Introduction

The family Chitinophagaceae, belonging to the order Chitinophagales, class Chitinophagia, phylum Bacteroidetes, was proposed by Kämpfer et al. (2011) with Chitinophaga as the type genus. The members of the family Chitinophagaceae are usually characterized as thin, rod-shaped, sometimes filamentous, and non-motile or sometimes motile by gliding. The predominant respiratory quinone is MK-7, and iso-C15:0, iso-C17:0 3-OH and iso-C15:1 G are the typical fatty acids of the family described so far. At the time of writing, the family Chitinophagaceae consists of 48 genera with validly published names (https://lpsn.dsmz.de/family/chitinophagaceae). According to the phylogenetic trees based on the 16S rRNA gene or genome sequences, three genera named Paraflavitalea, Niastella, and Pseudoflavitalea are close neighbors. The genus Paraflavitalea was recently proposed by Heo et al. (2020), and includes two species, Paraflavitalea soli (Heo et al. 2020) and Paraflavitalea devenefica (Hou et al. 2021), of which both type strains were isolated from soil. The genus Niastella was first proposed by Weon et al. (2006) with two species, Niastella koreensis and Niastella yeongjuensis. Later, six more species, Niastella populi (Zhang et al. 2010), Niastella gongjuensis (Kim et al. 2015), Niastella vici (Chen et al. 2016), Niastella hibisci (Yan et al. 2016), Niastella caeni (Sheng et al. 2020) and Niastella soli (Akter et al. 2021) were subsequently proposed. The genus Pseudoflavitalea was proposed by Kim et al. (2016b) with two species, Pseudoflavitalea rhizosphaerae, as the type species of the genus, and the other species Pseudoflavitalea soli, which was reclassified from Flavitalea soli (Kim et al. 2016a). Furthermore, one other species of this genus has been added, namely Pseudoflavitalea ginsenosidimutans (Lawson et al. 2020), which was reclassified from Pseudobacter ginsenosidimutans (Siddiqi and Im 2016). Notably, none of the members of these three genera mentioned above were reported to have any flagellar structures.

Here, we described the polyphasic characterization of two novel bacterial strains, SYSU D01084T and SYSU D00799T, belonging to a novel genus of the family Chitinophagaceae, isolated from a sandy soil sample collected from Gurbantunggut Desert in Xinjiang, north-west China.

Materials and methods

Sample collection and isolation

The soil sample (5–10 cm in depth) for the isolation was collected from the Gurbantunggut Desert in Xinjiang, north-west China (45° 27′ N, 86° 40′ E) in September 2019 using a small shovel, and kept in a sterilized plastic bag at 4 °C until the isolation procedure was carried out. The moisture content and pH of the sample were 0.4% and 8.5, respectively. The sample also depicted the following chemical parameters: electronic conductivity (61.3 μs/cm), total carbon (0.29%), total nitrogen (0.03%). The pretreatment method of soil sample and isolation procedure were described in the previous studies (Li et al. 2021a, b). The strains SYSU D01084T and SYSU D00799T were isolated from R2A (Reasoner’s 2A) agar (tryptone 0.5 g L−1, yeast extract 0.5 g L−1, glucose 0.5 g L−1, casamino acids 0.5 g L−1, soluble starch 0.5 g L−1, magnesium sulfate 0.05 g L−1, dipotassium hydrogen phosphate 0.3 g L−1, pyruvic acid sodium 0.3 g L−1, agar 18 g L−1, distillated water 1000 mL, pH 7.5 ± 0.2) and 1/10 strength NA agar (peptone 1.0 g L−1, beef extract 0.3 g L−1, NaCl 0.5 g L−1, agar 18.0 g L−1, distillated water 1000 mL, pH 7.5 ± 0.2), respectively. After incubated at 28 °C under aerobic condition for 5 days, strains SYSU D01084T and SYSU D00799T both grew well and presented light-yellow colonies on R2A agar. The purified strains were maintained on R2A slants at 4 °C and as glycerol suspensions (20%, v/v) at − 80 °C for long-term preservation.

