Introduction

The family Sphingomonadaceae, proposed by Kosako et al. (2000), belongs to the order Sphingomonadales of the Alphaproteobacteria. At the time of writing, the family Sphingomonadaceae comprises of 22 validly named genera (https://www.namesforlife.com/10.1601/tx.1165) that were isolated from various environments, such as freshwater, pristine or contaminated soils, the rhizosphere and clinical specimens (Balkwill et al. 2006; Gich and Overmann 2006; Glöckner et al. 2000; Kim et al. 2007; Takeuchi et al. 2001; Zwart et al. 2002). Members of the family Sphingomonadaceae are typically Gram-stain negative, non-spore-forming, ovoid to rod-shaped, non-motile or motile, aerobic [with the exception of the facultative anaerobe Zymomonas (Kalnenieks 2006)] and chemoorganotrophic. Members of the family contain sphingoglycolipid as a major polar lipid component, ubiquinone-10 as the main respiratory quinone, C18:1ω7c as the predominant fatty acid and homospermidine as the major polyamine. The DNA G+C content is in the range of 59–68.5 mol% (Kosako et al. 2000). In this study, we describe the taxonomic position of strain SYSU M10002T by using a polyphasic taxonomic approach.

Materials and methods

Isolation and culture conditions

Water samples were collected from a conjunct point of freshwater and seawater, located along the coastal region of Pearl River estuary (113°35′42.00″E, 23°6′35.25″N), Guangdong Province, southern China. The sample was diluted 103-fold with sterile water and 0.2 ml aliquots spread on Reasoner’s 2A (R2A; BD) agar plates adjusted to pH 7.0. The isolation plates were incubated at 28 °C for 14 days. Single colonies were picked, purified and cultivated on the same medium. The strain SYSU M10002T was maintained on R2A agar and preserved as glycerol suspensions (20%, v/v) at −80 °C. The basal growth condition of the strain for all experiments was maintained at pH 7.0 and 28 °C, and the biomass for chemical and molecular studies were obtained by cultivating in R2A media, unless otherwise stated.

Phenotypic characterization

Cell morphology was observed using a light microscope (BH2; Olympus) and a transmission electron microscope (JEM-100CX-II; JEOL), on R2A agar at 28 °C after 3 days of incubation. Samples for transmission electron microscopy were prepared as described by Ming et al. (2012). Gram reaction was tested by the Gram Stain Solution kit (Shanghai Yeasen Biotechnology). Motility was determined by the development of turbidity in a tube containing semi-solid medium (Leifson 1960). Growth temperature range (4, 14, 20, 28, 37, 40, 45, 50, 55, and 60 °C) and NaCl tolerance (up to 5.0%, at intervals of 0.5%, w/v) were observed in R2A agar following 2 weeks of incubation. For pH tolerance, R2A agar was prepared between pH 4.0–10.0 (with an interval of 1.0 pH unit) using the buffer system described by Nie et al. (2012), and growth was observed after 2 weeks of incubation. Oxidase and catalase activities were determined by assessing the oxidation of 1% (w/v) tetramethyl-p-phenylenediamine (Kovacs 1956), and the production of gas bubbles on the addition of a drop of 3% H2O2 (v/v), respectively. Hydrolysis of cellulose, gelatin and starch, milk peptonization and coagulation, nitrate reduction, Tweens (20, 40, 60 and 80) degradation and activity of urease were examined as described previously (Gonzalez et al. 1978; Mac Faddin 1976; Tindall et al. 2007). H2S production and indole formation were carried out as described by Cui et al. (2007). Additional physiological and biochemical tests were further performed using API 20NE (bioMérieux), API ZYM (bioMérieux), and GenIII MicroPlate systems (Biolog). All the commercial phenotypic tests were performed according to the manufacturer’s recommendations.

