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

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.

INTRODUCTION

A variety of microorganisms are used in industrial fermentation for production of enzymes, medicines, and other organic compounds. Those microorganisms are generally grown in high‐nutrient medium containing large amounts of sugar, nitrogen, phosphorus, minerals, and other nutrients that are considered essential for their growth. Consequently, nutrient cost is an important factor when trying to achieve cost‐effective fermentation. This has led to the development of several related technologies, including bioengineering of microorganisms so as to enhance their productivity and yield (Min, Hwang, Lim, & Jung, 2017), use of agricultural byproducts as carbon and mineral sources (Thomsen, 2005), and production of chemicals such as bioemulsifiers (Banat, Satpute, Cameotra, Patil, & Nyayanit, 2014), biofuels (Ho, Ngo, & Guo, 2014), and biosurfactants (Banat et al., 2014) from renewable substrates.

Oligotrophs are organisms that grow under conditions of low levels of nutrients but grow more slowly at high levels (Kuznetsov, Dubinina, & Lapteva, 1979). Consequently, oligotrophs have not been applied for industrial use. We suggest that production costs 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.

MATERIALS AND METHODS

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 OD600, 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. Voges–Proskauer (VP) test was carried out using RapiD 20E (bioMérieux). Enzyme activities were evaluated using API ZYM (bioMérieux).

Chemotaxonomic analyses

The cellular fatty acid composition of strain CCA6 was determined using Sherlock Microbial Identification System Version 6.0 (MIDI, Newark, DE) with TSBA6 database (MIDI). Using the method of Bligh and Dyer (1959), lipids were extracted from lyophilized cells of strain CCA6 and loaded onto a Sep‐Pak Plus Silica cartridge (Waters, Milford, MA). The cartridge was then washed and the quinones were eluted. The quinones were quantified using an ACQUITY UPLC system (Waters) with an Eclipse Plus C18 column (Agilent technologies, Santa Clara, CA). The chromatographic conditions were as follows: mobile phase, methanol/isopropanol (3:1 v/v); flow rate, 0.5 ml/min; column oven temperature, 35°C. The quinone forms were identified as previously described (Tamaoka, Katayama‐Fujimura, & Kuraishi, 1983).

Phylogenetic analysis based on 16S rRNA gene

After strain CCA6 was cultured aerobically for 6 hr at 37°C in Nutrient Broth, the cells were harvested by centrifugation, and their genomic DNA was extracted and purified using an illustra bacteria genomicPrep Mini Spin Kit (GE Healthcare, Chicago, IL) according to the manufacturer's instructions. The 16S rRNA gene was amplified 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). After purifying the amplified PCR product using a Wizard SV Gel and PCR Clean‐up System (Promega, Madison, WI), the purified product was cloned into pTA2 vector (TOYOBO) and sequenced. Sequence was then compared with reference sequences available in the GenBank/EMBL/DDBJ databases using BLAST. Multiple alignment and construction of a maximum‐likelihood tree were performed using MEGA‐X (Kumar, Stecher, Li, Knyaz, & Tamura, 2018) with Tamura and Nei model (1993).

Multilocus sequence analysis based on housekeeping genes

Multilocus sequence analysis (MLSA) was performed using the method of Brady et al. (2008), Brady, Cleenwerck, Venter, Coutinho, and De Vos (2013) with some modifications. A phylogenetic tree of concatenated sequences (2,637 bp), including partial sequences of four housekeeping genes [atpD (β subunit of ATP synthase; 642 bp), gyrB (DNA gyrase; 743 bp), infB (translation initiation factor 2; 615 bp), and rpoB (β subunit of RNA polymerase; 637 bp)] from strain CCA6, was also reconstructed using the maximum‐likelihood method with Tamura and Nei model (1993). The housekeeping genes of strain CCA6 and the related type strains are available in the GenBank/DDBL/EMBL databases.