Phenotypic characterization

To examine cell morphological characteristics, strains SYSU D01084T and SYSU D00799T were grown on R2A agar for 3 days at 28 °C and studied by transmission electron microscopy (JEM-1400FLASH; JEOL). Colony characteristics were observed after incubated at 28 °C for 3–7 days on R2A agar, nutrient agar (NA, Oxoid), Luria–Bertani agar (LB, Oxoid) and trypticase soy agar (TSA, BD). Gram-staining reaction was performed by a commercial kit according to the manufacturer’s instruction (Solarbio). Cell motility was checked by inoculating the strain in a tube containing R2A broth supplemented with 0.4% agar. The growth under anaerobic condition was assessed using R2A agar supplemented with 5% cysteine and 5% Na2S·9H2O in a Whitley A45 anaerobic workstation at 28 °C for 7 days. Oxidase and catalase activities were determined using the oxidase reagent (bioMérieux) and the formation of bubbles on addition of 3% (v/v) H2O2 (Sigma), respectively. The range and optimum of temperature (4, 15, 28, 30, 37, 45, 50, 55 and 60 °C) and NaCl concentrations (up to 10.0%, at intervals of 0.5% units, w/v) for growth were tested on R2A agar plates. The pH range for growth was tested from pH 4.0 to 10.0 (at intervals of 1.0 pH unit). The pH values were adjusted using the following buffers: C6H8O7·H2O/Na3C6H5O7·2H2O (pH 4.0–5.0), NaOH/KH2PO4 (pH 6.0–8.0) and Na2CO3·10H2O/NaHCO3 (pH 9.0–10.0). Hydrolysis of aesculin, casein, tyrosine, gelatin, CM-cellulose, starch and Tweens (20, 40, 60 and 80), Nitrate reduction, H2S production, and urease activity were tested as previously described (Gonzalez et al. 1978; Tindall et al. 2007). Some other physiological characteristics and enzyme activities were determined using API 20NE, API 50CH, API ZYM strips and Biolog GEN III MicroPlates according to the instructions provided by the manufacturers.

Chemotaxonomic characterization

Chemotaxonomic characteristics of two strains were determined using standard methods under similar conditions unless otherwise mentioned. Biomass of strains SYSU D01084T and SYSU D00799T were harvested after incubation on R2A agar at 28 °C for 5 days. The respiratory quinones of strains were extracted from freeze-dried cell biomass, then purified and identified by HPLC (Kroppenstedt 1982; Tamaoka et al. 1983). The cellular fatty acids were determined according to the standard MIDI procedure (Microbial Identification System, Sherlock) and a gas chromatography (Agilent Technologies 6850). The resulting profiles were identified using the TSBA6 database (version 6.1) of MIDI (Sasser 1990). The Polar lipids were extracted, separated and identified by two-dimensional thin-layer chromatography (TLC) on silica gel G60 plates (Merck) according to previously described methods (Collins and Jones 1980; Minnikin et al. 1979). The reagents for detecting polar lipids were as follows: molybdatophosphoric acid (5%, w/v), molybdenum blue (0.2%, w/v), ninhydrin and α-naphthol, which were used to detect total polar lipids, phospholipids, aminolipids and glycolipids, respectively.

