Introduction

The genus Hymenobacter is a member of the family Cytophagaceae in the phylum Bacteroidetes, which was first described by Hirsch et al. (1998) and then emended by Buczolits et al. (2006). The genus is differentiated from other members of the family by having high DNA G+C contents (55–65 mol%). Members of the genus Hymenobacter are Gram-negative, pink to red-pigmented and rod shaped. Species of this genus are well known to survive under unfavourable conditions such as desiccation and radiation, and can tolerate high levels of oil and heavy metals. At the time of writing, the genus is comprised of 31 validly named (http://www.bacterio.net/hymenobacter.html.2014.; Parte 2014), including the recently described species Hymenobacter arcticus (Chang et al. 2014), Hymenobacter kanuolensis (Su et al. 2014) and Hymenobacter qilianensis (Han et al. 2014). During an investigation into the biodiversity of microorganisms from freshwater sediment collected from a tourism cave (Jiuxiang) located in Yiliang country, Yunnan province, south-west China, two strains designated as YIM 77920T and YIM 77921T were isolated. The two strains showed many characters similar to the members of the genus Hymenobacter, such as Gram-negative, rod shaped and high DNA G+C contents (55–65 mol%); their 16S rRNA gene sequences similarities were found to below 97 % compared to other validly named species, which inspired us to identify their phenotypic, chemotaxonomic and molecular characters in order to classify them.

Materials and methods

Strains and culture conditions

Strains YIM 77920T and YIM 77921T were isolated on Reasoner’s 2A agar (R2A) medium from the freshwater sediment of Jiuxiang tourist cave (E103°22.791′, N25°04.270′) located in Yiliang county, Yunnan province, south-west China, by serial dilution followed by incubation for 7–10 days at 28 °C. Pure colonies of strains YIM 77920T and YIM 77921T were obtained by repeatedly re-streaking on R2A medium at 28 °C and routinely cultivated on the same medium. Both the strains were stored as glycerol suspensions (20 % v/v) in R2A broth at −80 °C for further use. Biomass for chemical and molecular studies was obtained by cultivation on R2A medium or broth at 28 °C. The type strain Hymenobacter xinjiangensis JCM 23206T was obtained from Japan collection of microorganisms (JCM) and cultured under the same conditions as appropriate for specific comparative tests.

Morphological, physiological and biochemical characterization

Cell motility was studied by the development of turbidity throughout a tube of semi-solid medium (Leifson 1960). For morphological studies, strains YIM 77920T and YIM 77921T were observed using light microscopy (BH-2; Olympus) and scanning electron microscopy (QUANTA200; FEI). For scanning electron microscopy, harvested cells were suspended with sterilized water and were fixed with 3 % glutaraldehyde for two hours. Subsequently the fixed cells were dehydrated through a gradient series of alcohol (30, 50, 70, 90 and 100 %, respectively). The cell specimens were sputter coated with gold and observed with a scanning microscope. Gram staining was carried out by using the standard Gram reaction and was confirmed by using the KOH lysis test method (Cerny 1978). Strains YIM 77920T and YIM 77921T were examined for physiological and biochemical characteristics. Growth at different temperatures (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 °C) and NaCl tolerance at various concentrations (0, 0.5, 1, 1.5, 2, 2.5, 3 and 5.0 % w/v) was determined using R2A medium. The pH range (4.0–10.0, at intervals of 1.0 pH unit) for growth was tested in R2A broth using the buffer system described by Xu et al. (2005). Growth on several media such as nutrient agar (NA), trypticase soy agar (TSA) and Luria–Bertani (LB) at 28 °C were also evaluated. Oxidation of carbon sources was tested using the Biolog GEN III MicroPlate according to the manufacturer’s instructions. Oxidase activity was determined by the oxidation of tetramethyl-p-phenylenediamine (Kovacs 1956). Catalase activity was detected by assessing the production of bubbles on addition of a drop of 3 % (v/v) H2O2. H2S production, nitrate reduction and hydrolysis of cellulose, gelatin, starch, urea and Tweens (20, 40, 60 and 80) were performed as described by Gonzalez et al. (1978). Other enzyme activities and biochemical characteristics were additionally determined by using API ZYM and API 20 NE kits according to the manufacturer’s instructions (bioMérieux, France). Susceptibility of strains YIM 77920T, YIM 77921T and H. xinjiangensis JCM 23206T to antibiotics was investigated by the agar-diffusion method using 0.5 McFarland bacterial suspensions plated onto R2A agar medium for 2 days at 28 °C. The following antibiotics were tested : amikacin (30 µg), ampicillin (30 µg), bacitracin (10 IU), cefoperazone (10 µg), ceftriaxone (10 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), clindamycin (15 µg), erythromycin (15 µg), gentamicin (10 µg), norfloxacin (10 µg), ofloxacin (10 µg), penicillin (10 IU), piperacillin (100 µg), tetracycline (30 µg) and vancomycin (30 µg). The antimicrobial susceptibility was determined by measuring the zone of inhibition.

