Phylogenetic analysis based on 16S rRNA gene indicated that the strain LST-1T 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-1T 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.
The genome of Lelliottia sp. LST-1T 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, Kyoto Encyclopedia of Genes and Genomes, Swiss-Prot, and Non-Redundant Protein Database. The whole genome sequence was uploaded to NCBI (https://www.ncbi.nlm.nih.gov/), and the accession number is CP063663.
ANI analysis, DDH analysis, and G + C content calculation (Yuk, Kim, Huh, & Lee, 2018) were conducted for strains with similarities > 98.5%. ANI values < 95% correspond to DDH values < 70% for identification of a novel bacterial species (Lawrence, G., & Wayne %J Zentralblatt für Bakteriologie, 1988; Wayne, 1988). The results showed that the ANI values between strain LST-1T and L. jeotgali and L. nimipressuralis were 89.21% and 83.11%, respectively. The DDH values between strain LST-1T 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-1T 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-1T lies between L. nimipressuralis and L. jeotgali (Table 1).
The cells of strain LST-1T were Gram-negative, aerobic, catalase-positive, rod-shaped, and without flagella, and the surface of the bacteria was rough. The colony of strain LST-1T was milky white, round, and smooth. Compared with two reference strains, LST-1T could grow in a wide temperature range from 4 °C to 45 °C. The salt tolerance and pH growth range of strain LST-1T 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-1T from other Lelliottia species strains. In accordance with the analysis of the MIDI Microbial Identification System, the phenotypic differences between the strain LST-1T and other Lelliottia species are shown in Table 3. Only LST-1T could ferment with capric acid, and adipic acid could only be used for LST-1T and L. nimipressuralis DSM 18955.
LST-1T 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 unidentified aminolipids, whereas LST-1T does not have one. In addition, DPG and GL were detected from L. nimipressuralis DSM 18955, but it was absent in LST-1T.
The overall fatty acids profile of LST-1T 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-1T 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 C13:0, C11:0 2-OH, C14:1 w5c, and C19:0 cyclo w8c. The three strains have their own unique fatty acid composition.
Combination of the analysis of physiological and biochemical characteristics provided evidence that LST-1T 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.
L. rebaudianalis sp. nov. (re’bau.dia.na.lis N.L.; L. fem. suff. -alis, suffix denoting pertaining to, from which the type strain was isolated from S. rebaudiana Bertoni).
The cells of Lelliottia sp. LST-1T are Gram-stain-negative, aerobic, and chemoheterotrophic. SEM results (Fig. 3) showed that it was rod-shaped and without flagella, and the surface of the bacteria was rough. After incubation for 24 h on LB solid medium, the colonies were circular and flat, 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, D-mannose, 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.
The type strain LST-1T (= CGMCC 1.19175T = JCM 34938T) was isolated from the stevia planting and production base in Dongtai City, Yancheng City, Jiangsu Province (32°84′ N, 120°31′ E).
The GenBank accession numbers for the 16S rRNA gene and genome sequences of strain LST-1T are MZ497264 and CP063663, respectively.