INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

Some morphologically conspicuous bacteria were observed in the 19th century but still have not been isolated in pure culture. In 1892, Famintzin (7) described Nevskia ramosa from the water surface of an aquarium in the botanical garden of St. Petersburg, Russia. The typical microcolonies consist of flat rosettes with a bush-like appearance on the water surface. The rosettes are colonies of dichotomously branched slime stalks with rod-shaped, slightly bent cells in the tips. The cells contain refractile globules, which were presumed to be ethereal oil (7), sulfur globules (12), or fat droplets (3). The slime stalks consist of polysaccharides (3) and sometimes appear to contain iron and aluminum encrustations (11).

Enrichments of Nevskia-like cells in lake water supplied with lactate were described by Babenzien (1-4). He observed the following life cycle of N. ramosa. Young motile cells develop submersed, then adsorb to the water surface, lose the polar flagellum, and form a hyaline slime stalk on the concave side of the cell. When a cell multiplies by binary fission, branching of the stalk occurs. The resulting flat rosette can reach a size of 70 µm in diameter.

Since pure cultures have not been available, little is known about the physiology, phylogeny, and ecology of Nevskia. It was assumed that Nevskia is oligocarbophilic (14). Tests with the nitrification inhibitor nitrapyrin gave no indications that the cells oxidize ammonia (16). N. ramosa was assumed to be related to the stalk-forming genera Caulobacter and Gallionella or to the sulfur-oxidizing Thiobacterium. In Bergey's manual (4) N. ramosa was affiliated with the budding and/or appendaged bacteria.

In addition to its conspicuous morphology, the typical habitat of N. ramosa prompted us to initiate the present investigation. The water-air interface is a very special environment, characterized by high surface tension and a relatively high hydrophobicity. Organic compounds and various typical bacteria are enriched in this zone. The living community is called the neuston (18, 21). Depending on whether they adsorb to the underside or the top of the water surface, organisms belong to the hyponeuston or epineuston, respectively. This habitat requires special adaptations with respect to adsorption, substrate uptake, and UV tolerance.

In our study we have isolated N. ramosa in pure culture and carried out ecophysiological and phylogenetic characterizations. We found several adaptions to life in the epineuston in this interesting bacterium.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Sources of inoculum. Samples were taken with a sterile loop needle from surface films of an enrichment culture from Lake Soelkensee, a small bog lake near Greifswald (Germany), which had been subcultivated for over 30 years (1). Additional sampling was done in a ditch near our institute in Oldenburg and from a watering can containing stagnant tap water.

Media and cultivation. Media for enrichments were prepared from filtered (Nalgene polycarbonate filter; 0.2-µm pore size) and autoclaved surface water (pH 5.9) from a bog lake near Oldenburg (Lake Theikenmeer), supplemented with 5 mM sodium lactate. Pure cultures were cultivated as surface films in a synthetic medium of the following composition: sodium lactate, 5 mM; MgSO₄ · 7H₂O, 0.2 mM; CaCl₂ · 2H₂O, 0.1 mM; KH₂PO₄, 25 mM; trace element solution SL 9 (26), 0.5 ml · liter¹; and vitamin solution (20), 0.5 ml · liter¹. The pH was adjusted to 7.0. To obtain submersed cultures, the medium was supplemented with 5 mM NH₄Cl and 10 mM lactate. The cultures were incubated at room temperature without shaking.

Isolation of pure cultures. From enrichments, pure cultures were obtained by repeated streaking of diluted samples on agar plates (1.0% [wt/vol] Difco agar) with lake water medium. Purity was proven microscopically and by 16S ribosomal DNA (rDNA) analysis. Stock cultures were deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) (Braunschweig, Germany) as N. ramosa Soe1, DSMZ 11499^T, and N. ramosa OL1, DSMZ 11500.

UV tolerance experiments. Freshly grown submersed cultures were diluted to approximately 10⁸ cells · ml¹ and plated on agar plates with medium containing 10 mM lactate and 5 mM NH₄Cl. Open petri dishes were exposed to UV radiation from a transilluminator (wavelength, 254 nm; high dosage; 8 W; six tubes; distance, 25 cm) (Herolab, Wiesloch, Germany). After UV exposure, one series was incubated in the dark and the other was exposed to light at 350 nm for 1 h (portable lamp; 6 W; distance, 25 cm) (Vetter, Wiesloch, Germany).

Analytical methods. Cytochromes were identified by a redox difference spectrum (UV/Vis spectrophotometer, model Lambda 2S; Perkin-Elmer, Überlingen, Germany). Gram staining and analysis of catalase and cytochrome oxidase activities were done as described by Süßmuth et al. (25). The hydrophobicity of the surface film was determined qualitatively by sprinkling fine droplets of distilled water over it. Nitrogenase was analyzed by means of the acetylene reduction test, using a culture of Azotobacter vinelandii as a positive control (23). The composition of the exopolysaccharide slime capsules was kindly determined by Ingmar Janse (University of Groningen, Groningen, The Netherlands).