Molecular characterization

After growth on R2A agar at 28 °C for 5 days, biomass of strains SYSU D01084T and SYSU D00799T were harvested to extract the genomic DNA. The almost full-length 16S rRNA gene sequences of strains SYSU D01084T (1453 bp) and SYSU D00799T (1470 bp) were amplified and sequenced as previously described (Li et al. 2021a). The sequences obtained were quality-checked and assembled via SeqMan program (DNAStar v7.1.0) and then the 16S rRNA gene sequence similarities among SYSU D01084T, SYSU D00799T and their relatives were calculated using the EzBioCloud server (www.ezbiocloud.net) (Yoon et al. 2017). Multiple alignments of the 16S rRNA gene sequences were analyzed by CLUSTAL_X (Thompson et al. 1997). The phylogenetic trees were reconstructed using neighbor-joining (NJ) (Saitou and Nei 1987) with Kimura’s two-parameter model (Kimura 1980), maximum-likelihood (ML) (Felsenstein 1981) with Tamura-Nei model (Tamura and Nei 1993) and maximum-parsimony (MP) (Fitch 1971) methods by MEGA X software (Kumar et al. 2018). To evaluate the topology stability of each phylogenetic tree, bootstrap analysis was performed with 1000 replications (Felsenstein 1985). Flavobacterium aquatile LMG 4008T (JRHH01000003) was used as the outgroup.

The draft genome sequences of strains SYSU D01084T and SYSU D00799T were sequenced by Illumina NovaSeq 6000 platform at Novagene (Beijing, China). Reads of each data sets were assembled using SPAdes v3.13 (Anton et al. 2012). Only contigs with length more than 500 bp were retained for the following analyses. Genome annotation was conducted through the pipelines of Global Catalogue of Type Strain (gcType) platform (http://gctype.wdcm.org) (Shi et al. 2021). The average nucleotide identity (ANI), amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values were calculated using OrthoANI Tool (https://www.ezbiocloud.net/tools/orthoani) (Lee et al. 2016), EzAAI tool v1.0 (Kim et al. 2021) and Genome-to-Genome Distance Calculator (GGDC) v3.0 (https://ggdc.dsmz.de/ggdc.php#) (Meier-Kolthoff et al. 2013), respectively. The DNA G + C content was determined from the genome sequence. Biosynthetic gene clusters (BGCs) of secondary metabolites were predicted by antiSMASH v6.0.1 (Blin et al. 2021). 22 genomes of strains SYSU D01084T, SYSU D00799T, some related type species of the family Chitinophagaceae and one outgroup were selected for the genome-based phylogenomic tree reconstruction. A total of 29 conserved marker genes (frr, infC, nusA, pgk, rplA, rplB, rplC, rplD, rplE, rplF, rplK, rplL, rplM, rplN, rplP, rplS, rplT, rpmA, rpoB, rpsB, rpsC, rpsE, rpsI, rpsJ, rpsK, rpsM, rpsS, smpB, tsf) were extracted using AMPHORA2 (Wu and Scott 2012). Sequences of each marker gene were aligned separately by MUSCLE (Edgar 2004). Poorly aligned regions were removed from the datasets using Gblocks (Castresana 2000). Cleaned alignments were then concatenated using perl script (https://github.com/nylander/catfasta2phyml) and phylogenomic tree was generated through RAxML method (Stamatakis 2014).

Results and discussions

Phenotypic characteristics

Both strains SYSU D01084T and SYSU D00799T were Gram-stain-negative, aerobic, oxidase- and catalase-negative, and appeared as circular, translucent, convex, smooth and light-yellow clones (1–3 mm in diameter) on R2A agar. Cells of strain SYSU D01084T were long-rod-shaped (0.2–0.3 µm wide and 5.8–9.5 µm long) (Fig. S1a, b), motile and amphitrichous, yet cells of strain SYSU D00799T were long-rod-shaped (0.2–0.3 µm wide and 5.5–12.3 µm long) (Fig. S1c, d) and non-motile. Strain SYSU D00799T showed growth on R2A, TSA, NA and LB agar. However, strain SYSU D01084T was only found to grow on R2A agar. Strain SYSU D01084T grew at 4–50 °C (optimum, 28–30 °C) and in the presence of 0–1.0% (w/v) NaCl (optimum, 0%). Strain SYSU D00799T grew at 4–50 °C (optimum, 30–37 °C) and in the presence of 0–1.0% (w/v) NaCl (optimum, 0–0.5%). In addition, both two strains grew at pH 6.0–8.0 (optimum, 7.0). The major differences between strains SYSU D01084T and SYSU D00799T with other closely related genera in the family Chitinophagaceae were given in Table 1. Detailed phenotypic characteristics of strains SYSU D01084T and SYSU D00799T determined by API ZYM, API 20NE, API 50CH and Biolog GEN III MicroPlates were provided in the Tables S1, S2, S3 and S4, respectively.

