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1 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7b, 30124 Braunschweig, Germany
2 Mugla University, Egitim Fakültesi, TR-48170 Kötekli, Mugla, Turkey
Correspondence
Elke Lang
ela{at}dsmz.de
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ABSTRACT |
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7c. Strain NS11T also contained high proportions of C10 : 0 3-OH and C18 : 17c. This pattern is typical for members of the genus Herminiimonas. The results of DNA–DNA hybridization experiments and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain NS11T from the three recognized Herminiimonas species. It is therefore concluded that strain NS11T represents a novel species of the genus Herminiimonas, for which the name Herminiimonas saxobsidens sp. nov. is proposed. The type strain is NS11T (=DSM 18748T=CCM 7436T).
A figure showing the diversity of normalized ribotype patterns and a dendrogram showing the fatty-acid relationships among the type strains of species of the genus Herminiimonas are available as supplementary material with the online version of this paper.
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MAIN TEXT |
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). Two additional species in the genus, Herminiimonas aquatilis isolated from drinking water (Kämpfer et al., 2006) and Herminiimonas arsenicoxydans isolated from arsenic-contaminated activated sludge (Muller et al., 2006), were described shortly thereafter. In contrast to the aqueous habitat of these three species, strain NS11T, determined herein to represent a novel species of the genus Herminiimonas, was isolated from the lichen–rock interface of a limestone bedrock colonized by lichen at Mugla, Turkey, from enrichment cultures with potassium oxalate as the sole source of carbon and energy (Sahin et al., 2002). The lichen–rock interface contains a zone with high oxalate content. Oxalic acid produced by the lichen communities accelerates weathering of the rock by solubilizing the cement between the rock grains (Johnston & Vestal, 1993).
The following DSMZ strains were used as reference: H. aquatilis DSM 18803T, H. arsenicoxydans DSM 17148T and H. fonticola DSM 18555T. Strain NS11T was grown routinely on nutrient agar (per litre: 5 g peptone, 3 g beef extract, 15 g agar; Difco), and the type strains of the three recognized Herminiimonas species on R2A medium (Difco; Reasoner & Geldreich, 1985) at 28 °C.
From enrichment cultures with 4 g potassium oxalate l–1 as the sole source of carbon and energy in mineral medium (Aragno & Schlegel, 1992), a Gram-negative, rod-shaped bacterium, designated strain NS11T, was isolated (Sahin et al., 2002). Cells were motile, non-sporulating and strictly aerobic. Colonies of strain NS11T were cream coloured and convex. Addition of oxalate (2 g l–1) to nutrient agar did not enhance growth.
16S rRNA gene sequences were produced and aligned as described by Somvanshi et al. (2006). Phylogenetic dendrograms were constructed by using the neighbour-joining algorithm (De Soete, 1983). Analysis of the almost-complete 16S rRNA gene sequence of strain NS11T grouped it within the family Oxalobacteraceae. Highest sequence similarities were found with members of the genus Herminiimonas (Fig. 1), namely with H. arsenicoxydans ULPAs1T (98.8 %), H. aquatilis CCUG 36956T (98.0 %) and H. fonticola S-94T (98.0 %).
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). The fragment patterns of strain NS11T and of the type strains of the three recognized Herminiimonas species varied, resulting in a unique pattern for each of the strains (see Supplementary Fig. S1 in IJSEM Online). This dissimilarity between the ribopatterns for strain NS11T and reference Herminiimonas type strains excludes the possibility that strain NS11T is affiliated to any of the three recognized Herminiimonas species.
For DNA–DNA hybridization experiments, DNA was isolated using a French pressure cell (Thermo Spectronic) and was purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). Hybridization was carried out in SSC buffer at 69 °C as described by De Ley et? al. (1970), with the modifications given by Huß et al. (1983), by using a model Cary 100 Bio Uv/vIS spectrophotometer equipped with a Peltier-thermostatted 6x6 multicell changer and a temperature controller with in situ temperature probe (Varian). The level of DNA–DNA relatedness between strain NS11T and H. arsenicoxydans DSM 17148T, sharing a 16S rRNA gene sequence similarity of 98.8 %, was 22.4 %, which confirms that strain NS11T does not belong to the genospecies H. arsenicoxydans. The level of DNA–DNA relatedness between H. aquatilis CCUG 36956T and H. fonticola S-94T, sharing a 16S rRNA gene sequence similarity value of 99.3 %, was similarly low (25.7 %) (Kämpfer et al., 2006). These low hybridization values support a previous report of the lack of high levels of DNA–DNA reassociation even at 16S rRNA gene sequence similarities as high as around 99 % (Stackebrandt & Ebers, 2006).
For analysis of fatty acids, cells were grown on R2A agar for 48 h at 28 °C. This growth medium, rather than the trypticase soy agar recommended for analysis according to the MIDI system, was used as strain NS11T and the Herminiimonas reference strains did not grow well on the latter medium. Fatty acid methyl esters were obtained by saponification, methylation and extraction as described by Kämpfer & Kroppenstedt (1996) and separated by GC (model 5898A; Hewlett Packard). Peaks were automatically integrated and fatty-acid components and their proportions were determined by using the Microbial Identification standard software package MIDI (Sasser, 1990). Fatty acids of strain NS11T were dominated by C16 : 0 (36.4 %), C17 : 0 cyclo (22.3 %) and C16 : 17c (18.7 %) (Table 1). The presence of C10 : 0 3-OH as a significant component was also characteristic. The fatty acid patterns generated in the present study only partially agree with those provided in the species descriptions previously given for H. aquatilis, H. fonticola and H. arsenicoxydans. Although some components differed slightly only in quantity, a significant deviation was the absence of C17 : 16c in H. aquatilis DSM 18803T, which is described to be present in H. aquatilis CCUG 36956T by Kämpfer et al. (2006). C17 : 16c is absent in four H. fonticola strains (Fernandes et al., 2005) and in H. arsenicoxydans ULPAs1T (Muller et al., 2006). Possibly, the choice of the growth medium and age of cells used has influenced the fatty-acid composition. A dendrogram of Euclidian distances depicts the separate position of strain NS11T, confirming the result of 16S rRNA gene sequence analysis and riboprinting, i.e. the distinct position of strain NS11T among the type strains of recognized Herminiimonas species (see Supplementary Fig. S2 in IJSEM Online).
