Introduction

The genus Aeromicrobium was first proposed by Miller in 1991 [15]. According to the original, the genus comprised non-mycelial, non-sporulating actinomycetes that produced the macrolide antibiotic erythromycin; the type strain is Aeromicrobium erythreum. In 1994, Nocardioides fastidiosa was transferred to the genus as Aeromicrobium fastidiosum [22]. Members of the genus Aeromicrobium are Gram-reaction-positive, non-endospore-forming rods or cocci and are characterized chemotaxonomically by having a tetrahydrogenated menaquinone with nine isoprene units [MK-9(H4)] as the predominant respiratory quinone, and 10-methyl C18:0, C16:0 and both or either of C18:1 ω9c and C16:0 2-OH as the major cellar fatty acids [25]. At the time of writing, the genus Aeromicrobium comprises 12 recognized species (http://www.bacterio.net). Strains of the genus are commonly isolated from various sources, including soil [4, 10], marine environment [11], Pu’er tea [17], human stools [18], and air [23].

In this study, we describe the taxonomic characterization of new Gsoil 137T which appeared to be a member of the genus Aeromicrobium.

Materials and Methods

Isolation of Bacterial Strain

Strain Gsoil 137T was isolated from soil of a ginseng field of Pocheon province in South Korea. The soil samples were collected from different places and thoroughly suspended with 50 mM phosphate buffer (pH 7.0), and the suspensions were spread on R2A agar plates after serial dilution with 50 mM phosphate buffer (pH 7.0). The plates were aerobically incubated at 30 °C for two weeks. Single colony was purified by transferring onto new R2A agar plates and was incubated at 30 °C. Gsoil 137T was routinely cultured on R2A agar at 30 °C and maintained as a glycerol suspension (20%, v/v) at − 80 °C. The strain Gsoil 137T was deposited to the Korean Collection for Type Cultures (= KCTC 19130T), German Collections of Microorganisms and Cell Cultures (= DSM 17940T), and the Culture Collection of the University of Gothenburg (= CCUG 52475T).

Physiological, Morphological, and Biochemical Characteristics

The Gram reaction was determined using the non-staining method using 3% KOH, as described previously [2]. Cell morphology was examined by a scanning electron microscope (Hitachi SU-3500), using cells grown for 2 days at 30 °C on R2A agar medium. Catalase and oxidase tests were performed as outlined by Cappuccino and Sherman [3]. Biochemical phenotypic tests were carried out using API 20E, API ID 32GN, and API ZYM test kits according to the instructions of the manufacturer (bioMérieux, France). Tests for degradation of DNA (using DNase agar from Scharlau, with DNase activity by flooding plates with 1M HCl), casein, and starch were performed and evaluated after 5 days [1]. Growth at different temperatures (4, 10, 18, 30, 37, 42, and 45 °C) and various pH values (pH 3.5–10.0 at intervals of 0.5 pH units) was assessed after 5 days of incubation. The following buffers (final concentration, 20 mM) were used to adjust the pH of R2A broth: acetate buffer was used for pH 3.5–5.5, phosphate buffer was used for pH 6.0–8.0, and Tris buffer was used for pH 8.5–10.0. Salt tolerance was tested on nutrient medium supplemented with 1–10% (w/v at intervals of 1% unit) NaCl after 5 days of incubation. Growth on trypticase soy agar (TSA, BD) and MacConkey agar (BD) was also evaluated at 30 °C.

Phylogenetic Tree Construction and Determination of DNA G+C Content (mol%)

For phylogenetic analysis of strain Gsoil 137T, DNA was extracted using a genomic DNA extraction kit (Solgent Co. Ltd, Korea). The 16S rRNA gene was amplified from the chromosomal DNA using the universal bacterial primer set (800R, 1492R, 27F, and 518F) and the purified PCR products were sequenced by Solgent Co. Ltd. (Daejeon, South Korea) as described previously [9]. Almost full length of the 16S rRNA gene was compiled using SeqMan software (DNASTAR). The 16S rRNA gene sequences of related taxa were obtained from the GenBank and EzTaxon-e server (http://www.ezbiocloud.net/eztaxon). Multiple sequence alignments were performed by Clustal X program [24]. Gaps were edited in the BioEdit program [7]. Evolutionary distances were calculated using the Kimura two-parameter model [12]. Phylogenic trees were constructed using a neighbor-joining method [19] and maximum-parsimony [6] using the MEGA 6 Program [21] with bootstrap values based on 1000 replications [5].

