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Research Collection Journal Article Characterization of Methylobacterium strains isolated from the phyllosphere and description of Methylobacterium longum sp nov Author(s): Knief, Claudia; Dengler, Vanina; Bodelier, Paul L.E.; Vorholt, Julia A. Publication Date: 2012-01 Permanent Link: https://doi.org/10.3929/ethz-b-000042009 Originally published in: Antonie van Leeuwenhoek 101(1), http://doi.org/10.1007/s10482-011-9650-6 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library Antonie van Leeuwenhoek (2012) 101:169–183 DOI 10.1007/s10482-011-9650-6 ORIGINAL PAPER Characterization of Methylobacterium strains isolated from the phyllosphere and description of Methylobacterium longum sp. nov Claudia Knief • Vanina Dengler • Paul L. E. Bodelier • Julia A. Vorholt Received: 18 June 2011 / Accepted: 24 September 2011 / Published online: 11 October 2011 Ó Springer Science+Business Media B.V. 2011 Abstract Methylobacterium strains are abundantly acids and sugars, while others could only metabolize a found in the phyllosphere of plants. Morphological, restricted number of organic acids. The strains that physiological and chemotaxonomical properties of 12 were most distinct from existing type strains based on previously isolated strains were analyzed in order to 16S rRNA gene sequence analysis were selected for obtain a more detailed overview of the characteristics DNA–DNA hybridization experiments to analyze of phyllosphere colonizing Methylobacterium strains. whether they are sufficiently different at the genomic All strains showed the typical properties of the genus level from existing type strains to justify their classi- Methylobacterium, including pink pigmentation, fac- fication as new species. This resulted in the delineation ultative methylotrophy, a fatty acid profile dominated of strain 440 and its description as Methylobacterium by C18:1 x7c, and a high G?C content of 65 mol % or longum sp. nov. strain 440 (=DSM 23933T = CECT more. However, some strains showed only weak 7806T). A main characteristic of this species is the growth on methanol and pigmentation varied from formation of relatively long rods compared to other pale pink to red. Strains grew best under mesophilic, Methylobacterium species. neutrophilic conditions and low salt (B1%) concen- trations, but variation was seen with respect to the Keywords Phyllosphere Á Methylobacterium temperature and pH range under which growth longum sp. nov occurred. Likewise, differences were seen with respect to carbon source utilization. Some strains were versatile and utilized diverse organic acids, amino Introduction Electronic supplementary material The online version of this article (doi:10.1007/s10482-011-9650-6) contains The alphaproteobacterial genus Methylobacterium supplementary material, which is available to authorized users. comprises aerobic, Gram-negative and pink pig- mented bacteria with a facultative methylotrophic & C. Knief ( ) Á V. Dengler Á J. A. Vorholt lifestyle (Green 2006). Members of this genus can Institute of Microbiology, ETH Zurich, Wolfgang-Pauli- Strasse 10, 8093 Zurich, Switzerland grow on diverse one-carbon compounds such as e-mail: [email protected] methanol, formaldehyde or formate and some of them on methylated amines or halogenated one-carbon P. L. E. Bodelier compounds (Kayser et al. 2002; Scha¨fer et al. 2007), Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Nieuwersluis, Utrecht, but none of them on methane (Dedysh et al. 2004). The Netherlands Methylobacterium strains occur widespread in nature; 123 170 Antonie van Leeuwenhoek (2012) 101:169–183 e. g., they have been isolated from freshwater, soil, different enrichment strategies in that previous study, dust, sediments, air, or hospital environments (Green strains 108, 109, 113, 189, 440, 679 and 812 were 2006). Remarkable is their consistent occurrence in initially cultivated on a fivefold diluted mineral salt association with plants (Corpe and Rheem 1989; Knief medium, but were shown to grow equally well or et al. 2010b). Methylobacterium strains colonize the better at the higher substrate concentration. Thus, all aerial and below ground parts of plants as epi- and strains were cultivated on normal strength media in endophytes (e. g., Andreote et al. 2009; Elbeltagy et al. this study. All strains characterized in this study were 2000; Mano et al. 2007; Pirttila¨ et al. 2000; Schauer deposited in the German and Spanish culture collec- and Kutschera 2008). Upon plant colonization, they tion; their deposition numbers are included in Table 1. express genes involved in methylotrophy (Delmotte Reference strains Methylobacterium aquaticum et al. 2009; Gourion