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Int J Syst Evol Microbiol 57 (2007), 1966-1969; DOI  10.1099/ijs.0.64978-0
© 2007 International Union of Microbiological Societies

Loktanella atrilutea sp. nov., isolated from seawater in Japan

Shoichi Hosoya and Akira Yokota

Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032, Japan

Correspondence
Shoichi Hosoya
shouichi.hosoya{at}mbio.jp


   ABSTRACT
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A Gram-negative, rod-shaped bacterium, IG8T, was isolated from seawater off the Sanriku coast, Japan. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain IG8T represented a separate lineage within the genus Loktanella; the highest 16S rRNA gene sequence similarity values were found with the type strains of Loktanella salsilacus (98.6 %) and Loktanella fryxellensis (98.4 %). DNA–DNA hybridization values between strain IG8T and the type strains of L. salsilacus (27.9–36.1 %) and L. fryxellensis (11.3–31.0 %) were clearly below 70 %, the generally accepted limit for species delineation. The DNA G+C content of strain IG8T was 66.3 mol%. On the basis of DNA–DNA hybridization, some biochemical characteristics and 16S rRNA gene sequence comparison, it is proposed that the isolate represents a novel species, Loktanella atrilutea sp. nov. The type strain is IG8T (=IAM 15450T=NCIMB 14280T).

Abbreviations: ASW, artificial seawater

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain IG8T is AB246747.


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The genus Loktanella was first described by Van Trappen et al. (2004)Go and contained three species, Loktanella salsilacus, Loktanella fryxellensis and Loktanella vestfoldensis. Recently, four more species, Loktanella hongkongensis (Lau et al., 2004Go), Loktanella agnita, Loktanella rosea (Ivanova et al., 2005Go) and Loktanella koreensis (Weon et al., 2006Go), have been reported.

In this study, the taxonomic position of strain IG8T, isolated from seawater off Masaki on the Sanriku coast, Iwate Prefecture, Japan, was determined. The seawater sample (0.05 ml) was spread onto a plate containing modified gelatin agar [0.75x artificial seawater (ASW; 1x ASW consists of 3 % NaCl, 0.07 % KCl, 1.08 % MgCl2 . 6H2O, 0.54 % MgSO4 . 7H2O and 0.1 % CaCl2 . 2H2O), 0.4 % gelatin, 0.025 % peptone, 0.025 % yeast extract, 0.001 % FeSO4 . 7H2O, 0.001 % Na2HPO4 and 1 % agar] and incubated at 25 °C for a week. Strain IG8T was purified and maintained at 25 °C on marine agar 2216 (MA; Difco).

16S rRNA gene sequences were obtained by direct sequencing of PCR-amplified DNA as described by Hosoya et al. (2006)Go. The most closely related sequences were found using the BLAST program from the GenBank database (Altschul et al., 1990Go). Multiple alignments were performed by using the CLUSTAL_X program (version 1.83; Thompson et al., 1997Go). Nucleotide substitution rates (Knuc; Kimura, 1980Go) were determined and a phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987Go). Alignment gaps and unidentified base positions were not taken into consideration in the calculation. Bootstrap analysis was based on 1000 trials. The results of the phylogenetic analysis based on 16S rRNA gene sequences showed that strain IG8T belonged to the genus Loktanella (Fig. 1Go). The highest 16S rRNA gene sequence similarity values were found with the type strains of L. salsilacus (98.6 %) and L. fryxellensis (98.4 %); similarity values with the type strains of L. vestfoldensis, L. hongkongensis, L. agnita, L. rosea and L. koreensis were 95.5, 94.6, 94.6, 94.5 and 93.9 %, respectively. L. salsilacus LMG 21507T and L. fryxellensis LMG 22007T, which are the closest relatives of strain IG8T, were obtained from the BCCM/LMG Bacteria Collection (Belgium). Genomic DNA was extracted by the method of Saito & Miura (1963)Go. For analysis of genetic relatedness, DNA–DNA hybridization was carried out at 49 °C for 4 h and measured fluorometrically using the method of Ezaki et al. (1989)Go. Strain IG8T showed relatively low DNA–DNA relatedness values to L. salsilacus LMG 21507T (27.9–36.1 %) and L. fryxellensis LMG 22007T (11.3–31.0 %). These values are significantly lower than that accepted as the phylogenetic definition of a species (Wayne et al., 1987Go). The DNA G+C content was determined by HPLC according to the method of Mesbah et al. (1989)Go. The DNA G+C content of strain IG8T was 66.3 mol%; the DNA G+C contents of the reference strains L. salsilacus and L. fryxellensis are given in Table 1Go.