Table 1 Phenotypic differences between strains SYSU D01084T and SYSU D00799T with other closely related genera in the family Chitinophagaceae
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Chemotaxonomical characteristics

The major fatty acids of strains SYSU D01084T and SYSU D00799T were iso-C15:0 (26.1–28.9%), iso-C17:0 3-OH (24.9–26.1%), and iso-C15:1 G (14.1–15.5%), which were consistent with the predominant fatty acid components in close members of the family Chitinophagaceae, but differences in the presence or absence of several components or percentage content were observed (Table S5). The polar lipid profiles of strains SYSU D01084T and SYSU D00799T were similar, containing phosphatidylethanolamine (PE), unidentified aminoglycolipids (AGL), unidentified aminolipids (AL) and unidentified polar lipids as major components (L) (Fig. S2), but lacking diphosphatidylglycerol (DPG), phosphatidylmethylethanolamine (PME), phosphatidylcholine (PC), phosphatidylglycerol (PG), unidentified glycolipid (GL), unidentified aminophospholipid (APL) and unidentified phospholipid (PL) (Table 1). The predominant respiratory quinone of strains SYSU D01084T and SYSU D00799T was identified as MK-7, which is characteristic of the family Chitinophagaceae.

Molecular characteristics

Phylogenetic analyses based on 16S rRNA gene sequences showed strains SYSU D01084T and SYSU D00799T belonged to the family Chitinophagaceae, and shared 98.1% similarity to each other, which was clearly below the cut-off value of 98.7% proposed for novel species (Stackebrandt and Ebers 2006; Meier-Kolthoff et al. 2013; Kim et al. 2014). On the basis of the comparison results with EzBioCloud database, the similarity values (lower than 95.4%) determined for the two isolates with members of neighboring genera were also below the 98.7% threshold. NJ, ML, and MP phylogenetic trees revealed that strains SYSU D01084T and SYSU D00799T clustered into a stable clade with each other, forming an independent branch in the phylogenetic tree reconstructions, and closely related with the genus Niastella (Figs. 1, S3, S4).

Fig. 1
figure 1

Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences of SYSU D01084T, SYSU D00799T and other close relatives. Filled circles at nodes indicate generic branches that were also recovered using the ML and MP algorithms. Bootstrap values (≥ 50%) based on 1000 replications are shown at branch nodes. Flavobacterium aquatile LMG 4008T (JRHH01000003) is used as the outgroup. Bar, 0.01 substitutions per nucleotide position

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Confirming the results of the 16S rRNA gene-based analysis, the phylogenomic tree based on the concatenated alignment of 29 marker genes showed that both strains formed a monophyletic group, with Niastella caeni HX-16-21T being the closest neighbor (Fig. S5). The assembled genomes sequences of strains SYSU D01084T and SYSU D00799T have been deposited in the GenBank database under the accession numbers JAFFJX000000000 and JAFIQX000000000, respectively. The draft genome sequences of strains SYSU D01084T and SYSU D00799T were 7,284,291 bp with a G + C content of 46.0%, and 7,299,432 bp with a G + C content of 45.6%, respectively. The detailed comparison of genomic features between strains SYSU D01084T, SYSU D00799T and other closely related taxa were shown in Tables 2, S6. The genome size of strains SYSU D01084T and SYSU D00799T were lower than those of members of Paraflavitalea (8.4–8.7 Mb) and Niastella (8.1–10.1 Mb). The genomic G + C content of two strains were higher than Niastella (43.5–45.4%), but lower than Paraflavitalea (47.4–47.5%).

