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1 Departamento de Microbiología y Genética, Edificio Departamental, Lab. 209, Campus Miguel de Unamuno, Universidad de Salamanca, 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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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA sequences of isolates Lupac 06, Lupac 07, Lupac 08, Lupac 09T, Lupac 13 and Lupac 14NT are respectively AJ783990–AJ783993, AJ783995 and AJ783996.
Scanning electron micrographs of spores of strains Lupac 09T and Lupac 14NT, an extended phylogenetic tree, cultural characteristics of the novel strains and detailed fatty acid profiles are available as supplementary material with the online version of this paper.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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, a member of the family Micromonosporaceae (Stackebrandt et al., 1997), forms a stable group based on phylogenetic, chemotaxonomic and morphological data (Lechevalier & Lechevalier, 1970; Kroppenstedt, 1985; Lechevalier et al., 1977; Stackebrandt et al., 1997). Members of Micromonospora are widely distributed in aquatic, marine and terrestrial environments (Kawamoto, 1989; Zhao et al., 2004; Maldonado et al., 2005) but, recently, members of this genus have also been recovered from diverse plant tissues, especially nitrogen-fixing root nodules (Valdés et al., 2005; Trujillo et al., 2006b; M. E. Trujillo, unpublished observations). During an ecological study of nitrogen-fixing bacterial communities from Lupinus angustifolius collected in Spain, different orange-pigmented actinomycete colonies were isolated from surface-sterilized nodules on yeast extract-mannitol agar (YMA) (Vincent, 1970). The results of a detailed taxonomic study of six isolates indicate that these strains represent two novel species.
Strains Lupac 06, Lupac 07, Lupac 08, Lupac 09T, Lupac 13 and Lupac 14NT were isolated from surface-sterilized nitrogen-fixing nodules of Lupinus angustifolius collected in Saelices el Chico (Salamanca, Spain) following the procedure described previously (Trujillo et al., 2006a). Isolation plates were incubated for 2 weeks at 28 °C, after which colonies were picked under a stereoscopic microscope. All strains were Gram-stained using the procedure described by Doetsch (1981). Morphological and cultural characteristics of the strains were determined using various agar media: ISP 2 and ISP 3 (Shirling & Gottlieb, 1966), SA1 (Trujillo et al., 2005), Bennett's (Jones, 1949) and N-Z amine, which contained (l–1) 10.0 g glucose, 20.0 g soluble starch, 5.0 g yeast extract, 5.0 g N-Z amine, 1.0 g CaCO3, and 15.0 g agar. (DSM medium 554; DSMZ catalogue). Cell morphology and motility were observed by phase-contrast microscopy using 5-day-old cultures on SA1 agar (Leica; CTR MIC). Isolates Lupac 09T and Lupac 14NT were fixed, dehydrated, critical-point-dried and coated with gold before examination by scanning electron microscopy (Zeiss; DSM 940).
All strains grew well on all media tested, except that isolates Lupac 13 and Lupac 14NT grew poorly or did not grow, respectively, on Bennett's agar. Colonies of all strains ranged from light orange to brown on the different media and were folded and raised, and some of them turned dark brown to black after spore production. Orange–brown and brown diffusible pigments were produced by strains Lupac 09T, Lupac 06 and Lupac 07 on ISP 3 agar. All strains were Gram-positive, produced branched hyphae (0.3–0.6 µm diameter) with terminal spores and lacked aerial mycelium. Strains Lupac 09T and Lupac 14NT formed single spores (0.6–0.8 µm diameter), mainly on long hyphae. The spores had a ‘lemon-shaped’ form and presented a smooth surface (Supplementary Fig. S1, available in IJSEM Online). Cultural characteristics of the six strains in all tested media are presented in Supplementary Table S1.
DNA extraction and PCR amplification of the 16S rRNA gene were performed using the REDExtract-N-Amp Plant PCR kit (Sigma) following the manufacturer's instructions but with an additional purification step using phenol/chloroform. Amplification and sequencing conditions followed methods described by Rivas et al. (2003).
The six sequences obtained were aligned manually and compared with corresponding sequences of all species of the genus Micromonospora with validly published names downloaded from GenBank/EMBL. Phylogenetic distances were calculated with Kimura's two-parameter model (Kimura, 1980) and tree topologies were inferred using the maximum-parsimony (Fitch, 1971) and neighbour-joining (Saitou & Nei, 1987) methods. Branch support based on 1000 replications was calculated with the bootstrap test. All analyses were carried out using the MEGA3 program (Kumar et al., 2004).
