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1 DSMZ German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstr. 7b, 38124 Braunschweig, Germany
2 Helmholtz Centre for Infection Research, Inhoffenstr. 7, 38124 Braunschweig, Germany
Correspondence
Erko Stackebrandt
erko{at}dsmz.de
| ABSTRACT |
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7c. Polyhydroxybutyrate and polyphosphate granules as well as unidentified enterosomes and a polar organelle are visible by electron microscopy. Comparative 16S rRNA gene sequence analysis indicated that the isolates were placed within the class Betaproteobacteria, remotely related to Chitinibacter tainanensis DSM 15459T, Silvimonas terrae KCTC 12358T, Formivibrio citricus DSM 6150T and Iodobacter fluviatilis DSM 3764T. On the basis of phylogenetic and phenotypic distinctness, we propose a novel genus, Deefgea gen. nov., with Deefgea rivuli sp. nov. as the type species. The type strain of Deefgea rivuli is strain WB 3.4-79T (=DSM 18356T=CIP 109326T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain WB 3.4-79T is AM397080.
Results of EELS and a 16S rRNA gene sequence-based maximum-likelihood tree are available as supplementary material in IJSEM Online.
| MAIN TEXT |
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40 %) and many of the slime-producing bacteria were members of the genus Flavobacterium (Brambilla et al., 2007
Strains WB 3.4-79T and WB 3.3-25 were isolated from sampling site 3 (Brambilla et al., 2007
) of the Westerhöfer Bach on the western slopes of the Harz Mountains in Lower Saxony, Germany. Creek water was spread on R2A plates (Difco) which were incubated at 10 or 18 °C for 4 days. Single slimy colonies on the plates were purified by transferring them onto new plates and incubating again under the same conditions. The isolates was cultured routinely on R2A agar at 28 °C and maintained as a glycerol suspension (20 %, w/v) at 70 °C. Strains were preserved in the long term as freeze-dried cultures or in liquid nitrogen.
Ultrastructural analysis
Mid-exponential phase cells of strain WB 3.4-79T were prepared as whole-mount samples either shadow-cast (Fig. 1a, b
) or negatively stained (Fig. 1c
), following a general protocol (Yakimov et al., 1998
; Golyshina et al., 2000
). Cells were also embedded in epoxy resin (Spurr, 1969
) and ultrathin-sectioned. Energy-filtered transmission electron microscopy (EFTEM) was done with a CEM 902 instrument (Zeiss) at 80 kV in the elastic bright-field mode (Lünsdorf et al., 2001
, 2006
).
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Analysis of ultrathin sections showed the cell wall of strain WB 3.4-79T to be outlined by an outer and cytoplasmic membrane (Fig. 1e
), characteristic of Gram-negative bacteria. In contact with the cytoplasmic membrane at the cytoplasmic face, a regularly structured layer was occasionally found, which is isomorphic to polar organelles. These have been described in Sphaerotilus natans (and other bacteria), and have been assumed to play a role in energy supply in this polytrichous monopolarly flagellated bacterium, based on their ATPase and cytochrome oxidase activities (Tauschel, 1985
). Additionally, three morphotypes of intracellular inclusions, polyhydroxybutyrate (phb), polyphosphate (pp) and enterosomes (es) (Fig. 1d, e
), are present. Electron-transparent polyhydroxybutyrate inclusions filled most of the cellular volume and were often torn out during the sectioning process. Polyphosphate granules, intensely stained and small in size, were interspersed as small clusters within the cytoplasm or were associated with the surface of large, electron-dense inclusions, 170200 nm in size. Electron energy loss spectroscopy (EELS) revealed distinctly the presence of phosphorus in both small and large dark cellular inclusions (see Supplementary Fig. S1 in IJSEM Online). EELS also revealed the electron density to be based mainly on uranium, added as a stain during the dehydration process. A closer view of these large inclusions revealed the interior to be filled with a fine particulate matrix that, after low-frequency filtering or fast Fourier transformation, revealed a faint structural order, indicative of residual crystallinity (see Supplementary Fig. S1fh). Residual crystallinity can also be deduced from the polyhedral contours of these inclusions. EELS and high-resolution elemental mapping showed the presence of phosphorus and nitrogen as the main constituents of these inclusions, supplemented by small amounts of calcium (Supplementary Fig. S1be). Nitrogen, as an indicator element of proteins, and the weak structural order of the particulate matrix indicate that these dark inclusions can be considered enterosomes' in the general sense (Cannon et al., 2001
). The presence of a distinct outer proteinaceous shell could not be observed and thus they differ at the structural level from typical polyhedral bodies. Further biochemical and physiological analysis has to be done to differentiate whether these inclusions represent carboxysomes' in a specific sense (Cannon et al., 2001
) or, because of the presence of calcium and phosphate, analogues to the acidocalcisomes' of Agrobacterium tumefaciens (Seufferheld et al., 2003
) or general metabolosomes' (Brinsmade et al., 2005
).
