Molecular Identification and Ultrastructural and Phylogenetic Studies of Cyanobacteria from Association with the White Sea Hydroid Dynamena pumila (L., 1758)

Three new cyanobacterial strains, that have been previously purified from the hydroid Dynamena pumila (L., 1758), isolated from the White Sea, were studied using scanning and transmission electron microscopy methods and were characterized by using almost complete sequence of the 16S rRNA gene, internal transcribed spacer 16S-23S rRNA, and part of the gene for 23S rRNA. The full nucleotide sequences of the rRNA gene clusters were deposited to GenBank (HM064496.1, GU265558.1, JQ259187.1). Comparison of rRNA gene cluster sequences of Synechococcus cyanobacterium 1Dp66E-1, Oscillatoriales cyanobacterium 2Dp86E, and Nostoc sp. 10Dp66E with all sequences present at the GenBank shows that these cyanobacterial strains do not have 100% identity with any organisms investigated previously. Furthermore, for the first time heterotrophic bacterium, associated with Nostoc sp. 10Dp66E, was identified as a member of the new phylum Gemmatimonadetes, genus of Gemmatimonas (GenBank accession number is JX437625.1). Phylogenetic analysis showed that cyanobacterium Synechococcus sp. 1Dp66E-1 forms the unique branch and belongs to a cluster of Synechococcus, including freshwater and sea strains. Oscillatoriales cyanobacterium 2Dp86E belongs to a cluster of Leptolyngbya strains. Isolate Nostoc sp. 10Dp66E forms unique branch and belongs to a cluster of the genus Nostoc, with the closest relative of Nostoc commune isolates.


Introduction
Cyanobacterial symbioses and associations with eukaryotes are widely distributed in aquatic and terrestrial environments. Various cyanobacterial strains form associations with sponges, hydroids, corals, dinoflagellates, radiolarians, and tintinnids [1][2][3][4][5][6][7][8]. Mostly, the studies of cyanobacterial associations are limited to subtropical and tropical marine water systems. Symbioses and associations between cyanobacteria and marine animals of high latitudes are poorly studied. Recently we isolated the several cyanobacteria from the associations with White Sea hydroid Dynamena pumila (L., 1758) [7]. Purification of the symbiotic cyanobacteria from the associations with animals is rarely successful. Four cyanobacteria were isolated and purified into culture from native samples of hydroid D. pumila and were identified based on morphological characteristics as representatives of the I, II, and IV subsections of phylum BX Cyanobacteria [7].
The goal of this study was the identification of the new cyanobacteria from association with the hydroid Dynamena pumila and their characterization on the basis of ultrastructural and molecular features.

Light Microscopy.
The isolated cyanobacteria have been studied under Leitz Laborlux D microscope (Ernst Leitz Wetzlar GmbH, Germany).

Transmission Electron Microscopy (TEM).
For negative staining, drop of cyanobacteria suspension was placed onto grids and treated with 1% (w/v) sodium phosphotungstate solution and dried. For the preparation of ultrathin sections, cyanobacteria were fixed in 2% (w/v) glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7,2) for 0.5 h and then postfixed in 1% (w/v) osmium tetroxide in the same buffer for 4 h, dehydrated by incubation in dilutions of ethanol, including absolute ethanol saturated with uranyl acetate, and embedded in araldite. Thin sections were prepared on an LKB-8800 (Sweden) ultratome and stained with lead citrate [10].

Scanning Electron Microscopy (SEM).
Cyanobacterial samples were fixed as described above and dehydrated through an ethanol series, with an overnight exposure in absolute acetone followed by critical-point drying in a Dryer HCP-2 (Hitachi, Japan), coated with Au-Pd alloy in an IB-3 Ion Coater (Eiko, Japan), and examined with a JSM-6380LA scanning electron microscope (JEOL, Japan).
PCR was carried out on a Tercik DNA amplifier (DNA Technology, Russia) by using DreamTaq PCR Master Mix (Fermentas, EU), under the following conditions: 1 cycle at 94 ∘ C for 10 min, 25 cycles at 94 ∘ C for 45 sec, 54 ∘ C for 45 sec, 68 ∘ C for 2 min, 1 cycle at 68 ∘ C for 7 min, and a final soak step at 4 ∘ C. PCR products were resolved in 1.5% agarose gel containing ethidium bromide at 5 microgram mL −1 . All experiments were repeated at least three times.     significance of the obtained dendrograms was calculated by bootstrap analysis by generation of 1000 permutations.

