Mobilicoccus Pelagius Gen. Nov., Sp. Nov. and Piscicoccus Intestinalis Gen

J. Gen. Appl. Microbiol., 56, 427‒436 (2010) Full Paper

Mobilicoccus pelagius gen. nov., sp. nov. and Piscicoccus intestinalis gen. nov., sp. nov., two new members of the family Dermatophilaceae, and reclassiﬁ cation of Dermatophilus chelonae (Masters et al. 1995) as Austwickia chelonae gen. nov., comb. nov.

Moriyuki Hamada,* Takao Iino, Takahiro Iwami, Shigeaki Harayama, Tomohiko Tamura, and Ken-ichiro Suzuki

NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation, Kisarazu, Chiba 292‒0818, Japan

(Received May 7, 2010; Accepted July 30, 2010)

Two Gram-positive bacteria, designated strains Aji5-31T and Ngc37-23T, were isolated from the intestinal tracts of ﬁ shes. 16S rRNA gene sequence analysis indicated that both strains were related to the members of the family Dermatophilaceae, with 95.6‒96.9% 16S rRNA gene sequence similarities. The family Dermatophilaceae contains 2 genera and 3 species: Dermatophi- lus congolensis, Dermatophilus chelonae and Kineosphaera limosa. However, it has been sug- gested that the taxonomic position of D. chelonae should be reinvestigated using a polyphasic approach, because the chemotaxonomic characteristics are not known (Stackebrandt, 2006; Stackebrandt and Schumann, 2000). Our present study revealed that strains Aji5-31T, Ngc37-23T and D. chelonae NBRC 105200T should be separated from the other members of the family Der- matophilaceae on the basis of the following characteristics: the predominant menaquinone of T T strain Aji5-31 is MK-8(H2), strain Ngc37-23 possesses iso- branched fatty acids as major components, and the menaquinone composition of D. chelonae is MK-8(H4), MK-8 and MK-8(H2) (5：3：2, respectively). On the basis of these distinctive phenotypic characteristics and phylogenetic analysis results, it is proposed that strains Aji5-31T and Ngc37-23T be classiﬁ ed as two novel genera and species of the family Dermatophilaceae. The names are Mobilicoccus pelagius gen. nov., sp. nov. and Piscicoccus intestinalis gen. nov., sp. nov., and the type strains are Aji5- 31T (=NBRC 104925T =DSM 22762T) and Ngc37-23T (=NBRC 104926T =DSM 22761T), respectively. In addition, D. chelonae should be reassigned to a new genus of the family Dermatophi- laceae with the name Austwickia chelonae gen. nov., comb. nov.

Key Words—Austwickia chelonae gen. nov., comb. nov.; Dermatophilaceae; Mobilicoccus pelagius gen. nov., sp. nov.; Piscicoccus intestinalis gen. nov., sp. nov.