Chemotaxonomy

Chemotaxonomic characteristics of strains YIM 77920T, YIM 77921T and the reference strain H. xinjiangensis JCM 23206T were observed using several standard methods under the same conditions. The respiratory quinones were extracted and purified as described by Collins et al. (1977) and analysed by HPLC (Kroppenstedt 1982). Polar lipids were extracted as described by Minnikin et al. (1979) and identified by two-dimensional TLC (Collins and Jones 1980). To standardise for physiological age, the biomass for fatty acid analysis was harvested on R2A broth at 28 °C when the population quantity was half of maximum value. Cellular fatty acids were prepared and analysed according to the standard protocol of the microbial identification system (Sherlock Version 6.1; MIDI database: TSBA6). The G+C content of the genomic DNAs were determined by using reversed-phase HPLC (Mesbah et al. 1989) with Escherichia coli DH5α as the reference strain.

Molecular analysis and Molecular analysis and DNA–DNA hybridizations

Extraction of genomic DNAs and PCR amplification of the 16S rRNA genes were performed as described by Li et al. (2007). The sequences obtained were compared with available 16S rRNA gene sequences of validly named species from the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al. 2012). Multiple alignments with sequences of the most closely related taxa and calculations of levels of sequence similarity were carried out using CLUSTAL_X program (Thompson et al. 1997). Phylogenetic analyses were performed by using three tree-making algorithms, the Neighbour-joining (Saitou and Nei 1987), Maximum-likelihood (Felsenstein 1981) and Maximum-parsimony (Fitch 1971) trees were constructed by using the MEGA version 5.0 software package (Tamura et al. 2011). Kimura’s two parameter model was used to calculate evolutionary distance matrices of the phylogenetic trees (Kimura 1980). Bootstrap analysis was performed with 1,000 replications (Felsenstein 1985). Flavobacterium aquatile ATCC 11947T (M62797) was used as an outgroup.

DNA–DNA relatedness between strains YIM 77920T and YIM 77921T was studied using the fluorometric micro-well method (Ezaki et al. 1989; Christensen et al. 2000; He et al. 2005), using eight replications for each hybridization reaction.

Results and discussion

Morphological, physiological and biochemical characterization

Cells of strains YIM 77920T and YIM 77921T were observed to be Gram-stain negative, aerobic and non-motile. Scanning electron microscopy results showed strain YIM 77920T is rod-shaped, about 0.6–0.8 µm in width and 1.3–3.5 µm in length, and strain YIM 77921T is also rod-shaped, about 0.6–0.8 µm in width and 1.4–3.3 µm in length (Fig. S1). Strains YIM 77920T and YIM 77921T shared common phenotypic characters but differed in producing brick-red and orange-red coloured colonies, respectively, on R2A medium. Both strains were found to grow on R2A and NA but not on TSA or LB, with a temperature range for growth of 5–35 °C and salt tolerance up to 0.5 % (w/v). The strains were found to differ in their pH ranges for growth. Strain YIM 77920T was found to grow at a pH range of 5.0–8.0 with an optimum pH 7.0, while strain YIM 77921T was found to have a pH range of 6.0–8.0 with an optimum pH 7.0. Both strains were found to be positive for catalase, oxidase and hydrolysis of starch and Tweens (40, 80) but to be negative for H2S production, nitrate reduction and hydrolysis of cellulose, Tweens (20, 60) and urea. Strain YIM 77920T was found to be able to hydrolyse gelatine, while strain YIM 77921T could not. For the antibiotics, strains YIM 77920T and YIM 77921T were found to be sensitive to amikacin, ampicillin, cefoperazone, ceftriaxone, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, norfloxacin, ofloxacin, piperacillin, tetracycline and vancomycin, while YIM 77920T is sensitive to penicillin but strain YIM 77921T is not. The major differential characteristics between strains YIM 77920T, YIM 77921T and the most closely related type strain H. xinjiangensis JCM 23206T are shown in Table 1. The detailed physiological characteristics of strains YIM 77920T and YIM 77921T are given in the species description.