Genomic and phylogenetic characterization
Genomic DNA was extracted using the DNeasy Tissue kit (Qiagen) following the manufacturer's instructions. The 16S rRNA gene was amplified as described by Rainey et al. (1996)
using the primers 1030 forward and 1500 reverse. PCR products were purified with the QIAquick PCR purification kit (Qiagen) and sequenced directly by using the CEQ Dye Terminator cycle sequencing kit and on a CEQ 8000 Genetic Analysis system. 16S rRNA gene sequences were aligned with corresponding sequences from the DSMZ database using the ae2 editor (Maidak et al., 1997
). Evolutionary distances were calculated by the method of Jukes & Cantor (1969)
. A distance analysis dendrogram was reconstructed by the neighbour-joining and maximum-likelihood algorithms (Felsenstein, 1993
) and by the algorithm of De Soete (1983)
. Bootstrap analysis was used to evaluate the tree topology by performing 500 resamplings (Felsenstein, 1985
).
Nearly complete 16S rRNA gene sequences (1508 nt) were obtained from strains WB 3.4-79T and WB 3.3-25. As the two sequences were identical, only that of strain WB 3.4-79T was analysed further. Preliminary comparison against the 16S rRNA gene sequences in GenBank by BLAST (Altschul et al., 1997
) indicated that strain WB 3.4-79T belonged to the ChitinibacterIodobacter branch of the family Neisseriaceae, class Betaproteobacteria. The sequence of strain WB 3.4-79T was then included in the DSMZ database of 16S rRNA gene sequences and similarity values were determined. On the basis of these values, the closest described type strains were Chitinibacter tainanensis BCRC 17254T (93.8 %), Formivibrio citricus DSM 6150T (92.6 %), Silvimonas terrae KCTC 12358T (91.9 %) and Iodobacter fluviatilis ATCC 33051T (92.3 %). The 16S rRNA gene sequence similarity between strain WB 3.4-79T and other members of the order Neisseriales was less than 90 %. Strain WB 3.4-79T and the four type strains indicated above formed a monophyletic clade with a high bootstrap value (99 %), which was in accordance with the different treeing algorithms employed. While strain WB 3.4-79T branched adjacent to Chitinibacter tainanensis BCRC 17254T according to the neighbour-joining and De Soete analyses (Fig. 2
), maximum-likelihood analysis showed strain WB 3.4-79T and Formivibrio citricus DSM 6150T to be phylogenetic neighbours (Supplementary Fig. S2).