Introduction

The family Dermatophilaceae was first proposed by * Address reprint requests to: Dr. Moriyuki Hamada, NITE Bi- Austwick (1958) and later emended by Stackebrandt ological Resource Center (NBRC), National Institute of Technol- et al. (1997), Stackebrandt and Schumann (2000) and ogy and Evaluation, 2‒5‒8 Kazusakamatari, Kisarazu, Chiba 292‒0818, Japan. Zhi et al. (2009). This family currently contains two Tel: +81‒438‒20‒5763 Fax: +81‒438‒52‒2329 genera; Dermatophilus and Kineosphaera. The genus E-mail: [email protected] Dermatophilus was proposed by Gordon (1964) as or- 428 HAMADA et al. Vol. 56 ganisms that form branching mycelia with several coast of Tokyo Bay, Japan. The procedure employed transverse and longitudinal divisions which leads to for isolating the bacteria from the intestinal tracts was the formation of packets or clusters of cuboid cells or described by Hamada et al. (2009). NBRC medium coccoid. Presently, two species are included in this 802 (1.0% Polypepton (Wako), 0.2% yeast extract (Dif- genus. Dermatophilus congolensis (the type species) co), 0.1% MgSO4･7H2O and 1.5% agar if required; was first described as the causative organism of a skin pH 7.0) was used for general laboratory cultivation, disease (Van Saceghem, 1915) and was reported to morphological study and determination of optimal affect a wide variety of mammalian species (Zaria, growth parameters. Biomass for chemotaxonomic and 1993). The other species, Dermatophilus chelonae molecular systematic studies was prepared by incu- (Masters et al., 1995), was originally obtained from a bating the strains in shake flasks containing NBRC nose scab on a snapping turtle. The assignment of D. medium 802 for 48 h at 28°C at 100 rpm. Dermatophi- chelonae to the genus Dermatophilus was mainly lus congolensis NBRC 105199T, Dermatophilus che- based on morphological characteristics. However, its lonae NBRC 105200T and Kineosphaera limosa NBRC chemotaxonomic features were not reported. The ge- 100340T were included in the study. nus Kineosphaera, proposed by Liu et al. (2002), con- Morphological physiological, and biochemical tests. tained a single species: Kineosphaera limosa. This Colony appearance was examined after incubation at was based on its morphological (coccoid), phenotypi- 28°C for 3 days on NBRC medium 802. Morphological cal (including its polyhydroxyalkanoate-accumulating change was observed with age (up to 7 days) under a ability) and genetic characteristics (G+C content). The light microscope (BX-51; Olympus) and a scanning type strain of K. limosa was isolated from an inefficient electron microscope (JSM-6060; JEOL). For the biological phosphorus removal-activated sludge reac- morphological observation of D. congolensis NBRC tor, and its pathogenicity was not reported. Phyloge- 105199T and D. chelonae NBRC 105200T, nutrient netically, it is known that D. chelonae and D. congolen- agar (Difco), Nissui sheep blood agar (Nissui Pharma- sis do not form a coherent clade. Additionally, D. ceutical) and R agar (NBRC medium 264 containing chelonae shares a 96.6% 16S rRNA gene sequence 1% Bacto peptone (Difco), 0.5% yeast extract (Difco), similarity with K. limosa, indicating a closer relation- 0.5% malt extract (Difco), 0.5% Bacto casamino acids ship to K. limosa than to D. congolensis (94.9% simi- (Difco), 0.2% beef extract (Difco), 0.2% glycerol, larity). The necessity of phylogenetic and chemotaxo- 0.005% Tween 80, 0.1% MgSO4･7H2O and 1.5% agar; nomic studies for the assignment of D. chelonae at the pH 7.0) were used. Cell motility was determined by genus level has previously been raised (Stackebrandt, observing cells suspended in a saline solution under a 2006; Stackebrandt and Schumann, 2000). light microscope. Flagella were observed using a While isolating bacteria from marine samples, we transmission electron microscope (H-7600; Hitachi) obtained two novel actinobacteria (strains Aji5-31T and after negative staining with 1% phosphotungstic acid. Ngc37-23T) from the intestinal tracts of fish species Growth parameters, namely, temperature, pH and collected from Tokyo Bay, Japan. Comparative analy- NaCl tolerance, were determined by measuring the sis of the 16S rRNA gene sequence revealed that turbidity of 5 ml of the culture medium in test tubes strains Aji5-31T and Ngc37-23T were related to the after 1‒3 days of incubation at 610 nm. The tempera- members of the family Dermatophilaceae. By using a ture for growth was determined by incubating the cul- polyphasic approach, we aimed to clarify the taxo- tures at 5, 10, 15, 20, 25, 28, 37, 45 and 60°C. The pH nomic positions of strains Aji5-31T and Ngc37-23T with for growth was determined using a medium-adjusted members of the family Dermatophilaceae. pH between 4 and 10 with either 4 N HCl or 5 N KOH and incubation at 28°C. NaCl tolerance (1%, 3%, 5%, Materials and Methods 7%, 10% and 15%, w/v) was examined at 28°C. Growth under anaerobic conditions was determined by incu-