et al. 2006) and benefit from DSM 16371T, Methylobacterium variabile DSM methanol utilization (Abanda-Nkpwatt et al. 2006; 16961T, Methylobacterium brachiatum DSM 19569T, Schmidt et al. 2010; Sy et al. 2005). Potential Methylobacterium mesophilicum DSM 1708T, Methy- advantages of this association for the plant host, in lobacterium tardum DSM 19566T, Methylobacterium particular with regard to growth stimulation, have radiotolerans DSM 1819T, Methylobacterium oryzae been discussed in different reports (e. g., Fedorov et al. DSM 18207T, Methylobacterium fujisawaense DSM 2011; Holland et al. 2002; Idris et al. 2004; Koenig 5686T, Methylobacterium phyllosphaerae DSM et al. 2002; Kutschera 2007; Lidstrom and Chistoser- 19779T and Methylobacterium adhaesivum DSM dova 2002; Madhaiyan et al. 2007; Omer et al. 2004). 17169T were obtained from the German Collection of Diverse members of the genus Methylobacterium are Microorganisms and Cell Cultures (DSMZ). They found in association with plants and the Methylobac- were cultured under the conditions described above. terium community composition has been shown to vary on different plant species and in dependence on Morphological characterization the location of the plant (Knief et al. 2010a; Knief et al. 2010b). Among the retrieved Methylobacterium iso- Morphology of the isolates is described for cultures lates from the plant phyllosphere are phylogenetic grown on mineral salt medium agar plates with lineages that are not well described (Knief et al. methanol as sole carbon source after 4 days of 2010a). In order to obtain a more detailed picture incubation at 28°C. For microscopical observations about the physiology and morphology of the diverse and cell size measurements an Axiophot micro- plant associated Methylobacterium lineages, we char- scope and the Axiovision 4.7 software (Zeiss) were acterized different strains that were isolated from the used. Motility of the strains was tested in swarming phyllosphere of plants. assays using tenfold diluted R2A medium (Difco) soft agar plates (0.15% agar), which were inocu- lated with cell material in the center of the plate. Methods Swarming of the cells was evaluated after 5 days of incubation. Enrichment and isolation of Methylobacterium strains Analysis of growth characteristics Methylobacterium strains were isolated from the Growth at different temperatures, pH and NaCl phyllosphere of Arabidopsis thaliana, Cardamine concentrations was tested on mineral salt medium hirsuta or Medicago truncatula (Table 1) in previous agar plates with methanol as carbon source. Cells from studies (Knief et al. 2008; Knief et al. 2010a). These plate-grown cultures were suspended in medium to an plants grew at different natural sampling sites in Spain OD600 of 1 and a tenfold serial dilution was done in a or France, except the greenhouse grown A. thaliana microtiter plate. 5 ll of each dilution was spotted onto plant from which strain F15 was isolated (Table 1). agar plates. Each test was performed twice based on All strains were enriched and isolated on a mineral salt independently grown precultures. Growth under the medium supplemented with 120 mM methanol at different conditions was evaluated after 1 week and up 28°C (Knief et al. 2010b). Due to the application of to 4 weeks in case of slow growth. Growth was tested 123 Antonie van Leeuwenhoek (2012) 101:169–183 171 Table 1 Origin of the Methylobacterium strains Strain Sampling site Location Ecosystem Type of sample Sampling Culture date collection identifier 31 Menasalbas, 39°39.5360N Mediterranean xeric Leaf of April 2005 DSM 23928 Spain 4°20.6540W grassland and scrubland Arabidopsis CECT 7804 thaliana 32 Menasalbas, 39°39.5360N Mediterranean xeric Leaf of April 2005 DSM 23929 Spain 4°20.6540W grassland and scrubland Arabidopsis CECT 7805 thaliana 85 Ciruelos de 41°12.8820N Sandy site at the edge of a Leaf of April 2005 DSM 23930 Coca, Spain 4°32.6640W pine forest Arabidopsis CECT 7807 thaliana 108 Marjaliza, 39°34.9510N Trackside in a Mediterranean Leaf of April 2005 DSM 23934 Spain 3°55.5260W forest of evergreen Quercus Arabidopsis CECT 7808 species thaliana 109 Marjaliza, 39°34.9510N Trackside in a Mediterranean Leaf of April 2005 DSM 23977 Spain 3°55.5260W forest of evergreen Quercus Arabidopsis CECT 7812 species thaliana 113 Marjaliza, 39°34.9510N Trackside in a Mediterranean Leaf of April 2005 DSM 23935 Spain 3°55.5260W forest of evergreen Quercus Cardamine CECT 7814 species hirsuta 189 La Chopera, 40°30.8780N Trackside adjacent to a xeric Fruit surface of April 2005 DSM 23931 Spain 3°53.8100W grassland and nearby a Arabidopsis CECT 7810 residential area thaliana 337 Polan, Spain 39°49.5150N Mediterranean scrubland