Molecular biological techniques. Extraction, purification, PCR amplification, and sequencing of the 16S rDNA; construction of a phylogenetic tree; and comparison of enterobacterial repetitive intergenic consensus (ERIC)-PCR patterns were performed as described by Overmann and Tuschak (19). The guanine-plus-cytosine content of the DNA was determined by the DSMZ.

Nucleotide sequence accession numbers. The two 16S rDNA sequences obtained have been deposited with EMBL (accession no. AJ001010 and AJ001011 for strains Soe1 and OL1, respectively).

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Enrichments and isolation of pure cultures. Enrichments of Nevskia-like microcolonies on the surface of lactate-supplemented lake water medium developed from various sources within 3 weeks. The development of a microcolony with 25 cells in a drop of enrichment culture hanging under a sealed coverslip is shown in Fig. 1. The cells had doubling times of 2 to 4 days (Fig. 1A to D). The flat rosettes of the various enrichments showed slightly different shapes but always consisted of dichotomously branched slime stalks with single cells in the tips (Fig. 1E). On agar plates, no typical flat rosettes were formed. Instead, geometrical patterns of slime-coated cells were observed (Fig. 1F). After repeated streaking on agar, pure cultures were obtained from the Lake Soelkensee enrichment (strain Soe1 [Fig. 1G]) and from the ditch in Oldenburg (strain OL1 [Fig. 1H]). These cultures formed the typical flat rosettes if they were returned to ammonia-free liquid medium with lactate.

View larger version (149K): [in this window] [in a new window]   FIG. 1.   Phase-contrast photomicrographs of N. ramosa. (A to D) A drop of an enrichment culture hanging under a sealed coverslip was incubated at room temperature for 2 (A), 5 (B), 6 (C), and 10 (D) days. (E) An enrichment of flat rosettes of Nevskia-like cells from stagnant water in a watering can. (F) Geometrical patterns often formed on agar plates. (G) Safranine-stained flat rosettes of N. ramosa Soe1. (H) Pure culture of strain OL1. Bars, 25 µm (A to D and F to H) and 10 µm (E).

Physiological characterization. Cells of N. ramosa strains were slightly bent rods that stained gram negative. Cells of strain Soe1 had a size of 0.7 to 1.1 by 1.5 to 2.3 µm; cells of strain OL1 had a size of 1.0 by 2.5 to 5.5 µm. Often the cells contained two to five refractile globules, probably polyhydroxyalkanoates (4). The exopolysaccharide of the stalks consisted mainly of rhamnose, with small amounts of glucose and mannose. The strains showed weak catalase activity and no cytochrome oxidase activity. Cytochromes of the b and c types were present. Both isolates could be grown on a synthetic medium. They had a strictly aerobic metabolism and grew with a broad range of organic compounds (Table 1).

Adaptations to life at the air-water interface. As indicated by the dull appearance of the surface films, N. ramosa is a member of the epineuston. Droplets of distilled water which were sprayed on the film remained visible for at least a minute, indicating a high hydrophobicity.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Effect of UV radiation without () and with () exposure to light at 350 nm for 1 h after UV treatment. (A) N. ramosa OL1; (B) E. coli K-12. The counts (± standard deviations) were obtained from duplicate plates.

Phylogenetic relatedness and DNA base composition. The 16S rDNA sequences of the isolates showed 99.2% similarity (Table 2). Genomic fingerprinting by ERIC-PCR showed different banding patterns on an agarose gel (Fig. 3). The base composition of the DNA was 67.8 ± 0.1 mol% G+C for strain Soe1 and 69.0 ± 0.2 mol% G+C for strain OL1. The value reported by Babenzien (4) for a surface film was 60.4 mol%.

View larger version (48K): [in this window] [in a new window]   FIG. 3.   Genomic fingerprints of N. ramosa strains and E. coli generated by ERIC-PCR.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Since its initial description by Famintzin (7), N. ramosa has been rediscovered and enriched several times (1, 11-14, 16). The first pure cultures, isolated in the present work from two different sources, enabled us to study the physiology, phylogeny, and life in the neuston layer of this interesting microorganism.

N. ramosa as a well-adapted neuston organism. N. ramosa is a representative of the epineuston, as indicated by the dull appearance and hydrophobicity of the surface films. Thus, the flat rosettes live essentially outside of the water phase. A morphological adaptation to life in a two-dimensional environment can be seen in the cell shape. Unlike a straight rod, a slightly bent cell is forced into a defined horizontal position. At the same time, a concave side excreting the slime stalk is defined.

Phylogenetic classification of N. ramosa. On the basis of morphology, different stalked bacteria have previously been combined in a special group (4). The phylogenetic analysis of the 16S rRNA gene indicated no close relationships of Nevskia with those organisms (Table 2). For example, Caulobacter is a member of the alpha-subclass Proteobacteria and Gallionella is a member of the beta-subclass Proteobacteria. Our study indicates that N. ramosa has no known near relatives. Its lineage branches very deeply, at the root of the gamma-subclass Proteobacteria.