J. Gen. Appl. Microbiol., 55, 201‒205 (2009) Full Paper

Ilumatobacter fl uminis gen. nov., sp. nov., a novel actinobacterium isolated from the sediment of an estuary

Atsuko Matsumoto,1 Hiroki Kasai,2 Yoshihide Matsuo,2 Satoshi Ōmura,1 Yoshikazu Shizuri,2 and Yōko Takahashi1,*

1 Kitasato Institute for Life Sciences, Kitasato University, Minato-ku, Tokyo 108‒8641, Japan 2 Marine Biotechnology Institute, Kitasato University, Kamaishi, Iwate 026‒0001, Japan

(Received December 1, 2008; Accepted February 2, 2009)

Bacterial strain YM22-133T was isolated from the sediment of an estuary and grew in media with an artifi cial seawater base. Strain YM22-133T was Gram-positive, aerobic, non-motile and rod shaped. The cell-wall peptidoglycan contained LL-DAP, glycine, alanine and hydroxyglutamate. The predominant menaquinone was MK-9 (H8), with MK-9 (H0), MK-9 (H2), MK-9 (H4) and MK-9 (H6) present as minor menaquinones. The G+C content of the genomic DNA from the strain was 68 mol%. Phylogenetic analysis of the 16S rRNA gene sequence showed that the strain is near- est to ferrooxidans DSM 10331T. However, the similarity is relatively low (87.1%) and the physiological characteristics are also different: Acidimicrobium ferrooxidans is thermo- tolerant and acidophilic. Therefore, strain YM22-133T can be classifi ed as a novel genus and species, Ilumatobacter fl uminis gen. nov., sp. nov. (type strain YM22-133T =DSM 18936T=MBIC 08263T).

Key Words—Acidimicrobium; ; artifi cial sea water; Ilumatobacter fl uminis gen. nov., sp. nov.

Introduction isolated as part of this study. Phylogenetic analysis on the basis of 16S rRNA gene sequence analysis showed Recently, isolated from marine environ- that the strain is most closely related to the genus Aci- ments have attracted attention due to the recognition dimicrobium (Clark and Norris, 1996). The genus Aci- of great diversity among marine bacteria (Fiedler et al., dimicrobium branches near the presumed root of the 2005; Gontang et al., 2007; Jensen et al., 2005). Ma- class Actinobacteria (Stackebrandt et al., 1997). At rine bacteria also appear to be a rich source for the present the class Actinobacteria consists of four sub- identifi cation and isolation of new bioactive com- classes: Actinobacteridae, Acidimicrobidae, Coriobac- pounds and indeed, we have identifi ed new com- teridae and Rubrobacteridae. But the majority of gen- pounds from metabolites of the marine bacteria era contained in this class belong to subclass (Hayakawa et al., 2007; Kanoh et al., 2005; Matsuo et Actinobacteridae and few genera and species in the al., 2007; Shindo et al., 2007). Strain YM22-133T was other three subclasses are known. Acidimicrobium fer- rooxidans is also located outside of subclass Acti- nobacteridae, and is the sole family, genus and spe- * Address reprint requests to: Dr. Yōko Takahashi, Kitasato cies in order Acidimicrobiales. In this paper we describe Institute for Life Sciences, Kitasato University, 5‒9‒1 Shirokane, Minato-ku, Tokyo 108‒8641, Japan. the taxonomic characterization and classifi cation of T Tel: 81‒3‒5791‒6133 Fax: 81‒3‒5791‒6133 strain YM22-133 , which is a rare strain that branches E-mail: [email protected] near the presumed root of the class Actinobacteria. 202 MATSUMOTO et al. Vol. 55

