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Int J Syst Evol Microbiol 57 (2007), 2106-2109; DOI  10.1099/ijs.0.65040-0
© 2007 International Union of Microbiological Societies

Terrabacter aerolatus sp. nov., isolated from an air sample

Hang-Yeon Weon1, Peter Schumann2, Reiner M. Kroppenstedt2, Byung-Yong Kim3, Jaekyeong Song3, Soon-Wo Kwon3, Seung-Joo Go3 and Erko Stackebrandt2

1 Applied Microbiology Division, National Institute of Agricultural Science and Technology, Rural Development Administration, Suwon 441-707, Republic of Korea
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7b, 38124 Braunschweig, Germany
3 KACC – Korean Agricultural Culture Collection, Microbial Genetics Division, National Institute of Agricultural Biotechnology, Rural Development Administration, Suwon 441-707, Republic of Korea

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


   ABSTRACT
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A Gram-positive, strictly aerobic, motile, rod- or coccoid-shaped bacterium, strain 5516J-36T, was isolated from an air sample from Jeju region, Korea, and its taxonomic position was investigated by a polyphasic approach. The organism grew optimally at 30 °C and pH 7.0–8.0. Comparative 16S rRNA gene sequencing studies demonstrated that this strain was highly related phylogenetically to Terrabacter terrae PPLBT and Terrabacter tumescens DSM 20308T, showing 98.9 % sequence similarity to both strains. However, the DNA–DNA reassociation values between 5516J-36T and the type strains of Terrabacter terrae and Terrabacter tumescens were low (51 and 48 %, respectively). The peptidoglycan type was A3{gamma}, the predominant menaquinone was MK-8(H4), the polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol and an unidentified phosphoglycolipid and the whole-cell sugars were glucose, ribose, rhamnose, xylose and galactose. Mycolic acids were absent. The major fatty acids (>5 % of total fatty acids) were iso-C15 : 0, iso-C16 : 0, iso-C14 : 0, iso-C17 : 0 and anteiso-C15 : 0. The DNA G+C content was 71.7 mol%. On the basis of the above data, it is proposed that strain 5516J-36T represents a novel species, Terrabacter aerolatus sp. nov. The type strain of Terrabacter aerolatus is 5516J-36T (=KACC 20556T =DSM 18562T).

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 5516J-36T is EF212039.


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The genus Terrabacter, with the type species Terrabacter tumescens, was created by the transfer of Pimelobacter tumescens on the basis of phylogenetic and chemotaxonomic data (Collins et al., 1989Go). Recently, another species Terrabacter terrae was proposed for an isolate from soil in Spain (Montero-Barrientos et al., 2005Go). The genus was placed in the family Intrasporangiaceae and is characterized by MK-8(H4) as the predominant isoprenoid quinone, A3{gamma} as the peptidoglycan type, iso-C15 : 0 as the major fatty acid and high DNA G+C contents (71–73 mol%).

Strain 5516J-36T was isolated from an air sample from the Jeju region of Korea. The air sample was collected using an MAS-100 air sampler (Merck; single-stage multiple-hole impactor) which contained Petri dishes with R2A agar (BBL) amended with 200 µg cycloheximide ml–1 (Sigma). After sampling, plates were incubated at 30 °C for 5 days, and strain 5516J-36T was recovered. Terrabacter tumescens DSM 20308T and Terrabacter terrae CECT 3379T were used as reference strains. These strains were cultivated on R2A medium (pH 7.0; Difco) at 30 °C and maintained on R2A medium.

The 16S rRNA gene of the isolate was amplified by a PCR and sequenced directly using an ABI Prism 310 Genetic Analyzer (Applied Biosystems). Phylogenetic analysis was carried out using MEGA3 (Kumar et al., 2004Go) after multiple alignment of the sequences by CLUSTAL W (Thompson et al., 1994Go). Distances using distance options according to the Kimura two-parameter model (Kimura, 1983Go) and clustering using neighbour-joining were determined by using bootstrap values based on 1000 replicates. DNA–DNA hybridization was carried out as described by Seldin & Dubnau (1985)Go. Probe labelling was conducted by using the non-radioactive DIG High Prime DNA labelling and detection starter kit II (Roche Molecular Biochemicals). Reassociation was conducted at 65 °C. The hybridized DNA was visualized using the DIG luminescent detection kit (Roche). DNA–DNA relatedness was quantified by using a densitometer (Bio-Rad).