Table 2 General genomic features of strains SYSU D01084T, SYSU D00799T and related type strains of neighboring genera in the family Chitinophagaceae
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The ANI/AAI/dDDH values between SYSU D01084T, SYSU D00799T and related taxa are shown in Table S7. The low ANI (83.8%) and dDDH (51.4%) values between SYSU D01084T and SYSU D00799T were below the threshold values for species delineation (Goris et al. 2007; Richter and Rosselló-Móra 2009; Meier-Kolthoff et al. 2013), thus confirming that SYSU D01084T and SYSU D00799T were distinct from each other and belonged to different species. The AAI value between SYSU D01084T and SYSU D00799T was 89.2%, which was higher than the maximum threshold value of 85% proposed for classification of a novel genus (Konstantinidis and Tiedje 2005; Luo et al. 2014), indicating they belonged to the same genus. However, the AAI values between these two isolates and their reference genomes ranged from 68.1 to 82.9%, which were below 85% threshold, indicating that these two isolates might belong to a novel genus.

In addition, five BGCs matched known clusters were found in strain SYSU D01084T, including one 20.8 kb-long terpene cluster, one 41.1 kb-long type III polyketide synthase (T3PKS) cluster, one 15.6 kb-long siderophore cluster, one 22.1 kb-long lanthipeptide-class-IV cluster and one 12.3 kb-long RiPP-like cluster; and seven BGCs matched known clusters were obtained in strain SYSU D00799T, including one 20.8 kb-long terpene cluster, one 41.1 kb-long T3PKS cluster, one 15.7 kb-long siderophore cluster, one 27.7 kb-long lanthipeptide-class-I cluster, one 13.7 kb-long lanthipeptide-class-IV cluster and two RiPP-like clusters with 12.3 kb- and 11.1 kb-long, respectively. However, some of the characteristic BGC gene clusters associated with the genera Paraflavitalea and Niastella, including non-ribosomal peptide synthetase (NRPS), microviridin and type I polyketide synthase (T1PKS), etc., were not predicted in strains SYSU D01084T and SYSU D00799T (Table S6).

Combined phenotypic, biochemical, chemotaxonomic, phylogenetic and genomic results enabled the differentiation of strains SYSU D01084T and SYSU D00799T from other closely related genera of the family Chitinophagaceae. The most notable difference is that SYSU D1084T is amphitrichous, while SYSU D00799T and all members of the three neighboring genera (Paraflavitalea, Niastella and Pseudoflavitalea) are not. As shown in Table 1, strains SYSU D01084T and SYSU D00799T could grow at 4–50 ℃, while members of related genera could only grow at 10–42 ℃. In addintion, negative for naphthol-AS-BI-phosphohydrolase differentiated strains SYSU D01084T and SYSU D00799T from almost all the related species, except for N. caeni HX-16-21T. In the fatty acid profile comparison (Table S5), the absence of C14:0 and presence of anteiso-C15:1 A distinguished the two strains from Paraflavitalea. Besides, the presence of C12:0, anteiso-C17:0 and C18:1 ω9c could also differentiated them from members of related genera. Furthermore, unidentified aminoglycolipids, which were not found in any members of Paraflavitalea, Niastella and Pseudoflavitalea, were detected in strains SYSU D01084T and SYSU D00799T. Also, the phylogenetic and genomic results allow the differentiation of strains SYSU D01084T and SYSU D00799T as well, indicating that these two distinct strains represent two novel species within a new genus, for which the names Longitalea arenae gen. nov., sp. nov., and Longitalea luteola sp. nov. were proposed.

Description of Longitalea gen. nov.

