUI 15^T, P. rubra TT 97-42^T and Micromonospora pattaloongensis TJ2-2^T, supported by a 100 % bootstrap value. These results demonstrated that strain GUI 15^T is a member of the genus Micromonospora.

Chemotaxonomic analyses were carried out to support the above results. For these analyses strain GUI 15^T was grown in ISP 2 broth in flasks on a rotary shaker at 125 r.p.m. and 28 °C for 1 week. Biomass was harvested, washed in distilled water and freeze-dried. Isomers of diaminopimelic acid in whole-cell hydrolysates were determined by TLC on cellulose (modified method of Hasegawa et al., 1983; Rhuland et al., 1955). Whole-cell sugars were analysed according to Staneck & Roberts (1974). Menaquinones were extracted and purified by using the method of Minnikin et al. (1984) and were analysed by HPLC (Hewlett Packard 1100). Methyl esters of cellular fatty acids were prepared from cells grown for 24 h on trypticase soy agar and ISP 2 agar (28 °C) and were analysed by GLC (Schröder et al., 1997). Polar lipids were extracted and identified by using two-dimensional TLC (Minnikin et al., 1984). For whole-cell sugar and fatty acid analyses, P. rubra TT 97-42^T was also studied under the same growth conditions. The DNA G+C content of strain GUI 15^T was 71 mol% as determined by using the thermal melting method (Mandel & Marmur, 1968).

Strain GUI 15^T contained meso-diaminopimelic acid, which is the characteristic diamino acid of peptidoglycan type A1γ (Schleifer & Kandler, 1972), as also reported for the genera Micromonospora and Polymorphospora. Galactose, glucose, mannose, ribose, xylose, rhamnose (trace) and arabinose (trace) were detected as whole-cell sugars in strain GUI 15^T. This sugar profile is identical to that reported for Micromonospora pattaloongensis TJ2-2^T (Thawai et al., 2008). The sugar profile of P. rubra TT 97-42^T (Tamura et al., 2006) was similar to that of strain GUI 15^T except that arabinose and ribose were absent, and 3-O-methyl mannose was present. In the present sugar analyses, P. rubra contained galactose, glucose and xylose but not 3-O-methyl mannose or mannose; instead ribose was detected. A summary of these results is given in Table 1⇓. Strain GUI 15^T contained MK-10(H₄) as the main menaquinone with MK-10(H₆) and MK-10(H₈) as minor components. These compounds were also reported for Micromonospora pattaloongensis TJ2-2^T and P. rubra TT 97-42^T, in addition to other menaquinones (Table 1⇓). Similarly, the diagnostic lipid phosphatidylethanolamine, which corresponds to pattern II (Lechevalier et al., 1977), was found in strain GUI 15^T, Micromonospora pattaloongensis TJ2-2^T and P. rubra TT 97-42^T. Additionally, the polar lipid profile of strain GUI 15^T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and an unknown phosphoglycolipid; some unidentified glycolipids and phospholipids were also detected. The major fatty acids of strain GUI 15^T were iso-C_16 : 0 (27.4 % of the total), anteiso-C_15 : 0 (22.9 %), anteiso-C_17 : 0 (15.4 %) and iso-C_15 : 0 (13.7 %); a trace of 10-methyl C_17 : 0 (0.29 %) was also detected. This profile corresponds to fatty acid type 3b (Kroppenstedt, 1985). The major fatty acids of P. rubra TT 97-42^T under the same conditions were iso-C_16 : 0 (30.4 %), C_17 : 1ω8c (23.3 %), C_17 : 0 (7.7 %), anteiso-C_15 : 0 (7.7 %), anteiso-C_17 : 0 (7.6 %) and iso-C_15 : 0 (5.7 %); 10-methyl C_17 : 0 was also detected (0.3 %). According to our results, the profile of P. rubra is also of type 3b (Kroppenstedt, 1985). The full fatty acid profiles of the two strains are given in Supplementary Table S2 (in IJSEM Online). Chemotaxonomic characteristics of strain GUI 15^T, Micromonospora pattaloongensis and P. rubra are given in Table 1⇓.

Physiological tests were carried out according to standard methods and included determination of catalase and oxidase activities (Trujillo et al., 2006a), degradation of various organic compounds (Trujillo et al., 2005), carbon substrate utilization (Williams et al., 1983), and growth at various temperatures (4–45 °C), pH (4.5–9) and NaCl concentrations (1–5 %). Enzyme activity tests by using API ZYM and API Coryne kits (bioMérieux) were also carried out for strain GUI 15^T, Micromonospora pattaloongensis TJ2-2^T, Micromonospora olivasterospora DSM 43868^T and P. rubra TT 97-42^T.

Strain GUI 15^T presented a phenotypic profile that clearly distinguished it from its closest phylogenetic neighbours (Table 2⇓). In particular, it could be differentiated based on substrate mycelium colour, NaCl tolerance, temperature growth range, and production of pyrazinamidase, β-galactosidase, β-glucosidase and pyrrolidonyl arylamidase. A full description of the physiological properties determined for strain GUI 15^T is provided in the species description.

In summary, the phenotypic and genotypic data presented indicate that strain GUI 15^T represents a novel species of the genus Micromonospora, for which we propose the name Micromonospora pisi sp. nov. Further investigation will be necessary to determine whether P. rubra TT 97-42^T is sufficiently different to warrant representing a species distinct from members of the genus Micromonospora.