Introduction

The family Chitinophagaceae was described by Kämpfer et al. [1] with Chitinophaga as the type genus, which also indicated that the genera Sediminibacterium, Lacibacter, Flavihumibacter, Flavisolibacter, Niabella, Niastella, Segetibacter, Parasegetibacter, Terrimonas, Ferruginibacter, Filimonas, and others are phylogenetically reclassified in the same family. At the time of writing, the family Chitinophagaceae contained 46 genera (https://www.ncbi.nlm.nih.gov). The members of this family are widespread microorganisms commonly isolated from various sources, including rhizosphere soil, subtropical rainforest compost, freshwater sediment, estuarine water, and human peritoneal tumors [2,3,4,5,6,7].

During the course of a study on the diversity of bacterial communities associated with activated sludge in Hanam City, Korea, a bacterial strain, designated BO-59T, which form yellowish-pigmented colonies, was isolated. The results of 16S rRNA gene sequence and phylogenetic analyses indicated that BO-59T is closely related to members of the family Chitinophagaceae within the phylum Bacteroidetes. Based on taxonomic data obtained in this study, we established the putative taxonomic position of strain BO-59T as the type strain of a new genus in the family Chitinophagaceae, for which the name Hanamia caeni gen. nov., sp. nov. is proposed.

Materials and Methods

Strain Isolation

During the course of a study on cultivable aerobic bacterial strains, we screened an activated sludge sample collected from a wastewater treatment plant in Hanam City, South Korea (37° 31ʹ 14.5ʺ N, 127° 10ʹ 21.2ʺ E). The sample was processed using a standard dilution plating technique by spreading on R2A agar using sterile saline (0.85% NaCl). The isolates were purified by transferring onto an R2A agar plate and incubating at 30 °C for 3 days. A novel bacterium, designated BO-59T, was isolated and assessed to be closely related to members in the family Chitinophagaceae within the phylum Bacteroidetes. Strain BO-59T was routinely cultured on R2A agar and maintained in a glycerol suspension (20%, v/v in R2A broth), at − 80 °C.

Phylogenetic and Genomic Analysis

Genomic DNA of strain BO-59T was obtained using a genomic DNA extraction kit (Macrogen Co. Ltd, Korea) for subsequent genome and 16S rRNA sequence analyses. The 16S rRNA gene was amplified using a set of universal bacterial primers (800R, 1492R, 27F, and 518F) [8], and the purified PCR products were sequenced commercially by Macrogen, Inc. (Seoul, South Korea). The sequence of the 16S rRNA gene was compiled using SeqMan software (DNASTAR, USA) and for the purposes of phylogenetic analysis, we obtained the 16S rRNA gene sequences of related taxa from the GenBank database and EzTaxon-e server (http://www.ezbiocloud.net) [9]. Multiple alignments were performed by Clustal_X program with gaps edited using the BioEdit program [10, 11]. Neighbor joining (NJ), maximum likelihood (ML), and maximum parsimony (MP) trees were constructed using Molecular Evolutionary Genetics Analysis 7 (MEGA X) software with bootstrap analysis based on 1,000 replications. Kimura two-parameter model was used for ML and NJ tree construction with pairwise deletion of gaps, whereas the MP tree was constructed based on the Subtree–Pruning–Regrafting heuristic method with pairwise deletion of gaps [12,13,14,15,16].

The draft genomic sequencing for strain BO-59T was performed based on Illumina HiSeq X Ten analysis and the sequences were assembled using the SOAPdenovo v. 3.10.1 de novo assembler. The draft genome sequence has been submitted to the GenBank database (www.ncbi.nlm.nih.gov) and annotated using the NCBI Prokaryotic Genomes Annotation Pipeline (PGAP) [17]. The genomic DNA G + C content was determined directly from the draft genome sequence, and pairwise genome-based relatedness between strain BO-59T and closely related strains was estimated based on the average nucleotide identity (ANI) using the ANI calculator employing the OrthoANIu algorithm [18] available from the EzBioCloud service. Digital DNA–DNA hybridization (dDDH) values were determined using the online Genome-to-Genome Distance Calculator (GGDC) based on GGDC’s formula 2 (http://ggdc.dsmz.de/ggdc.php) [19]. To calculate the genus-level average amino acid identity (AAI) value of the phylum “Bacteroidetes,” AAI calculation was performed using the Lab AAIrCalculator (http://lycofs01.lycoming.edu/*newman/AAI/) [20].