Bacterial strains and isolation. Two actinobacteria bation in an anaerobic chamber with an O2-absorbing T T strains, Aji5-31 and Ngc37-23 , were isolated from and CO2-generating agent (Anaero-Pack; Mitsubishi the intestinal tracts of two species of fishes, Trachurus Gas Chemical). Gram staining was performed using japonicus and Repomucenus richardsonii, respective- Hucker’s modification (Gerhardt et al., 1994). Oxidase ly, which were collected from Kyonan Beach on the activity was determined using a cytochrome oxidase 2010 Three new genera of the family Dermatophilaceae 429 paper (Nissui Pharmaceutical). Other physiological pids were visualized by spraying the TLC plates with and biochemical tests were performed using API ZYM, Dittmer-Lester reagent (Dittmer and Lester, 1964). Anis- API Coryne, API 20E and API 50CH systems (bio- aldehyde (sugar), ninhydrin (amino groups) and Mérieux) according to the manufacturer’s instruc- Schiff’s reagent (glycol) were also used as specific tions. spray reagents for polar lipids. Isoprenoid quinones 16S rRNA sequence determination and phylogenetic were extracted using chloroform-methanol (2：1, v/v) analysis. The 16S rRNA gene was amplified by PCR from approximately 300 mg of dry cells. The menaqui- using TaKaRa Ex Taq (TaKaRa Bio) with the following none fractions were separated by TLC using hexane/ pair of primers: 9F (5′-GAGTTTGATCCTGGCTCAG) diethyl ether (8.5：1.5, v/v) as a solvent and detected and 1541R (5′-AAGGAGGTGATCCAGCC). The ampli- under UV light. The menaquinones were extracted fied 16S rRNA gene was subjected to cycle sequenc- with acetone, dried using a nitrogen stream, and sub- ing using a BigDye Terminator v3.1 Cycle Sequencing sequently analyzed using a liquid chromatograph- Kit (Applied Biosystems) with the following primers: mass spectrometer (LC/MS; model LCMS-QP8000α 9F, 785F (5′-GGATTAGATACCCTGGTAGTC), 802R apparatus; Shimadzu). (5′-TACCAGGGTATCTAATCC) and 1541R. The resul- G+C content of DNA. DNA was obtained using the tant products were analyzed using an automated method outlined by Saito and Miura (1963). The G+C DNA sequencer (ABI PRISM 3730 Genetic Analyzer; content of the DNA was determined using the method Applied Biosystems). The 16S rRNA gene sequences outlined by Tamaoka and Komagata (1984) using a determined in this study were aligned with reference model 2695 HPLC apparatus (Waters). sequences of related taxa using the CLUSTAL X pro- Nucleotide sequence accession numbers. The gram (Thompson et al., 1997). A phylogenetic tree GenBank/EMBL/DDBJ accession numbers for the 16S was reconstructed using the neighbor-joining (Saitou rRNA gene sequence of strains Aji5-31T and Ngc37- and Nei, 1987), maximum-parsimony (Fitch, 1971) and 23T are AB550798 and AB550799, respectively. maximum-likelihood methods (Felsenstein, 1981). The resultant neighbor-joining tree topology was evaluated Results and Discussion by bootstrap analysis based on 1,000 replicates (Felsenstein, 1985). Strains Aji5-31T and Ngc37-23T were determined to Chemotaxonomic characterization. Cell-wall sam- be facultatively anaerobic, Gram-positive and non- ples were prepared from approximately 1 g of wet cells sporulating. The cells of strain Aji5-31T were coccus- by mechanical disruption with an ultrasonic oscillator shaped (approximately 0.7‒1.2 μm in diameter, Fig. 1A) and glass beads and were purified as described by and actively motile. Electron microscopy demonstrat- Schleifer and Kandler (1972). Molar ratios of the amino ed the presence of peritrichous flagella (Fig. 1B). This acids in cell-wall hydrolysates (4 N HCl, 16 h at 100°C) strain developed clusters of coccoid in early phase were determined as their phenyl isothiocyanate deriv- of growth, and then the clusters were gradually dis- atives (Wako) by high-performance liquid chromatography (HPLC; model LC-20AB apparatus; Shimadzu). Amino acid isomers present in cell-wall hydrolysates were analyzed according to the method outlined by Nozawa et al. (2007) using the HPLC apparatus described above. Analysis of cell-wall sugars was carried out using the method described by Mikami and Ishida (1983) using HPLC with a model LC-10AD apparatus (Shimadzu). The preparation and analysis of cellular fatty acid methyl esters was performed using the pro- tocol of the MIDI Sherlock Microbial Identification Sys- Fig. 1. (A) Scanning electron micrograph of strain Aji5-31T tem (Sasser, 1990). Free lipids were extracted from grown on NBRC medium 802 for 1 day at 28°C. (B) Transmis- 100 mg of dry cells, purified using the method outlined sion electron micrograph of strain Aji5-31T. by Minnikin et al. (1975) and examined by two-dimen- Negatively stained cells cultivated for 2 days. Bars, 5 μm (A), sional thin-layer chromatography (TLC). Phospholi- 1 μm (B). 430 HAMADA et al. Vol. 56 rupted. Strain Ngc37-23T also developed clusters of tained the clusters or aggregates until stationary cuboid cells or coccoid (single cell was approximately phase. Motile cells were not observed. Dermatophilus 0.7‒1.0 μm in diameter, Fig. 2A, B). This strain main- congolensis NBRC 105199T developed branching mycelia (Fig. 3A). Eventually, these mycelia became thick branching filaments with several transverse and longitudinal division which leads to the formation of packets or clusters of cuboid cells or coccoid (Fig. 3B, C, D). In addition, motile coccoid cells were observed. Dermatophilus chelonae NBRC 105200T grown on sheep blood agar also developed branching mycelia in early phase of growth (Fig. 4A). These mycelia were eventually divided into cocci to short rods (Fig. 4B, C).

Fig. 2. Scanning electron micrographs of strain Ngc37-23T Motile coccoid cells were also observed. However, T grown on NBRC medium 802 for 2 days at 28°C. type of division between D. congolensis NBRC 105199 T Bars, 5 μm (A), 20 μm (B). and D. chelonae NBRC 105200 was clearly different. Also, this strain grown on R agar (NBRC medium 264) formed bulb-tipped mycelia, and germination-like and/ or budding-like cells from these bulges were observed (Fig. 4D). The bulb-tipped mycelia were not observed in D. congolensis NBRC 105199T. After 3 days’ incubation at 28°C on NBRC medium 802, strain Aji5-31T formed smooth, orange-yellow colonies (approximately 1‒2 mm in diameter) and strain Ngc37-23T formed rough, orange-yellow colonies (approximately 0.2‒0.5 mm in diameter). The results of other physiological and biochemical analyses are summarized in the species descriptions below. The almost-complete 16S rRNA gene sequences of strains Aji5-31T and Ngc37-23T were compared with those of related taxa. Phylogenetic analysis, based on