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Analyses of chemotaxonomic properties
Except for fatty acid analysis, biomass for chemotaxonomic studies was prepared by growing the strain in shake flasks in marine broth 2216 (Difco) at 28 °C for 2 days. Cultures were checked for purity, harvested by centrifugation and freeze-dried. Polar lipids and isoprenoid quinones were extracted following the procedure of Minnikin et al. (1984)
. The polar lipids were separated by TLC using the standard method (Minnikin et al., 1984
), whereas the quinone extract was filtered and reduced to dryness by a stream of dry nitrogen. The dried preparation was dissolved in 200 µl isopropanol and filtrated using a Dyna Gard 0.2 µl syringe filter. Aliquots (5 µl) were separated by HPLC following a described method (Kroppenstedt & Mannheim, 1989
). Fatty acids of Chitinibacter tainanensis DSM 15459T, Formivibrio citricus DSM 6150T and Silvimonas terrae DSM 18233T were extracted and analysed (Miller, 1982
; Sasser, 1990
) according to the standard protocol of the Microbial Identification System (MIDI Inc., 1999
). As Formivibrio citricus DSM 6150T does not grow anaerobically on TSBA medium, medium DSM 505 (DSMZ, 2001
) was used to obtain biomass for fatty acid extraction. The same protocol was also reported for Silvimonas terrae KCTC 12358T (Yang et al., 2005
). Extracts were analysed by using a Hewlett Packard model HP6890A gas chromatograph equipped with a flame-ionization detector as described by Kämpfer & Kroppenstedt (1996)
.
Ubiquinone 8 was the only isoprenoid quinone present. Phosphatidylethanolamine and phosphatidylglycerol were the major polar lipids. Diphosphatidylglycerol occurred in smaller amounts. The fatty acid composition is compiled in Table 1
.
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-hydroxybutyrate was determined as described by Ostle & Holt (1982)
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The pH range for growth was tested in buffered R2A medium at 28 °C between pH 5.1 and 9.5, with steps of about 0.3 pH. Growth of strain WB 3.4-79T occurred at pH 5.88.5, with an optimum around pH 7.37.6.
Salt tolerance of strain WB 3.4-79T was tested on R2A supplemented with 0.5, 1, 2, 4, 6, 8 and 10 % (w/v) NaCl at 28 °C. Good growth was observed after 3 days incubation at 0.5 %, while growth was weak on agar containing 1 % NaCl. No growth occurred at 2 % NaCl or above.
Tests for chemolithoautotrophic growth were done in mineral medium 81 (DSMZ, 2001
) at 28 °C for 7 days under reduced (H2/N2/CO2/air; 70 : 10 : 10 : 10 by vol.) and aerobic conditions. Hydrogen and oxygen served as electron donor and acceptor, respectively. Growth was positive under both conditions and cells grew to similar densities.
Antibiotic-sensitivity tests were performed by using filter-paper discs containing 15 different antibiotics and concentrations. Discs were placed on R2A plates spread with WB 3.4-79T culture and were then incubated at 28 °C for 3 days. Susceptibility was scored as positive at zone diameters above 13 mm, intermediate susceptibility at 1012 mm and resistance at less than 10 mm. The reactions are indicated in the species description.
Strains WB 3.4-79T and WB 3.3-25 are virtually identical in all taxonomic criteria used. They were isolated from the same site and may be considered clones, enriched on different growth media. The novel taxon can be distinguished from all neighbours by its significantly lower G+C content and in addition from Chitinibacter tainanensis (Chern et al., 2004
) by the lack of chitin and gelatin hydrolysis, lack of growth at 40 °C, lack of C16 : 0 3-OH and C19 : 0 methyl fatty acids and by some phenotypic reactions. It differs from Silvimonas terrae (Yang et al., 2005
) by the lack of hydrolysis of chitin, starch and cellulose, lack of growth at 40 °C, lack of C17 : 0 cyclo and C14 : 0 3-OH fatty acids and in the quantities of C16 : 0 fatty acid and those summed in features 4 and 7. While strains WB 3.4-79T and WB 3.3-25 are facultatively anaerobic, Formivibrio citricus (Tanaka et al., 1991
) is strictly anaerobic. Due to this feature, only the API 20A (anaerobe) tests could be performed on the latter organism. Based on phylogenetic, cultural and physiological distinctness, we propose the genus Deefgea gen. nov. and Deefgea rivuli sp. nov. as new taxa, with strain WB 3.4-79T as the type strain of Deefgea rivuli.