Table 1 Different characteristics within strains YIM 77920T, YIM 77921T and H. xinjiangensis JCM 23206T
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Chemotaxonomy

The respiratory quinone identified in strains YIM 77920T and YIM 77921T was MK-7. The major fatty acids (>10 %) of strains YIM 77920T and YIM 77921T were identified as C16:1 ω5c, iso-C15:0 and summed feature 4 (C17:1 anteiso B/iso I). The major fatty acid profiles of the two strains were consistent with that of H. xinjiangensis JCM 23206T, but there were also some differences compared with the latter (Table 2). The polar lipids profile of strain YIM 77920T was found to consist of phosphatidylethanolamine, four unidentified polar lipids, three unidentified aminophospholipids, two unidentified phospholipids and two unidentified aminolipids, while that of strain YIM 77921T consisted of phosphatidylethanolamine, four unidentified polar lipids, two unidentified aminolipids, one unidentified phospholipid and four unidentified aminophospholipids. The results showed that both strains share similar profiles for quinone and fatty acids but they are different in their polar lipids. The G+C contents of strains YIM 77920T and YIM 77921T were determined to be 57.5 and 59.6 mol%, respectively, while that of H. xinjiangensis JCM 23206T was reported to be 54 mol% (Zhang et al. 2007).

Table 2 Cellular fatty acid compositions of strains YIM 77920T, YIM 77921T and Hymenobacter xinjiangensis JCM 23206T
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Phylogenetic analysis and DNA–DNA relatedness

The almost complete 16S rRNA gene sequences of strains YIM 77920T (1,540 bp) and YIM 77921T (1,545 bp) were obtained; the GenBank accession numbers are AB859260 and AB859261, respectively. Sequence analysis of the almost complete 16S rRNA gene sequences of the two strains using the EzTaxon-e server showed that they both had highest similarities to members of the genus Hymenobacter and that both strains are closely related to H. xinjiangensis X2-1T (96.66 and 96.59 % similarity), Hymenobacter rigui WPCB131T (95.54 and 95.54 %) and Hymenobacter perfusus A1-12T (95.42 and 95.28 %), and the two strains are 99.40 % similar to each other. The Neighbour-Joining phylogenetic tree based on 16S rRNA sequences showed the two strains form a clade with H. xinjiangensis X2-1T well separated with other members of the genus Hymenobacter (Fig. 1). The stabilities of trees were further confirmed by Maximum Likelihood and Maximum Parsimony methods (Fig. S3, S4). As the 16S rRNA gene sequence similarities of strains YIM 77920T and YIM 77921T with the type strains of members of the genus Hymenobacter were below 97 %, DNA–DNA hybridizations between them were not carried. However, the sequence similarity between YIM 77920T and YIM 77921T was 99.40 %, so DNA–DNA hybridization between strains YIM 77920T and YIM 77921T was carried out and the value was 56.55 ± 2.72 %, which is less than cut-off point (70 %) for the delineation of genomic species (Stackebrandt and Goebel 1994).

Fig. 1
figure 1

Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences of strains YIM 77920T, YIM 77921T and their closest relatives. Bootstrap values (expressed as percentages of 1,000 replications) of above 50 % are shown at the branch points. Asterisks denote nodes that were also recovered using the maximum-parsimony and maximum-likelihood methods. The sequence of Flavobacterium aquatile ATCC 11947T was used as the outgroup. Bar 0.01, represents substitutions per nucleotide position

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Therefore, on the basis of phylogenetic analysis, phenotypic and chemotaxonomic characteristics (Fig. 1, Tables 1 and 2, and Figs S1, S2), strains YIM 77920T and YIM 77921T should be affiliated to the genus Hymenobacter. However, strain YIM 77920T and YIM 77921T could be distinguished from the type strain of H. xinjiangensis JCM 23206T by differences in several properties, such as colony colour, pH range for growth, hydrolysis of gelatin and Tween 40, G+C content, oxidation of carbon sources, as well as the proportions of some fatty acids. In addition, analyses of 16S rRNA gene sequences, DNA–DNA relatedness values and their different properties, notably colony colour, enzyme activities, oxidation of carbon sources, assimilation of adipic acid, malic acid and trisodium citrate, indicates that strains YIM 77920T and YIM 77921T represent two novel species within the genus Hymenobacter, for which the names Hymenobacter latericoloratus sp. nov and Hymenobacter luteus sp. nov. are proposed, respectively.

Description of Hymenobacter latericoloratus sp. nov

Hymenobacter latericoloratus (la.te.ri.co.lo.ra’tus. L. n. latus brick; L. part. adj. coloratus coloured; N.L. part. adj. latericoloratus brick-coloured).

