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Int J Syst Evol Microbiol 56 (2006), 389-392; DOI  10.1099/ijs.0.63905-0
© 2006 International Union of Microbiological Societies

Janibacter anophelis sp. nov., isolated from the midgut of Anopheles arabiensis

Peter Kämpfer1, Olle Terenius2, Jenny M. Lindh2 and Ingrid Faye2

1 Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, IFZ–Heinrich-Buff-Ring 26–32, D-35392 Giessen, Germany
2 Department of Genetics, Microbiology and Toxicology, Stockholm University, S-106 91 Stockholm, Sweden

Correspondence
Peter Kämpfer
peter.kaempfer{at}agrar.uni-giessen.de


   ABSTRACT
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A Gram-positive, aerobic, non-motile strain, H2.16BT, isolated from the midgut of the mosquito Anopheles arabiensis was investigated using a polyphasic approach. On the basis of 16S rRNA gene sequence similarity studies, strain H2.16BT was shown to belong to the genus Janibacter, being most closely related to Janibacter melonis (98·3 %), Janibacter terrae (98·5 %) and Janibacter limosus (98·5 %). Chemotaxonomic data (meso-diaminopimelic acid as the diagnostic diamino acid in the cell wall and major fatty acids of iso-C16 : 0, C17 : 1{omega}8c and C17 : 0) supported the allocation of the strain to the genus Janibacter. The results of DNA–DNA hybridization and physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strain H2.16BT from closely related species. Thus, H2.16BT represents a novel species of the genus Janibacter, for which the name Janibacter anophelis sp. nov. is proposed. The type strain is H2.16BT (=CCUG 49715T=CIP 108728T).

Published online ahead of print on 7 October 2005 as DOI 10.1099/ijs.0.63905-0.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Janibacter anophelis sp. nov. H2.16BT is AY837752.


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The genus Janibacter was originally proposed by Martin et al. (1997)Go as a member of the family Intrasporangiaceae. At present, the genus comprises three species, Janibacter limosus (Martin et al., 1997Go), Janibacter terrae (Yoon et al., 2000Go) and Janibacter melonis (Yoon et al., 2004Go). Janibacter brevis, originally described by Imamura et al. (2000)Go, was shown to be a heterotypic synonym of J. terrae (Lang et al., 2003Go).

Strain H2.16BT was isolated during the characterization of organisms from the midgut of Anopheles arabiensis mosquitoes originating from Kenya (Lindh et al., 2005Go). Subcultivation was done on nutrient agar (Oxoid) at 30 °C for 24 h. Gram-staining (Hucker method) was performed as described by Gerhardt et al. (1994)Go. Cell morphology was observed under a Zeiss light microscope at x1000 using cultures that had been grown for 10 h or for 3 days at 30 °C on nutrient agar. The growth ability test was performed in Luria–Bertani broth at 10–50 °C (at 5 °C intervals) with shaking at 160 r.p.m.

The 16S rRNA gene was analysed as described previously (Kämpfer et al., 2003Go). Phylogenetic analysis was performed by using the ARB (Ludwig et al., 2004Go) and MEGA version 2 (Kumar et al., 2001Go) software packages, after multiple alignment of the data by CLUSTAL_X (Thompson et al., 1997Go). Distances were obtained (using distance options according to the Kimura two-parameter model; Kumar et al., 2001Go) and clustering was performed using the neighbour-joining (Fig. 1Go) and maximum-parsimony methods by using bootstrap values based on 1000 replications. The 16S rRNA gene sequence of strain H2.16BT was a continuous stretch of 1483 bp. Sequence similarity calculations after neighbour-joining analysis indicated that the closest relatives of strain H2.16BT were J. melonis (98·3 %), J. terrae (98·5 %) and J. limosus (98·5 %). Both the neighbour-joining tree (Fig. 1Go) and the maximum-parsimony tree (not shown) revealed that strain H2.16BT clustered most closely with these species.


Figure 1
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  		Fig. 1. Phylogenetic analysis, based on 16S rRNA gene sequences available from the EMBL database (accession numbers are given in parentheses), constructed after multiplealignment of data by CLUSTAL_X (Thompson et al., 1997Go). Distances were obtained with distance options according to the Kimura-2 model and clustering was performedwith the neighbour-joining method using the software package MEGA, version 2.1 (Kumar et al., 2001Go). Bootstrap values based on 1000 replications are given as percentages at branching points. Bar, 0·01 Knuc unit.