Genome sequencing and ANI value analysis

The concentration and purity of the genomic DNA were measured using a NanoDrop ND‐1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and a Quant‐iT dsDNA BR assay kit (Invitrogen, Waltham, MA), respectively. After fragmenting the genomic DNA (20.9 μg) into approximately 20‐kb pieces using g‐TUBE (Covaris, Brighton, UK), the resultant fragments were ligated to SMRTbell sequencing adapters using a SMRTbell Template Prep Kit 1.0 (Pacific Biosciences, Menlo Park, CA), yielding SMRTbell libraries. The library size was measured using Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA). The SMRTbell libraries were then bound to polymerases and sequencing primers using a DNA/Polymerase binding kit P6 v2 (Pacific Biosciences), yielding the sequencing templates. The concentration of the sequencing templates was calculated using Binding Calculator v2.3.1.1 (Pacific Biosciences), after which the templates were bound to MagBeads using a MagBead kit (Pacific Biosciences) and loaded onto SMRT Cells 8Pac v3 (Pacific Biosciences). Sequencing was then performed using PacBio RS II (Pacific Biosciences).

The raw data included 100,771 reads with 330 coverage and were assembled de novo using SMRT Analysis v2.3.0 (Pacific Biosciences; Chin et al., 2013) to filter the subreads. Genome annotation was performed using CRITICA (Badger & Olsen, 1999) and Glimmer2 (Delcher, Harmon, Kasif, White, & Salzberg, 1999). The tRNA and rRNA genes were detected using tRNAScan‐SE (Lowe & Eddy, 1997) and BLASTN (Altschul, Gish, Miller, Myers, & Lipman, 1990), respectively. ANI values were calculated through pairwise genome comparison of whole‐genome sequences of strain CCA6 and its related Enterobacter type strains using the ANI algorithm (Goris et al., 2007) implemented within OrthoANIu tools (Yoon, Ha, Lim, Kwon, & Chun, 2017).

The genome properties of type strains of Enterobacter, Klebsiella, Kosakonia, Lelliottia, Pluralibacter, Pseudescherichia, Pseudomonas, and Raoultella species are presented in Table A1.

RESULTS AND DISCUSSION

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 resistant to ampicillin, but chloramphenicol and kanamycin inhibited its growth.

Figure 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 19061T as the outgroup

Strain CCA6 showed a broad range of enzyme activities, including acid phosphatase, N‐acetyl‐β‐d‐glucosaminidase, alkaline phosphatase, cystine aminopeptidase, esterase lipase (C8), α‐galactosidase, β‐galactosidase, α‐glucosidase, β‐glucosidase, leucine aminopeptidase, α‐mannosidase, naphthol AS‐BI phosphate, trypsin, and valine aminopeptidase. By contrast, strain CCA6 did not exhibit α‐chymotrypsin, esterase (C4), α‐fucosidase, β‐glucuronidase, or lipase (C14) activity. These results suggest that strain CCA6 is capable of catabolizing a variety of different carbon sources. Culture with different carbon sources revealed that CCA6 was able to utilize the following compounds as a carbon source for growth: inulin, amygdalin, arbutin, esculin ferric citrate, 2‐nitrophenyl β‐d‐galactopyranoside, d‐cellobiose, d‐trehalose, d‐maltose, d‐lactose, d‐galactose, d‐glucose, l‐sorbose, d‐fructose, d‐mannose, l‐rhamnose, dulcitol, d‐sorbitol, d‐mannitol, N‐acetyl‐glucosamine, methyl‐β‐d‐xylopyranoside, d‐arabinose, l‐arabinose, d‐xylose, l‐xylose, l‐fucose, d‐lyxose, d‐ribose, adonitol, erythritol, glycerol, l‐arginine, l‐lysine, l‐ornithine, l‐tryptophane, 2‐keto gluconate, citrate, gluconate, pyruvate, and urea. By contrast, no growth occurred on glycogen, gelatin, starch, salicin, d‐melezitose, d‐raffinose, gentiobiose, d‐sucrose, d‐melibiose, d‐turanose, d‐tagatose, inositol, methyl‐α‐d‐glucopyranoside, methyl‐α‐d‐mannopyranoside, d‐fucose, d‐arabitol, l‐arabitol, xylitol, 5‐keto gluconate, or thiosulfate. Differences in phenotypic characteristics of strain CCA6 and its related type species are shown in Table 1.

