Ecophysiological and Phylogenetic Studies of Nevskia ramosa in Pure Culture

Abstract

During the last 100 years, the neuston bacterium Nevskia ramosa has been described several times. This bacterium forms conspicuous rosette-like microcolonies at the air-water interface. In this study, pure cultures of Nevskia ramosa were obtained for the first time, from a bog lake (strain Soe1, DSMZ 11499^T) and a freshwater ditch (strain OL1, DSMZ 11500). The isolates showed special adaptations to life in the epineuston. They formed hydrophobic surface films with a dull appearance. N. ramosa is sensitive to UV radiation but revealed a very effective photorepair mechanism. Exposure to light at a wavelength of 350 nm after UV treatment raised the number of surviving cells by several orders of magnitude. The isolates grew with a broad range of organic substrates. Surface films were formed only in the absence of combined nitrogen; however, nitrogenase activity was not detected. It appears that during growth at the air-water interface the cells benefit from trapping ammonia from the air. The G+C content of the DNA was 67.8 and 69.0 mol% for strains Soe1 and OL1, respectively. The slight difference was confirmed by enterobacterial repetitive intergenic consensus PCR. The 16S rRNA sequences revealed 99.2% similarity. Thus, both isolates belong to the same species. The phylogenetic analysis indicated that Nevskia is a member of the gamma-subclass Proteobacteria that has no known close relatives.

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

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 · liter⁻¹; and vitamin solution (20), 0.5 ml · liter⁻¹. 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 · ml⁻¹ 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

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.

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).

TABLE 1.

Utilization of organic substrates by N. ramosa^a

Component	Concn (mM)	Growth	Rosette formation
Ribose	5	+	+
Glucose	5	+	+
Fructose	5	−	−
Sucrose	5	+	+
Starch	0.2%	+	−
Cellulose	0.2%	+	−
Formate	5	+	+
Acetate	5	+	+
Lactate	5	+	+
Lactate + ammonium chloride	5	+	−
Butyrate	8	−	−
Palmitate	2	−	−
Malate	10	−	−
Pyruvate	10	+	+
Citrate	5	−	−
Fumarate	10	+	+
Succinate	10	−	−
Ethanol	5	+	+
Glycerol	5	+	+
Alanine	5	−	−
Arginine	5	+	−
Cysteine	5	−	−
Glutamate	5	+	−
Benzoate	2	+	−
Tween 20	0.001%	+	−
Tween 80	0.001%	+	−

^aStrains Soe1 and OL1 used the same substrates. Anaerobic growth with nitrate or sulfate and fermentation of glucose were not observed. +, present; −, absent. 

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.

As mentioned above, the cells did not grow obligately in the surface film. Some motile cells were always present. The development of a film depended on the availability of combined nitrogen. If ammonia, nitrate, or amino acids were supplied, the cultures grew submersed. In the absence of these compounds, surface films developed with most substrates. Only with polymers and benzoate was submersed growth observed even in the absence of ammonia. Nitrogenase activity was not detected.

N. ramosa was more sensitive to UV radiation at a wavelength of 254 nm than Escherichia coli (Fig. 2). However, the cells had a very effective photorepair mechanism. The number of cells surviving 10 s of UV radiation increased by 7 orders of magnitude if the cells were exposed to light at 350 nm after the UV treatment.

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%.

TABLE 2.

Evolutionary distance matrix and percent similarity of 16S rDNA sequences of N. ramosa and 10 reference species