Materials and Methods lyophilization with liquid nitrogen. The motility of cells was observed by light microscopy after 4 to 6 days of Isolation. Strain YM22-133T was isolated from a culture on Marine Agar 2216 (Difco). sediment sample collected at the mouth of the Kuira- Cultural and physiological characteristics. Gram- gawa River, Iriomote, Okinawa Prefecture, Japan staining was performed using a Gram’s reagent kit (depth: 1 m, GPS location: N 24°19′23.9″, E 123° (Nacalai Tesque). The pH range (pH 3‒11) and tem- 44′44.7″) in October 2005. The sample (0.5 cm3) was perature range for growth (5‒50°C) were determined homogenized with a glass rod in 5 ml of sterile sea on Marine Agar 2216. General biochemical character- water. The homogenate (50 μl) was used to isolate a istics were determined using API ZYM test strips (bio- bacterium on medium “R” (Table 1) after cultivation at Mérieux). 25°C for 30 days, and then single isolates were ob- Chemotaxonomic characteristics. Cells for chemo- tained on Marine Broth 2216 (Difco) containing 1.5% taxonomic analysis were obtained by culturing in agar. MPY2. The N-acyl types of muramic acid were deter- Morphology. Morphological characteristics of the mined using the method of Uchida and Aida (1977). strain were observed by scanning electron microsco- Purifi ed cell wall was obtained as described by Kawa- py (SEM; model JSM-5600, JEOL). For SEM, cells moto et al. (1981), and 1 mg of purifi ed cell wall pre- grown in MPY2 (1% peptone, 0.5% yeast extract in paration was hydrolyzed at 100°C with 1 ml of 6 M HCl Daigo’s Artifi cial Seawater SP) for 11 days at 27°C for 15 h. The hydrolysate was dissolved in 100 μl of were fi ltered onto a polycarbonate membrane and water and used for amino acid analysis. The amino fi xed using 4% osmium tetroxide vapor, followed by acid composition was determined by HPLC using the Pico-Tag method (Waters). The presence of mycolic Table 1. The composition of “R” medium. acids was assayed by TLC as described by Tomiyasu (1982). Menaquinones were extracted and purifi ed as NaCl 25 g in Collins et al. (1977) then analyzed by HPLC (Tama- MgSO ･7H O9 g 4 2 oka et al., 1983) and liquid chromatography/mass spec- CaCl 2H O 0.14 g 2･ 2 φ KCl 0.7 g trometry (LC/MS) with a PEGASIL ODS column (2 × 50 mm) using methanol/2-propanol (7 3). Analysis of Na2HPO4･12H2O 0.25 g ： Na2‒EDTA 30 mg fatty acids was performed according to the procedures H3BO3 34 mg for the Sherlock Microbial Identifi cation System (Mi- FeSO4･7H2O 10 mg crobial ID) using cells grown on Marine Agar 2216. FeCl3･6H2O 1.452 mg DNA base composition. DNA was isolated as de- MnCl2･4H2O 4.32 mg scribed by Saito and Miura (1963). DNA base compo- ZnCl 0.312 mg 2 sition was estimated by the HPLC method of Tamaoka CoCl ･6H O 0.12 mg 2 2 and Komagata (1984). NaBr 6.4 mg Analysis of 16S rRNA gene sequence. DNA for Na2MoO･2H2O 0.63 mg SrCl2･6H2O 3.04 mg analysis of the 16S rRNA gene sequence was pre- RbCl 0.1415 mg pared using InstaGene matrix (Bio-Rad, CA, USA). The LiCl 0.61 mg 16S rRNA gene was amplifi ed by PCR using a forward KI 0.00655 mg primer corresponding to positions 8‒27 and a reverse V2O5 0.001785 mg primer corresponding to 1492‒1510 (Escherichia coli Cycloheximide 50 mg numbering system; Weisburg et al., 1991). The ampli- Griseofl uvin 25 mg fi ed fragment was sequenced with an automatic se- Nalidixic acid 20 mg Aztreonam 40 mg quence analyzer (ABI 3730 DNA Analyzer; Applied ® RPMI1640 500 mg Biosystems, CA, USA) using a BigDye Terminator Eagle Medium 500 mg v3.1 Cycle Sequencing Kit (Applied Biosystems). The L-Glutamine 15 mg CLUSTAL X version 1.83 (Thompson et al., 1997) was NaHCO3 100 mg used for multiple alignment with selected sequences Agar 20 g to calculate evolutionary distances (Kimura, 1980), Distilled water 1 L and similarity values and a phylogenetic tree were 2009 Ilumatobacter fl uminis gen. nov., sp. nov. 203 based on the neighbor-joining method (Saitou and glycolyl. The predominant menaquinone was MK-9