Table 1

Differential characteristics of strain CCA6 and phylogenetically related species

Characteristic	1	2	3	4	5	6	7	8	9	10
Carbon source utilization
d‐Sucrose	‐	++	++	++	++	++	++	++	++	++
d‐Melibiose	‐	+	++	++	W	++	++	++	++	++
d‐Turanose	‐	+	+	‐	W	+	‐	+	W	+
l‐Rhamnose	++	+	++	‐	++	++	++	++	++	++
Inositol	‐	+	++	++	‐	W	W	+	++	++
Dulcitol	+	+	W	‐	++	‐	‐	+	+	+
d‐Sorbitol	++	++	++	++	W	++	++	++	++	++
Methyl‐α‐d‐glucopyranoside	‐	++	++	++	++	+	+	++	++	++
d‐Arabinose	++	+	++	++	++	++	++	+	++	+
l‐Fucose	++	+	+	‐	++	+	++	+	‐	W
d‐Lyxose	++	++	W	++	+	++	++	++	+	++
Adonitol	+	+	+	‐	W	W	++	+	‐	+
d‐Arabitol	‐	+	+	‐	W	W	++	+	‐	+
2‐Keto gluconate	+	+	++	+	W	‐	+	+	W	++
Enzyme activity
Arginine dihydrolase	+	++	++	++	++	++	++	‐	++	++
Ornithine decarboxylase	+	++	++	++	++	++	++	‐	++	++
Lysine decarboxylase	+	‐	‐	‐	‐	‐	‐	‐	‐	W
Esculin hydrolysis	+	++	+	++	‐	W	W	+	W	+
Voges–Proskauer test	‐	++	++	++	++	++	++	++	++	++

Strains: 1, strain CCA6; 2, E. asburiae ATCC 35953T; 3, E. cloacae subsp. cloacae ATCC13047T; 4, E. cloacae subsp. dissolvens ATCC 23373T; 5, E. hormaechei subsp. hormaechei ATCC 49162T; 6, E. hormaechei subsp. oharae DSM 16687T; 7, E. hormaechei subsp. steigerwaltii DSM 16691T; 8, E. hormaechei subsp. xiangfangensis LMG 27195T; 9, E. kobei ATCC BAA‐260T; 10, E. ludwigii EN‐119T. ++, strong positive; +, positive; W, weak positive; —, not detected.

Nearly all Enterobacter species produce acetoin as the end product of glucose metabolism, which yields a red complex in the VP test medium. The related Enterobacter type strains show a positive VP test; however, strain CCA6 was negative (Table 1).

Chemotaxonomic characterization

When strain CCA6 was cultured aerobically in Nutrient Broth, the major fatty acids were C16:0 and summed feature 8 (comprising C18:1ω6c and/or C18:1ω7c). The overall fatty acid profile of strain CCA6 was similar to that of E. hormaechei subsp. hormaechei ATCC 49162T (Table 2). Respiratory quinone analysis showed the presence of ubiquinone‐7 (4.2%), ubiquinone‐8 (87.2%), and menaquinone‐8 (8.6%).

Table 2

Comparative fatty acid contents (%) of strain CCA6 and phylogenetically related reference strains

