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Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-Ku, Tokyo 113-0032, Japan
Correspondence
Cheng-Hui Xie
aa37116{at}mail.ecc.u-tokyo.ac.jp
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), 16S rRNA gene sequence analysis (Nakagawa et al., 2002) and chemotaxonomic studies (Takeuchi & Yokota, 1992) had shown that [Flavobacterium] ferrugineum IAM 15098T was not related to other members of the genus Flavobacterium and represented an independent taxon within the phylum Bacteroidetes. Therefore, [Flavobacterium] ferrugineum IAM 15098T should be reclassified (Sly et al., 1999; Nakagawa & Yamasato, 1996; Bernardet et al., 1996). New data [sequences of the 16S rRNA and gyrB (B subunit of DNA gyrase) genes, respiratory quinone system, cellular fatty acids and phenotypic characteristics] indicated that the soil isolate DYT and [Flavobacterium] ferrugineum IAM 15098T represent two separate species of a novel genus.
[Flavobacterium] ferrugineum IAM 15098T and strain DYT grew well in nutrient broth (NB; Difco), IAM medium 802 [polypeptone (Nihon Pharmaceutical Co.), 10 g; yeast extract, 2.0 g; MgSO4.7H2O, 1 g; distilled water, 1.0 l] and nitrogen-free medium (Xie & Yokota, 2005) at 29 °C. API 20E, API 50 CH and API ZYM kits (bioMérieux) were used to determine physiological and biochemical characteristics. The API strips were incubated for 2 days at 30 °C, except for API ZYM, the results of which were recorded after 4 h incubation at 37 °C, according to the manufacturer's instructions. Acid production from carbohydrates was determined using O/F basal medium (peptone, 2.0 g; NaCl, 5.0 g; K2HPO4, 0.3 g; bromothymol blue, 0.03 g; agar, 3.0 g; distilled water, 1 l; pH 7.1). A carbohydrate solution (sterilized by filtration) was added at a concentration of 1 % (w/v) and the results were recorded after 7 days of incubation. Tolerance of salinity was determined by inoculating the strains into NB supplemented with 0–4.0 % NaCl (w/v). Hydrolysis of chitin was assessed using the following medium: colloidal chitin, 4.0 g; (NH4)2SO4, 1.0 g; KH2PO4, 1.36 g; MgSO4.7H2O, 0.3 g; NaCl, 1.0 g; and distilled water, 1 l; pH 7.3. The colloidal chitin was prepared as follows: 4.0 g chitin was dissolved in 40 ml of 36 % HCl by stirring for 30 min, precipitated in cold water and washed with water until the pH reached 3.5. Chitosan degradation was assessed using the method described by Park et al. (1999) and Mitsuaria chitosanitabida IAM 14711T was used as a chitosanase-positive control. Cells grown on trypticase soy agar (TSA; Becton Dickinson) for 3 days at 29 °C were used for cellular fatty acid analysis. Fatty acid methyl esters were prepared and identified using the Microbial Identification System (MIDI; Sasser, 1990). Cells grown in NB for 5 days at 29 °C were used for the determination of the respiratory quinone system and of the DNA G+C content using HPLC (Shimadzu), as described by Xie & Yokota (2003). Genomic DNA was extracted using the method of Marmur (1961) from cells cultured for 2 days in NB.
Since strain DYT was able to grow well in nitrogen-free medium, the presence of nitrogenase activity was assessed. The sensitive, rapid and inexpensive acetylene reduction assay (ARA) (Hardy et al., 1968) was first used. [Flavobacterium] ferrugineum IAM 15098T and strain DYT were grown in 7 ml LB broth for 24 h at 30 °C. The cells were centrifuged and washed three times with nitrogen-free medium and 0.1 ml suspensions (OD600=1.0) were transferred to gas vials (20 ml) containing 2 ml semi-solid nitrogen-free medium (0.3 % agar), in an atmosphere of 2 % oxygen, 10 % acetylene and 88 % nitrogen. After incubation at 30 °C for 18–24 h, 0.1 ml gas was withdrawn from the vials and analysed by gas chromatography (GC-17A; Shimadzu; equipped with a Porapak N column of 30 m in length) at 50 °C. Helium was used as a carrier gas at a flux of 3.4 ml min–1 and H2 flame ionization was used for detection. As ethylene was not produced from acetylene in the ARA test, nitrogenase activity was further assessed by attempting to amplify the nifH gene (encoding the iron protein of nitrogenase) using the primers IGK-AQE, PolF-PolR or IGK-R750 (Xie & Yokota, 2005). However, the nifH gene could not be amplified from strain DYT or [Flavobacterium] ferrugineum IAM 15098T. Hence, the fact that nitrogenase activity could not be detected using two different tests demonstrated that the two strains did not have nitrogen-fixing ability, although they could grow well on nitrogen-free medium. They may have used atmospheric nitrogen for growth or may possess some unusual nitrogen-fixing system, as reported for Streptomyces thermoautotrophicus (Gadkari et al., 1992).
