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Int J Syst Evol Microbiol 58 (2008), 93-96; DOI  10.1099/ijs.0.65047-0
© 2008 International Union of Microbiological Societies

Methylobacterium iners sp. nov. and Methylobacterium aerolatum sp. nov., isolated from air samples in Korea

Hang-Yeon Weon1, Byung-Yong Kim2, Jae-Ho Joa3, Jung-A Son1, Myung-Hee Song2, Soon-Wo Kwon2, Seung-Joo Go2 and Sang-Hong Yoon2

1 Applied Microbiology Division, National Institute of Agricultural Science and Technology, Rural Development Administration (RDA), Suwon 441-707, Republic of Korea
2 Korean Agricultural Culture Collection (KACC), Microbial Genetics Division, National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, Republic of Korea
3 National Institute of Subtropical Agriculture, RDA, Jeju 690-150, Republic of Korea

Correspondence
Soon-Wo Kwon
swkwon{at}rda.go.kr


   ABSTRACT
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Two bacterial strains isolated from air samples, 5317S-33T and 5413S-11T, were characterized by determining their phenotypic characteristics, cellular fatty acid profiles and phylogenetic positions based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that these isolates belonged to the genus Methylobacterium. Strain 5317S-33T was most closely related to Methylobacterium adhaesivum AR27T (97.9 % sequence similarity). Strain 5413S-11T was most closely related to Methylobacterium fujisawaense DSM 5686T (97.3 % sequence similarity), Methylobacterium oryzae CBMB20T (97.1 % similarity) and Methylobacterium radiotolerans JCM 2831T (97.0 % similarity). Cells of both strains were strictly aerobic, Gram-negative, motile and rod-shaped. The major fatty acid was C18 : 1{omega}7c. The G+C contents of the genomic DNA were 68.0 mol% for strain 5317S-33T and 73.2 mol% for strain 5413S-11T. According to DNA–DNA hybridization data, strain 5317S-33T showed a level of DNA–DNA relatedness of 33 % with M. adhaesivum DSM 17169T, and strain 5413S-11T showed low levels of DNA–DNA relatedness (<35 %) with M. fujisawaense DSM 5686T, M. oryzae CBMB20T and M. radiotolerans DSM 1819T. On the basis of this polyphasic analysis, it was concluded that strains 5317S-33T and 5413S-11T represent two novel species within the genus Methylobacterium, for which the names Methylobacterium iners sp. nov. (type strain 5317S-33T =KACC 11765T =DSM 19015T) and Methylobacterium aerolatum sp. nov. (type strain 5413S-11T =KACC 11766T =DSM 19013T) are proposed.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains 5317S-33T and 5413S-11T are EF174497 and EF174498, respectively.

Cellular fatty acid compositions of strains 5317S-33T and 5413S-11T and related Methylobacterium species are detailed as supplementary material available with the online version of this paper.


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The genus Methylobacterium was originally proposed by Patt et al. (1976)Go. This genus was characterized as containing strictly aerobic, Gram-negative and pink-pigmented facultatively methylotrophic bacteria (Green, 1992Go). Members of the genus Methylobacterium have been isolated mainly from soil and water (Corpe & Rheem, 1989Go; Lidstrom & Chistoserdova, 2002Go).

In the course of study of the bacterial population in air samples, two pink-coloured bacterial strains were isolated. Air samples were collected in the Suwon region, Republic of Korea, using an MAS-100 air sampler (single-stage multiple-hole impactor; Merck). The sampler contained Petri dishes with R2A agar (BBL) amended with 0.02 % cycloheximide (Sigma). After sampling, plates were incubated at 28 °C for 5 days and strains 5317S-33T and 5413S-11T were recovered. Routine cultivation was conducted at 28 °C with R2A media.

Cell morphology was examined by light microscopy (AXIO; Zeiss). The test for motility was performed on one-tenth-strength R2A broth 2216 (Difco) supplemented with 0.2 % agar. Anaerobic growth was tested by incubating cultures on R2A plates in a GasPak jar (BBL) for 14 days, with aerobically incubated plates as controls. Growth at different temperatures (5–45 °C) and pH values (4, 5, 6, 7, 8, 9 and 10) was monitored in R2A broth. The requirement for NaCl was tested in R2A broth supplemented with 0, 2, 3, 5 and 7 % (w/v) NaCl. Gram-staining, catalase and oxidase activities and hydrolysis of aesculin, casein, CM-cellulose, DNA, hypoxanthine, tyrosine, Tween 80, starch and xanthine were determined as described by Smibert & Krieg (1994)Go. For the various physiological tests, API 20NE and API ZYM test strips (bioMérieux) were used according to the manufacturer's instructions. Biolog GN2 plates were used to test activity against 95 carbon substrates. Both strains were strictly aerobic, Gram-negative, motile and rod-shaped. Strain 5317S-33T grew on R2A (Difco) and R2A plus 0.5 % methanol, but did not grow on nutrient agar (NA; Difco), NA plus 0.5 % methanol, trypticase soy agar (Difco) or MacConkey agar (Difco). Strain 5413S-11T grew on R2A (Difco), R2A plus 0.5 % methanol, NA and NA plus 0.5 % methanol, but did not grow on trypticase soy agar or MacConkey agar. Both strains could be differentiated clearly from their close relatives by several phenotypic characteristics such as nitrate reduction, urease, gelatin hydrolysis, substrate assimilation and enzyme activities (Table 1Go).


