Enterobacter oligotrophica sp. nov., a novel oligotroph isolated from leaf soil

Abstract A novel oligotrophic bacterium, designated strain CCA6, was isolated from leaf soil collected in Japan. Cells of the strain were found to be a Gram‐negative, non‐sporulating, motile, rod‐shaped bacterium. Strain CCA6 grew at 10–45°C (optimum 20°C) and pH 4.5–10.0 (optimum pH 5.0). The strain was capable of growth in poor‐nutrient (oligotrophic) medium, and growth was unaffected by high‐nutrient medium. The major fatty acid and predominant quinone system were C16:0 and ubiquinone‐8. Phylogenetic analysis based on 16S rRNA gene sequences indicated strain CCA6 presented as a member of the family Enterobacteriaceae. Multilocus sequence analysis (MLSA) based on fragments of the atpD, gyrB, infB, and rpoB gene sequences was performed to further identify strain CCA6. The MLSA showed clear branching of strain CCA6 with respect to Enterobacter type strains. The complete genome of strain CCA6 consisted of 4,476,585 bp with a G+C content of 54.3% and comprising 4,372 predicted coding sequences. The genome average nucleotide identity values between strain CCA6 and the closest related Enterobacter type strain were <88.02%. Based on its phenotypic, chemotaxonomic and phylogenetic features, strain CCA6 (=HUT 8142T =KCTC 62525T) can be considered as a novel species within the genus Enterobacter with the proposed name Enterobacter oligotrophica.

could be reduced if oligotrophs could be used for industrial fermentation, and therefore screened for oligotrophs that are unaffected by a high-nutrient condition. Here, we report the screening, isolation, and characterization of an oligotrophic bacterium from leaf soil, which is one kind of the compost and is accrued by fermenting the dry leaves. The isolate was named strain CCA6. This bacterium was capable of growth on poor-nutrient medium, and its growth was unaffected by high-nutrient mixtures. Moreover, physiological, chemotaxonomic, and phylogenetic analyses as well as average nucleotide identity (ANI) value analysis were performed to characterize strain CCA6. Based on the results of these analyses, we propose that strain CCA6 represents a novel species within the genus Enterobacter, for which the name E. oligotrophica sp. nov. is proposed.

| Bacterial isolation
Soil samples were collected from Higashi-Hiroshima city in Hiroshima prefecture, Japan. A 1.5% agar (Nacalai tesque, Kyoto, Japan) plate (pH 7.2), which contained sulfates (>0.4%), calcium (>0.1%), iron (>0.01%), and a few fatty acids and/or other minerals at concentrations <0.01% was used for isolation. After 1 ml of a 10% (w/v) soil wash solution was inoculated onto a plate, the plate was incubated for 2 days at 37°C. Thereafter, a single colony was successively re-streaked onto a new 1.5% agar plate at least three times to obtain a pure colony. The purified strain was then grown aerobically at 37°C in Nutrient Broth (Kyokuto, Tokyo, Japan) and preserved at −20°C as a suspension in Nutrient Broth supplemented with glycerol (30%, w/v).

| Physiological characterization
Growth of strain CCA6 in Nutrient Broth was evaluated at various temperatures (4-50°C), pH (4.0-10.5), and NaCl concentrations (1-7%, w/v), and in the presence of selected antibiotics (ampicillin, chloramphenicol, and kanamycin). The OD 600 , which reflects cell growth, was measured by monitoring the difference between cellular and cell-free turbidity values using an Eppendorf BioSpectrometer (Eppendorf, Hamburg, Germany). Carbon source utilization was assessed using API 20E (bioMérieux, Marcy-l'Etoile, France) and API 50 CHE (bioMérieux) according to the manufacturer's instructions.
TA B L E 1 Differential characteristics of strain CCA6 and phylogenetically related species Voges-Proskauer (VP) test was carried out using RapiD 20E (bioMérieux). Enzyme activities were evaluated using API ZYM (bioMérieux).