Morphological and Ultrastructural Characteristics and
Identification of Cyanobacterial Isolates. Cyanobacterium 1Dp66E-1 was characterized by short rods (Table 2). These cells contained 2-3 thylakoids located in parallel to a cell surface. In the center of the cell there were a nucleoid, ribosomes, and carboxysomes ( Figure 1). Visible polymers have been identified as granules of poly--hydroxybutyrate (Phb) and polyphosphate. Cyanophycin granules were absent. As it was possible to see after negative staining, cells of 1Dp66E-1 were characterized by a rugulose relief of the cell surface. Similar surface type has been observed in cases of no phototrophic Gram-negative bacteria [15] and of cyanobacteria Synechococcus sp. PCC 6301 and Synechococcus sp. PCC 7942 [16]. On ultrathin sections the outer leaflet of the outer membrane of the cell wall looked like a structure of high electron density, and it was merged with an additional layer consisting of orderly packed subunits. This additional layer was similar to bacterial S-layer glycoprotein. The cyanobacterium 1Dp66E-1 had characteristics typical to genus of Synechococcus.
The study by light and electron microscopy revealed that cyanobacterium 2Dp86E was filamentous (Table 2; Figure  2). Trichomes were often bound into bundles and were surrounded by layered common sheathes, which had in local    areas sleeve-like bulges (Figure 2). In the structure of many sheaths, there were revealed electron transparent sections. The sheaths of neighboring trichomes could be closely contacted. The end cells in trichomes usually are smaller and narrower at the terminal parts. Cells of this isolate had 4-6 thylakoids, located on the periphery of the cytoplasm and in parallel to a cell surface. Nucleoid, carboxysomes, polyphosphate, and Phb-like granules were located in the cell center ( Figure 2). Clusters of ribosomes were concentrated around the nucleoid. Cyanophycin granules were rare.  Despite the fact that this strain was able to greatly accumulate phycocyanin [7], phycobilisomes on ultrathin sections were not detected due to the high density of the surrounding cytoplasm. From the combination of morphological and ultrastructural signs of cyanobacteria 2Dp86E had similarities with the representatives of the genus Leptolyngbya, which includes groups of filamentous cyanobacteria in LPP-group B that exhibit very thin trichomes (<3 m) [17,18].
Isolate 10Dp66E was a filamentous cyanobacterium (Figure 3, Table 2). Depending on the phase of the culture development in the colonies of cyanobacteria dominated hormogonia, long-row vegetative trichomes with intercalary heterocysts and akinetes, or rounded clusters formed by twisted trichomes. Many chains of cells were "dressed" by one-or multilayer fibrillar sheaths (Figure 3). Colonial mucus and extracellular matrix of clusters were composed from loosely packed reticular-fibrous and granular materials. Peripheral layer of mucous extracellular matrix was compacted and was common for the entire cluster. Bacteria of different morphotypes were localized in colonial mucus, in the intercellular matrix, and in sheaths of trichomes. A feature of this cyanobacterium is the differentiation of heterocysts even in presence of combined nitrogen in the medium. Photosynthetic apparatus of cyanobacterium 10Dp66E was represented by randomly distributed thylakoids in the cytoplasm. Intrathylakoid space could be increased and in some cases was filled by mesh substance. Nucleoid, ribosomes, and carboxysomes are located between thylakoids. In the 10Dp66E, cells were accumulated with glycogen, Phb, cyanophycin, protein granules, and lipid globules ( Figure 3). By morphological criteria and ultrastructural features, cyanobacterium 10Dp66E was similar to representatives of the genus Nostoc.