Phenotypic and Biochemical Characteristics

Gram staining was performed using the procedure described by Buck [21]. The optimum growth of BO-59T was examined on R2A (BD, USA), nutrient (NA, BD), trypticase soy (TSA, BD), LB (BD), and MacConkey (BD) agars at 30 °C for 7 days. Given that BO-59T was found to grow optimally on R2A agar, we monitored cell growth on R2A agar (BD) over a range of different temperatures between 4 and 50 °C (4, 10, 15, 18, 20, 25, 30, 35, 37, 40, 42, 45, and 50 °C). Growth was also assessed over a range of pH values from 4 to 10 (at intervals of 0.5 pH units) adjusted using the following buffers: acetate buffer for pH 4.0–5.5, phosphate buffer for pH 6.0–8.0, and Tris buffer for pH 8.5–10.0. Salt tolerance was assessed on R2A medium supplemented with 0.5% and 1% to 10% (w/v at intervals of 1% unit) NaCl after 7 days of incubation. Cell shape, size, and the presence of flagella were examined by scanning electron microscopy (Hitachi SU-3500) and Nikon light microscopy (× 1,000 magnification), after cells have been grown for 3 days at 30 °C on R2A agar medium. Motility was examined on R2A broth supplemented with 0.2% agar [22]. Catalase activity was determined as previously described [23] by assessing bubble production by cells in 3% (v/v) H2O2, and oxidase activity was determined using 1% (w/v) N,N,N,N,-tetramethyl-1,4-phenylenediamine reagent. An anaerobic growth test was conducted over 2 weeks using the GasPak™ EZ anaerobe pouch system (BD). Tests for the hydrolysis of starch, casein, carboxylmethyl cellulose, DNA, and Tween-60 [24, 25] were carried out after incubating for 5 days at 30 °C. Production of a flexirubin-type pigment was determined based on a reversible color shift to red, purple, or brown when yellow or orange colonies are covered with a 20% aqueous KOH solution [26]. Biochemical tests were carried out using API (API 20NE, API ID 32GN, and API ZYM) kits (BioMérieux) according to the manufacturer’s instructions. API ZYM test strip was read after 5 h of incubation at 37 °C, whereas strips for the other API kits were examined after 3 days at 30 °C.

Chemotaxonomic Analysis

Isoprenoid quinones were extracted using chloroform/methanol (2:1, v/v), evaporated under vacuum conditions, and reextracted in n-hexane/water (1:1, v/v). The crude n-hexane–quinone solution thus obtained was purified using Sep-Pak Vac cartridge silica (Waters, USA) and subsequently analyzed by HPLC as previously described [27]. Strain BO-59T was examined for polar lipid contents as described previously [28]. For the extraction of cellular fatty acids, strain BO-59T was cultured on R2A for 48 h at 30 °C, harvested at the exponential phase, and subjected to saponification, methylation, and extraction according to the protocol of the Sherlock Microbial Identification System (MIDI). The resulting fatty acid methyl esters were analyzed via gas chromatography (model 6890; Hewlett Packard) using the Microbial Identification software package [29].

Results and Discussion

Phylogenetic and Genomic Analysis

The 16S rRNA gene sequence (1451 bp) of strain BO-59T was determined and subjected to a comparative analysis. The novel isolate, identified as a member of the family Chitinophagaceae, showed 16S rRNA gene sequence similarities of less than 92.8% compared with Ferruginibacter alkalilentus HU1-GD23T (92.8%) and Ferruginibacter lapsinanis HU1-HG42T (92.6%) and less than 92% compared with other assessed members of the Chitinophagaceae. Moreover, in our phylogenetic tree reconstructions, we established that strain BO-59T forms an independent branch with the genus Ferruginibacter (Fig. 1, Supplementary Fig. S1). Based on these finding, we thus propose that strain BO-59T should be considered as a new genus with the family Chitinophagaceae. Furthermore, the phylogenetic tree based on the core/house-keeping gene sets in the family Chitinophagaceae was also reconstructed with whole-genome sequencing using automated multi-locus species tree analysis (Supplementary Fig. S2) [30].