Fatty acids	1	2	3	4	5	6
Saturated fatty acids
C10 : 0	0.04	‐	‐	‐	‐	‐
C11 : 0	0.11	‐	‐	0.08	‐	‐
C12 : 0	3.7	3.03	2.47	2.50	2.26	3.96
C13 : 0	1.4	0.72	0.61	1.18	0.37	0.90
C14 : 0	5.6	6.46	6.74	8.39	9.73	6.18
C16 : 0	22.7	27.89	29.27	21.75	30.16	25.71
C17 : 0	4.0	3.73	3.43	4.94	2.02	3.18
C18 : 0	0.3	0.47	0.53	0.38	0.47	0.39
Branched‐chain fatty acids
iso‐C15 : 0 3‐OH	‐	‐	‐	‐	‐	‐
anteiso‐C19 : 0	‐	‐	‐	‐	‐	‐
iso‐C19 : 0	0.2	‐	0.11	‐	‐	‐
Unsaturated fatty acids
C15 : 1ω6c	0.1	‐	‐	‐	‐	‐
C15 : 1ω8c	0.3	‐	0.09	0.14	‐	‐
C16 : 1ω5c	‐	0.21	0.21	0.22	‐	0.21
C17 : 1ω8c	0.4	‐	‐	0.44	‐	‐
11‐methyl‐C18 : 1ω7c	‐	0.26	‐	‐	‐	‐
C18 : 1ω5c	‐	‐	0.18	0.19	‐	‐
Hydroxy fatty acids
C15 : 0 3‐OH	‐	0.16	0.15	0.36	‐	0.21
Cyclopropane acids
cyclo‐C17 : 0	14	26.01	20.4	21.08	21.69	25.17
cyclo‐C19 : 0ω8c	3.3	7.04	6.10	4.35	5.79	5.99
Summed feature
1	1.3	0.93	0.64	1.46	0.34	0.74
2	8.1	6.14	6.85	6.21	6.79	6.79
3	12.6	3.51	6.50	5.71	4.49	4.9
8	21.3	13.44	14.27	20.62	15.88	15.68

Strains: 1, strain CCA6; 2, E. asburiae ATCC 35953T; 3, E. cloacae subsp. cloacae ATCC 13047T; 4, E. hormaechei subsp. hormaechei ATCC 49162T; 5, E. hormaechei subsp. xiangfangensis LMG 27195T; 6, E. ludwigii EN‐119T. Data from 2 to 6 are from Gu, Li, Yang and Huo (2014). —, not detected/not reported. Summed feature 1 consists of iso‐C15 : 1 H and/or C13 : 0 3‐OH; Summed feature 2 consists of iso‐C16:1 I and/or C14:0 3‐OH and/or C12:0 unidentified aldehyde or an unidentified fatty acid with an equivalent chain length of 10.928; Summed feature 3 consists of C16:1ω6c and/or C16:1ω7c; summed feature 8 consists of C18:1ω6c and/or C18:1ω7c.

Phylogenetic affiliation of strain CCA6

The genus Enterobacter was first proposed by Hormaeche and Edwards (1960), and was classified as Gram‐negative, rod‐shaped, motile bacteria. To date, more than 18 Enterobacter species have been reported, and the Enterobacter cloacae complex has been rearranged in E. cloacae, E. asburiae, E. hormaechei, E. kobei, E. ludwigiii, and their subspecies based on whole‐genome DNA–DNA hybridizations and phenotypic characteristics (Mezzatesta, Gona, & Stefani, 2012).

To confirm the phylogenetic position of strain CCA6, the 16S rRNA gene sequence (1,294 bp) was determined. In the maximum‐likelihood tree based on almost complete sequences of the 16S rRNA gene, strain CCA6 fell inside the cluster comprising members of the genus Enterobacter and Kosakonia (Figure 1). The sequences of the following E. cloacae complex species showed similarity to that of strain CCA6: E. cloacae subsp. dissolvens LMG 2683T (98.3%), E. cloacae subsp. cloacae ATCC 13047T (98.0%), E. sichuanensis WCHECL1597T (97.8%), E. chengduensis WCHECl‐C4T (97.7%), K. oryzendophytica LMG 26432T (97.7%), E. ludwigii EN‐119T (97.6%), E. roggenkampii DSM 16690T (97.6%), and E. mori LMG 25706T (97.4%).