Figure 1
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  		Fig. 1. Neighbour-joining phylogenetic tree of Loktanella atrilutea and other related genera of the class Alphaproteobacteria based on 16S rRNA gene sequence analysis. Bootstrap values greater than 50 % are given at branch points. Bar, 0.01 nucleotide substitutions per site.

 

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  		Table 1. Differential characteristics of strain IG8T, L. salsilacus LMG 21507T and L. fryxellensis LMG 22007T

Strains: 1, L. atrilutea sp. nov. IG8T; 2, L. salsilacus LMG 21507T; 3, L. fryxellensis LMG 22007T.

 
The following phenotypic characteristics were determined. Cell morphology was observed by using transmission electron microscopy (TEM) after negative staining with phosphotungstic acid. Gram staining was performed using the Hucker staining method as described by Smibert & Krieg (1994)Go. Spore staining was performed using the Schaeffer & Fulton staining method as described by Barrow & Feltham (1993)Go. Salt tolerance was tested on R2A agar (Difco) supplemented with 0–10 % NaCl. In addition, the growth of colonies on nutrient agar (NA; Difco) and trypticase soy agar (TSA; BBL) was tested. Growth at different temperatures (8–37 °C) and pH values (range pH 5.0–9.0), cytochrome oxidase and catalase activities, and degradation of alginate were determined by previously described methods (Hosoya et al., 2006Go). Hydrolysis of DNA was determined on DNA agar (Nissui) supplemented with 0.5x ASW. Degradation of casein was tested on NA supplemented with 0.5x ASW by the method of Smibert & Krieg (1994)Go. Degradation of starch was tested on SP5 medium containing 0.2 % soluble starch by the method of Smibert & Krieg (1994)Go. Hydrolysis of Tweens (20, 40, 60 and 80) was tested on modified Tween 80 medium [containing 0.1 % peptone, 0.5x ASW, 1 % (v/v) Tweens and 2 % agar] according to the method of Barrow & Feltham (1993)Go. Degradation of L-tyrosine was tested on NA containing 0.5 % L-tyrosine, supplemented with 0.5x ASW using the method of Barrow & Feltham (1993)Go. Acid production from carbon sources was assessed using Leifson modified O/F medium incubated for 10 days (Smibert & Krieg, 1994Go). Tests with the commercial API ZYM and API 20E systems (bioMérieux) were generally performed according to the manufacturer's instructions. The API ZYM tests were read after 4 h incubation at 37 °C and API 20E tests were read after 48 h at 25 °C. Cells for inoculation onto the API test strips were suspended in 0.5x ASW. For analysis of cellular fatty acids, cells were grown for 48 h at 25 °C on MA and were analysed using the GC-based Microbial Identification system (MIDI).