Fig. 1
figure 1

Neighbour-joining phylogenetic tree constructed from a comparative analysis of 16S rRNA gene sequences showing the relationships of BO-59T with related species of the family Chitinophagaceae. Filled circles indicate that the corresponding nodes were also recovered in trees generated with the maximum-likelihood and maximum-parsimony algorithm. Bootstrap values expressed as percentages of 1000 replications) greater than 60% are shown at the branch points. Bar, 0.050 substitutions per nucleotide position

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The genome of strain BO-59T consists of a circular chromosome of 4.96 Mb in length comprising 49 contigs with an N50 value of 275,585 bp and G + C content of 39.1 mol%. Among the 4254 predicted genes, there are 4209 protein-coding genes (CDSs), three rRNAs, 39 tRNAs, three other RNA genes, and 52 pseudogenes. In addition, two CRISPR arrays were also identified. The draft genome sequence of BO-59T has been deposited in the DDBJ/EMBL/NCBI GenBank database under the accession number RJJR00000000. The digital DDH analysis between strain BO-59T and related type species showed 22.2–22.9%, which were below the proposed thresholds for species delineation of 60–70%. [31]. Strain BO-59T shared highest AAI (59.8%) with Ferruginibacter lapsinanis KCTC 22305 T and the AAI values among strain BO-59T and other family Chitinophagaceae were 54.7–55.6%. These values are lower than the genus describing threshold AAI value of 60%, which supports the new genus proposal [20]. In addition, the average nucleotide identity values of strain BO-59T compared with members of the genera Flavisolibacter and Pseudoflavitalea were between 66.5% and 66.9%, which indicates that strain BO-59T is not a strain of any of the genome-sequenced species of these genera (Table 2) [32]. These findings thus tend to indicate that the isolated strain BO-59T represents a novel genus.

Phenotypic and Biochemical Characteristics

When cultured on R2A agar for 3 days, strain BO-59T grew as yellowish-pigmented, convex, circular colonies with entire margins. The cells were Gram-negative, aerobic, non-motile, and long rod-shaped with dimensions in the range of 3–4 × 11–45 μm (Supplementary Fig. S3). As shown in Table 1, the physiological and biochemical characteristics of BO-59T differentiate this bacterium from related genera in the family Chitinophagaceae (Table 2).

Table 1 Differential characteristics of strain BO-59T and type species of most closely related genera of the family Chitinophagaceae
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Table 2 Genome characteristics between strain BO-59T and other type species in the family Chitinophagaceae
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Chemotaxonomic Analysis

Strain BO-59T contains MK-7 as the only respiratory quinone. The major polar lipids were identified as phosphatidylethanolamine (PE), an unidentified aminophospholipid (APL1), and three unidentified polar lipids (PL1, PL2, and PL3). The minor polar lipids were two unidentified polar lipids (PL4 and PL5) and two unidentified aminolipids (AL1 and AL2) (Supplementary Fig. S4). Based on the polar lipid analysis, we established that strain BO-59T has major polar lipids profile similar to that of recently designated members of the family Chitinophagaceae [33,34,35,36], whereas the suite of minor polar lipid distinguishes strain BO-59T from related genera in this family Chitinophagaceae. The predominant cellular fatty acids profiles of strain BO-59T mainly comprise iso-C15:0 (48.6%), iso-C17:0 3OH (17.1%), and iso-C15:1 G (10.9%), whereas the minor fatty acids are C16:1 ω7c and/or C16:1 ω6c (summed feature 3, 5.1%) and other fatty acids (< 5.0%) (Supplementary Table S1). The strain BO-59T strain can be distinguished from the related neighboring genera within the family Chitinophagaceae primarily by the presence of hydroxyl fatty acids. Although other members of the family Chitinophagaceae have similar major fatty acid profiles (iso-C15:0, iso-C17:0 3OH, and iso-C15:1 G), the relative proportions tend to differ. Strain BO-59T has larger relative amounts of iso-C15:0 and iso-C15:1 G that are 15.5% higher and 7.1% lower, respectively, than those in members of the genus Ferruginibacter, which is the closest neighboring taxon based on 16S rRNA gene analysis [33]. The absence of iso-C16:0 3OH and C16:0 3OH is mostly unique to the four-related strains when compared with the fatty acid profiles of representatives of related genera. Although strain BO-59T does not produce iso-C16:0 3OH and C16:0 3OH, these fatty acids are produced by Ferruginibacter alkalilentus HU1-GD23T and also detected four-related genera. Similarly, strain BO-59T does not appear to produce C15:0 2OH, C15:0 3OH, or C17:0 3OH. Thus, the qualitative and quantitative differences between fatty acid profiles of representatives of each genus (Table 3) could also be used for differentiation at the genus level.