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). API 20NE, API ZYM and API 50 CH strips (bioMérieux) were used according to the manufacturer's instructions, except that nitrate reduction and indole production from tryptophan were read after 2 days, whereas other reactions of the API 20NE and API 50 CH strips were observed after 7 days. Utilization and assimilation of carbohydrates was determined on API 50 CH strips with modified AUX medium in which growth factors and amino acids were replaced by 0.1 g yeast extract l–1. Biolog GN plates (AES) were incubated for 48 h before being read. Cavities showing a photometric value above 20 or 100 were scored as weak or positive, respectively.
Physiologically, strain NS11T was characterized by poor reactivity. Several organic acids but no carbohydrates or sugar alcohols were metabolized in Biolog GN plates (Table 2). None of the carbohydrates offered in the API 20NE or API 50 CH strips was utilized. The enzyme reactions found for strain NS11T with API ZYM strips as given in the species description below are in good agreement with those reported for strains of H. fonticola (Muller et al., 2006). These characteristics of strain NS11T are generally in accordance with those described for the three recognized Herminiimonas species and differentiate these species from members of the closely related genus Janthinobacterium, which utilize carbohydrates (Lincoln et al., 1999). Nevertheless, strain NS11T was more versatile than the type strains of the three recognized Herminiimonas species in the Biolog GN plates. Under the given conditions, strain NS11T was able to assimilate, among others, acetate, DL-malate, propionate and succinate. Utilization of these organic acids by strain NS11T as the sole source of carbon and energy was confirmed in mineral medium (Table 2).
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and Kämpfer et al. (2006) for the respective species reveals that results are reproducible from laboratory to laboratory if the same method (microplates or mineral medium in tubes) is applied.
The specific growth rate (µ) of strain NS11T with oxalate as the sole source of carbon and energy was 0.112 h–1 (td=6.2 h) at 4 g potassium oxalate l–1 and 30 °C. No growth was observed in the presence of 20 g potassium oxalate l–1. Resistance to antibiotics as well as resistance to heavy metal ions were determined as described by Sahin et al. (2002). The results are given in the species description below. Both strain NS11T and H. arsenicoxydans were tolerant to heavy metals, although different metal elements were tested for the two taxa (Sahin et al., 2002; Muller et al., 2006).
The inability to utilize carbohydrates, the utilization of short-chain organic acids and the fatty-acid profile were in agreement with the placement of strain NS11T within the genus Herminiimonas as suggested by the phylogenetic analysis. On the other hand, some traits distinguished strain NS11T from the type strains of the three recognized species in the genus Herminiimonas (assimilation reactions with acetic acid, propionic acid, 
-hydroxybutyric acid, succinic acid, bromosuccinic acid, succinamic acid; utilization of malate but not of citrate; and lack of C14 : 0 fatty acid). Therefore, strain NS11T is considered to represent a novel species of the genus Herminiimonas, for which the name Herminiimonas saxobsidens sp. nov. is proposed.
Description of Herminiimonas saxobsidens sp. nov.
Herminiimonas saxobsidens (sax.ob'si.dens. L. n. saxum rock; L. v. obsideo to occupy; N.L. part. adj. saxobsidens rock-occupying).
Cells are Gram-negative, small ovoid rods 0.8x0.4 µm, motile by means of polar flagella. Cells occur singly or in pairs. No spores are found. Non-pigmented. Forms round, translucent, cream-coloured, convex colonies with flat margins, reaching 1.5 mm in diameter on nutrient agar after 3 days incubation. Growth occurs at 4–37 °C. Optimum growth occurs at pH 7.0–7.5. Weak growth occurs in media containing 2 % NaCl. No acid is produced from glucose. Oxidase- and catalase-positive. Nitrate is reduced to nitrite but not further to dinitrogen. Alkaline phosphatase, C4-esterase, C8-esterase lipase, leucine arylamidase, trypsin and phosphohydrolase are produced. Negative for indole production, arginine dihydrolase, urease, aesculin, casein and gelatin hydrolysis, and 
-galactosidase. Has a very limited substrate spectrum. Does not utilize carbohydrates or polyols. Utilizes acetate, propionate, oxalate, succinate and malate. Does not utilize adipate, citrate, gluconate, caprate, malonate or ethanol. Predominant fatty acids are C16 : 0, C17 : 0 cyclo and C16 : 17c; C18 : 17c and C10 : 0 3-OH are present in smaller amounts. Does not contain C14 : 0. Resistant to ampicillin, bacitracin and streptomycin (10 µg per disc each), but susceptible to erythromycin (15 µg), chloramphenicol (30 µg) and gentamicin (10 µg). Sensitive to HgCl2 (2.5 µg per disc), but resistant to ZnSO4 . 7H2O, NiCl2 . 6H2O, CoCl2 . 6H2O, CuSO4 . 5H2O, lead acetate and K2CrO7 (2.5 µg per disc each).
The type strain, NS11T (=DSM 18748T=CCM 7436T), was isolated from limestone covered by lichen after enrichment with oxalate in mineral medium.
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ACKNOWLEDGEMENTS |
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