Strain IG8T was a Gram-negative, aerobic, rod-shaped, non-sporulating bacterium. Cells were approximately 1.8–2.0 µm long by 0.5–1.0 µm wide and motile by means of a subpolar flagellum (Fig. 2Go). The isolate formed beige-coloured colonies that turned dark orange in the stationary phase. Phenotypic characteristics are given in Table 1Go and in the species description. Phenotypically, strain IG8T, L. salsilacus LMG 21507T and L. fryxellensis LMG 22007T differed from each other in their beta-galactosidase and {alpha}-glucosidase activities and in their ability to produce acid from arabinose, cellobiose, glycerol, inositol, rhamnose, sorbitol and trehalose. The major cellular fatty acid of strain IG8T, L. salsilacus LMG 21507T and L. fryxellensis LMG 22007T was 18 : 1{omega}7c; however, strain IG8T also contained the fatty acid 11-methyl 18 : 1{omega}7c, which was not detected in L. salsilacus or L. fryxellensis (Table 2Go).


Figure 2
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  		Fig. 2. TEM of a negatively stained cell of strain IG8T. Bar, 2 µm.

 

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  		Table 2. Fatty acid profiles of strain IG8T, L. salsilacus LMG 21507T and L. fryxellensis LMG 22007T

Strains: 1, L. atrilutea sp. nov. IG8T; 2, L. salsilacus LMG 21507T; 3, L. fryxellensis LMG 22007T. Values are percentages (w/w) of total fatty acids. –, <1 % total fatty acid or not detected.

 
On the basis of DNA–DNA hybridization, phylogenetic analysis and phenotypic characteristics, strain IG8T is proposed as a novel species of the genus Loktanella, Loktanella atrilutea sp. nov.

Description of Loktanella atrilutea sp. nov.
Loktanella atrilutea (at.ri.lu'te.a. L. adj. ater -tra -trum dark; L. adj. luteus -a -um orange; N.L. fem. adj. atrilutea dark orange).

Cells are Gram-negative, aerobic and rod-shaped, approximately 1.8–2.0x0.5–1.0 µm. Motile by means of a subpolar flagellum. Colonies on MA plates are round and beige-coloured; older colonies are dark orange. Cytochrome oxidase- and catalase-positive. Optimal growth temperature is 25–30 °C; able to grow at 8 °C, but not at 37 °C. The pH range for growth is 6.0–8.0. Growth occurs at NaCl concentrations of 0–8 % on R2A agar, but no growth is observed on NA or TSA. Positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, {alpha}-galactosidase, {alpha}-glucosidase and beta-glucuronidase. Positive for degradation of Tweens (20, 40, 60 and 80). Acid is produced from arabinose, cellobiose, fructose, galactose, glucose, lactose, maltose, mannose, raffinose, rhamnose, sorbitol, sucrose and xylose. Does not decompose alginate, citrate, DNA, starch, tyrosine or urea. Does not reduce nitrate. Does not produce acetoin, H2S or indole. Negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, lipase (C4), valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, beta-galactosidase, beta-glucosidase, N-acetyl-beta-glucosamidase, {alpha}-mannosidase and {alpha}-fucosidase. Does not produce acid from dulcitol, glycerol, inositol, mannitol or trehalose. The major fatty acid is 18 : 1{omega}7c. The DNA G+C content of the type strain is 66.3 mol%.

The type strain, IG8T (=IAM 15450T=NCIMB 14280T), was isolated from seawater off the Sanriku coast, Japan.


   ACKNOWLEDGEMENTS
 
We thank K. Uematsu (Japan Agency for Marine-Earth Science & Technology, JAMSTEC) for operating the electron microscopic system.


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Barrow, G. I. & Feltham, R. K. A. (1993). Cowan and Steel's Manual for the Identification of Medical Bacteria, 3rd edn. Cambridge: Cambridge University Press.

Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.[Abstract/Free Full Text]

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Ivanova, E. P., Zhukova, N. V., Lysenko, A. M., Gorshkova, N. M., Sergeev, A. F., Mikhailov, V. V. & Bowman, J. P. (2005). Loktanella agnita sp. nov., and Loktanella rosea sp. nov., from the north-west Pacific Ocean. Int J Syst Evol Microbiol 55, 2203–2207.[Abstract/Free Full Text]

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