Description of Deefgea gen. nov.
Deefgea [De.ef.ge'a. N.L. fem. n. Deefgea arbitrary name derived from the acronym DFG for Deutsche Forschungsgemeinschaft (German Science Foundation)].
Cells are Gram-negative, rod-shaped (0.70.8x34 µm) and facultatively anaerobic. They occur mostly singly (Fig. 1a
) and are motile by means of a single flagellum (Fig. 1b
) or rarely two polar flagella. Colonies on R2A are circular, convex and pearl white. Catalase and oxidase are positive. Contain polyhydroxybutyrate and polyphosphate granules. The predominant quinone is Q-8. Polar lipids are phosphatidylethanolamine and phosphatidylglycerol. Major cellular fatty acids (>10 %) are C16 : 0 and C16 : 1
7c. The G+C content of the DNA is 49 mol%. Based on 16S rRNA gene sequence analysis, Deefgea belongs to the class Betaproteobacteria, family Neisseriaceae, showing a distant relatedness to Chitinibacter tainanensis, Silvimonas terrae and Formivibrio citricus. The type species is Deefgea rivuli.
Description of Deefgea rivuli sp. nov.
Deefgea rivuli (ri'vu.li. L. gen. masc. n. rivuli of/from a rivulet, a small brook).
In addition to the characters that define the genus, it has the following characteristics. Cells have one round end and one pointed end; in older cultures, larger cells (47 µm) are occasionally present. Cells are highly motile at temperatures below 10 °C. Colonies on R2A agar are 12.5 mm in diameter, opaque with a smooth, shiny surface and butyrous with entire margins. Similar characteristics define colonies formed on Columbia agar (greywhite, umbonate, 3.24 mm in diameter), H3P (creamwhite, 0.50.7 mm) and BTS (creamwhite, 1.02.2 mm). Unspecified enterosomes and a polar organelle are present in the cytoplasm. No acid production in OF test under oxic or anoxic conditions. Grows at 4 °C but not above 32 °C. No growth above 2 % NaCl (w/v). pH optimum is between pH 7.3 and 7.6. Grows chemolithoautotrophically under aerobic and reduced conditions. Positive in API 20NE strips for reduction of nitrate to nitrite and utilization of glucose, N-acetylglucosamine and gluconate (all substrates utilized weakly). Tests positive in API 20E strips for acidification of glucose (only transiently after 24 h; realkalinization after 48 h) and acid production from sucrose. Substrates utilized in API 50CH with AUX medium are ribose, glucose, fructose, N-acetylglucosamine, sucrose and gluconate. Substrates positive in Biolog plates are dextrin, N-acetylglucosamine, D-fructose and
-D-glucose; weakly positive for D-mannose, methylpyruvate and
-hydroxybutyric acid. No degradation of chitin, cellulose, casein, starch or xylan. Resistant (per disc) to penicillin G (10 IU), oxacillin (5 µg), ampicillin (10 µg), cephalothin (30 µg), cefotaxime (30 µg), pipemidic acid (20 µg), erythromycin (15 µg), vancomycin (30 µg) and lincomycin (15 µg); intermediately susceptible to norfloxacin (10 µg), ofloxacin (5 µg) and colistin (10 µg); susceptible to tetracycline (30 µg), chloramphenicol (30 µg), imipenem (10 µg) and doxycycline (30 µg).
The type strain, WB 3.4-79T (=DSM 18356T=CIP 109326T), was isolated from water of the Westerhöfer Bach, near Westerhof, Lower Saxony, Germany (51° 45' 49'' N 10° 05' 31.7'' E).
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