Longitalea (Lon.gi.ta'le.a. L. masc. adj. longus, long; L. fem. n. talea, a slender staff, a rod; N.L. fem. n. Longitalea, a long rod.).

Cells are Gram-stain-negative, aerobic, long-rod-shaped, oxidase- and catalase-negative, motile by means of a pair of polar flagella or non-motile without flagella. The predominant quinone is MK-7. The major cellular fatty acids are iso-C15:0, iso-C17:0 3-OH, and iso-C15:1 G. The polar lipid profile consists of phosphatidylethanolamine (PE), unidentified aminolipid (AL), unidentified aminoglycolipid (AGL) and unidentified lipid (L). The genus is phylogenetically located within the family Chitinophagaceae. The genomic G + C content of the DNA is in the range of 45.6–46.0%. The type species is Longitalea arenae.

Description of Longitalea arenae sp. nov.

Longitalea arenae (a.re'nae. L. fem. gen. n. arenae, of sand, referring to the isolation source of the type strain).

Exhibits the following characteristics in addition to those given in the genus description. Cells are Gram-stain-negative, aerobic, oxidase- and catalase-negative, long-rod-shaped (0.2–0.3 µm wide and 5.8–9.5 µm long), motile with a pair of polar flagella. Good growth on R2A agar, and absent on NA, TSA and LB agar. Colonies on R2A agar for 5 days are circular, translucent, convex, smooth and light-yellow with a diameter of 1–3 mm. Growth occurs at 4–50 ºC (optimum 28–30 ºC), pH 6.0–8.0 (optimum 7.0) and in the presence of 0–1.0% NaCl (optimum 0%, w/v). Positive for hydrolysis of aesculin, gelatin, casein, tyrosine and Tween 20. Negative for nitrate reduction, H2S production, urease activity and hydrolysis of CM-cellulose, starch and Tweens (40, 60 and 80). In the API ZYM system, the activities of alkaline phosphate, leucine arylamidase, valine arylamidase, α-chymotrypsin, acid phosphatase, β-galactosidase and N-acetyl-β-glucosaminidase are positive. The genomic DNA G + C content of the type strain is 46.0%.

The type strain SYSU D01084T (= CGMCC 1.18641T = MCCC 1K05006T = KCTC 82283T) was isolated from a soil sample collected from Gurbantunggut Desert in Xinjiang, north-west China.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and draft genome are MT527704 and JAFFJX000000000, respectively.

Description of Longitalea luteola sp. nov.

Longitalea luteola (lu.te.o'la. L. fem. adj. luteola, light yellow).

Exhibits the following characteristics in addition to those given in the genus description. Cells are Gram-stain-negative, aerobic, oxidase- and catalase-negative, long-rod-shaped (0.2–0.3 µm wide and 5.5–12.3 µm long), non-motile without flagella. Good growth on R2A agar, moderate on NA agar, poor on TSA and LB agar. Colonies on R2A agar for 5 days are circular, translucent, convex, smooth and light-yellow with a diameter of 1–3 mm. Growth occurs at 4–50 ºC (optimum 30–37 ºC), pH 6.0–8.0 (optimum 7.0) and in the presence of 0–1.0% NaCl (optimum 0–0.5%, w/v). Positive for hydrolysis of aesculin, gelatin, casein and tyrosine. Negative for nitrate reduction, H2S production, urease activity and hydrolysis of CM-cellulose, starch and Tweens (20, 40, 60 and 80). In the API ZYM system, the activities of alkaline phosphate, leucine arylamidase, valine arylamidase, α-galactosidase, β-glucosidase and N-acetyl-β-glucosaminidase are positive. The genomic DNA G + C content of the type strain is 45.6%.

The type strain SYSU D00799T (= MCCC 1K04987T = KCTC 82282T = NBRC 114888T) was isolated from a soil sample collected from Gurbantunggut Desert in Xinjiang, north-west China.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and draft genome are MT527658 and JAFIQX000000000, respectively.