Nei, 1987). The data were resampled with 1,000 boot- (H8), and MK-9 (H0), MK-9 (H2), MK-9 (H4) and MK-9 strap replications (Felsenstein, 1985). The PHYLIP (H6) were detected as minor menaquinones. Mycolic software package (Felsenstein, 1981) was used to cre- acids were not detected. The major cellular fatty acids ate a phylogenetic tree using the maximum-likelihood were iso-C16:0 (17.9%), iso-C17:1ω9c (14.7%), iso-C17:0 method. Sequence similarity was determined by visual (11.3%), iso-C16:1 (7.6%), C17:1ω8c (7.6%), C18:1ω7c comparison and manual calculation. (7.5%), iso-C18:1 (7.2%) and anteiso-C17:0 (3.7%). The Nucleotide sequence accession number. The G+C content of the DNA was 68 mol%. DDBJ accession number for the 16S rRNA gene se- quence of strain YM22-133T is AB360343. Phylogenetic analysis An almost complete 16S rRNA gene sequence Results (1,430 nt) was determined for strain YM22-133T and compared to 16S rRNA sequences deposited in Gen- Morphological, cultural and physiological characteris- Bank using the BLAST program. The validly described tics species that shows the highest similarity to strain Strain YM22-133T grew on Marine Agar 2216 but did YM22-133T is Acidimicrobium ferrooxidans DSM not grow on 1/5 diluted Nutrient Agar (Difco) contain- 10331T. Figure 2 shows a phylogenetic tree construct- ing NaCl (0‒5%). The colonies were round, under ed among genera belonging to a clade of deeply 1 mm in diameter, and colorless. In liquid cultivation, branching lineage within class Actinobacteria and growth did not occur in Nutrient Broth (Difco), 1/5 di- strain YM22-133T was placed in the same branch as luted Nutrient Broth, TSB (Difco) or YD broth (1% yeast Acidimicrobium ferrooxidans. extract, 1% glucose), but did occur in Marine Broth 2216 or MPY2 containing artifi cial sea water. The strain Discussion and Conclusion was aerobic. Cells were rod-shaped, about 0.4‒0.5× 1.3‒1.6 μm (Fig. 1), and non-motile. The strain grew at Although Acidimicrobium ferrooxidans is closest to 26‒31°C and in a pH range of pH 7 to pH 11. strain YM22-133T, the thermotolerant and acidophilic physiological characteristics of this species are quite Chemotaxonomy different from strain YM22-133T. Furthermore, as the The cell-wall peptidoglycan contained LL-diamino- similarity value between them is relatively low (87.1%), pimelic acid (LL-DAP), glycine, alanine and hydroxy- it is clear that YM22-133T belongs to a new taxon. glutamate in a molar ratio of 1.4：2.4：1.1：1.0 ap- Therefore we propose Ilumatobacter fl uminis gen. nov., proximately. The acyl type of muramic acid was sp. nov. for the strain YM22-133T. Genus Ilumatobacter is placed in the position of deeply branching lineage within the class Actinobacteria as shown by the phylo- genetic tree. Though the phylogenetic trees construct- ed by more than one method are often different from each other, it is certain that Acidimicrobium is far from the order Actinomycetales cluster (Rudi et al., 2006). Excepting order Actinomycetales from class Acti- nobacteria, not only are there 24 genera in 8 families but also most genera consist of the sole species al- though there are 6 orders: Acidimicrobiales, Bifi do- bacteriales, Coriobacteriales, Rubrobacterales Soliru- brobacterales and Thermoleophilales (Reddy and Garcia-Pichel, 2009). In other words, very few species exist in the 6 orders. The closest species, Acidimicro-

Fig. 1. Scanning electron micrograph of cells of strain bium ferrooxidans, is also a unique species in genus YM22-133T cultured in MPY2 for 11 days at 27°C. Acidimicrobium, in family and in or- Bar, 1 μm. der Acidimicrobiales. On the basis of these observa- 204 MATSUMOTO et al. Vol. 55

Fig. 2. Phylogenetic tree based on 16S rRNA gene sequences and created using the neighbor-joining method and Knuc values. Only bootstrap values above 50% (percentages after 1,000 replications) are indicated. Solid circles indicate that the corresponding nodes are also recovered in a maximum-likelihood tree. Sphaerobacter thermophilum DSM20745T which was transferred from the phylum Actinobacteria to the phylum Chlorofl exi (Hugenholtz and Stackebrandt, 2004) was used as an outgroup. tions, we fi rmly believe that a large number of living growth characteristics are as given above in the genus bacteria have not yet been found, and we assume that description. The colonies are colorless. The cells are bacteria belonging to these groups will be isolated in about 0.4‒0.5×1.3‒1.6 μm. Sea water or artifi cial sea the near future. Consequently the higher taxon of this water is needed for growth. The temperature range for genus, that is, the family and the order should be dis- growth is 26‒31°C. Growth occurs at pH 7‒11. Alkaline cussed after phylogenetically close strains are added phosphatase, esterase (C4), esterase lipase (C8), leu- by considerable efforts. cine arylamidase, valine arylamidase, cystine arylami- dase, trypsin, chymotrypsin, acid phosphatase, naph- Description of Ilumatobacter gen. nov. thol-AS-BI-phosphohydrolase, α-glucosidase, and Ilumatobacter (I.lu.ma.to.bac’ter. Gr. n. iluma -atos, β-glucosidase are present, but α-galactosidase, sediment deposited in water; N.L. masc. n. bacter, a β-glucuronidase, N-acetyl-β-glucosaminidase, α-man- rod, a bacterium; N.L. masc. n. Ilumatobacter a rod nosidase, and α-fucosidase are absent. The G+C isolated from a sediment). content of the genomic DNA of the type strain is Cells are Gram-positive, aerobic, non-motile. Cells 68 mol%. Habitat is marine. The type strain is YM22- are rod-shape. The cell-wall peptidoglycan contains 133T (=DSM 18936 T =MBIC 08263 T). LL-DAP, glycine, alanine and hydroxyglutamate (molar ratio, ca. 1.4：2.4：1.1：1.0). The acyl type is glyco- Acknowledgments lyl. Predominant menaquinone is MK-9 (H8). Mycolic acids are not detected. The major cellular fatty acids This work was fi nancially supported by the Institute for Fer- mentation, Osaka (IFO), Japan. We thank Professor Jean P. Eu- are iso-C16:0, iso-C17:1ω9c, iso-C17:0, iso-C16:1, C17:1ω8c, zéby for his help with the nomenclature. C18:1ω7c, iso-C18:1 and anteiso-C17:0. The type species is Ilumatobacter fl uminis. References

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