Introduction

Members of the genus Meiothermus originally belonged to the genus Thermus and were referred to as the “low temperature” group based on their optimum growth temperature (Loginova et al. 1984; Tenreiro et al. 1995). Later, Nobre et al. (1996a) found that these members formed a separate lineage from the species of the genus Thermus based on 16S rRNA gene sequences. Therefore they proposed a new genus Meiothermus, distinguished from the genus Thermus by their low optimum growth temperature, red or yellow-pigmented colonies and the presence of moderate levels of 2-hydroxy fatty acids. Subsequently, eight species of the genus Meiothermus were isolated from geyser or hot springs world-wide, including Meiothermus cerbereus (Chung et al. 1997), Meiothermus taiwanensis (Chen et al. 2002), Meiothermus timidus (Pires et al. 2005), Meiothermus rufus and Meiothermus granaticius (Albuquerque et al. 2009, 2010), Meiothermus cateniformans (Zhang et al. 2010), Meiothermus hypogaeus (Mori et al. 2012) and Meiothermus terrae (Yu et al. 2014). The species of the genus Meiothermus have low growth temperature ranges from 35 to 68 °C whereas other members of the family Thermaceae grow at 40–83 °C. All of the species in this genus share chemotaxonomic characteristics, such as having MK-8 as the predominant menaquinone, iso- and anteiso-branched fatty acids as major fatty acids and an uncharacterised phospholipid and glycolipids as major polar lipids (da Costa et al. 2006).

During a study on thermophilic bacteria from hot springs, strains YIM 71031T and YIM 71039 were isolated from a hot spring in Tengchong county, Yunnan province, south-western China. A preliminary phylogenetic analysis based on 16S rRNA gene sequences indicated that strains YIM 71031T and YIM 71039 might be candidates for inclusion in a novel species belonging to the genus Meiothermus. The aim of the present study was to determine the taxonomic status of these two isolates, by using a polyphasic approach. The two strains were characterised by phenotypic, chemotaxonomic and phylogenetic analysis. The results of phenotypic, chemotaxonomic and phylogenetic analyses indicated that strains YIM 71031T and YIM 71039 represent a novel species in the genus Meiothermus, here named Meiothermus roseus sp. nov.

Materials and methods

Strains and culture conditions

Geothermally heated sediment with a few water mixture samples were collected from a hot spring. The sediment sample was sandy, a black-green colour with bacterial mats, and the temperature and pH was 67 °C and 7.0, respectively. For the isolation of thermophilic bacteria, 2 g of sediment samples collected from the hot spring were taken into flasks with 18 ml sterile hot spring water and several glass beads. After mixing, samples were shaken at 50 °C with 200 rpm min−1 for 1 h, 1 ml of the mixtures were then diluted 10 and 100 fold with sterile water and 0.2 ml of the diluted samples were spread on R2A medium (BD; Becton, Dickinson and Company), Thermus medium (Williams and da Costa 1992) and modified T5 medium (tryptone 0.5 g, yeast extract 2 g, glucose 1 g, lotus root starch 1 g, CaSO4, 0.04 g, 0.01 M Fe citrate, 0.5 ml, KH2PO4 0.01 g, K2HPO4 0.01 g, NaHCO3 0.1 g, NaCl 0.2 g, CaCl2 0.02 g, MgSO4 0.02 g, water 1000 ml, agar 20 g, pH 7.2). These isolation plates were incubated at 45, 50 and 60 °C for 7 days. Strains YIM 71031T and YIM 71039 were selected on modified T5 medium at 50 °C. These two strains were purified and cultured on modified T5 medium adjusted to pH 7.2 at 50 °C. The pure cultures were preserved as glycerol suspensions (20 %, v/v) at −80 °C. The type strain M. timidus DSM 17022T was obtained from the DSMZ (German Collection of Microorganisms and Cell Cultures) and used as a reference strain in the present study cultured under comparable conditions.