Nearly complete 16S rRNA gene sequences (1460 nt) were obtained for the six isolates and confirmed their phylogenetic affiliation to the genus Micromonospora. Identification of the closest phylogenetic neighbours and calculation of pairwise 16S rRNA gene sequence similarities were achieved using the EzTaxon server (http://www.eztaxon.org/; Chun et al., 2007). Sequence similarities between the new isolates and currently described Micromonospora species ranged from 96.7 to 99.0 %. Lupac 14NT showed 98.5 % sequence similarity to Micromonospora mirobrigensis DSM 44830T, while Lupac 09T shared 98.7 % sequence similarity with Micromonospora purpureochromogenes DSM 43821T and Micromonospora matsumotoense DSM 44110T. Two different lineages were revealed by the phylogenetic analysis: isolates Lupac 06, Lupac 07 and Lupac 09T (group A), sharing sequence similarity of 99.6–99.7 %, formed a compact cluster, while strains Lupac 08, Lupac 13 and Lupac 14NT (group B) formed an independent branch and shared sequence similarity of 99.6–100 %. Members of the two clusters showed sequence similarity between 98.8 and 99.1 % and the clusters were supported by a bootstrap value of 99 %. The phylogenetic tree based on the neighbour-joining method (Fig. 1) shows the distribution of the six isolates and the phylogenetically closest Micromonospora species. An extended tree including all Micromonospora species with validly published names is available as Supplementary Fig. S2.
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) were clearly distinguished and divided the strains into two homogeneous clusters which corresponded to the phylogenetic groups (A and B) obtained by 16S rRNA gene sequence analyses.
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) and reassociation measurements were obtained by spectrophotometry (De Ley et al., 1970; Huß et al., 1983). These studies revealed low levels of hybridization among the strains tested. Specifically, Lupac 09T and Lupac 14NT shared 27.9 % and 33.2 % relatedness, respectively, with M. matsumotoense DSM 44100T, while Lupac 09T and Lupac 14NT showed 26 % DNA–DNA relatedness. The G+C contents of strains Lupac 09Tand Lupac 14NT were 71.6 and 70.9 mol%, respectively, as determined by the thermal melting method (Mandel & Marmur, 1968).
Methyl esters of cellular fatty acids for all study strains were prepared from cells grown for 24 h on trypticase soy agar (Schröder et al., 1997) and analysed using the MIDI system (Microbial ID, Inc.). Diaminopimelic acid (DAP) determination was achieved by cellulose TLC using whole-cell hydrolysates (modified method of Hasegawa et al., 1983; Rhuland et al., 1955). Biomass for additional chemotaxonomic analyses of strains Lupac 09T and Lupac 14NT was obtained in tryptic soy broth in flasks on a rotary shaker at 90 r.p.m. and 28 °C. Whole-cell sugars were analysed according to Staneck & Roberts (1974). Phospholipids and menaquinones were prepared according to the method of Minnikin et al. (1984) and respectively analysed by two-dimensional TLC and HPLC.
Differences in fatty acid composition divided the strains into two groups which corresponded to those defined by the 16S rRNA gene sequence and riboprint analyses. The main differences between the two groups were in the amounts of iso-15 : 0, iso-16 : 0, iso-16 : 1 and iso-17 : 0. The detailed fatty acid patterns of the study strains are presented in Supplementary Table S2. All strains contained meso-DAP. Whole-cell hydrolysates of Lupac 09T and Lupac 14NT contained glucose, mannose, arabinose, xylose and ribose; in addition, Lupac 09T contained rhamnose. Galactose, which has been reported for M. matsumotoense DSM 44100T and M. mirobrigensis DSM 44830T, was not found in the new isolates. The menaquinones of strain Lupac 09T were made up of MK-10(H4) (>90 %) and traces of MK-10(H6), while Lupac 14NT contained these two quinones (83 and 10 %, respectively) in addition to MK-9(H4) (7 %). The two strains contained phosphatidylglycerol, phosphatidylinositol and phosphatidylethanolamine, which corresponds to phospholipid pattern II. Overall chemotaxonomic differences found between Lupac 09T and Lupac 14NT and related species are given in Table 1.
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), degradation of various organic compounds (Trujillo et al., 2005), carbon substrate utilization (Williams et al., 1983) and growth at and tolerance of various temperatures (4–45 °C) and NaCl concentrations (1–5 %). Enzyme activities were also determined using API ZYM and API Coryne kits (bioMérieux) for the six isolates and M. matsumotoense DSM 44100T, M. mirobrigensis DSM 44830T and Micromonospora carbonacea DSM 43168T.
All strains were catalase- and oxidase-positive with the exception of Lupac 06, which did not produce oxidase. All strains tolerated 1 % NaCl and grew at 20–37 °C. All strains were negative for nitrate reduction and urease production and were sensitive to 0.01 % sodium azide. Differentiating metabolic profiles of isolates Lupac 09T, Lupac 14NT and phylogenetically related Micromonospora type strains are given in Table 1
. The two groups represented by strains Lupac 09T (group A) and Lupac 14NT (group B) could be differentiated by the use of arabinose, trehalose, alanine and sucrose. Pigment production was also considered an important difference between the two groups. Additional physiological data are presented in Table 1 and the species descriptions.