For strain 5516J-36T, 1400 bp of the 16S rRNA gene sequence was determined. Comparative 16S rRNA gene sequence analysis showed that strain 5516J-36T was most closely related to members of the genus Terrabacter. In a phylogenetic tree based on the neighbour-joining algorithm, strain 5516J-36T joined the clade comprising Terrabacter species at a bootstrap confidence value of 100 % (Fig. 1Go). Strain 5516J-36T exhibited 16S rRNA gene sequence similarity values of 98.9 % to both Terrabacter terrae PPLBT and Terrabacter tumescens DSM 20308T. It showed 97.4 and 96.9 % sequence similarity, respectively, to Terracoccus luteus DSM 44267T and Intrasporangium calvum DSM 43043T. DNA–DNA relatedness data demonstrated that strain 5516J-36T represents a genomic species that is different from recognized Terrabacter species (Wayne et al., 1987Go). DNA–DNA relatedness values between strain 5516J-36T and Terrabacter tumescens DSM 20308T, Terrabacter terrae CECT 3379T and Terracoccus luteus DSM 44267T were respectively 51, 48 and 32 %.


Figure 1
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  		Fig. 1. 16S rRNA gene sequence-based dendrogram obtained by distance matrix (neighbour-joining) analysis showing the position of strain 5516J-36T. Numbers at nodes indicate levels of bootstrap support based on a neighbour-joining analysis of 1000 resampled datasets. Bootstrap values below 50 % are not indicated. Bar, 0.01 substitutions per position.

 
Cell morphology was examined by phase-contrast microscopy (AXIO; Zeiss). The isolate was tested for a number of key characteristics, such as Gram staining, catalase, oxidase and hydrolysis of casein, DNA, hypoxanthine, gelatin, starch, Tween 80 and xanthine, by using standard procedures (Smibert & Krieg, 1994Go). Growth was assessed at 5, 10, 20, 25, 30, 35 and 40 °C, at pH 4–10 (increments of 1 pH unit) and at 0, 1, 3, 5, 7 and 10 % NaCl. Hydrolysis of CM-cellulose (Sigma) (0.1 %), chitin from crab shells (1 %, w/v), pectin (0.5 %, w/v) and tyrosine (0.5 %, w/v) was checked. Motility was examined on 1/10-strength R2A medium. Anaerobic growth was checked using a BBL anaerobic jar (Becton Dickinson). Other physiological properties and enzyme activities were tested using the API 20NE, API ID 32GN and API ZYM systems (bioMérieux). The results of API 20NE and API ID 32GN were recorded after 5 days incubation.

Strain 5516J-36T formed visible colonies on R2A agar after 48 h at 28 °C. The strain grew on R2A, nutrient agar (Difco) and tryptic soy agar (TSA; Difco), but did not grow on MacConkey agar (Difco). The colonies were white, convex and round. Cells were strictly aerobic, Gram-positive, motile, catalase-positive and oxidase-negative. Cells are cocci (diameter 0.9–1.0 µm) that occur in pairs, or rods (0.9x1.5–3.5 µm). The physiological and biochemical characteristics are summarized in Table 1Go.


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  		Table 1. Differential characteristics between strain 5516J-36T and type strains of Terrabacter species

Strains: 1, 5516J-36T; 2, Terrabacter tumescens DSM 20308T; 3, Terrabacter terrae CECT 3379T. Data were obtained in this study unless indicated. According to API 20NE and API ZYM strips, all strains are positive for aesculin hydrolysis, gelatin hydrolysis and beta-galactosidase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, {alpha}-galactosidase and {alpha}-glucosidase activities. All strains are negative for indole production, glucose fermentation and arginine dihydrolase, urease, alkaline phosphatase, cystine arylamidase, trypsin, {alpha}-chymotrypsin, beta-glucuronidase, N-acetyl-beta-glucosaminidase and {alpha}-fucosidase activities. According to API 20NE and API ID32GN strips, all strains assimilate D-glucose, D-mannose, N-acetylglucosamine, maltose, potassium gluconate, malic acid, inositol, sucrose, sodium malonate, sodium acetate, L-alanine, glycogen, propionic acid, valeric acid, L-histidine and 3-hydroxybutyric acid. None of the strains assimilate capric acid, trisodium citrate, phenylacetic acid, L-rhamnose, itaconic acid, suberic acid, potassium 5-ketogluconate, salicin, L-fucose, potassium 2-ketogluconate or 4-hydroxybenzoic acid. +, Positive; (+), weakly positive; –, negative.

 
Biomass for chemotaxonomic studies was grown in shake flasks of R2A broth for 4 days at 28 °C, checked for purity, harvested by centrifugation and freeze-dried. The peptidoglycan was analysed as described by Schleifer & Kandler (1972)Go. The phospholipids and menaquinones were analysed by the method of Minnikin et al. (1984)Go. Sugar analysis of whole cells and mycolic acid determinations were carried out as described by Staneck & Roberts (1974)Go and Minnikin et al. (1975)Go, respectively. For fatty acid methyl ester analysis, cell mass of strain 5516J-36T was harvested from TSA plates after incubation for 3 days at 28 °C. Analyses of the whole-cell fatty acid pattern followed described methods using the MIDI system (Microbial ID, Inc.) (Kroppenstedt, 1985Go; Sasser, 1990Go). The G+C content of the DNA was determined according to Mesbah et al. (1989)Go using a reversed-phase column (Supelcosil LC-18S; Supelco).