Phenotypic characteristics

For observation of phenotypic characteristics, strains YIM 71031T and YIM 71039 were cultured on Thermus, R2A and modified T5 media adjusted to pH 7.2 at 50 °C unless mentioned. Growth and microscopic morphological characterisation were performed by light microscopy (Olympus BH2, Olympus) and scanning electron microscopy (XL30 ESEM-TMP, Philips-FEI). The procedure for SEM sample preparation was described previously by Ming et al. (2014). Gram-staining was carried out using 3 % KOH by the non-staining method (Buck 1982). Cell motility was studied depending on turbidity development throughout a tube of semi-solid medium (Leifson 1960).

Growth at different temperatures was tested at over a range of 4, 15, 28, 30, 37, 40, 45, 50, 55, 60, 65, 70 and 75 °C described by Chung et al. (2000) and Bjornsdottir et al. (2009). The tolerance of sodium levels for growth was observed in the presence of 0–5 % (w/v) NaCl concentration (0, 0.5, 1.0, 1.5 2, 2.5, 3.0, 4.0 and 5.0 % w/v, respectively). The pH for growth was tested from 4.0 to 10.5 (at intervals of 0.5 pH units) at 50 °C for 7 days in Thermus medium broth, by using the buffer system as described by Ming et al. (2014). The ability to utilise single carbon sources was tested in a minimal medium composed of Thermus basal salts with yeast extract (0.1 g per liter) supplemented with 2 g/l filter sterilized (0.22 μm, Millipore) test carbon source. Nitrogen source utilisation was observed in the minimal Thermus medium described above supplemented with 2 g/l filter sterilised (0.22 μm, Millipore) test compounds. Determination of oxidase activity was carried out by using 1 % (w/v) tetramethyl p-phenylenediamine as described by Kovacs (1956). Catalase activity was tested using 3 % (w/v) H2O2 by assessing bubble production as the positive result. Tween (20, 40, 60 and 80) degradation, H2S production and nitrate reduction, hydrolysis of gelatin, starch, xylan, aesculin, pectin and cellulose, activity of urea, milk peptonization and coagulation were observed as described by Smibert and Krieg (1994), Gonzalez et al. (1978) and MacFaddin (1980). Antibiotic susceptibility was observed by the disc (Himedia) diffusion plate method (Groth et al. 2004). API galleries (API 20NE and API ZYM) were used to determined metabolic properties and some enzyme activities according to the instructions of the manufacturer (bioMérieux, France). The Biolog Gen III microplate™ system was used for carbon source utilisation and chemical sensitivity tests according to the manufacturer’s instructions.

Chemotaxonomy

Biomass for chemical and molecular studies of strains YIM 71031T, YIM 71039 and M. timidus DSM 17022T were obtained by cultivation on Thermus medium adjusted to pH 7.2 at 50 °C for 3 days. Cells were checked for purity by spreading on Thermus agar plates, harvested and washed twice with distilled water by centrifugation and then freeze-dried.

Quinones were extracted from lyophilized cells as described by Collins et al. (1977) and Minnikin et al. (1984) and then purified and analysed by HPLC (Tamaoka et al. 1983; Hu et al. 2001). Polar lipids were extracted and examined by two-dimensional thin-layer chromatography (TLC) method on silica gel G 60 plates (Merck; Germany). The profile of the lipids was identified using previously described procedures (Collins and Jones 1980; Minnikin et al. 1979, 1984). Cells for cellular fatty acids analysis were obtained by culturing strains YIM 71031T, YIM 71039 and M. timidus DSM 17022T in Thermus and modified T5 media for 3 days. The cellular fatty acids were extracted, methylated and analysed following the instructions of version 6.1 of the Sherlock Microbial Identification System (MIDI) and the TSBA6 databse (Sasser 1990).

The genomic DNA G+C contents of the novel strains were determined by HPLC after enzymatic degradation (Mesbah et al. 1989), with Escherichia coli DH5α as the reference strain.