Table 3 Characteristics between strain BO-59T and other related genera in the family Chitinophagaceae
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Taxonomic Conclusion

On the basis of our phylogenetic, phenotypic, and chemotaxonomic characterization of the novel isolate BO-59T, we were unable to assign this strain BO-59T to any of the known taxa. Certain phenotypic features (Table 1), fatty acids profiles (Supplementary Table S1), and 16S rRNA gene sequences clearly differentiate the strain BO-59T from the phylogenetically related taxa and provide convincing evidence to indicate that this bacterium is a novel species of a novel genus in the family Chitinophagaceae, for which the name Hanamia caeni gen. nov., sp. nov. is proposed.

Description of Hanamia gen. nov.

Hanamia (Ha.nam'i.a. N.L. fem. n. Hanamia, named after the city of Hanam).

Cells are Gram-stain-negative, non-motile, aerobic, and long rods. The major fatty acids are iso-C15:0, iso-C17:0 3-OH, and C15:1 G. The major quinone is MK-7. The major polar lipids are phosphatidylethanolamine (PE), an unidentified aminophospholipid, and three unidentified polar lipids. Phylogenetically, the genus is affiliated to the family Chitinophagaceae in the phylum Bacteroidetes. The type species is Hanamia caeni.

Description of Hanamia caeni sp. nov.

Hanamia caeni (cae'ni. L. gen. n. caeni, of sludge).

The long non-motile rod-shaped cells (3–4 × 11–45 μm) are Gram-stain-negative, oxidase, and catalase positive, and there is an absence of the flexirubin-type pigment production. Colonies grown on R2A are yellowish-pigmented, convex, and circular with entire. Growth occurs well at temperatures between 15 and 37 °C (optimum, 25–30 °C) and pH 6.0 to 9.0 (optimum pH 7.0) without NaCl supplementation and in the presence of 1% NaCl (w/v, optimum 0%). Colonies develop on R2A, NA, and TSA agars (optimally on R2A agar), although no growth are detected on LB and MacConkey agars. Negative for the hydrolysis of casein, DNA, starch, Tween-60, and cellulose. On API ZYM strips, positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α- and β-galactosidase, α- and β-glucosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase activities and negative for lipase (C14). On API ID 32GN and API 20NE strips, positive for arginine dihydrolase, urease, and esculin hydrolysis and negative for nitrate reduction, indole production, and glucose acidification. Utilizes the sugars such as L-arabinose, N-acetyl-β-glucosaminidase, d-maltose, salicin, d-melibiose, d-sorbitol, and d-sucrose, while negative for the other. The predominant quinone is MK-7. The predominant fatty acids are iso-C15:0, iso-C17:0 3-OH, and C15:1 G. In addition to the major polar lipids phosphatidylethanolamine (PE), an unidentified aminophospholipid (APL1), and three unidentified polar lipids (PL1, PL2, and PL3), unidentified polar lipids (PL4 and PL5) and unidentified aminolipids (AL1 and AL2) are also present. The size of the strain BO-59T draft genome was determined to be 4.96 Mbp and the DNA G + C content of the type strain is 39.1 mol%.

The type strain, BO-59T (= KACC 19646T = LMG 30865 T), was isolated from activated sludge in Hanam city, South Korea.

The 16S rRNA gene and draft genome sequence accession numbers are MH094634 and RJJR00000000, respectively.