| Phylogenetic analysis based on 16S rRNA gene
F I G U R E 1 Phylogenetic tree constructed from analysis of 16S rRNA gene sequences showing the relationships between strain CCA6 and the related type strains. The bar indicates a 0.02% nucleotide substitution rate. The tree was rooted using Xenorhabdus nematophila ATCC 19061 T as the outgroup F I G U R E 2 Phylogenetic tree reconstructed from analysis of the sequences of four housekeeping genes (atpD, gyrB, infB, and rpoB) and showing the relationships between strain CCA6 and the related type strains. The bar indicates a 0.1% nucleotide substitution rate. The tree was rooted using X. nematophila ATCC 19061 T as the outgroup using KOD plus DNA Polymerase (TOYOBO, Osaka, Japan) with the bacterial universal primers 27f (5′-AGAGTTTGATCMTGGCTCAG-3′; Lane, 1991) and 1391r (5′-GACGGGCGGTGTGTRCA-3′; Turner, Pryer, Miao, & Palmer, 1999

| Isolation of strain CCA6
To obtain oligtrophic microorganisms, filtrates were prepared from several soil samples and plated onto 1.5% agar (pH 7.2) without a carbon source or other medium components. After incubation for 2 days at 37°C, a single colony was obtained from the leaf soil filtrate. A purified colony was then obtained through standard dilution plating on the same plates and was named strain CCA6. Although high-nutrient mixtures suppress the growth of some oligotrophic bacteria (Ohta, 2000;Ohta & Taniguchi, 1988), strain CCA6 showed a higher rate of growth, similar to that of Escherichia coli MG1655, when cultured in Nutrient Broth or LB media ( Figure A1). By contrast, E. coli MG1655 did not grow on a 1.5% agar (pH 7.2). These results suggest we had successfully isolated the desired oligotroph.

| Morphological and physiological characterization
Cells of strain CCA6 were Gram-negative, motile, rod-shaped and non-sporulating. Colonies grown on Nutrient Broth plates were circular, smooth, glistening, light yellow, and 5.0 mm in diameter after incubation overnight at 37°C. When we examined the effect of culture temperature and pH, we found that the strain was capable of growing at temperatures between 10 and 45°C, but no growth was seen at 4 or 50°C ( Figure A2a). The strain also grew effectively at pHs between 4.5 and 10.0, but growth rates were sharply lower at pHs below 4.0 or above 10.5 ( Figure A2b). The strain was tolerant to 6% (w/v) NaCl ( Figure A2c) and was resist-

| Chemotaxonomic characterization
When strain CCA6 was cultured aerobically in Nutrient Broth, the major fatty acids were C 16:0 and summed feature 8 (comprising

| Phylogenetic affiliation of strain CCA6
The genus Enterobacter was first proposed by Hormaeche and Edwards (1960), and was classified as According to Brady et al. (2008Brady et al. ( , 2013, MLSA is also use- Enterobacter species. (Figure 2).

| Genome properties and ANI values
The genome sequence of strain CCA6 was 4,476,585 bp. The G+C content was 54.3%, which fell within range of those of Enterobacter type strains (Table S1). Within the genomic DNA of strain CCA6, 4,372 predicted coding sequences were identified. In addition, 85 tRNA genes and 25 rRNA genes were detected.
To carry out a phylogenetic comparison of strain CCA6 and the related species in the family Enterobacteriaceae, ANI values were calculated (

| CON CLUS ION
We have isolated a Gram-negative, non-sporulating, rod-shaped bacterium from leaf soil collected in Japan, which was designated strain CCA6. 16S rRNA gene sequence analysis revealed that strain CCA6 presented as a member of the family Enterobacteriaceae. (Figure 1).
Moreover, MLSA based on partial sequences of the atpD, gyrB, infB, and rpoB gene showed clear separation between strain CCA6 and the related Enterobacter type strains ( Figure 2). The ANI values between strain CCA6 and its closely related type strains were <88.02% (Table S1). Interesting features of strain CCA6 were its growth potential in oligotrophic medium and the fact that its growth was unaffected by high-nutrient media. Strain CCA6 therefore has potential for utilization as a host bacterium for industrial fermentation of valuable compounds. Although the related Enterobacter type strains are capable of utilizing disaccharides such as d-sucrose and d-turanose, strain CCA6 did not catabolize those disaccharides (Table 1).
When cellular fatty acids were compared between strain CCA6 and the related Enterobacter type strains, we found that fatty acids C 16:0 and summed feature 8 occur in most members of the related Enterobacter type strains. By contrast, the ratio of C 11:0 , C 15:1 ω8c, C 17:1 ω8c, and iso-C 19:0 in strain CCA6 was significantly higher than in the close relatives, and the fatty acid C 15:1 ω6c was only detected in strain CCA6 (Table 2).
Based on its phylogenetic, phenotypic, and chemotaxonomic fea-

CO N FLI C T O F I NTE R E S T S
None declared.

AUTH O R CO NTR I B UTI O N S
HA an ZK designed, carried out the experiments, and wrote the manuscript. AM revised the manuscript.

E TH I C S S TATEM ENT
None required.

DATA ACCE SS I B I LIT Y
The 16S