An approximately 1500-bp fragment of the 16S rRNA gene sequence was amplified by PCR and sequenced as described by Xie & Yokota (2003). The gyrB gene sequences were amplified and sequenced using a set of primers described by Yamamoto & Harayama (1995) and a newly designed primer, gyrB840 (CTTCACCAACAACATCCC). The sequences obtained in this study and from GenBank were aligned using the CLUSTAL W software package (Thompson et al., 1994) and evolutionary distances and the Knuc value (Kimura, 1980) were generated. Alignment gaps and ambiguous bases were excluded from the calculations. A phylogenetic tree based on a comparison of 1189 bases was constructed using the neighbour-joining method (Saitou & Nei, 1987). The topology of the tree was evaluated by using the bootstrap resampling method of Felsenstein (1985) with 1000 replicates, whereas similarity values were calculated using PAUP 4.0b1 (Swofford, 1998). Using the same method, 399 aa sequences of GyrB were also aligned and a phylogenetic tree was constructed.
Comparison of the 16S rRNA gene sequences showed that strain DYT and [Flavobacterium] ferrugineum IAM 15098T had a sequence similarity of 94.8 %, forming a branch that shared less than 92 % sequence similarity with the cluster comprising [Cytophaga] arvensicola, [Flexibacter] japonensis, [Flexibacter] sancti, [Flexibacter] filiformis and Chitinophaga pinensis (Oyaizu et al., 1982; Fujita et al., 1996; Sly et al., 1999) (Fig. 1).
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) based on 399 aa of GyrB was consistent with the tree based on 16S rRNA gene sequences. The level of 16S rRNA (94.8 %) and gyrB (77.5 %) gene sequence similarity shared by strain DYT and [Flavobacterium] ferrugineum IAM 15098T suggests that they represent two separate species in the same genus, which can be easily differentiated from each other by several phenotypic characteristics (Table 1).
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7c, which could not separated by the MIDI system) (Table 2). This fatty acid composition differed significantly from that of [Cytophaga] arvensicola (major fatty acids 15 : 0 iso, 15 : 0 iso 2-OH, 15 : 0 iso 3-OH, 16 : 111c and 17 : 0 iso 3-OH; Oyaizu et al., 1982). Several characteristics (non-filamentous cell morphology; absence of gliding motility, chitin degradation and urease activity and presence of catalase activity) distinguished strain DYT and [Flavobacterium] ferrugineum IAM 15098T from [Flexibacter] japonensis, [Flexibacter] sancti, [Flexibacter] filiformis and Chitinophaga pinensis (Table 3). On the other hand, strain DYT and [Flavobacterium] ferrugineum IAM 15098T possessed MK-7 as the major menaquinone and MK-6 as a minor component, similar to [Cytophaga] arvensicola (Oyaizu et al., 1982) and [Flexibacter] japonensis (Fujita et al., 1996). The G+C content of the DNA of strain DYT (48.9 mol%) and [Flavobacterium] ferrugineum IAM 15098T (47.2 mol%) was similar to that of their close relatives. Our data confirmed that [Flavobacterium] ferrugineum differs considerably from other members of the genus Flavobacterium, where the major menaquinone is MK-6 and the G+C content is 32–37 mol% (Bernardet et al., 1996).
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Description of Terrimonas gen. nov.
Terrimonas (Ter.ri.mo'nas. L. n. terra soil; L. fem. n. monas a unit, monad; N.L. fem. n. Terrimonas soil monad).