Molecular analysis

Genomic DNA of strains YIM 71031T and YIM 71039 was extracted, and the 16S rRNA genes were amplified and sequenced by using the universal bacterial primers, as described by Li et al. (2007). The amplicons were purified by using a PCR purification kit (Sangon Biotech, China). The obtained almost complete 16S rRNA gene sequences of the novel strains were compiled using the SeqMan program (DNAStar software). To determine the phylogenetic relationships of strains YIM 71031T and YIM 71039, the 16S rRNA gene sequences of species in genus Meiothermus were retrieved from a BLAST search (Altschul et al. 1990) and the EzTaxon-e server database (Kim et al. 2012). Multiple sequence alignment was performed using the CLUSTAL_X software package (Thompson et al. 1997). Evolutionary distances were calculated by use of the Kimura two-parameter model (Kimura 1980, 1983). Phylogenetic trees were constructed with the neighbour-joining (Saitou and Nei 1987), maximum-parsimony (Fitch 1971) and maximum-likelihood (Felsenstein 1981) tree-making algorithms. Phylogenetic and molecular evolutionary analyses were carried out by using the software packages MEGA version 6.0 (Tamura et al. 2013) and PHYML (Guindon and Gascuel 2003). The topology of each tree with 1000 replications was evaluated by bootstrap analysis (Felsenstein 1985). The 16S rRNA gene from strain Thermus scotoductus SE-1T (AF032127) was used as outgroup. DNA–DNA hybridizations between strains YIM 71031T, YIM 71039 and M. timidus DSM 17022T were performed at the optimal hybridization temperature (46 °C) using the fluorometric micro-well method (Ezaki et al. 1989; Christensen et al. 2000). The DNA probes labeled with photobiotin (A1935; Sigma) and 96-well microdilution plates (Greiner BioOne) were used; each sample was set with eight replications.

Results and discussion

Phenotypic characteristics

Strains YIM 71031T and YIM 71039 were both found to grow well on Thermus, R2A and modified T5 media. Colonies of the two strains were observed to be regular circular, opaque, convex and yellow-pigmented on all tested media, and rose-coloured diffusible pigment was produced on R2A medium. Cells of strains YIM 71031T and YIM 71039 were observed to be Gram-negative, aerobic and non-motile. Cells were found to be rod shaped with 0.3–0.5 μm diameter and 2.0–4.0 μm length, and no spores were formed (Fig. S1). Strains YIM 71031T and YIM 71039 were found to grow at 37–65 °C and pH 6.0–8.0, with optimal growth at 50 °C and pH 7.0–7.5, and at low NaCl concentrations (up to 1.0 % w/v). The strains were found to be positive for catalase and urease activities, milk coagulation and peptonization. Oxidase activity was found to be negative. The strains can hydrolyse gelatin, pectin and aesculin. The degradation of xylan, cellulose, starch, Tweens 20, 40, 60 and 80 was found to be negative. Nitrate was found to be reduced and H2S not to be produced. The strains were observed to be resistant to amikacin (30 μg/disc), cefurosime sodium (30 μg), ciprofloxacin (5 μg), chloroamphenicol (30 μg), erythromycin (5 μg), gentamicin (10 μg), norfloxacin (10 μg), novobiocin (5 μg), penicillin (10 IU), piperacillin (100 μg), tetracycline (30 μg) and vancomycin (30 μg) but sensitive to ethylhydrocupreine (5 μg), oxacillin (1 μg), polymyxin B (300 IU) and sulfamethoxazole/trimethoprim (23.75/1.25 μg). Phenotypic properties useful for distinguishing strains YIM 71031T and YIM 71039 from related members of the genus Meiothermus are shown in Table 1. The detailed physiological characteristics of strains YIM 71031T and YIM 71039 are given in the species description below.

Table 1 Differential physiological characteristics among strains YIM 71031T, YIM 71039 and their close neighbour in the genus Meiothermus
Full size table