The 16S rRNA gene sequences, cultural characteristics and chemotaxonomic data clearly demonstrate that the six strains isolated from nitrogen-fixing nodules of L. angustifolius belong to the genus Micromonospora. Furthermore, riboprint analyses, fatty acid compositions and metabolic profiles indicate that the strains can be divided into two groups which, according to DNA–DNA hybridization data, represent novel species. Thus, we propose to classify strains Lupac 09T, Lupac 06, and Lupac 07 (group A) within the novel species Micromonospora saelicesensis sp. nov. and strains Lupac 14NT, Lupac 08 and Lupac 13 (group B) as representatives of the species Micromonospora lupini sp. nov.
Description of Micromonospora saelicesensis sp. nov.
Micromonospora saelicesensis (sae.li.ces.en'sis. N.L. fem. adj. saelicesensis pertaining to Saelices, the place where the plants were collected from which the first strains were isolated).
Gram-positive, chemo-organotrophic and aerobic actinomycete. Well-developed branched hyphae (0.3–0.6 µm in diameter). Colonies are orange on ISP 2 agar. Orange–brown to brown diffusible pigments are produced on ISP 3 agar. Smooth-surfaced spores are produced at the tip of the hyphae. Growth occurs at 20–37 °C but not at 15 or 45 °C; optimum growth at 28 °C. Catalase-positive and oxidase-variable. Nitrate is not reduced. Growth is observed in 2 % NaCl but not in the presence of 0.001 % crystal violet or 0.01 % sodium azide. Arbutin, casein, aesculin, gelatin and xylan are degraded, but not tyrosine. Starch degradation is variable. Carbon sources assimilated include arabinose, cellobiose, galactose, glutarate, histidine, inositol, maltose, mannose, melibiose, raffinose and sucrose. Alanine, arginine, gluconate, lysine, proline, rhamnose, serine, sorbitol, sorbose, sucrose, trehalose, valine and xylitol are not assimilated. Enzyme tests with the API ZYM and API Coryne systems are positive for acid and alkaline phosphatases, esterase (C4), esterase lipase (C8), lipase (C15), leucine arylamidase, cystine arylamidase, trypsin, 
-chymotrypsin, naphthol-AS-BI-phosphohydrolase, -galactosidase, β-galactosidase, -glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase and pyrazinamidase and negative for β-glucuronidase, pyrrolidonyl arylamidase, urease and β-glucuronidase. Variable reactions are observed for -mannosidase and -fucosidase. Acid is produced from glucose. Peptidoglycan contains meso-DAP; whole-cell sugars are glucose, mannose, arabinose, xylose, ribose and rhamnose. Major menaquinone is MK-10(H4). Diagnostic phospholipid is diphosphatidylethanolamine. The G+C content of the type strain is 71.6 mol%.
The type strain, Lupac 09T (=DSM 44871T =LMG 24056T), and two other strains, Lupac 06 (=DSM 44868) and Lupac 07 (=DSM 44869), were isolated from root nodules of Lupinus angustifolius.
Description of Micromonospora lupini sp. nov.
Micromonospora lupini (lu'pin.i. L. gen. n. lupini of a lupin, referring to the isolation of the first strains from Lupinus angustifolius).
Gram-positive, chemo-organotrophic and aerobic actinomycete. Well-developed branched hyphae (0.3–0.6 µm in diameter). Colonies are light orange on ISP 2 agar. No diffusible pigments are observed. Smooth-surfaced spores are produced at the tip of the hyphae. Growth occurs at 20–37 °C but not at 15 or 45 °C; optimum growth at 28 °C. Catalase- and oxidase-positive. Nitrate is not reduced. Growth is observed at 1 % NaCl but variable at 2 % NaCl. No growth in 0.001 % crystal violet or 0.01 % sodium azide. Arbutin, casein, aesculin, gelatin, starch and xylan are degraded but not tyrosine. Carbon sources assimilated include alanine, cellobiose, galactose, maltose, mannose, melibiose, raffinose and trehalose. Arginine, histidine, lysine, proline, rhamnose, serine, sorbitol, sorbose, tyrosine, valine and xylitol are not assimilated. Enzyme tests with the API ZYM and API Coryne systems are positive for acid and alkaline phosphatases, esterase (C4), esterase lipase (C8), lipase (C15), leucine arylamidase, cystine arylamidase, trypsin, -chymotrypsin, naphthol-AS-BI-phosphohydrolase, -galactosidase, β-galactosidase, -glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase and pyrazinamidase and negative for β-glucuronidase, -mannosidase, -fucosidase and urease. Acid is not produced from glucose. Peptidoglycan contains meso-DAP; whole-cell sugars are glucose, mannose, arabinose, xylose and rhamnose. Major menaquinone is MK-10(H4). Diagnostic phospholipid is diphosphatidylethanolamine. The G+C content of the type strain is 70.9 mol%. Strain Lupac 08 produces the antitumoral compounds lupinadicins A and B.
The type strain, Lupac 14NT (=DSM 44874T =LMG 24055T), and two other strains, Lupac 08 (=DSM 44870) and Lupac 13 (=DSM 44873), were isolated from root nodules of Lupinus angustifolius.
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ACKNOWLEDGEMENTS |
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