Figure 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 19061T as the outgroup

According to Brady et al. (2008, 2013), MLSA is also useful for identification of bacterial species. The MLSA showed that strain CCA6 exhibited similarities of 96.0%, 96.0%, 96.0%, 95.9%, 95.9%, 95.8%, 95.8%, 95.7%, and 95.6% to its closest relatives, E. bugandensis EB‐247T, E. hormaechei subsp. xiangfangensis LMG 27195T, E. ludwigii EN‐119T, E. hormaechei subsp. hormaechei ATCC 49162T, E. hormaechei subsp. steigerwaltii DSM 16691T, E. asburiae ATCC 35953T, E. hormaechei subsp. oharae DSM 16687T, E. tabaci CCUG 72520T, and E. mori LMG 25706T, respectively. Moreover, strain CCA6 clusters on its own branch separately from other 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 (Table S1). The ANI values between strain CCA6 and the related type strains belonging to the genera Klebsiella, Kosakonia, Pluralibacter, Pseudescherichia, and Raoultella were all <79.70%. Moreover, the ANI values between strain CCA6 and the related Enterobacter type strains were in the range of 79.75–88.02%, which was clearly below the cutoff of 95–96% for prokaryotic species delineation as established by Richter and Rosselló‐Móra (2009).

CONCLUSION

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 C16:0 and summed feature 8 occur in most members of the related Enterobacter type strains. By contrast, the ratio of C11:0, C15:1ω8c, C17:1ω8c, and iso‐C19:0 in strain CCA6 was significantly higher than in the close relatives, and the fatty acid C15:1ω6c was only detected in strain CCA6 (Table 2).

Based on its phylogenetic, phenotypic, and chemotaxonomic features, strain CCA6 can be considered as a novel species in the genus Enterobacter, which we propose to name E. oligotrophica sp. nov.

Description of E. oligotrophica sp. nov

Enterobacter oligotrophica (o.li.go.tro'phi.ca. Gr. adj. oligos little; Gr. adj. trophikos nursing, tending or feeding; N.L. fem. adj. oligotrophica eating little, referring to a bacterium living on low‐nutrient media).

Cells are aerobic, Gram‐negative, non‐sporulating, and rod‐shaped (1.0–2.0 μm × 4.0–5.0 μm). Colonies are circular, smooth, glistening, light yellow, and grow to 5.0 mm in diameter on Nutrient Broth plates after incubation for 24 hr at 37°C. Growth is observed in poor‐nutrient medium, and growth is unaffected by high‐nutrient medium. The VP test is negative. The major cellular fatty acids are C16:0 and sums of C16 : 1ω6c and/or C16 : 1ω7c or C18 : 1ω6c and/or C18 : 1ω7c. The predominant quinone system is ubiquinone‐8. Growth is observed in Nutrient Broth at 10–45°C and pH 4.5–10.0, with optimal growth at 20°C and pH 5.0. Growth occurs in the presence of 0–6% (w/v) NaCl as well as ampicillin. Strain CCA6 is positive for lysine decarboxylase. No growth occurs on d‐sucrose, d‐melibiose, d‐turanose, d‐tagatose, inositol, or methyl‐α‐d‐glucopyranoside. Strain CCA6 is clearly separated from the related Enterobacter type strains by MLSA based on partial sequences of the atpD, gyrB, infB, and rpoB gene. The genome size of the type strain is 4,476,585 bp, which has a G+C content of 54.3%.

CONFLICT OF INTERESTS

None declared.

AUTHOR CONTRIBUTIONS

HA an ZK designed, carried out the experiments, and wrote the manuscript. AM revised the manuscript.

ETHICS STATEMENT

None required.