Fig. 4. Scanning electron micrographs of Dermatophilus Fig. 3. Scanning electron micrographs of Dermatophilus chelanae NBRC 105200T grown on sheep blood agar for 2 days congolensis NBRC 105199T grown on Nutrient agar for 2 days (A) and 6 days (B, C), and R agar (NBRC Medium 264) for (A, B, C) and 6 days (D) at 37°C. 6 days (D) at 28°C. Bars, 20 μm (A), 5 μm (B, C, D). Bars, 10 μm (A, C), 20 μm (B), 5 μm (D). 2010 Three new genera of the family Dermatophilaceae 431 the 16S rRNA gene sequence, revealed that strains the cell-wall peptidoglycans of strains Aji5-31T and T T T Aji5-31 and Ngc37-23 are related to the members of Ngc37-23 were of the A1γ type, with meso-A2pm as the family Dermatophilaceae (Fig. 4). Strains Aji5-31T the diagnostic cell-wall diamino acid, as described by and Ngc37-23T exhibited 16S rRNA gene sequence Schleifer and Kandler (1972). Similarly, the purified similarities as follows: D. congolensis NBRC 105199T; peptidoglycans of D. congolensis NBRC 105199T and 95.6% and 96.0%, D. chelonae NBRC 105200T; 96.8% D. chelonae NBRC 105200T contained Ala, Glu and T and 95.6%, and Kineosphaera limosa NBRC 100340 ; meso-A2pm in molar ratios of 2.2：1.0：0.8 and 96.3% and 96.9%. The similarity values between strains 2.0：1.0：0.7, respectively. Thus, it was revealed that Aji5-31T and Ngc37-23T, D. congolensis NBRC 105199T the peptidoglycans of these species were also of the and D. chelonae NBRC 105200T, D. congolensis NBRC A1γ type. Ribose and mannose were detected as the 105199T and K. limosa NBRC 100340T, and D. che- cell-wall sugars in strains Aji5-31T and Ngc37-23T. The lonae NBRC 105200T and K. limosa NBRC 100340T menaquinone detected in strain Aji5-31T was exclu- were 96.1%, 94.9%, 95.3% and 96.6%, respectively. sively MK-8(H2), whereas the menaquinone composi- T T The purified peptidoglycan of strains Aji5-31 and tion of strain Ngc37-23 was MK-8(H4) and MK-8(H6) Ngc37-23T contained alanine (Ala), glutamic acid (96：4). The menaquinone patterns of D. congolensis T T (Glu) and diaminopimelic acid (A2pm) in molar ratios NBRC 105199 and D. chelonae NBRC 105200 were of 1.6：1.0：0.7 and 1.7：1.0：0.7, respectively. composed of MK-8(H4), MK-8(H6) and MK-8(H2) Enantiomeric analysis of the peptidoglycan amino ac- (96：3：1), and MK-8(H4), MK-8 and MK-8(H2) ids of both strains revealed the presence of D-Ala, L- (51：29：20), respectively. The polar lipids of strain T Ala, D-Glu and meso-A2pm. These data indicated that Aji5-31 were diphosphatidylglycerol, phosphatidyl-