Chemotaxonomy

Biomass for chemical and molecular studies of strain SYSU M10002T were obtained from cultures grown in R2A broth for 3 days. Extraction and analysis of polyamines were carried out as described earlier (Busse et al. 1997; Busse and Auling 1988). For this, cells were cultivated for 3 days, homogenized in 0.2 M perchloric acid (HClO4) and centrifuged. Polyamines in the resultant supernatant were treated with dansyl chloride solution (7.5 μg ml−1 in acetone), and analyzed by HPLC, using UV–Vis detector L-7420. Polar lipids were extracted, separated and examined by a two-dimensional TLC procedure on silica gel G60 plates using the method of Minnikin et al. (1979). Respiratory quinones were extracted from lyophilized cells (Collins et al. 1977), purified and analyzed by HPLC (Groth et al. 1996). The fatty acid profile of strain SYSU M10002T from cultures grown on R2A agar for 3 days (stationary phase) under optimal growth conditions [Note: Cells of strain SYSU M10002T show no growth on tryptic soy agar (Difco)]. Cellular fatty acids were extracted, methylated and analyzed following the instructions of the Sherlock Microbial Identification System (MIDI) version 6.1 and the TSBA6 database (Sasser 2001).

Molecular characterization

Genomic DNA extraction, PCR amplification of the 16S rRNA gene and sequencing of the purified products from strain SYSU M10002T were carried out as described by Li et al. (2007). The whole genome sequencing of strain SYSU M10002T was performed using paired-end sequencing method with Hiseq X platform (Illumina, San Diego, CA, USA) at Magigene Company (Guangzhou, China). Reads of each data set were filtered, and high-quality paired-end reads were assembled using Spades (Harrison and Strulo 2000). Contigs with length greater than 500 bp were kept for gene prediction by applying Prodigal (Hyatt et al. 2010). The predicted coding sequences of the genome were translated and annotated in the COG, KEGG, GO and Pfam databases (Ashburner et al. 2000; Finn et al. 2016; Kanehisa et al. 2017; Tatusov et al. 2001). The phylogenetic relationship of strain SYSU M10002T was determined after BLAST searches (Altschul et al. 1990) of the 16S rRNA gene sequences in NCBI and the EzBioCloud server (Yoon et al. 2017) databases. Analysis of the sequence data was performed by using the software package BioEdit (Hall 1999) and MEGA 7 (Kumar et al. 2016), after multiple alignments of the data by CLUSTAL X software package (Thompson et al. 1997). Phylogenetic analyses were carried out with three tree-making algorithms: neighbour-joining (NJ; Saitou and Nei 1987), maximum-likelihood (ML; Felsenstein 1981) and maximum-parsimony (MP; Fitch 1971) methods. Evolutionary distances in the NJ and ML dendrograms were calculated by the Kimura two-parameter model (Kimura 1983) and the topologies of the phylogenetic trees were evaluated by the bootstrap analysis of Felsenstein (1985) with 1000 replicates. Genome trees based on the concatenated sequences of 29 protein marker genes were generated using RAxML (Stamatakis 2014). Overall genomic relatedness indices between strain SYSU M10002T and related strains in the family Sphingomonadaceae were calculated based on the percentage of conserved proteins (POCP; Qin et al. 2014) and Average amino acid identities (AAI; Hua et al. 2018).