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Table A1

Genome properties of type strains of Enterobacter, Klebsiella, Kosakonia, Lelliottia, Pluralibacter, Pseudescherichia, Pseudomonas, and Raoultella species used in this study

Strains	Size (bp)	G+C (mol%)	Protein	Accession no.
Enterobacter
E. asburiae ATCC 35953T	4,713,742	55.4	4,436	CP011863
E. bugandensis EB‐247T	4,971,744	56.0	4,344	FYBI00000000
E. cancerogenus ATCC 33241T	4,879,939	55.6	4,521	FYBA00000000
E. chengduensis WCHECl‐C4T	5,138,130	55.7	4,745	MTSO00000000
E. cloacae subsp. cloacae ATCC 13047T	5,551,574	54.6	5,393	JPPR00000000
E. hormaechei subsp. hormaechei ATCC 49162T	4,890,213	55.2	4,522	MKEQ00000000
E. hormaechei subsp. oharae DSM 16687T	4,724,316	55.6	4,390	CP017180
E. hormaechei subsp. steigerwaltii DSM 16691T	4,782,480	55.6	4,424	CP017179
E. hormaechei subsp. xiangfangensis LMG 27195T	4,661,849	55.3	4,306	CP017183
E. kobei ATCC BAA‐260T	4,700,329	55.5	4,424	FTNJ00000000
E. ludwigii EN‐119T	4,952,770	54.6	4,459	JTLO00000000
E. mori LMG 25706T	4,953,765	55.3	4,496	AEXB00000000
E. muelleri JM‐458T	4,695,678	55.9	4,423	FXLQ01000000
E. roggenkampii DSM 16690T	4,748,414	56.0	4,451	CP017184
E. sichuanensis WCHECL1597T	4,897,201	55.2	4,634	POVL01000000
E. soli ATCC BAA‐2102T	4,960,767	53.8	4,571	LXES00000000
E. tabaci CCUG 72520T	4,927,887	55.5	4,627	QZDQ00000000
Klebsiella
K. aerogenes KCTC 2190T	5,280,350	54.8	4,912	CP002824
K. grimontii 06D021T	6,168,876	55.4	5,986	FZTC00000000
K. michiganensis DSM 25444T	6,193,009	56.0	5,732	PRDB00000000
K. pneumoniae subsp. ozaenae ATCC 11296T	4,925,250	57.2	4,458	CDJH00000000
K. pneumoniae subsp. pneumoniae ATCC 13883T	5,544,684	57.0	5,205	JOOW00000000
K. pneumoniae subsp. rhinoscleromatis ATCC 13884T	5,280,675	56.9	5,671	ACZD00000000
K. quasipneumoniae subsp. quasipneumoniae 01A030T	5,465,736	58.0	5,287	CCDF00000000
K. quasipneumoniae subsp. similipneumoniae 07A044T	5,109,717	58.2	4,927	CBZR000000000
K. variicola DSM 15968T	5,521,203	57.6	5,200	CP010523
Kosakonia
K. arachidis Ah‐143T	5,135,597	52.5	4,861	FPAU00000000
K. oryzae CGMCC 1.7012T	5,380,462	54.0	4,980	FOKO00000000
K. oryzendophytica LMG 26432T	4,878,776	53.7	4,459	FYBE00000000
K. oryziphila REICA_142T	4,814,900	52.7	4,667	FMBC00000000
K. pseudosacchari JM‐387T	4,956,546	53.9	4,638	FXWP00000000
K. radicincitans DSM 16656T	5,817,639	53.7	5,660	AKYD00000000
K. sacchari SP1T	4,902,027	53.7	4,545	CP007215
Lelliottia
L. amnigena DSM 4486T	4,370,208	52.9	4,070	PDDA01000000
L. jeotgali PFL01T	4,603,334	54.2	4,243	CP018628
Pluralibacter
P. gergoviae NBRC 105706T	5,662,775	58.6	5,176	BCZS00000000
Pseudescherichia
P. vulneris NBRC 102420T	4,374,581	56.4	4,196	BBMZ00000000
Raoultella
R. ornithinolytica NBRC 105727T	5,533,930	55.7	5,099	BCYR00000000
R. planticola ATCC 33531T	5,668,028	55.8	5,237	JMPP00000000
Xenorhabdus
X. nematophila ATCC 19061T	4,587,917	44.3	3,754	FN667742

Data are from the GenBank/EMBL/DDBJ databases.