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  		Table 1. Differential properties of strains 5317S-33T and 5413S-11T and closely related Methylobacterium species

Strains: 1, 5317S-33T; 2, 5413S-11T; 3, M. adhaesivum DSM 17169T; 4, M. fujisawaense DSM 5686T; 5, M. mesophilicum DSM 1708T; 6, M. oryzae KACC 11585T; 7, M. radiotolerans DSM 1819T. According to the API 20NE test strip, all strains are negative for indole production, glucose fermentation, arginine dihydrolase, aesculin hydrolysis and β-galactosidase. None of the strains assimilate D-mannose, D-mannitol, N-acetylglucosamine, maltose, capric acid or phenylacetic acid. According to the API ZYM test strip, all strains are positive for esterase (C4), esterase lipase (C8), leucine arylamidase and naphthol-AS-BI-phosphohydrolase, but negative for lipase (C14), cystine arylamidase, {alpha}-chymotrypsin, {alpha}-galactosidase, β-galactosidase, β-glucuronidase, {alpha}-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, {alpha}-mannosidase and {alpha}-fucosidase. +, Positive; (+), weakly positive; –, negative.

 
Whole-cell fatty acids were analysed according to the standard protocol of the MIDI/Hewlett-Packard Microbial Identification system (Sasser, 1990Go). DNA G+C contents were determined according to the method of Mesbah et al. (1989)Go using a reversed-phase column (Supelcosil LC-18 S; Supelco). Both strains had C18 : 1{omega}7c as the major fatty acid, which was in agreement with the fatty acid profiles of other Methylobacterium species (Supplementary Table S1, available in IJSEM Online). The DNA G+C content of strain 5317S-33T was 68.0 mol%; that of strain 5413S-11T was 73.2 mol%.

The 16S rRNA gene was amplified by using PCR with primers fD1 and rP2 (Weisburg et al., 1991Go) on colonies; the entire PCR fragment was sequenced directly (Hiraishi, 1992Go). The resultant 16S rRNA gene sequences were compared with available sequences from GenBank using the program BLAST (http://www.ncbi.nlm.nih.gov/blast/) to determine an approximate phylogenetic affiliation and gene sequences were aligned with those of closely related strains using the software CLUSTAL W (Thompson et al., 1994Go). Phylogenetic trees were constructed by the neighbour-joining method with the program package MEGA3 (Kumar et al., 2004Go). Bootstrap confidence values were obtained using 1000 resamplings. Phylogenetic trees were also constructed by using the maximum-parsimony method. DNA–DNA hybridization was carried out as described by Seldin & Dubnau (1985)Go. Probe labelling was conducted using a non-radioactive DIG High Prime DNA Labelling and Detection Starter kit II (Roche Molecular Biochemicals). Hybridized DNA was visualized using a DIG luminescent detection kit (Roche Molecular Biochemicals). The 16S rRNA gene sequence similarities between strain 5317S-33T and the type strains of all recognized Methylobacterium species were in the range 93.6–97.9 %; its closest relative was Methylobacterium adhaesivum AR27T (97.9 % sequence similarity). Strain 5413S-11T showed sequence similarity levels of 91.6–97.3 % with all type strains of the genus Methylobacterium. It revealed >97 % sequence similarity with Methylobacterium fujisawaense DSM 5686T (97.3 %), Methylobacterium oryzae CBMB20T (97.1 %) and Methylobacterium radiotolerans JCM 2831T (97.0 %). According to the neighbour-joining tree (Fig. 1Go), strain 5317S-33T formed a compact cluster with M. adhaesivum AR27T (99 % bootstrap support). Strain 5413S-11T clustered with M. fujisawaense, M. oryzae, M. radiotolerans and Methylobacterium mesophilicum. The topologies of phylogenetic trees built using the maximum-parsimony algorithm also supported the notion that these isolates belong to the genus Methylobacterium (data not shown). Strain 5317S-33T showed a level of DNA–DNA relatedness of 33 % with M. adhaesivum DSM 17169T. Strain 5413S-11T showed low levels of DNA–DNA relatedness with M. fujisawaense DSM 5686T (34 %), M. oryzae CBMB20T (28 %) and M. radiotolerans DSM 1819T (35 %). On the basis of this polyphasic taxonomic analysis, the names Methylobacterium iners sp. nov. and Methylobacterium aerolatum sp. nov. are proposed to accommodate these two novel strains.