Fig. 5. Phylogenetic tree derived from the 16S rRNA gene sequences of strains Aji5-31T and Ngc37-23T and their taxonomic neighbors. The tree was constructed using the neighbor-joining method. The 16S rRNA gene sequence of Brevibacterium linens DSM 20425T (X77451) was used as the outgroup. The numbers at the branch nodes are bootstrap percentages (from 1,000 replicates); only values of 50% or above are shown. Dots (●) indicate that the corresponding nodes were also recov- ered in the trees generated with both the maximum-likelihood (ML) and maximum-parsimony (MP) algorithms. The supporting nodes of either ML or MP trees were indicated by squares (■) and circles (○), respectively. Bar, 0.01 Knuc. 432 HAMADA et al. Vol. 56 glycerol and phosphatidylinositol, while those of strain of D. congolensis NBRC 105199T with a bootstrap re- Ngc37-23T were phosphatidylglycerol, phosphati- sampling value of 56%. Since these values are low, it is dylinositol and lyso-phosphatidylethanolamine. Gly- unlikely that both strains can be accommodated in ei- colipids were not detected in either strain. The results ther genera within the family Dermatophilaceae. Fur- of cellular fatty acid analysis are shown in Table 1. The thermore, D. chelonae NBRC 105200T did not form a DNA G+C contents of Aji5-31T, Ngc37-23T, D. congol- clade with D. congolensis NBRC 105199T, the type ensis NBRC 105199T, D. chelonae NBRC 105200T, species of the genus Dermatophilus (Fig. 4). The se- and K. limosa NBRC 100340T were 71.6, 71.5, 59.8, quence similarity between these strains was, at most, 66.5 and 71.1 mol%, respectively. 94.9%. Phylogenetic analysis on the basis of the 16S rRNA This study revealed that strains Aji5-31T and Ngc37- gene sequence revealed that strains Aji5-31T and 23T, D. congolensis NBRC 105199T, D. chelonae NBRC Ngc37-23T are related to the type strains of D. congol- 105200T and K. limosa NBRC 100340T had several dif- ensis, D. chelonae and K. limosa (Fig. 4). This result ferentiating phenotypic characteristics (Table 2). The supports an affiliation of both strains to the family Der- predominant menaquinone of strain Aji5-31T is MK- T matophilaceae. The highest sequence similarity values 8(H2). The major fatty acids of strain Ngc37-23 are T T to strains Aji5-31 and Ngc37-23 were observed in D. monounsaturated fatty acids (C17:1) as well as iso- T chelonae NBRC 105200 (96.8%) and K. limosa NBRC branched fatty acids (iso-C16:0 and iso-C14:0). In addi- 100340T (96.9%), respectively. In the neighbor-joining tion, this strain develops clusters of coccoid, but my- tree, strain Aji5-31T joined the phylogenetic lineage of celia are not developed. Also, only strain Ngc37-23T is D. chelonae NBRC 105200T with a bootstrap resam- non-motile in the family Dermatophilaceae. The DNA pling value of 51%, while strain Ngc37-23T joined that G+C content of D. congolensis NBRC 105199T is rela- tively low (59.8 mol%) compared with other members of the family. The menaquinone composition of D. che- Table 1. Cellular fatty acid compositions (%) of strains Aji5-31T T lonae NBRC 105200 is MK-8(H4), MK-8 and MK-8(H2) and Ngc37-23T and related taxa of the family Dermatophilaceae. (approximately 5：3：2); the DNA G+C content is Fatty acid 1 2 3 4 5 66.5 mol%. In addition, although D. chelonae NBRC T Straight-chain saturated fatty acids 105200 develops branching mycelia as with D. con- T C13:0 1.2 ― 6.3 2.9 ― golensis NBRC 105199 in early stage of growth, it C14:0 ― 1.3 5.7 15.8 ― does not become thick branching filaments with C15:0 18.9 7.4 26.4 24.8 13.5 several transverse and longitudinal division which C 1.9 7.4 11.5 13.0 9.5 16:0 leads to the formation of packets of coccoid (Fig. 3, 4). C17:0 7.9 6.0 16.2 5.1 19.4 T Branched saturated fatty acids K. limosa NBRC 100340 is strictly aerobic, whereas iso-C14:0 ― 11.3 ――― the other members of the family are facultatively an- iso-C16:0 1.4 23.1 ――― aerobic. Furthermore, the combination of phenotypic Mono-unsaturated fatty acids characteristics of K. limosa NBRC 100340T differs from C 2.9 ― 3.6 1.6 15:1 those of the other members of the family (Table 2). C16:1 ω9c 2.2 3.2 4.0 6.9 2.7 It should be considered that strains Aji5-31T and C17:1 ω9c 57.6 23.9 20.4 25.1 48.4 T C18:1 ω9c 1.8 6.7 2.3 4.8 6.5 Ngc37-23 reside in independent genera on the basis Hydroxy fatty acids of phylogenetic analysis results and their distinctive 2-OH C16:0 4.2 ― 1.0 ―― phenotypic characteristics. Therefore, it is proposed 2-OH C 3.1 16:1 ― ――― that strains Aji5-31T and Ngc37-23T be classified as 10-Methyl fatty acid two novel genera and species of the family Dermato- 10-Methyl C17:0 ― 5.2 ――― philaceae, with the names Mobilicoccus pelagius gen. T T Taxa: 1, strain Aji5-31 ; 2, strain Ngc37-23 ; 3, Dermatophilus nov., sp. nov. and Piscicoccus intestinalis gen. nov., T chelonae NBRC 105200 ; 4, Dermatophilus congolensis NBRC sp. nov., respectively. In addition, Dermatophilus che- 105199T; 5, Kineosphaera limosa NBRC 100340T. lonae should be reassigned to a new genus of the fam- Components comprising less than 1% of the total fatty acids in all strains were omitted. Bold type shows the major compo- ily Dermatophilaceae, with the name Austwickia nents (＞10%). -, Not detected or not described. All data are chelonae gen. nov., comb. nov. Furthermore, the de- from the present study. scription of the genus Dermatophilus Gordon (1964) 2010 Three new genera of the family Dermatophilaceae 433

Table 2. Phenotypic characteristics of strains Aji5-31T and Ngc37-23T and related taxa of the family Dermatophilaceae.

Characteristic 1 2 3 4 5 Morphology Coccoid Coccoid in clusters Branching mycelia, coccoid, Branching mycelia Coccoida bulb-tipped mycelia with cuboidal packets Peptidoglycan type A1γ A1γ A1γ A1γ A1γ a Predominant MK-8(H2)MK-8(H4)MK-8(H4), MK-8, MK-8(H2)MK-8(H4) MK-8(H4) menaquinone(s) Major fatty acids C17:1, C15:0 C17:1, iso-C16:0, C15:0, C17:1, C17:0, C16:0 C17:1, C15:0, C14:0, C17:1, C17:0, (＞10 %) iso-C14:0 C16:0 C15:0 Phospholipidsb PG, DPG, PI Lyso-PE, PG, PI Lyso-PE, PG, DPG, PI Lyso-PE, PG, PI Lyso-PE, PG DNA G+C content 71.6 71.5 66.5 59.8 71.1 (mol%) Oxygen relationship Facultatively Facultatively Facultatively Facultatively Strictly anaerobic anaerobic anaerobic anaerobic aerobica Motility + - +++a