Cells are strictly aerobic, Gram-negative, non-motile, non-gliding, non-filamentous single rods. Growth is inhibited by >1.0 % NaCl. Oxidase-positive and weakly positive for catalase. Positive for gelatin liquefaction, Voges–Proskauer test and nitrate reduction, but negative for urease production and chitin degradation. Major cellular fatty acids are 15 : 0 iso, 15 : 1 iso, 17 : 0 iso 3-OH and summed feature 3. MK-7 is the predominant respiratory quinone and MK-6 is a minor component. The G+C content of the DNA is 47.2–48.9 mol%. The type species is Terrimonas ferruginea.
Description of Terrimonas ferruginea comb. nov.
Terrimonas ferruginea (fer.ru.gi'ne.a. L. fem. adj. ferruginea rust-coloured).
Basonym: Flavobacterium ferrugineum Sickles and Shaw 1934.
The characteristics are the same as those given in the genus description. Cells are single rods, 0.3–0.5 µm in width and 1–3 µm in length. Colonies are circular, about 1 mm in diameter and non-spreading. Growth occurs at 10–37 °C, with optimum growth at 25–32 °C. Colonies on nutrient agar and IAM medium 802 are salmon red. Grows well on TSA and nitrogen-free medium. Chitin and chitosan are not degraded on conventional media. Acid is produced from glucose, cellobiose, maltose, mannose, melibiose, rhamnose and xylose, but not from sucrose, dulcitol, glycerol, inositol, mannitol, sorbitol, raffinose, fructose or galactose. Glucose, mannose, aesculin, xylose, maltose, cellobiose, rhamnose, lactose, 5-ketogluconate, starch and glycogen are assimilated, but sucrose, trehalose, dulcitol, arabinose, galactose, fructose, glycerol, inositol, mannitol, methyl 
-D-mannoside, raffinose, sorbitol and gentiobiose are not. Activities of 
-galactosidase, alkaline phosphatase, leucine arylamidase, valine arylamidase, acid phosphatase, -chymotrypsin, naphthol phosphohydrolase, C4 esterase, C8 esterase lipase and N-acetyl--glucosaminidase are strongly positive; activities of cystine arylamidase, trypsin, -glucosidase and -fucosidase are weakly positive. -Galactosidase, -glucuronidase, -glucosidase, -mannosidase and C14 lipase activities are absent. The G+C content of the DNA of the type strain is 48.9 mol%.
The type strain is IAM 15098T (=NBRC 14992T=LMG 4021T=ATCC 13524T), which was isolated from soil from an unknown locality.
Description of Terrimonas lutea sp. nov.
Terrimonas lutea (lu.te'a. L. fem. adj. lutea golden-yellow).
The characteristics are the same as those given in the genus description. Cells are single rods, 0.3–0.5 µm in width and 1–3 µm in length. Colonies are circular, about 1 mm in diameter and non-spreading. Growth occurs at 10–37 °C, with optimum growth at 25–32 °C. Colonies are yellow on nutrient agar and IAM medium 802. Grows well on TSA and nitrogen-free medium. Chitin and chitosan are not degraded on conventional media. Acid is produced from glucose, cellobiose, maltose, mannose, fructose, galactose, raffinose and sucrose, but not from dulcitol, glycerol, inositol, mannitol, melibiose, rhamnose, xylose or sorbitol. Glucose, mannose, aesculin, maltose, lactose, 5-ketogluconate, arabinose, galactose, fructose, methyl -D-mannoside, cellobiose, N-acetylglucosamine, sucrose, raffinose, gentiobiose and melibiose are assimilated, but starch, D-xylose, glycogen, glycerol, inositol, mannitol, sorbitol, rhamnose and dulcitol are not. Alkaline phosphatase, leucine arylamidase, valine arylamidase, acid phosphatase, naphthol phosphohydrolase, C4 esterase, C8 esterase lipase and N-acetyl--glucosaminidase activities are strongly positive; C14 lipase, cystine arylamidase, - and -glucosidase and trypsin activities are weakly positive; -glucuronidase, - and -galactosidases, -mannosidase, -chymotrypsin and -fucosidase activities are absent. The fatty acid composition is only slightly different from that of the type species. The DNA G+C content of the type strain is 47.2 mol%.
The type strain is DYT(=IAM 15284T=CCTCC AB205006T), which was isolated from garden soil in Japan.
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