Results and discussion

Phenotypic characteristics

Cells of strain SYSU M10002T were Gram-stain negative, non-motile and aerobic. Colonies on R2A agar following incubation for 3 days were circular, opaque, convex and yellow. Cells of strain SYSU M10002T were short rods, with size measuring approximately 0.8–1.8 μm in length and 0.4–0.6 μm in width (Fig. S1). Strain SYSU M10002T grew at 14–37 °C, pH 6.0–10.0 and in the presence of up to 0.5% NaCl. Optimum growth was observed at 28 °C, pH 6.0–7.0 and in the absence of NaCl. The strain was positive for oxidase and catalase activities, but negative for H2S production, indole formation, milk coagulation and peptonization, nitrate reduction and urease activity tests. Strain SYSU M10002T could hydrolyse Tween 60, but not cellulose, gelatin, starch or Tweens 20, 40 or 80. The strain utilized d-turanose, N-acetyl-d-glucosamine, N-acetyl-β-d-mannosamine, l-fucose and glucuronamide as sole carbon source, but not l-alanine, d-fucose, d-galactose, d-galacturonic acid, d-glucose, stachyose, acetic acid, acetoacetic acid, N-acetyl-d-galactosamine, N-acetyl neuraminic acid, γ-amino-butyric acid, d-arabitol, l-arginine, d-aspartic acid, l-aspartic acid, bromosuccinic acid, d-cellobiose, citric acid, dextrin, formic acid, d-fructose, d-fructose-6-PO4, l-galactonic acid lactone, gelatin, gentiobiose, d-gluconic acid, d-glucuronic acid, d-glucose-6-PO4, glycerol, glycyl-l-proline, l-glutamic acid, l-histidine, α-hydroxy-butyric acid, β-hydroxy-d,l-butyric acid, p-hydroxy-phenylacetic acid, α-keto-butyric acid, α-keto-glutaric acid, inosine, l-lactic acid, d-lactic acid methyl ester, α-d-lactose, d-malic acid, l-malic acid, d-maltose, d-mannitol, d-mannose, d-melibiose, 3-methyl glucose, β-methyl-d-glucoside, methyl pyruvate, myo-inositol, mucic acid, pectin, propionic acid, l-pyroglutamic acid, quinic acid, d-raffinose, l-rhamnose, d-saccharic acid, d-salicin, d-serine, l-serine, d-sorbitol, sucrose, d-trehalose, or Tween 40. In the API ZYM system, strain SYSU M10002T was found to be positive for the activities of alkaline phosphatase, acid phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, chymotrypsin, naphthol-AS-BI phosphohydrolase and α-glucosidase, but not cystine arylamidase, trypsin, β-glucosidase, β-fucosidase, α-galactosidase, β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, lipase (C14) or α-mannosidase. In the API 20NE system, it could hydrolyse aesculin and assimilate glucose, arabinose, mannose, N-acetyl-d-glucosamine, maltose, gluconate, capric acid and malic acid. Differentiating characteristics of strain SYSU M10002T from members of related genera of the family Sphingomonadaceae are listed in Table 1. The detailed physiological characteristics of strain SYSU M10002T are given in the genus and species descriptions below.

Table 1 Key taxonomic characteristics differentiating strain SYSU M10002T from its closely related phylogenetic neighbours of the family Sphingomonadaceae
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Chemotaxonomic characteristics

The respiratory quinones of strain SYSU M10002T were identified as ubiquinone Q-9 (44.9%), Q-10 (43.2%) and Q-8 (11.9%). Strain SYSU M10002T contained homospermidine as its polyamine. The polar lipids of the strain comprised of diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingoglycolipid, two unidentified phospholipids and an unidentified aminolipid (Fig. S2). The major cellular fatty acids (> 10%) detected for strain SYSU M10002T were summed feature 3 (C16:1ω7c and/or C16:1ω6c, 37.4%), summed feature 8 (C18:1ω7c, 27.9%) and C14:0 2-OH (10.5%). A detailed fatty acid profile of strain SYSU M10002T is provided in Table S1.

Molecular characteristics

The DNA G+C content of strain SYSU M10002T was calculated at 64.0% (genome). Pairwise comparison with the almost-complete 16S rRNA gene sequence of strain SYSU M10002T showed sequence similarities of less than 96% with members of the family Sphingomonadaceae. The highest 16S rRNA gene sequence identity of 95.1% is determined with Sphingorhabdus buctiana T5T. Besides the low sequence identity, strain SYSU M10002T also formed a separate clade distinct from the members of the genera Novosphingobium, Blastomonas, Sphingopyxis, Sphingorhabdus or Sphingomonas in all three phylogenetic trees (Fig. 1; Figs. S3 and S4). These findings indicated that the topology of the strain SYSU M10002T is stable and that the strain should be affiliated to the family Sphingomonadaceae. A Similar finding was observed with the RAxML tree generated with related members of the family Sphingomonadaceae (Fig. 2). Strain SYSU M10002T showed low to moderately high POCP and AAI values with related members of the family Sphingomonadaceae (Table 2). Other general features of the genome of strain SYSU M10002T are listed in Table S2.