From quantitative analysis of the peptidoglycan amino acids by gas chromatography, strain 5516J-36T contained glycine, alanine, glutamic acid and LL-diaminopimelic acid in an approximate molar ratio of 4.0 : 1.8 : 1.0 : 1.0. From the two-dimensional TLC pattern (not shown) of peptides in the partial peptidoglycan hydrolysate, strain 5516J-36T showed peptidoglycan type A3{gamma} as described by Schleifer & Kandler (1972)Go (type A41.1 according to http://www.dsmz.de/species/murein.htm). The predominant menaquinone was MK-8(H4). The polar lipids phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol and a phosphoglycolipid of unknown structure were detected. The peptidoglycan structure, isoprenoid quinone and polar lipids of strain 5516J-36T were congruent with those of the two recognized species of the genus Terrabacter. The predominant fatty acids were iso-C15 : 0 (49.0 %), iso-C16 : 0 (14.5 %), iso-C14 : 0 (7.9 %), iso-C17 : 0 (5.8 %) and anteiso-C15 : 0 (5.2 %) (Table 2Go). The fatty acid profile of strain 5516J-36T was similar to that of Terrabacter terrae CECT 3379T, which was considerably different from that of Terrabacter tumescens DSM 20308T (Montero-Barrientos et al., 2005Go). The whole-cell sugar profile contained glucose, ribose, rhamnose, xylose and galactose (in decreasing order of abundance). Mycolic acids were absent. The DNA G+C content of strain 5516J-36T was 71.7 mol%.


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  		Table 2. Cellular fatty acid compositions of strain 5516J-36T and type strains of Terrabacter species

Strains: 1, 5516J-36T; 2, Terrabacter tumescens DSM 20308T; 3, Terrabacter terrae CECT 3379T. All strains were grown for 2 days on TSA. Values are percentages of total fatty acids; fatty acids that represented <0.5 % in all strains are omitted. –, Not detected.

 
On the basis of the data presented, strain 5516J-36T should therefore be placed in the genus Terrabacter within a novel species, for which the name Terrabacter aerolatus sp. nov. is proposed.

Description of Terrabacter aerolatus sp. nov.
Terrabacter aerolatus (ae.ro.la'tus. Gr. n. aer air; L. part. adj. latus carried; N.L. masc. part. adj. aerolatus airborne).

Cells are strictly aerobic, Gram-positive, motile rods or cocci (0.9–1.0x1.0–3.5 µm), catalase-positive and oxidase-negative. Colonies are white, convex and round. Optimal temperature for growth is 30 °C. Growth occurs in the range 5–35 °C. Optimal pH for growth is between 7.0 and 8.0; growth occurs in the range pH 4–9. Growth occurs in the presence of 0–5 % (w/v) NaCl. Casein, starch, tyrosine and Tween 80 are hydrolysed, but chitin, CM-cellulose, DNA, pectin, hypoxanthine and xanthine are not. LL-Diaminopimelic acid-containing peptidoglycan A3{gamma} has three glycine residues as the interpeptide bridge. Whole-cell sugars are glucose, ribose, rhamnose, xylose and galactose (in decreasing order of abundance). Mycolic acids are absent. The predominant menaquinone is MK-8(H4). The major fatty acids (>5 % of total fatty acids) are iso-C15 : 0 (49.0 %), iso-C16 : 0 (14.5 %), iso-C14 : 0 (7.9 %), iso-C17 : 0 (5.8 %) and anteiso-C15 : 0 (5.2 %). The major polar lipids are phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol and an unidentified phosphoglycolipid. The DNA G+C content of the type strain is 71.7 mol%.

The type strain, 5516J-36T (=KACC 20556T =DSM 18562T), was isolated from an air sample in Jeju, Korea.


   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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Kimura, M. (1983). The Neutral Theory of Molecular Evolution. Cambridge: Cambridge University Press.

Kroppenstedt, R. M. (1985). Fatty acid and menaquinone analysis of actinomycetes and related organisms. In Chemical Methods in Bacterial Systematics (SAB Technical Series no. 20), pp. 173–199. Edited by M. Goodfellow & D. E. Minnikin. London: Academic Press.

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]

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]

Minnikin, D. E., Alshamaony, L. & Goodfellow, M. (1975). Differentiation of Mycobacterium, Nocardia, and related taxa by thin-layer chromatographic analysis of whole-organism methanolysates. J Gen Microbiol 88, 200–204.[Abstract/Free Full Text]

Minnikin, D. E., O'Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, K. & Parlett, J. H. (1984). An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.[CrossRef]

Montero-Barrientos, M., Rivas, R., Velázquez, E., Monte, E. & Roig, M. G. (2005). Terrabacter terrae sp. nov., a novel actinomycete isolated from soil in Spain. Int J Syst Evol Microbiol 55, 2491–2495.[Abstract/Free Full Text]

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

Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36, 407–477.[Free Full Text]

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]

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