Cells are Gram-negative, aerobic and non-motile. Colonies are brick-red on R2A medium. Growth occurs on NA but not on TSA or LB. Has a temperature range for growth of 5–35 °C, with optimum at 25–30 °C. Cells can tolerate salt concentrations up to 0.5 % (w/v). pH range for growth is from 5.0 to 8.0, with optimum at pH 7.0. Positive for catalase, oxidase and hydrolysis of gelatine, starch and Tweens (40, 80) whereas negative for H2S production, nitrate reduction and hydrolysis of cellulose, Tweens (20, 60) and urea. According to the API ZYM system, positive for N-acetyl-β-glucosaminidase, acidic phosphatase, alkaline phosphatase, cystine arylamidase, esterase (C4), esterase lipase (C8), β-fucosidase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, leucine arylamidase, lipase (C14), α-mannosidase, naphthol-AS-BI-phosphohydrolase and valine arylamidase; negative for α-chymotrypsin, β-glucuronidase and trypsin. In the API 20NE system, assimilation of l-arabinose and trisodium citrate, hydrolysis of aesculin and gelatine are positive, while arginine dihydrolase, galactosidase, glucose fermentation, indole production, nitrate reduction, urease and assimilation of N-acetylglucosamine, adipic acid, capric acid, d-glucose, malic acid, maltose, d-mannose, d-mannitol, phenylacetic acid and potassium gluconate are negative. Positive reactions from the GEN III system for oxidation of aztreonam, citric acid, d-fucose, l-fucose, gelatin, glucuronamide, d-glucuronic acid, l-glutamic acid, glycyl-l-proline, l-histidine, lithium chloride, nalidixic acid, tolerance of pH 6 and pH 5, l-serine, tetrazolium violet, Tween 40 and vancomycin; negative reactions from the GEN III system for oxidation of others. The major fatty acids are C16:1 ω5c, iso-C15:0, and Summed Feature 4 (C17:1 anteiso B/iso I). The polar lipids consist of phosphatidylethanolamine, four unidentified polar lipids, three unidentified aminophospholipids, two unidentified phospholipids and two unidentified aminolipids. The respiratory quinone is MK-7. The G+C content of the DNA of the type strain is 57.5 mol%.

The type strain YIM 77920T (=JCM 30327T = CCTCC AB 2012949T) was isolated from the freshwater sediment of Jiuxiang tourist cave located in Yiliang country, Yunnan province, south-west China. The GenBank accession number for the 16S rRNA gene sequence of strain YIM 77920T is AB859260.

Description of Hymenobacter luteus sp. nov

Hymenobacter luteus (lu.te’us. L. masc. adj. luteus orange-yellow).

Cells are Gram-negative, aerobic, non-motile and produce orange-red coloured colonies on R2A medium. Growth occurs on NA but not on TSA and LB. The temperature range for growth is 5–35 °C, with optimum at 25–30 °C. Cells can tolerate salt concentrations up to 0.5 % (w/v). The pH range for growth is pH 6.0–8.0, with optimum at pH 7.0. Positive for catalase, oxidase and hydrolysis of starch and Tweens (40, 80) whereas negative for H2S production, nitrate reduction and hydrolysis of cellulose, gelatine, Tweens (20, 60) and urea. According to the API ZYM system, positive for N-acetyl-β-glucosaminidase, acidic phosphatase, alkaline phosphatase, cystine arylamidase, esterase lipase (C8), α-glucosidase, leucine arylamidase, lipase (C14), naphthol-AS-BI-phosphohydrolase, trypsin and valine arylamidase; negative for α-chymotrypsin, esterase (C4), β-fucosidase, α-galactosidase, β-galactosidase, β-glucosidase, β-glucuronidase and α-mannosidase. In the API 20NE system, assimilation of adipic acid, l-arabinose and malic acid, hydrolysis of aesculin are positive, while arginine dihydrolase, hydrolysis of gelatine, glucose fermentation, indole production, galactosidase, nitrate reduction, urease and assimilation of N-acetylglucosamine, capric acid, d-glucose, maltose, d-mannose, d-mannitol, phenylacetic acid, potassium gluconate and trisodium citrate are negative. Positive reactions from the GEN III system for oxidation of N-acetyl-β-dmannosamine, l-alanine, l-aspartic acid, aztreonam, bromo-succinic acid, l-fucose, d-galactose, gentiobiose, d-glucose-6-phosphate, glucuronamide, l-glutamic acid, glycyl-l-proline, l-histidine, d-malic acid, l-malic acid, d-lactic acid methyl ester, methyl pyruvate, tolerance of pH 6, quinic acid, d-saccharic acid, l-serine, tetrazolium violet, Tween 40, d-turanose and vancomycin; negative reactions from the GEN III system for oxidation of others. The major fatty acids are C16:1 ω5c, iso-C15:0, and summed feature 4 (C17:1 anteiso B/iso I). The polar lipids consist of phosphatidylethanolamine, four unidentified polar lipids, two unidentified aminolioids, one unidentified phospholipid and four unidentified aminophospholipids. The respiratory quinone is MK-7. The G+C content of the DNA of the type strain is 59.6 mol%.

The type strain YIM 77921T = JCM 30328T = CCTCC AB 2012947T was isolated from the freshwater sediment of Jiuxiang tourist cave located in Yiliang country, Yunnan province, south-west China. The GenBank accession number for the 16S rRNA gene sequence of strain YIM 77921T is AB859261.