Taxa: 1, strain Aji5-31T (Mobilicoccus pelagius gen. nov., sp. nov.); 2, strain Ngc37-23T (Piscicoccus intestinalis gen. nov., sp. nov.); 3, Dermatophilus chelonae (Austwickia chelonae gen. nov., comb. nov.); 4, Dermatophilus congolensis; 5, Kineosphaera. Data are from the present study, unless indicated. +, Positive; -, negative. aData are from Liu et al. (2002). bDPG, Diphoshatidylglycerol; Lyso-PE, lyso-phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol. should be emended for the addition of its chemotaxo- negative. Good growth occurs under both aerobic and nomic characteristics. anaerobic conditions. The temperature range for growth is 10‒37°C with an optimum 28°C. The pH Description of Mobilicoccus gen. nov. range for growth is 6.0‒9.0 with an optimal pH of 7.0. Mobilicoccus (Mo.bi.li.coc’cus. L. adj. mobilis mobile; Growth occurs at NaCl concentrations of 0‒7% (w/v), N.L. masc. n. coccus (from Gr. masc. n. kokkos grain, but not at concentrations at 10% and higher; the opti- seed) coccus; N.L. masc. n. Mobilicoccus a mobile mal NaCl concentration for growth is 1%. Acid is pro- coccus). duced from N-acetyl-glucosamine, D-fructose, D-ga- Cells are coccus-shaped, Gram-positive, facultative- lactose, D-glucose, inositol, D-maltose, D-mannose, ly anaerobic, non-sporulating and motile with peritri- D-ribose, D-sucrose and D-trehalose. Conversely, acid chous flagella. The cell-wall peptidoglycan is of the is not produced from D-adonitol, amygdalin, D- or

A1γ type. The predominant menaquinone is MK-8(H2), L-arabinose, D- or L-arabitol, arbutin, D-cellobiose, and the major polar lipids are diphosphatidylglycerol, dulcitol, erythritol, D- or L-fucose, gentiobiose, glu- phosphatidylglycerol and phosphatidylinositol. Cellu- conate, glycerol, inulin, 2-keto-gluconate, 5-keto-glu- lar fatty acids mainly comprise unsaturated and conate, D-lactose, D-lyxose, D-mannitol, D-melezitose, straight-chain saturated fatty acids. The major cell-wall D-melibiose, methyl-α-D-glucopyranoside, methyl-α-D- sugars are ribose and mannose. Phylogenetically, the mannopyranoside, methyl-β-D-xylopyranoside, D-raffino- genus Mobilicoccus belongs to the family Dermato- se, L-rhamose, salicin, D-sorbitol, L-sorbose, starch, D- philaceae within the suborder Micrococcineae. The tagatose, xylitol or D- or L-xylose. Alkaline phosphatase, type species is Mobilicoccus pelagius. α-glucosidase, leucine arylamidase and pyrazi- namidase are present, whereas acid phosphatase, Description of Mobilicoccus pelagius sp. nov. N-acetyl-β-glucosaminidase, arginine dihydrolase, α- Mobilicoccus pelagius (pe.la’gi.us. L. masc. adj. pela- chymotrypsin, cystine arylamidase, esterase (C4), es- gius of the sea, marine). terase lipase (C8), α-fucosidase, α-galactosidase, Shows the following characteristics in addition to β-galactosidase, β-glucosidase, β-glucuronidase, li- those given in the genus description. Cells are approx- pase (C14), lysine decarboxylase, α-mannosidase, imately 0.7‒1.2 μm in diameter. Colonies are circular, ornithine decarboxylase, phosphohydrolase, pyrrolid- smooth and orange-yellow. Catalase-positive, oxidase- onyl arylamidase, trypsin, tryptophan deaminase and 434 HAMADA et al. Vol. 56 valine arylamidase are not. Acetoin is produced, while binose, D- or L-arabitol, D-cellobiose, dulcitol, erythritol,

H2S and indole are not. Esculin, gelatin and urea are D- or L-fucose, gentiobiose, gluconate, glycogen, not hydrolyzed. Nitrate is reduced. The major cellular inositol, inulin, 2-keto-gluconate, 5-keto-gluconate, D- fatty acids are C17:1 ω9c and C15:0. The DNA G+C con- lactose, D-lyxose, D-melezitose, D-melibiose, methyl-α- tent of the type strain is 71.6 mol%. D-mannopyranoside, methyl-β-D-xylopyranoside, L- The type strain Aji5-31T (=NBRC 104925T =DSM rhamose, D-ribose, D-sorbitol, L-sorbose, starch, D- 22762T) was isolated from the intestinal tract of a fish tagatose, xylitol or D- or L-xylose. Acid phosphatase, collected from Kyonan Beach on the coast of Tokyo alkaline phosphatase, β-galactosidase, α-glucosidase, Bay, Japan. β-glucosidase, leucine arylamidase and pyrazinami- dase are present, whereas N-acetyl-β-glucosaminidase, Description of Piscicoccus gen. nov. arginine dihydrolase, α-chymotrypsin, cystine arylami- Piscicoccus (Pis.ci.coc’cus. L. n. piscis a fish; N.L. dase, α-fucosidase, α-galactosidase, β-glucuronidase, masc. n. coccus (from Gr. masc. n. kokkos grain, seed) lipase (C14), lysine decarboxylase, α-mannosidase, coccus; N.L. masc. n. Piscicoccus a coccus from a ornithine decarboxylase, phosphohydrolase, pyrrolid- fish). onyl arylamidase, trypsin, tryptophan deaminase, and