Figure 1
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  		Fig. 1. Phylogenetic tree showing the relationship between strains 5317S-33T and 5413S-11T and related species. The tree was constructed by using the neighbour-joining method based on 16S rRNA gene sequences. Numbers at nodes indicate percentage bootstrap values from 1000 replicates. Bootstrap values greater than 50 % are shown. Bar, 0.01 substitutions per nucleotide position.

 
Description of Methylobacterium iners sp. nov.
Methylobacterium iners (i'ners. L. neut. adj. iners inactive, lazy).

Cells are strictly aerobic, Gram-negative, motile and rod-shaped (1.0x1.5–2.5 µm). Colonies are pink-coloured, circular and convex with entire margins. Produces oxidase and catalase. Growth is observed at 10–30 °C (optimum 25–28 °C) and pH 6.0–8.0 (optimum 7.0). Cannot grow in the presence of NaCl concentrations of 2 % or more. Hydrolyses starch, but does not hydrolyse casein, CM-cellulose, DNA, hypoxanthine, Tween 80, tyrosine or xanthine. The following substrates produce positive results in Biolog GN2 plates: pyruvic acid methyl ester, acetic acid, formic acid, {alpha}-hydroxybutyric acid, β-hydroxybutyric acid, {alpha}-ketobutyric acid, DL-lactic acid, propionic acid, succinamic acid, L-asparagine, L-aspartic acid and L-glutamic acid. The major fatty acid is C18 : 1{omega}7c.

The type strain is 5317S-33T (=KACC 11765T =DSM 19015T), isolated from an air sample in Suwon city, Republic of Korea. The DNA G+C content of the type strain is 68.0 mol%.

Methylobacterium aerolatum sp. nov.
Methylobacterium aerolatum (ae.ro.la'tum. Gr. n. aer air; L. part. adj. latus carried; N.L. neut. part. adj. aerolatum airborne).

Cells are strictly aerobic, Gram-negative, motile and rod-shaped (1.0x1.5–3.0 µm). Colonies are pink-coloured, circular and convex with entire margins. Produces oxidase and catalase. Growth is observed at 10–35 °C (optimum 28 °C) and pH 5.0–9.0 (optimum 7.0). Cannot grow in the presence of NaCl concentrations of 2 % or more. Hydrolyses starch, hypoxanthine and xanthine, but does not hydrolyse casein, CM-cellulose, DNA, Tween 80 or tyrosine. The following substrates produce positive results in Biolog GN2 plates: pyruvic acid methyl ester, succinic acid monomethyl ester, formic acid, {alpha}-hydroxybutyric acid, β-hydroxybutyric acid, {gamma}-hydroxybutyric acid, {alpha}-ketoglutaric acid, DL-lactic acid, succinic acid, bromosuccinic acid, succinamic acid and glycerol. The major fatty acid is C18 : 1{omega}7c.

The type strain is 5413S-11T (=KACC 11766T =DSM 19013T), isolated from an air sample in Suwon city, Republic of Korea. The DNA G+C content of the type strain is 73.2 mol%.


   ACKNOWLEDGEMENTS
 
This study was supported by the National Institute of Agricultural Biotechnology (NIAB grant no. 06-4-11-19-1), Rural Development Administration, Republic of Korea.


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Corpe, W. A. & Rheem, S. (1989). Ecology of the methylotrophic bacteria on living leaf surfaces. FEMS Microbiol Ecol 62, 243–249.[CrossRef]

Green, P. N. (1992). The genus Methylobacterium. In The Prokaryotes, 2nd edn, pp. 2342–2349. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K. H. Schleifer. New York: Springer.

Hiraishi, A. (1992). Direct automated sequencing of 16S rDNA amplified by polymerase chain reaction from bacterial cultures without DNA purification. Lett Appl Microbiol 15, 210–213.[Medline]

Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.[Abstract/Free Full Text]

Lidstrom, M. E. & Chistoserdova, L. (2002). Plants in the pink: cytokinin production by Methylobacterium. J Bacteriol 184, 1818[Free Full Text]

Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.[Abstract/Free Full Text]

Patt, T. E., Cole, G. C. & Hanson, R. S. (1976). Methylobacterium, a new genus of facultatively methylotrophic bacteria. Int J Syst Bacteriol 26, 226–229.[Abstract/Free Full Text]

Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI.

Seldin, L. & Dubnau, D. (1985). Deoxyribonucleic acid homology among Bacillus polymyxa, Bacillus macerans, Bacillus azotofixans, and other nitrogen-fixing Bacillus strains. Int J Syst Bacteriol 35, 151–154.[Abstract/Free Full Text]

Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for General and Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.

Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.[Abstract/Free Full Text]

Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173, 697–703.[Abstract/Free Full Text]




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