Lelliottia Rebaudianalis Sp. Nov. Isolated From Wild Stevia Rebaudiana Bertoni

A Gram-stain-negative, aerobic, chemoheterotrophic bacterium, characterized with rod shape and mobility, designated as LST-1 T , was isolated from wild Stevia rebaudiana Bertoni and subjected to polyphasic taxonomic analysis. The LST-1 T strain grew optimally at 37 °C and pH 6.0–7.0 in the presence of 0.5 % (w/v) NaCl. Phylogenetic sequence analysis based on 16S rDNA from LST-1 indicated that it is close to Lelliottia jeotgali (99.85%), Lelliottia nimipressuralis (98.82%), and Lelliottia amnigena (98.54%). Multilocus sequence typing analysis of concatenated partial recA, atpD, and infB was performed to improve resolution, and clear distinctions between the closest related type strains were exhibited. Meanwhile, the results from average nucleotide identify analyses and DNA–DNA hybridization with four species (16S rDNA similarity > 98.65%) were less than 90% and 40% respectively, verifying the distinct characteristics from other species of Lelliottia, The cellular fatty acid prole of the strain consisted of C16:0, Summed Feature3, and Summed Feature8 (may be 16:1 w6c/16:1 w7c and 18:1 w6c) as major components. The major polar lipids included phosphatidylethanolamine, phosphatidylglycerol, aminophospholipid, three non-characteristic phospholipids, and a non-characteristic lipid. The genome of LST-1 T is 4,611,055 bp, with a DNA G + C content of 55.02%. Combination of several phenotypic, chemotaxonomic, and genomic characteristics proved that the LST-1 T strain does represent a novel genus, for which the name Lelliottia sp. LST-1 was proposed. The type strain is LST-1 T (= CGMCC 1.19175 T = JCM 34938 T ).


Introduction
Lelliottia cells are straight rods, motile by peritrichous agella, facultative anaerobic, and Gram-stainnegative. They have been identi ed in natural environments and foods. The taxonomy of Enterobacter has a complicated history, with several species transferred to and from this genus over the past 20 years.
Based on the results of multi-locus sequence typing (MLST) analysis, DNA-DNA hybridization (DDH), phenotypic carbohydrate fermentation characteristics, and fatty acid pro les in cell-wall membranes, the species E. amnigena and E. nimipressuralis were assigned to a novel genus, for which the name Lelliottia was proposed in 2013 (Brady, Cleenwerck, Venter, Coutinho, & De Vos, 2013). Although they are in the same genus, their habitats are quite different. L. nimipressuralis is often found in trees or plants, and L. amnigena has been isolated from onion, raw milk, cream, cheese, onion, and Spanish pork sausage. Both are notably suspected to have pathogenic possibilities (Chow et  This study focused on the description of strain LST-1 T , isolated from wild Stevia rebaudiana Bertoni obtained from the stevia planting and production base in Dongtai City, Yancheng City, Jiangsu Province. Lelliottia jeotgali JCM31901 and Lelliottia nimipressuralis DSM 18955 were used as reference strain. The new isolate represented a novel species of the genus Lelliottia on the basis of the taxonomical analysis and microbial/genomic characterizations presented in this study.