Chemotaxonomic characteristics

The predominant menaquinone of both novel isolates was identified as MK-8. The cellular fatty acids of strain YIM 71031T (>5 %) were identified as anteiso-C15:0 (21.6 %), iso-C15:0 (15.8 %), anteiso-C17:0 (13.5 %), iso-C16:0 (13.1 %), C16:0 (10.6 %) and iso-C17:0 (7.8 %). The cellular fatty acids of strain YIM 71039 were identified as anteiso-C15:0 (12.2 %), iso-C15:0 (15.4 %), anteiso-C17:0 (12.6 %), iso-C16:0 (10.9 %), C16:0 (11.7 %) and iso-C17:0 (13.6 %). Similar to the genus Thermus, iso- and anteiso-branched C15:0 and C17:0 fatty acids are the predominant fatty acids of most strains of the genus Meiothermus (Nobre et al. 1996b; da Costa et al. 2006). In the present study, strains YIM 71031T and YIM 71039 were found to not only contain anteiso-C15:0 and iso-C15:0 as the major fatty acids but also possessed iso-C16:0 and C16:0 reached levels >10 % of the total fatty acids. Among the present validly named species of the genus Meiothermus, only M. hypogaeus AZM34c11T had the similar results. Moreover, both strains YIM 71031T and YIM 71039 were found to contain <1 % hydroxy fatty acids, similar to M. rufus AZM34c11T, M. granaticius AF-68T and M. hypogaeus AZM34c11T. Modified T5 medium is widely used in the isolation and cultivation of thermophiles from extreme thermal ecological niches such as geothermal areas or hot springs, and many thermophilic bacteria and actinobacteria can grow well on this medium when they cannot be cultured on Thermus medium. Strains YIM 71031T and YIM 71039 exhibited similar fatty acid profiles regardless of which medium was used. Detailed fatty acid profiles of strains YIM 71031T and YIM 71039 are given in Table S1. The polar lipids profiles of the two strains were found to be comprised of one major unidentified phospholipid and two glycolipids (Fig. S2).

The genomic DNA G+C content of strains YIM 71031T and YIM 71039 were 64.0 and 65.4 mol%, respectively, which is consistent with the DNA G+C contents of members of the genus Meiothermus (60.8–69.6 mol%).

Molecular analysis

The almost-complete 16S rRNA gene sequences of strains YIM 71031T (1530 nt, GenBank accession number KP232921) and YIM 71039 (1533 nt, GenBank accession number KP232922) were determined. Comparison of the sequences with the corresponding 16S rRNA gene sequences retrieved from the GenBank/EMBL/DDBJ databases clearly demonstrated that strains YIM 71031T and YIM 71039 belong to the genus Meiothermus. Phylogenetic analyses (Fig. 1) showed that strains YIM 71031T and YIM 71039 form a monophyletic lineage (99.0 % similarity). The novel strains were found to be closely related to M. timidus DSM 17022T (98.6 % similarity) and formed a well separated branch with this type strain. This branch was also supported in the maximum-parsimony phylogenetic tree and maximum-likelihood phylogenetic tree (Figs. S3–S4). Strains YIM 71031T and YIM 71039 displayed low sequence similarity (<94.2 %) to all other currently recognised members of the genus Meiothermus.

Fig. 1
figure 1

Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, showing the position of strain YIM 71031T and YIM 71039 among members of the genus Meiothermus. Numbers at branch points refer to bootstrap percentages. Only bootstrap values above 50 % based on 1000 resampling are shown at branch points. Asterisks indicate branches that were also recovered using maximum-parsimony and maximum-likelihood tree. Bar 0.02 substitutions per nucleotide position. The 16S rRNA gene from strain Thermus scotoductus SE-1T (AF032127) was used as outgroup

Full size image

The DNA–DNA relatedness between strains YIM 71031T and YIM 71039 was 89.9 ± 2.3 %. The high DNA–DNA hybridization value supports the conclusion that these two novel strains belong to the same species, which is consistent with the phylogenetic analysis and their physiological and biochemical characteristics. The levels of DNA–DNA relatedness between strain YIM 71031T and strain YIM 71039 and M. timidus DSM 17022T were 53.9 ± 2.5 and 50.7 ± 3.1 %, respectively, values which are significantly lower than the threshold value of 70 % for prokaryotic species delineation (Wayne et al. 1987). Therefore, these data suggest that strains YIM 71031T and YIM 71039 represent a separate species of the genus Meiothermus.

In conclusion, chemotaxonomic characteristics (major fatty acids, polar lipids and predominant menaquinones) and phylogenetic trees demonstrated that strains YIM 71031T and YIM 71039 have many properties in common with species in the genus Meiothermus. However, the strains YIM 71031T and YIM 71039 possess a few physiological and biochemical properties and low DNA–DNA relatedness values that distinguish them from other described Meiothermus species (see Table 1 and the species description). Therefore, based on the phenotypic, phylogenetic and chemotaxonomic results, we conclude that strains YIM 71031T and YIM 71039 represent a novel species of the genus Meiothermus, for which the name Meiothermus roseus sp. nov. is proposed.