 
The results of chemotaxonomic analyses of diamino acid (as described by Kroppenstedt, 1985Go) and fatty acids (as described by Kämpfer & Kroppenstedt, 1996Go) are given in the species description. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid, essentially in accordance with the diamino acid type reported for other members of the genus (Martin et al., 1997Go; Yoon et al., 2000Go, 2004Go). The fatty acids (analysed from total cell hydrolysates grown on TSBA for 24 h at 28 °C) contained large amounts of iso-C16 : 0, C17 : 1{omega}8c and C17 : 0, in addition to other fatty acids (Table 1Go). The results of the physiological characterization, performed using previously described methods (Kämpfer et al., 1991Go), are given in Table 2Go and in the species description. DNA–DNA hybridizations between strain H2.16BT and the type strains of J. limosus, J. terrae and J. melonis were performed using the method described by Ziemke et al. (1998)Go, except that for nick translation 2 µg DNA was labelled during a 3 h incubation at 15 °C. Strain H2.16BT showed relatively low DNA–DNA relatedness to J. melonis DSM 16063T (12·0 %; reciprocal reaction, 9·8 %), J. terrae DSM 13876T (13·7 %; reciprocal reaction, 32·4 %) and J. limosus DSM 11140T (34·0 %; reciprocal reaction, 19·2 %). For these reasons, it is clear that strain H2.16BT represents a novel species of the genus Janibacter, for which we propose the name Janibacter anophelis sp. nov.


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  		Table 1. Fatty acid content of Janibacter anophelis H2.16BT and other Janibacter species

Taxa: 1, Janibacter anophelis H2.16BT; 2, J. terrae DPO strains (n=10); 3, J. terrae DPO 360 (=DSM 11214); 4, J. brevis DSM 13953T; 5,J.terrae DSM 13876T; 6, J. limosus DSM 11140T; 7, J. limosus DSM 11141; 8, J. melonis CM2104T. Data for taxa 2–7 are from Lang etal. (2003)Go; data for J. melonis CM2104T are from Yoon et al. (2004)Go. Values are percentages of total fatty acid content. Fatty acids C17 : 1{omega}6c, 10-methyl C16 : 0 and 10-methyl C19 : 0 were not present in any of the strains tested.

 

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  		Table 2. Biochemical properties of Janibacter anophelis H2.16BT and the type strains of other Janibacter species

Taxa: 1, Janibacter anophelis H2.16BT; 2, J. terrae DSM 13876T; 3, J. limosus DSM 11140T; 4, J. melonis CM2104T. All data are from this study. W, Weak reaction; D, delayed reaction.

 
Description of Janibacter anophelis sp. nov.
Janibacter anophelis (a.no'phe.lis. N.L. gen. n. anophelis from a mosquito of the genus Anopheles, as the type strain was isolated from the midgut of Anopheles arabiensis).

Gram-positive and oxidase-positive; shows oxidative metabolism. Non-motile, non-endospore-forming cocci of 1·0–1·5 µm in diameter. Very young cultures show rod-like cells. Good growth (visible colonies with a diameter >1 mm) occurs after 3 days incubation on nutrient agar at 25–30 °C. Grows in Luria–Bertani broth at 20–40 °C, with an optimum at 35 °C. Generation time at 35 °C is 44 min. meso-Diaminopimelic acid is the diagnostic diamino acid in the cell-wall peptidoglycan. The fatty acid content is mainly iso-C16 : 0, C17 : 1{omega}8c and C17 : 0. Different 10-methyl acids are present. Results of carbon source utilization tests (including differentiating characteristics) are shown in Table 2Go. The type strain does not produce acids from the following sugars: glucose, lactose, sucrose, D-mannitol, dulcitol, salicin, adonitol, inositol, sorbitol, L-arabinose, raffinose, L-rhamnose, maltose, D-xylose, trehalose, cellobiose, methyl D-glucoside, erythritol, melibiose or arabitol. The following carbon sources are utilized as a sole source of carbon (after 48 h incubation): D-gluconate, D-glucose, D-maltose, D-mannose, sucrose, D-trehalose, i-inositol, acetate, propionate, 4-aminobutyrate, fumarate, glutarate, 3-hyroxybutyrate, DL-lactate, L-malate, pyruvate, L-alanine, L-aspartate, L-histidine, L-leucine, L-proline and L-serine. The following carbon sources are not utilized: N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, L-arabinose, p-arbutin, D-cellobiose, D-fructose, D-galactose, melibiose, L-rhamnose, D-ribose, salicin, D-xylose, adonitol, maltitol, D-mannitol, sorbitol, putrescine, cis-aconitate, trans-aconitate, adipate, L-azelate, citrate, itaconate, mesaconate, 2-oxoglutarate, L-suberate, beta-alanine, L-ornithine, L-phenylalanine, L-tryptophan, DL-3-hydroxybenzoate, DL-4-hydroxybenzoate or L-phenylacetate.