Cells are coccus-shaped, Gram-positive, facultative- valine arylamidase are not. H2S and indole are not pro- ly anaerobic, non-motile and non-sporulating. Grows duced. Esculin is hydrolyzed, while gelatin and urea in clusters or aggregates of coccoid. The cell-wall pep- are not. Nitrate is reduced. The major cellular fatty ac- tidoglycan is of the A1γ type. The predominant me- ids are C17:1 ω9c, iso-C16:0 and iso-C14:0. The DNA naquinone is MK-8(H4), and the major polar lipids are G+C content of the type strain is 71.5 mol%. phosphatidylglycerol, phosphatidylinositol and lyso- The type strain Ngc37-23T (=NBRC 104926T =DSM phosphatidylethanolamine. Cellular fatty acids mainly 22761T) was isolated from the intestinal tract of a fish comprise unsaturated, iso- branched saturated and collected from Kyonan Beach on the coast of Tokyo straight-chain saturated fatty acids. The major cell-wall Bay, Japan. sugars are ribose and mannose. Phylogenetically, the genus Piscicoccus belongs to the family Dermatophi- Description of Austwickia gen. nov. laceae within the suborder Micrococcineae. The type Austwickia (Au.st.wi’c.kia. N.L. fem. n. Austwickia species is Piscicoccus intestinalis. named in honor of Peter K. C. Austwick, a botanist, who proposed the family Dermatophilaceae). Description of Piscicoccus intestinalis sp. nov. Cells are branching mycelia to cocci, Gram-positive Piscicoccus intestinalis (in.tes.ti.nal’is. L. n. intestinum and facultatively anaerobic. With time, the mycelia are gut, intestine; L. suff. -alis suffix denoting pertaining to; divided into cocci to short rods. Forms bulb-tipped N.L. masc. adj. intestinalis, pertaining to the intes- mycelia on R agar. Motile coccoid cells are present. tine). The cell-wall peptidoglycan is of the A1γ type. The pre-

Shows the following characteristics in addition to dominant menaquinones are MK-8(H4), MK-8 and those given in the genus description. Cells are approx- MK-8(H2), and the major polar lipids are diphosphati- imately 0.7‒1.0 μm in diameter. Colonies are rough dylglycerol, phosphatidylglycerol, phosphatidylinosi- and orange-yellow. Catalase-positive, oxidase-nega- tol and lyso-phosphatidylethanolamine. Cellular fatty tive. Good growth occurs under both aerobic and an- acids are mainly unsaturated and straight-chain satu- aerobic conditions. The temperature range for growth rated. Phylogenetically, the genus Austwickia belongs is 10‒37°C with an optimum at 28°C. The pH range for to the family Dermatophilaceae within the suborder Mi- growth is 5.0‒9.0 with an optimal pH of 6.0‒7.0. Growth crococcineae. The type species is Austwickia che- occurs at NaCl concentrations of 0‒5% (w/v), but not lonae. at concentrations at 7% and higher; the optimal NaCl concentration range for growth is 0‒1%. Acid is pro- Description of Austwickia chelonae (Masters et al. duced from arbutin, D-fructose, D-galactose, D-glucose, 1995) comb. nov. D-maltose, D-mannose, D-raffinose, D-sucrose and D- Austwickia chelonae (che.lo’na.e. N.L. gen. n. che- trehalose. Conversely, acid is not produced from N- lonae of a turtle or tortoise). acetyl-glucosamine, D-adonitol, amygdalin, D- or L-ara- Basonym: Dermatophilus chelonae Masters et al. 2010 Three new genera of the family Dermatophilaceae 435