Isolation and cultivation
For isolation of strain LST-1 T , inorganic salt medium (KH 2 PO 4 3 g/L, MgSO 4 0.1 g/L, K 2 HPO 4 1.5 g/L, CaCl 2 0.01 g/L, EDTA disodium 0.01 g/L, pH 7.5) containing 10 g/L stevioside as the only carbon source was adopted as the selection medium to screen a mass of endophytic bacterium from S. rebaudiana Bertoni that could use stevioside (Xu, Feng, Wang, & Lin, 2013). Then, esculin-MS medium was used for a second screening campaign to isolate β-glucosidase-producing endophytic bacteria in accordance with the method described by Xuan et al., with some modi cations (Xuan, 2012). MS medium with double agar was supplemented with 0.2% esculin (6,7-2-hydroxy-coumarin-β-D-glucoside) and 0.6% FeCl 3 of the same volume. Small wells (0.3-0.6 cm) were punched out in plates, loaded with 100 µL of bacterial broths, and incubated at 37°C until the wells were surrounded by black or dark brown (Pérez et al., 2011), and then a strain named LST-1 T was obtained. The puri ed strain culture was added with glycerol to the concentration of 25% (v/v) and frozen at −80°C for long-term storage.
Individual bacterial colonies were activated to the logarithmic stage, and 1 mL broths were taken out and centrifuged at 8000 rpm for 1 min. Then, 850 µL of the medium was discarded, and 150 µL of polypeptide was added. The mixtures were incubated at 37°C for 30 min. After centrifugation at 8000 rpm for 1 min, the supernatant was discarded, and 2.5% glutaraldehyde was added. The solutions were mixed, xed overnight at 4°C, and then centrifuged at 8000 rpm for 1 min to remove the supernatants. The precipitates were washed three times with PBS and then dehydrated by 30%, 50%, 70%, 80%, and 90% gradient ethanol. Finally, the precipitates were washed twice and suspended with 100% ethanol. After CO 2 critical point drying and ion sputtering were conducted, the bacteriological samples were observed by SEM.
Catalase activity was evaluated by assessing bubble formation in 3 % (v/v) hydrogen peroxide, and oxidase activity was tested by 1% N,N-dimethyl-p-phenylenediamine dihydrochloride, with observations on changes in color. Other physiological and biochemical tests were performed using API 20NE.

Chemotaxonomic analyses
Strain LST-1 T , L. jeotgali JCM31901, and L. nimipressuralis DSM 18955, which were grown on LB medium for 24 h at 37°C, were used for polar lipid and isoprenoid quinone analysis. Polar lipids were extracted using a chloroform/methanol system and analyzed using one-and two-dimensional TLC in accordance with Cui et al. (

16S rRNA phylogeny
The genomic DNA was extracted in accordance with the method described in the quick bacterial genomic DNA extraction kit. The 16S rRNA gene was ampli ed using the universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) ( MLST analysis MLST analysis with three housekeeping genes, namely, recombinase (recA), ATP synthase β subunit (atpD), and initiation translation factor 2 (infB), was performed to distinguish and further understand the taxonomic relationship between Lelliottia and Enterobacter and support the 16S rDNA phylogenetic trees of strain LST-1 T . The DNA sequences of the three genes were assembled in the order of recA-atpD-infB.

Results And Discussion
Phylogenetic analysis Phylogenetic analysis based on 16S rRNA gene indicated that the strain LST-1 T shared the highest similarity with L. jeotgali (99.85%), followed by L. nimipressuralis (98.82%) and Lelliottia amnigena (98.54%). Lelliottia and Enterobacter belong to the same family. However, the phylogenetic tree using 16S rRNA gene sequences (Fig. 1a) did not differentiate Lelliottia strains from Enterobacter strains, probably due to low resolution.
The MLST-based comparative phylogenetic tree analysis method based on recA-atpD-infB sequence was conducted using the same methods as the 16S rRNA gene sequence analysis. The strain LST-1 T was closest to L. jeotgali in a separate small group of the 16S rRNA gene and MLST-based phylogenetic trees (Figs. 1a and 1b). In accordance with the phylogenetic tree, two type strains were obtained from culture collections, used for comparison, and characterized in parallel with the novel isolates L. jeotgali JCM31901 and L. nimipressuralis DSM 18955.

Genomic features
The genome of Lelliottia sp. LST-1 T is composed of a chromosome with 4,611,055 base pairs and a plasmid with 39,319 base pairs. The total length of the predicted coding gene sequence is 4,140,156. Gene function annotation analysis was conducted after BLAST with functional databases, such as COG, nimipressuralis were 89.21% and 83.11%, respectively. The DDH values between strain LST-1 T and L. jeotgali and L. nimipressuralis were 38.80% and 25.80%, respectively (Table 1).
In addition, the G + C content of the genomic DNA from LST-1 T was 55.02%, which was different from that of L. jeotgali (54.2%) but close to that of L. nimipressuralis (55.2%). These differences indicated that the taxonomic status of LST-1 T lies between L. nimipressuralis and L. jeotgali (Table 1).