For the measurement of DNA G+C content, genomic DNA of the novel strain was extracted and purified as described by Moore and Dowhan [16], enzymatically degraded into nucleosides, and determined as described previously [14] using a reverse-phase HPLC.

Chemotaxonomic Analysis

Isoprenoid quinones were extracted with chloroform/methanol (2:1, v/v), evaporated under vacuum conditions, and reextracted in n-hexane/water (1:1, v/v). The crude n-hexane–quinone solution was purified using Sep-Pak Vac cartridges silica (Waters) and subsequently analyzed by HPLC as previously described [8]. Cellular fatty acid profiles were determined for strains grown on R2A agar for 48 h. The cellular fatty acids were saponified, methylated, and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI). The fatty acid methyl esters were then analyzed by gas chromatography (model 6890; Hewlett Packard) using the Microbial Identification software package [20]. The presence of diaminopimelic acid (DAP) isomers in the cell wall peptidoglycan was determined using thin-layer chromatography after hydrolysis with 6 N HCl at 100 °C for 18 h as previously described [13].

Nucleotide Sequence Accession Numbers

The 16S rRNA gene sequence of strain Gsoil 137T determined in this study was deposited in NCBI GenBank/EMBL/DDBJ under the accession number AB245395. The digital protologue database (DPD) number for the strain Gsoil 137T is TA00193.

Results and Discussion

Morphological and Phenotypic Characteristics

Colonies of strain Gsoil 137T grown on R2A agar plates for 2 days at 30 °C were convex and creamy colored, which grow well on R2A agar medium, whereas weakly grow on nutrient and Luria–Bertani (BD) agar media, but did not grow on trypticase soy agar (BD), potato-dextrose agar (BD), DNase agar (BD), and MacConkey agar. Strain Gsoil 137T was able to grow at 10–30 °C, but not at 4, 35, and 37 °C. Furthermore, the physiological characteristics of strain Gsoil 137T are summarized in the species description and comparison of selective characteristics with related type strains is shown in Table 1.

Table 1 Physiological and biochemical characteristics between strain Gsoil 137T and closely related species of the genus Aeromicrobium
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Phylogenetic and DNA G+C Content Analysis

The almost complete 16S rRNA gene sequence of strain Gsoil 137T (1470 nt) was determined and subjected to comparative analysis. Based on the EzTaxon-e analysis, the isolate was assigned to the genus Aeromicrobium with the highest sequence similarity to Aeromicrobium ginsengisoli KCTC 19207T (96.7%). The phylogenetic study based on the neighbor-joining, maximum-likelihood, and maximum-parsimony methods confirms that strain Gsoil 137T clustered within the genus Aeromicrobium and form a monophyletic clade with Aeromicrobium halocynthiae JCM 15749T (Fig. 1), well separated from the species of genera Nocardioides, Marmoricola, and others.

Fig. 1
figure 1

Phylogenetic tree constructed from a comparative analysis of 16S rRNA gene sequences showing the relationships of strain Gsoil 137T with other related species. The tree was made using the maximum-likelihood method with two-parameter distance matrix and complete deletion. Dots indicate generic branches that were also recovered using maximum-parsimony and neighbor-joining algorithms. Bootstrap values (expressed as percentages of 1000 replications) greater than 60% are shown at the branch points. Bar, 0.01 substitutions per single nucleotide position. Terrabacter tumescens KCTC 9133T (AF005023) was used as an outgroup

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On the basis of 16S rRNA gene sequence similarity analysis and phylogenetic inference, Aeromicrobium halocynthiae JCM 15749T, Aeromicrobium ginsengisoli KCTC 19207T, and Aeromicrobium panaciterrae KCTC 19131T were selected for comparative study.