1995. 88, 509‒ 522. The description is identical to those given for Der- Hamada, M., Iino, T., Iwami, T., Tamura, T., Harayama, S., and matophilus chelonae by Masters et al. (1995) with the Suzuki, K. (2009) Arsenicicoccus piscis sp. nov., a meso- philic actinobacterium isolated from the intestinal tract of a following addition: the major cellular fatty acids are fish. Actinomycetologica, 23, 40‒ 45. C , C ω9c, C and C , and the DNA G+C 15:0 17:1 17:0 16:0 Liu, W. T., Hanada, S., Marsh, T. L., Kamagata, Y., and Naka- content of the type strain is 66.5 mol%. The type strain mura, K. (2002) Kineosphaera limosa gen. nov., sp. nov., a T T T is W16 (=NBRC 105200 =ATCC 51576 =CCUG novel Gram-positive polyhydroxyalkanoate-accumulating 47447T =CIP 104541T =DSM 44178T =JCM 9706T). coccus isolated from activated sludge. Int. J. Syst. Evol. Mi- crobiol., 52, 1845‒ 1849. Emended description of the genus Dermatophilus Gor- Masters, A. M., Ellis, J. M., Carson, J. M., Sutherland, S. S., and don 1964 Gregory, A. R. (1995) Dermatophilus chelonae sp. nov., isolated from chelonids in Australia. Int. J. Syst. Bacteriol., 45, The description is as given by Gordon (1964) with 50‒ 56. the following modifications. Cells are branching myce- Mikami, H. and Ishida, Y. (1983) Post-column fluorometric de- lia, Gram-positive and facultatively anaerobic. Mature tection of reducing sugars in high-performance liquid chro- mycelia consist of several transverse and longitudinal matography using arginine. Bunseki Kagaku, 32, 207‒ 210 divisions which leads to the formation of packets or (in Japanese). clusters of cuboid cells or coccoid. Motile coccoid Minnikin, D. E., Alshamaony, L., and Goodfellow, M. (1975) Dif- cells are present. The cell-wall peptidoglycan is of the ferentiation of Mycobacterium, Nocardia and related taxa by thin-layer chromatographic analysis of whole-organism A1γ type. The predominant menaquinone is MK-8(H4), and the major polar lipids are phosphatidylglycerol, methanolysates. J. Gen. Microbiol., 88, 200‒ 204. Nozawa, Y., Sakai, N., Arai, K., Kawasaki, Y., and Harada, K. phosphatidylinositol, and lyso-phosphatidylethanol- (2007) Reliable and sensitive analysis of amino acids in the amine. Cellular fatty acids mainly comprise unsaturat- peptidoglycan of actinomycetes using the advanced Mar- ed and straight-chain saturated fatty acids. fey’s method. J. Microbiol. Methods, 70, 306‒ 311. Saito, H. and Miura, K. (1963) Preparation of transforming de- Acknowledgments oxyribonucleic acid by phenol treatment. Biochim. Bio- phys. Acta, 72, 619‒ 629. We would like to thank to Dr. J. P. Euzéby for his support with Saitou, N. and Nei, M. (1987) The neighbor-joining method: A nomenclature. This study, in part, was supported by a grant-in- new method for reconstructing phylogenetic trees. Mol. aid (Grant No. 04000182-0) from the New Energy Development Biol. Evol., 4, 406‒ 425. Organization (NEDO). Sasser, M. (1990) Identification of Bacteria by Gas Chromatog- raphy of Cellular Fatty Acids. MIDI Technical Note 101, References MIDI, Inc., Newark, DE. Schleifer, K. H. and Kandler, O. (1972) Peptidoglycan types of Austwick, P. K. C. (1958) Cutaneous streptotrichosis, mycotic bacterial cell walls and their taxonomic implications. Bacte- dermatitis and strawberry foot rot and the genus Dermato- riol. Rev., 36, 407‒ 477. philus Van Saceghem. Vet. Rev. Annot., 4, 33‒ 38. Stackebrandt, E. (2006) The family Dermatophilaceae. In The Dittmer, J. C. and Lester, R. L. (1964) A simple, specific spray for Prokaryotes; a Handbook on the Biology of Bacteria, Vol. the detection of phospholipids on thin-layer chromato- 3, 3rd ed., ed. by Dworkin, M., Falkow, S., Rosenberg, E., grams. J. Lipid Res., 15, 126‒ 127. Schleifer, K. H., and Stackebrandt, E., Springer, New York, Felsenstein, J. (1981) Evolutionary trees from DNA sequences: pp. 1002‒ 1012. A maximum likelihood approach. J. Mol. Evol., 17, 368‒ Stackebrandt, E., Rainey, F. A., and Ward-Rainey, N. L. (1997) 376. Proposal for a new hierarchic classification system, Acti- Felsenstein, J. (1985) Confidence limits on phylogenies: An ap- nobacteria classis nov. Int. J. Syst. Bacteriol., 47, 479‒ 491. proach using the bootstrap. Evolution, 39, 738‒ 791. Stackebrandt, E. and Schumann, P. (2000) Description of Bogo- Fitch, W. M. (1971) Toward defining the course of evolution: riellaceae fam. nov., Dermacoccaceae fam. nov., Rar- Minimum change for a specific tree topology. Syst. Zool., obacteraceae fam. nov. and Sanguibacteraceae fam. nov. 20, 406‒ 416. and emendation of some families of the suborder Micro- Gerhardt, P., Murray, R. G. E., Wood, W. A. and Krieg, N. R. coccineae. Int. J. Syst. Evol. Microbiol., 50, 1279‒ 1285. (1994) Methods for General and Molecular Bacteriology, Tamaoka, J. and Komagata, K. 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