No.	Species	Distance and similarityd
		1	2	3	4	5	6	7	8	9	10	11	12
1	N. ramosa Soe1T		99.2	88.7	90.0	89.1	88.9	89.3	84.5	88.4	86.6	87.5	82.9
2	N. ramosa OL1	0.0082		88.5	89.6	89.1	88.9	89.1	84.6	87.9	86.6	87.4	82.8
3	Nitrosococcus oceanus 107 (ATCC 19069T)	0.1225	0.1245		88.9	88.6	88.1	90.8	85.1	86.9	86.1	87.9	82.3
4	P. fluorescens ATCC 13525T	0.1071	0.1125	0.1207		98.0	97.4	91.1	87.6	86.8	85.3	87.7	83.8
5	Pseudomonas syringae A501 (ATCC 19310T)	0.1184	0.1184	0.1240	0.0208		97.1	90.5	87.5	86.3	85.4	86.7	83.0
6	Pseudomonas flavescens B61 (NCPPB 3063T)	0.1196	0.1201	0.1297	0.0266	0.0293		91.0	87.4	85.8	85.5	87.2	82.8
7	Methylococcus capsulatus bath (ATCC 19069T)	0.1149	0.1173	0.0983	0.0945	0.1018	0.0962		88.9	88.6	87.6	88.8	83.6
8	E. coli K-12 H105a	0.1736	0.1729	0.1666	0.1360	0.1369	0.1379	0.1203		85.4	83.1	84.1	81.6
9	Gallionella feruginea strain (stock Johan)b	0.1256	0.1320	0.1441	0.1448	0.1513	0.1575	0.1243	0.1619		91.1	92.7	82.7
10	Sphaerotilus natans 565c	0.1474	0.1474	0.1533	0.1638	0.1625	0.1606	0.1353	0.1913	0.0947		91.1	81.3
11	Zoogloea ramigera (ATCC 19544T)	0.1370	0.1375	0.1323	0.1345	0.1460	0.1410	0.1212	0.1785	0.0766	0.0949		82.6
12	C. bacteroides CB7 (ATCC 15254T)	0.1936	0.1951	0.2019	0.1826	0.1927	0.1953	0.1853	0.2109	0.1965	0.2148	0.1986

^aAccording to Dragon and Brakier-Gingras (6). 

^bAccording to Hallbeck et al. (10). 

^cAccording to Corstjens and Muyzer (5). 

^dValues on upper right are uncorrected percentages of similarity; on lower left are K_nuc values corrected for multiple base change by the method of Jukes and Cantor (15). 

FIG. 3.

Genomic fingerprints of N. ramosa strains and E. coli generated by ERIC-PCR.

A DNA distance matrix was constructed with the 10 16S rRNA sequences (obtained from the Ribosomal Database Project and the European Bioinformatics Institute) with the highest similarities to those of N. ramosa. In addition, four sequences of budding or appendaged bacteria were included in the analysis. Among these sequences, a maximum similarity of about 90% was found with Pseudomonas fluorescens ATCC 13525^T, a gamma-subclass proteobacterium. N. ramosa is only distantly related to Caulobacter bacteroides, an alpha-subclass proteobacterium (83% sequence similarity). Representatives of the beta and gamma subclasses of the class Proteobacteria showed 16S rDNA sequence homologies to N. ramosa of 83 to 88% (Table 2). A phylogenetic tree based on the K_nuc values indicated that N. ramosa represents a deeply branching member of the gamma-subclass Proteobacteria (data not shown).

DISCUSSION

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.

On the water surface Nevskia is exposed to sunlight and thus to harmful UV radiation. In spite of a high G+C content, which is known to protect DNA against damage by thymine dimerization (22), Nevskia was sensitive to UV radiation in the dark. However, the isolates overcame damage by UV radiation very effectively by photoreactivation (17). Since UV is combined with white light under natural conditions, Nevskia appears to be well prepared to survive sun radiation.

Our experiments indicate that rosette formation is controlled by the availability of combined nitrogen compounds. If ammonia, nitrate, or amino acids were supplied, the cells grew submersed. Extracellular polysaccharides and intracellular carbon reserve materials in the form of fat droplets or polyhydroxyalkanoates are typically produced under N limitation. We did not detect nitrogenase activity. However, since the atmosphere is a source of ammonia, the biofilm has a good chance to trap ammonia from the air before it is dissolved in the water. Thus, rosette formation appears to be caused by nitrogen deficiency, but at the same time it represents a mechanism to overcome it.

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.

The two isolates were closely related, as indicated by their 16S rDNA sequences (8). Different ERIC-PCR patterns (27) and G+C contents indicated differences between the isolates, which can be assigned to the same species (24). The 16S rDNA sequences fit well with those found in a culture-independent approach by Glöckner et al. (9).