Fig. 1
figure 1

Unrooted neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationships between strain SYSU M10002T and closely related members of the family Sphingomonadaceae. Asterisks indicate nodes that were supported in trees generated with maximum-likelihood and maximum-parsimony methods. Bootstrap values (≥ 50%) based on 1000 resamplings are given at the nodes. Bar, 0.005 substitutions per nucleotide position

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Fig. 2
figure 2

RAxML phylogenomic tree showing the phylogenetic relationships between strain SYSU M10002T and closely related members of the family Sphingomonadaceae. Bootstrap values (≥ 70%) based on 1000 resamplings are given at the nodes. Bar, 0.1 substitutions per nucleotide position

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Table 2 Overall genome relatedness indices between strain SYSU M10002T and related members of the family Sphingomonadaceae
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Discussion

On the basis of 16S rRNA gene sequences identity and chemotaxonomic features, strain SYSU M10002T can be affiliated to the family Sphingomonadaceae. While strain SYSU M10002T exhibited many characteristics similar to members of the family Sphingomonadaceae in having ovoid to rod-shaped morphology, optimum growth temperature of about 30 °C, optimum growth pH of about 7 and DNA G+C content within the range of the family, it could, however, be differentiated physiologically and phenotypically from the other closely related genera (Table 1). For example, strain SYSU M10002T could tolerate pH 10 which is obviously different from the other closely related genera. Unlike the other closely related genera, the major polyamine in strain SYSU M10002T is homospermidine. Further, on pairwise comparison with the closely related phylogenetic neighbor S. buctiana T5T, despite showing high 16S rRNA gene sequence similarity to S. buctiana T5T, strain SYSU M10002T differed from it on the basis of several distinct chemotaxonomic characteristics, such as (1) fatty acid profile [summed feature 3 (C16:1ω7c and/or C16:1ω6c), summed feature 8 (C18:1ω7c) and C14:0 2-OH for strain SYSU M10002T, and C18:1ω7c and C16:1ω7c as major fatty acids for Sphingorhabdus buctiana T5T]; and (2) respiratory quinone [ubiquinone Q-9 (44.9%), Q-10 (43.2%) and Q-8 (11.9%) for SYSU M10002T and ubiquinone Q-10 for S. buctiana T5T], and genomic DNA G+C content (64.0% for strain SYSU M10002T and 58.5 mol% for S. buctiana T5T) (Table 1). Observation of the POCP and AAI also indicate that strain SYSU M10002T was distinct from the related genera by low to moderate values ranging from 37 to 58 POCP values and 57–65 AAI values (Table 2). Based on the results of these differentiating characteristics (Tables 1 and 2) and phylogenetic analyses, strain SYSU M10002T is considered to represent a novel species in a new genus of the family Sphingomonadaceae, for which the name Aestuariisphingobium litorale gen. nov., sp. nov. is proposed.

Description of Aestuariisphingobium gen. nov.

Aestuariisphingobium [Aes.tu.a.ri.i.sphin.go’bi.um. L. neut. n. aestuarium an estuary; N.L. neut. n. Sphingobium, a bacterial genus; N.L. neut. n. Aestuariisphingobium, a Sphingobium-like organism from an estuary].

Cells are Gram-negative, non-motile, strictly aerobic, and short rods. Colonies are irregular, opaque, convex and yellow. No pigments are produced. Oxidase-positive and catalase-positive. The main respiratory isoprenologues are Q-9 and Q-10. Major polyamine is homospermidine. Major cellular fatty acids are summed feature 3, summed feature 8 and C14:0 2-OH. The known polar lipid profile comprises diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and sphingoglycolipid. The genomic DNA G+C content of the type strain of the type species is about 64.0 mol%. The type species is Aestuariisphingobium litorale.

Description of Aestuariisphingobium litorale sp. nov.

Aestuariisphingobium litorale (li.to.ra’le. L. neut. adj. litorale, of or belonging to the coast].

Displays the following properties in addition to those described for the genus. Cells measure 0.8–1.8 μm in length and 0.4–0.6 μm in width after 4 days of growth at 28 °C on R2A agar plates. Growth occurs at 14–37 °C (optimum, 28 °C), pH 6.0–10.0 (optimum, pH 6.0–7.0) and in the presence of up to 0.5% NaCl. Negative results for H2S production, indole formation, nitrate reduction, milk coagulation and peptonization, and urease activity. Hydrolyses Tween 60, but not cellulose, gelatin, starch, or Tweens 20, 40 or 80.

The type strain SYSU M10002T (= KCTC 52944T = NBRC 112961T) was isolated from a water sample collected along the coast of Pearl River estuary, Guangdong Province, southern China. The genomic DNA G+C content of the type strain is 64.0%. The GenBank accession number for the 16S rRNA gene sequence of strain SYSU M10002T is MH843155. The draft genome has been deposited under accession number RAGX00000000.