The G+C content of genomic DNA of strain Gsoil 137T was 70.3 mol%.

Chemotaxonomic Characteristics

Many species of the genus Aeromicrobium have MK-9(H4) as the predominant quinone; however, some species of the genus Aeromicrobium also possess menaquinone of the MK-7 (H4) and MK-8 (H4) types. The menaquinones possessed in strain Gsoil 137T were MK-8 (H4) and Mk-7 (H4) types. The cell wall peptidoglycan of strain Gsoil 137T contained LL-DAP. The major cellular fatty acids of strain Gsoil 137T were mainly composed of C14:0 (8.5%) C16:0 (34.4%), C18:1 ω9c (21.6%), and C16:1 ω7c/C15:0 [(summed feature 4) 11.3%], which were similar to those of Aeromicrobium species (Table 2). The high amount of C14:0 (8.5%) and summed feature 4 (11.3%) along with qualitative and quantitative differences in cellular fatty acid analysis distinguishes strain Gsoil 137T from those of Aeromicrobium species (Table 2).

Table 2 Fatty acid profiles of strain Gsoil 137T and related species of the genus Aeromicrobium
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Taxonomic Conclusions

In summary, the characteristics of strain Gsoil 137T are consistent with descriptions of the genus Aeromicrobium with regard to morphological, biochemical, and chemotaxonomic properties. However, on the basis of phylogenetic distance from type strains of Aeromicrobium species indicated by 16S rRNA gene sequence similarities and the combination of unique phenotypic characteristics (Table 1), strain Gsoil 137T represents a novel species, for which the name Aeromicrobium panacisoli sp. nov is proposed.

Description of Aeromicrobium Panacisoli sp. Nov

Aeromicrobium panacisoli (pa.na.ci.so´li. N.L. n. Panax-acis scientific name of ginseng; L. n. solum-i soil; N.L. gen. n. panacisoli of soil of a ginseng field, the source of isolation of the type strain).

Cells are Gram positive, strictly aerobic, non-spore-forming, non-motile, and longer rod shaped (1.2–2.0 μm in diameter and 2.0–5 μm in length). Colonies grown on R2A agar are circular, creamy colored, and 0.5–1.5 mm in diameter. Growth occurs at 10–30 °C in the presence of 0.5–4.5% NaCl (w/v) and at pH 6–9. Optimum growth occurs at 30 °C and pH 6.0–7.0 in the absence of NaCl. Catalase and oxidase activities are negative. Gsoil 137T grow well on R2A agar medium and grow weakly on nutrient agar (NA, BD) and Luria–Bertani agar (LB, BD). The isolate does not grow on potato-dextrose agar (PDA, BD), MacConkey agar (BD), and DNase agar (BD). In the API kit system, the strains were positive for l-rhamnose, N-acetyl-glucose, d-ribose, inositol, d-saccharose, d-maltose, lactic acid, l-alanine, glycogen, l-serine, d-mannitol, d-glucose, l-fucose, potassium 2-ketogluconate, esterase, esterase lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, arginine dihydrolase, lysine decarboxylase, citrate, acetoin production, gelatin hydrolysis, mannitol, sucrose, amygdalin, and arabinose. List of all negative traits of commercial kits is shown in Table S1. MK-8 (H4) predominant menaquinone and C14:0, C16:0, C18:1 ω9c and summed feature 4 (comprising C16:1 ω7c/C15:0 iso 2-OH) are the major components of cellular fatty acids. The DNA G+C content of genomic DNA is 70.3 mol%. The cell wall peptidoglycan of strains Gsoil 137T contains LL-DAP.

The type strain Gsoil 137T (= KCTC 19130T = DSM 17940T = CCUG 52475T) was isolated from soil of a ginseng field of Pocheon province, South Korea.