Actinomycetologica (2007) 21:53–58 Copyright Ó 2007 The Society for Actinomycetes Japan VOL. 21, NO. 2 Microbacterium flavum sp. nov. and Microbacterium lacus sp. nov., isolated from marine environments. Akiko Kageyama1, Yoko Takahashi1Ã, Yoshihide Matsuo2, Kyoko Adachi2, Hiroaki Kasai2, Yoshikazu Shizuri2 and Satoshi O¯ mura1;3 1Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan. 2Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan. 3The Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan. (Received Feb. 21, 2007 / Accepted Jul. 9, 2007 / Published Sep. 10, 2007) Strains YM18-098T and A5E-52T were both Gram-positive, aerobic, irregular rod-shaped bacteria, with lysine and ornithine as the diagnostic diamino acids of their peptidoglycans, respectively. The acyl type of the peptidoglycan in both cases was N-glycolyl. The major menaquinones were MK-11, -12 and -13. Mycolic acids were not detected. The G+C content of the DNA was 69–70 mol%. Comparative 16S rRNA studies revealed that the isolates belonged to the genus Microbacterium, that YM18-098T was closely related to the species Microbacterium lacticum and Microbacterium schleiferi, and that A5E-52T was closely related to the species Microbacterium aurum, Microbacterium aoyamense, Microbacterium deminutum and Microbacterium pumilum. DNA-DNA relatedness analysis showed that the isolated strains represented two separate genomic species. Based on both phenotypic and genotypic data, the following new species of the genus Microbacterium are proposed: Microbacterium flavum sp. nov. and Microbacterium lacus sp. nov., with the type strains YM18-098T (= MBIC08278T, DSM 18909T) and A5E-52T (= MBIC08279T, DSM 18910T), respectively. INTRODUCTION Didemnum moseleyi collected from 20 meters offshore and 1 meter in depth in Nomozaki at the tips of Nagasaki The genus Microbacterium was first proposed by Orla- Peninsula, Nagasaki Prefecture, Japan (GPS location: Jensen1) with the type species Microbacterium lacticum, and 3235031.500N, 12945023.200E), in July 2005. Samples was later emended by Takeuchi & Hatano2). The genus (0.5–1 cm3)ofD. moseleyi were homogenized with a glass Microbacterium is a Gram-positive, obligatory aerobic and rod in 5 mL of sterile seawater. The homogenate (50 mL) catalase-positive. The peptide subunit of peptidoglycan con- was used to isolate the bacterium on HSV agar medium. sists of alanine, D-glutamic acid (plus hydroxyglutamic acid), The components of medium HSV are shown in Table 1. and either L-lysine, L-ornithine or L-homoserine. Menaqui- Humic acid was purchased from ACROSORGANICS Inc. none are MK-10, 11, 12, 13 and 14. Muramic acid occurs in and purified by International Humic Substances Society the N-glycolyl form. The G+C content of the DNA ranges protocol: refer to http://www.ihss.gatech.edu/soilhafa.html. from 66 to 72 mol%. Members of the genus Microbacterium Colonies were picked after incubation for 30 days at 25C. are widespread and can be isolated from various environ- Strain A5E-52T was isolated from sediment (depth; roughly mental habitats3). The genus Microbacterium belongs to the 2 m) collected by using bottom sampler on March 2005 at family Microbacteriaceae in the order Actinomycetales. the mouth of Hiratafuna River to Lake Shinjiko, Shimane, In this paper, we report on the morphological, physio- Japan. Salinity around the sampling station was approx- logical, biochemical and chemotaxonomic characteristics, imately 0.5%4). The sediment was suspended in autoclaved DNA-DNA hybridization data and 16S rRNA gene se- seawater. The 1/10 diluents was applied to 1/10 diluted quences of two new isolates in comparison with related nutrient agar (Difco) supplemented with 2 mg/1 L carbonyl Microbacterium species. Based on the characteristics cyanide m-chlorophenylhydrazone. Colonies were picked studied, these isolates represent two novel species of the after incubation for 1 week at 25C. Biomass for bio- genus Microbacterium: Microbacterium flavum sp. nov. chemical and chemotaxonomic studies was prepared by and Microbacterium lacus sp. nov. culture in trypticase soy broth (Difco) at 27C. MATERIALS AND METHODS Morphological and biochemical tests Morphological observations were carried out using a Bacterial strains and isolation scanning electron microscope (model JSM-5600; JEOL) Strain YM18-098T was isolated from the ascidian and cultures grown on GPM agar medium (glucose 10 g, ÃCorresponding author and address: Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108- 8641, Japan., phone: +81-3-5791-6133, e-mail: [email protected], fax: +81-3-5791-6133. 