Description of Meiothermus roseus sp. nov.

Meiothermus roseus (ro’se.us. L. masc. adj. roseus, rose coloured, rosy, as a rose-coloured diffusible pigment is produced on R2A medium).

Cells are Gram-negative, aerobic, non-motile. The rod shaped cells are 0.3–0.5 μm in diameter and 2.0–4.0 μm in length, and no spores are formed. Good growth occurs on Thermus, R2A and modified T5 media. Colonies are regular circular, opaque, convex and yellow-pigmented on all tested media. A rose-coloured diffusible pigment is produced on R2A medium. Grows at 37–65 °C (optimal 50 °C). The pH range for growth is 6.0–8.0 (optimal pH 7.0–7.5) and the NaCl concentration range for growth is of 0–1 % (w/v). Activities of catalase and urease are positive but oxidase is negative. Positive for milk coagulation and peptonization, hydrolysis of gelatin, aesculin and pectin, negative for degradation of Tweens 20, 40, 60 and 80, cellulose, xylan and starch. Nitrate is reduced and H2S is not produced. Can utilise acetoacetic acid, d-cellobiose, dextrin, d-fructose, d-fructose-6-PO4, d-fucose, l-fucose, d-galactose, d-galacturonic acid, l-galactonic acid lactone, gentiobiose, α-d-glucose, d-glucuronic acid, α-hydroxy-butyric acid, α-keto-butyric acid, α-keto-glutaric acid, α-d-lactose, d-maltose, d-mannitol, d-mannose, d-melibiose, 3-methyl glucose, methyl pyruvate, d-raffinose, l-rhamnose, d-sorbitol, stachyose and d-turanose but not acetic acid, γ-amino-butryric acid, d-arabitol, bromo-succinic acid, citric acid, formic acid, d-gluconic acid, d-glucose-6-PO4, glycerol, p-hydroxy-phenylacetic acid, β-hydroxy-d,l-butyric acid, inosine, l-lactic acid, d-lactic acid methyl ester, l-malic acid, d-malic acid, β-methyl-d-glucoside, mucic acid, myo-inositol, propionic acid, quinic acid, d-saccharic acid, d-salicin, sucrose or d-trehalose as the sole carbon source. Able to use l-alanine, l-arginine, glucuronamide, l-glutamic acid, glycyl-l-proline and l-histidine as nitrogen source but not d-aspartic acid, l-aspartic acid, l-cystine, N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, glycine, l-isoleucine, l-lysine, N-acetyl-β-d-mannosamine, l-methionine, N-acetyl neuraminic acid, l-phenylalanine, l-proline, l-pyroglutamic acid, d-serine, l-serine, l-threonine, l-tryptophan, l-tyrosine or l-valine. Activities of acid phosphatase, alkaline phosphatase, arginine dihydrolase, α-chymotrypsin, cystine arylamidase, esterase (C4), esterase lipase (C8), α-galactosidase, β-galactosidase and β-glucosidase, α-glucosidase, β-glucuronidase, leucine arylamidase, naphthol-AS-BI-phosphohydrolase, trypsin and valine arylamidase are positive but activities of β-fucosidase, N-acetyl-β-glucosaminidase, lipase (C14) or α-mannosidase are negative. Positive results for assimilation of d-glucose, l-arabinose, d-mannitol, maltose and d-mannose but negative results for indole production, glucose acidification, assimilation of adipate, caprate, N-acetyl-glucosamine, phenylacetate, potassium gluconate or trisodium citrate. The predominant menaquinone is MK-8. The major cellular fatty acids consist of anteiso-C15:0, iso-C15:0, anteiso-C17:0, iso-C16:0 and C16:0. The polar lipid profile contains a major unidentified phospholipid and two glycolipids. The genomic DNA G+C content of the type strain is 64.0 mol%.

The type strain is YIM 71031T (=KCTC 42495T =NBRC 110900T), which was isolated from a hot spring in Tengchong county, Yunnan province, south-western China. The GenBank accession numbers for the 16S rRNA gene sequences of strains YIM 71031T and YIM 71039 are KP232921 and KP232922, respectively.