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

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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 se- quence 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 com- ponents, 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 phylo- genetic 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), respec- tively. 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.
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