53 ACTINOMYCETOLOGICA VOL. 21, NO. 2 Table 1. The components of medium HSV. HSV medium ÃMetal mix X Metal mix X 250 mLÃ NaCl 500 g ÃÃ Humic acid mix 100 mL MgSO4Á7H2O 180 g ÃÃÃ Vitamin mix A 4 ml CaCl2Á2H2O 2.8 g Vitamin B12 soln 1 mlÃÃÃÃ KCl 14 g Cycloheximide 50 mg Na2HPO4Á12H2O5g Gryseofluvin 25 mg FeSO4Á7H2O 200 mg Nalidixic acid 20 mg PII metals 600 mlÃÃÃÃÃ Aztreonam 40 mg S2 metals 100 mlÃÃÃÃÃÃÃ Agar 20 g DW 4300 ml/pH 7.6 DW 650 mL/pH 7.6 ÃÃHumic acid mix ÃÃÃÃÃPII metals Humic acid, purified 5 g Na2-EDTA 1 g DW 500 mL/pH 7.6 H3BO3 1.13 g ÃÃÃ Vitamin mix A Fe soln. 1 ml of FeCl3Á6H2O (2.42 g/50 ml) Thiamine HCl 500 mg Mn soln. 1 ml of MnCl2Á4H2O (7.2 g/50 ml) Riboflavin 500 mg Zn soln. 1 ml of ZnCl2 (0.52 g/50 ml (+HCl)) Niacin 500 mg Co soln. 1 ml of CoCl2Á6H2O (0.2 g/50 ml) Pyridoxin HCl 500 mg DW 996 ml/pH 7.5 Ca-panthothenate 500 mg ÃÃÃÃÃÃS2 metals p-Aminobenzonic acid 500 mg NaBr 1.28 g Biotin 250 mg Mo soln 10 ml of Na2MoO4Á2H2O (0.63 g/50 ml) DW 1000 ml/pH 7.5 Sr soln. 10 ml of SrCl2Á6H2O (3.04 g/50 ml) Filter sterile Rb soln. 10 ml of RbCl (141.5 mg/50 ml) ÃÃÃÃVitamin B12 soln Li soln. 10 ml of LiCl (0.61 g/50 ml) Cyanocovalamin 50 mg I soln. 10 ml of KI (6.55 mg/50 ml) DW 50 ml V soln. 10 ml of V2O5 (1.785 mg/50 ml (+NaOH)) Filter sterile DW 940 ml/pH 7.5 peptone 5 g, meat extract 5 g, NaCl 3 g and agar 12 g per Methyl esters of cellular fatty acids were prepared and were 1 L/pH 7.0) at 27C for 3 days. Assimilation of various sole analyzed by gas-liquid chromatography (GLC) (model carbon sources was assessed using Pridham-Gottlieb agar HP6890; Hewlett-Packard). medium (Nihon Pharmaceutical Co., Ltd.)5). NaCl toler- ance, pH and temperature ranges for growth were deter- G+C content of DNA and DNA-DNA hybridization mined on 1/5 nutrient agar. The two isolates were char- DNA was isolated as described by Saito & Miura11). acterized enzyme activity using API ZYM (bioMe´rieux) in The DNA base composition was determined by HPLC12). accordance with the manufacturer’s instructions. Levels of DNA-DNA relatedness were analyzed by using the method of Ezaki et al.13) using photobiotin and a micro- Chemotaxonomic tests plate format. The N-acyl type of muramic acid was determined by using the method of Uchida & Aida6). Purified cell walls 16S rRNA sequencing and phylogenetic analysis were obtained by using the method of Kawamoto et al.7). DNA was prepared using InstaGene matrix (Bio-Rad, One milligram of purified cell wall was hydrolyzed at CA, USA). The 16S rDNA was amplified by PCR using 100C with 1 mL of 6 N HCl for 16 h. The residue was a forward primer [name of primer was 27F, which is dissolved in 100 mL of water and was used for amino acid corresponding to positions 8-27] and a reverse primer analysis by thin layer chromatography (TLC). Whether [name of the primer was 1492R, which was correspond- mycolic acid was present was determined by using the TLC ing to positions 1492-1510 (Escherichia coli numbering method of Tomiyasu8). Menaquinones were extracted and system; Weisburg et al.14))] and was sequenced with an purified by using the method of Collins et al.9), and then automatic sequence analyzer (Applied Biosystems 3730 analyzed by high performance liquid chromatography DNA Analyzer; Applied Biosystems, CA, USA) using the (HPLC) (model 802-SC; Jasco) on a chromatograph equip- BigDyeÒ Terminator v3.1 Cycle Sequencing Kit (Applied ped with a Capcell Pak C18 column (Shiseido Co., Ltd.)10). Biosystems). Related sequences were identified by perform- 54 ACTINOMYCETOLOGICA VOL. 21, NO. 2 ing sequence database searches using BLAST15). Sequence menaquinones were MK-11, -12 and -13. The acyl types of data for related species were retrieved from GenBank. the peptidoglycans were N-glycolyl. The major cellular T Phylogenetic analysis was performed using CLUSTAL W fatty acids were anteiso-C15:0 for YM18-098 , and anteiso- 16) T software . Nucleotide substitution rates (Knuc values) were C15:0 and anteiso-C17:0 for A5E-52 (Table 2). calculated based on Kimura 2-parameter model17) and phylogenetic trees were constructed using the neighbor- 18) joining method . Sequence similarity values were deter- A B mined by visual comparison and manual calculation. RESULTS AND DISCUSSION Two novel actinobacteria strains were isolated from marine environment in Japan. Strain YM18-098T was isolated from the ascidian Didemnum moseleyi. Strain A5E- 52T was isolated from the estuarine sediment. Both strains were Gram-positive, aerobic, irregular rods (Fig. 1). The DNA base compositions of the two strains were 69–70 mol% G+C. The cell wall peptidoglycans of Fig. 1. Scanning electron micrographs of cells from 3-day-old YM18-098T and A5E-52T contained lysine and ornithine cultures of strains YM18-098T (A) and A5E-52T (B) grown on as the diagnostic diamino acids, respectively.