The type strain, H2.16BT (=CCUG 49715T=CIP 108728T), was isolated from the midgut of Anopheles arabiensis from Kenya.


   ACKNOWLEDGEMENTS
 
We thank Jean Euzéby for his help with the etymology of the species epithet.


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Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors) (1994). Methods for General and Molecular Bacteriology. Washington, DC: American Society for Microbiology.

Imamura, Y., Ikeda, M., Yoshida, S. & Kuraishi, H. (2000). Janibacter brevis sp. nov., a new trichloroethylene-degrading bacterium isolated from polluted environments. Int J Syst Evol Microbiol 50, 1899–1903.[Abstract]

Kämpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of fatty acid patterns of coryneform bacteria and related taxa. Can J Microbiol 42, 989–1005.

Kämpfer, P., Steiof, M. & Dott, W. (1991). Microbiological characterisation of a fuel-oil contaminated site including numerical identification of heterotrophic water and soil bacteria. Microb Ecol 21, 227–251.

Kämpfer, P., Dreyer, U., Neef, A., Dott, W. & Busse, H.-J. (2003). Chryseobacterium defluvii sp. nov., isolated from wastewater. Int J Syst Evol Microbiol 53, 93–97.[Abstract/Free Full Text]

Kroppenstedt, R. M. (1985). Fatty acid menaquinone analysis of actinomycetes and related organisms. In Chemical Methods in Bacterial Systematics, pp. 173–199. Edited by M. Goodfellow & D. E. Minnikin. London: Academic Press.

Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17, 1244–1245.[Abstract/Free Full Text]

Lang, E., Kroppenstedt, R. M., Swiderski, J., Schumann, P., Ludwig, W., Schmid, A. & Weiss, N. (2003). Emended description of Janibacter terrae, including ten dibenzofuran-degrading strains and Janibacter brevis as its later heterotypic synonym. Int J Syst Evol Microbiol 53, 1999–2005.[Abstract/Free Full Text]

Lindh, J. M., Terenius, O. & Faye, I. (2005). 16S rRNA gene-based identification of midgut bacteria from field-caught Anopheles gambiae sensu lato and A. funestus mosquitoes reveals new species related to known insect symbionts. Appl Environ Microbiol 71, 7217–7223.[Abstract/Free Full Text]

Ludwig, W., Strunk, O., Westram, R. & 29 other authors (2004). ARB: a software environment for sequence data. Nucleic Acids Res 32, 1363–1371.[Abstract/Free Full Text]

Martin, K., Schumann, P., Rainey, F. A., Schuetze, B. & Groth, I. (1997). Janibacter limosus gen. nov., sp. nov., a new actinomycete with meso-diaminopimelic acid in the cell wall. Int J Syst Bacteriol 47, 529–534.[Abstract/Free Full Text]

Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.[Abstract/Free Full Text]

Yoon, J.-H., Lee, K.-C., Kang, S.-S., Kho, Y. H., Kang, K. H. & Park, Y.-H. (2000). Janibacter terrae sp. nov., a bacterium isolated from soil around a wastewater treatment plant. Int J Syst Evol Microbiol 50, 1821–1827.[Abstract]

Yoon, J.-H., Lee, B. H., Yeo, S.-H. & Choi, J.-E. (2004). Janibacter melonis sp. nov., isolated from abnormally spoiled oriental melon in Korea. Int J Syst Evol Microbiol 54, 1975–1980.[Abstract/Free Full Text]

Ziemke, F., Höfle, M. G., Lalucat, J. & Rosselló-Mora, R. (1998). Reclassification of Shewanella putrefaciens Owen's genomic group II as Shewanella baltica sp. nov. Int J Syst Bacteriol 48, 179–186.[Abstract/Free Full Text]




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