Phenotypic characteristics
The cells of strain LST-1 T were Gram-negative, aerobic, catalase-positive, rod-shaped, and without agella, and the surface of the bacteria was rough. The colony of strain LST-1 T was milky white, round, and smooth. Compared with two reference strains, LST-1 T could grow in a wide temperature range from 4 °C to 45 °C. The salt tolerance and pH growth range of strain LST-1 T was similar to those of other strains. It could grow with the presence of 0.5% NaCl but not with 9% NaCl. It could also grow at pH 5.0-9.0 but not at pH 4.5 or 9.5.
API 20NE strips (bioMérieux) were used in conjunction with AUX medium (bioMérieux) for carbohydrate fermentation to further distinguish strain LST-1 T from other Lelliottia species strains. In accordance with the analysis of the MIDI Microbial Identi cation System, the phenotypic differences between the strain LST-1 T and other Lelliottia species are shown in Table 3. Only LST-1 T could ferment with capric acid, and adipic acid could only be used for LST-1 T and L. nimipressuralis DSM 18955.
Chemotaxonomic characterization LST-1 T respiratory quinones mainly include menaquinone (MK) 8 and coenzyme Q8, while polar lipids mainly include phosphatidylethanolamine (PE), phosphatidylglycerol (PG), aminophospholipid (APL), three non-characteristic phospholipids (PL1-3), and a non-characteristic lipid (PL), which is quite different from the other two strains ( Table 2 and Fig. 2). It is especially different from L. nimipressuralis DSM 18955, which has four types of unidenti ed aminolipids, whereas LST-1 T does not have one. In addition, DPG and GL were detected from L. nimipressuralis DSM 18955, but it was absent in LST-1 T .
The overall fatty acids pro le of LST-1 T was similar to those of the reference taxa of the genus Lelliottia, but the respective proportions of some fatty acid components had some differences ( Table 4). The data showed that LST-1 T has three main fatty acids, C16:0, Summed Feature3, and Summed Feature8 (may be 16:1 w6c, 16:1 w7c, and 18:1 w6c). In addition, many differences were observed in the fatty acid distribution, especially on C 13:0 , C 11:0 2-OH , C 14:1 w5c , and C 19:0 cyclo w8c . The three strains have their own unique fatty acid composition.

Taxonomic conclusion
Combination of the analysis of physiological and biochemical characteristics provided evidence that LST-1 T represents a novel species independent of genus L. jeotgali, for which the name Lelliottia rebaudianalis sp. nov. was proposed.
Description of L. rebaudianalis sp. nov.
The cells of Lelliottia sp. LST-1 T are Gram-stain-negative, aerobic, and chemoheterotrophic. SEM results (Fig. 3) showed that it was rod-shaped and without agella, and the surface of the bacteria was rough.
After incubation for 24 h on LB solid medium, the colonies were circular and at, the surface was smooth and moist, and the edges of the colonies were neat, milky white-pigmented, and opaque. Growth occurred at pH 5.0-9.0, with optimum growth at 6.0-7.0, but not at pH 4.5 or 9.5. Growth also occurred at 4 °C-45°C , with optimum growth at 37 °C, but no growth was detected at 48 °C. The NaCl range for growth was 0%-8 % (w/v, optimum of 0.5%) but not at 9 % NaCl (w/v) in LB medium. The cells were positive for catalase activity and weakly positive for oxidase activity. It assimilated with D-glucose, L-arabinose, Dmannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, malic acid, trisodium citrate, and phenylacetic acid. However, the assimilation of capric acid and adipic acid were weak and not clear. The major components of cellular fatty acids included C16:0, Summed Feature3, and Summed Feature8 (may be 16:1 w6c/16:1 w7c and 18:1 w6c). The polar lipids found in Lelliottia sp. LST-1 were PE, PG, APL, three non-characteristic phospholipids, and a non-characteristic lipid. MK8 and coenzyme Q8 were the predominant isoprenoid quinones detected in LST-1.

Statements & Declarations
Repositories: The GenBank accession numbers for the 16S rRNA gene and genome sequences of strain LST-1 T are MZ497264 and CP063663, respectively.

Con icts of interest
The authors declare that they have no con ict of interest.    Table 4 Composition of cellular fatty acids of LST-1 T and two species of genus Lelliottia Phylogenetic tree analysis of LST-1T. (a) Phylogenetic tree constructed based on 16S rRNA gene sequence using maximum likelihood method. (b) MLST analysis based on partial recA, atpD, and infB.  Scanning electron microscopic results of LST-1 T