Phylogenetic Analysis of the Ribosomal RNA Operons.
To identify and to determine the phylogenetic positions of the new cyanobacterial isolates purified from native samples of the sublittoral hydroid D. pumila, we used 16S ribosomal RNA gene cluster as a molecular marker. Cloning and sequencing of the DNA fragment, containing 16S ribosomal RNA gene and 16S-23S ribosomal RNA intergenic spacer sequences, have been performed for all three isolated strains using appropriate primers ( Table 1). Comparison of rRNA gene cluster sequence (2277 bp) of the cyanobacterium 1Dp66E-1 revealed that 1Dp66E-1 shows the highest similarity with several strains of Synechococcus (Table 3). Phylogenetic analysis ( Figure 4) revealed that the cyanobacterium 1Dp66E-1 forms the unique evolutionary branch and belongs to a cluster of picocyanobacteria Synechococcus, which includes freshand seawater strains. One group of closely related organisms had been isolated from freshwater European lakes (Table 3). The closest relatives of the 1Dp66E-1 are Synechococcus sp. BO981502, isolated from Lake Balaton, and Synechococcus sp. BGS171, isolated from Lake Constance (Bodensee). Another strain of the genus Synechococcus (Synechococcus sp. BS20) was isolated from the Baltic Sea near the Danish island of Bornholm. The strain Synechococcus sp. PCC 7009 was originated from the USA, whereas the Cyanobium sp. Suigetsu-CG3 was purified from a lake in Japan (Table 3). Thus, the majority of Synechococcus strains that are closely related to 1Dp66E-1 have been isolated from marine and freshwater habitats of Europe, as well of the USA and Japan. These organisms exist in the form of plankton. However, the strain of Synechococcus sp. PCC 7009 was clustered with chromatophore (photosynthetic endosymbiont) of the freshwater filose amoeba Paulinella chromatophora. This endosymbiont has cyanobacterial origin, and it is closely related to freeliving Prochlorococcus and Synechococcus species [19]. Here, we demonstrate that the picocyanobacterium Synechococcus sp. 1Dp66E-1 is associated with the invertebrate of White Sea. DNA fragment of 1732 bp (GU265558.1) containing almost complete sequence of the genes coding for 16S rRNA, internal transcribed spacer 16S-23S rRNA, and part of the gene for 23S rRNA of the cyanobacterium 2Dp86E (GeneBank accession no. GU265558.1) has the highest similarity with analogous sequences of different Leptolyngbya   [33] strains (Table 4), and it is clustered with the representatives of the genus Leptolyngbya ( Figure 5). The latter have been isolated from freshwater lakes in Italy and Poland (European lakes), estuaries of benthic temperate of Portugal, and from the desert soils of North America (Table 4). All these closely related organisms of the 2Dp86E have been isolated from freshwater and soil habitats. There is only one representative of the marine species which is related to the 2Dp86E. Until now, eight species of Leptolyngbya have been found in White Sea: they have been isolated from fouling stones, or, as Leptolyngbya mucicola-the endofit from colonies of Rivularia coadunata [20]. In addition, several Leptolyngbya strains have been identified in White Sea, which inhabit areas of varying salinity [21]. However, all mentioned strains have not been analyzed with molecular methods. Analysis of the nucleotide sequence (1902 bp) of the 16S rRNA gene cluster of the Nostoc sp. 10Dp66E (JQ259187.1) revealed that it has no identity to any of previously studied organisms. Cyanobacterium 10Dp66E has similarity with several strains of Nostoc commune (Table 5). It forms the unique branch and belongs to a cluster of Nostoc, with the closest relative of Nostoc commune isolates ( Figure 6). There are only two strains of Nostoc, namely, Nostoc commune Vaucher ex Bornet et Flahault and Nostoc zetterstedtii Aresch. ex Bornet et Flahault, that had been detected in White Sea so far [20]. No information is available on the molecular typing of these strains. However, it is possible that Nostoc commune Vaucher ex Bornet et Flahault might represent the same strain as Nostoc commune NC1 clone 10 and Nostoc commune NC5 clone 11, that were defined in the GenBank (Table 5). Nostoc commune NC1 (EU784149.1) was isolated from meadow soils, close to Vrbensky ponds, Ceske Budejovice, Czech Republic.
Cyanobacterial mucous sheaths provide the specific econiches-environments for numerous heterotrophic bacteria. In this work, using 16S rRNA gene, we first identified the heterotrophic bacterium associated with Nostoc sp. 10Dp66E. This bacterium is a member of the genus Gemmatimonas (JX437625.1). The Gemmatimonadetes represents recently described bacterial group whose members are widespread in nature. These bacteria which are phylogenetically novel Gram-negative aerobic heterotrophs are capable of accumulating polyphosphates [22]. Gemmatimonas was found in associations with sponge [6].

Conclusions
Based on phenotypic and genotypic characteristics, we identified new cyanobacteria from association with the hydroid Dynamena pumila living in White Sea. Two of them form the unique branches in the corresponding phylogenetic trees. Among species clustered with the new cyanobacterial isolates there are fresh-and seawater strains from Europe, America, and Japan. The identified strains are mostly photoautotrophic, but some of them are diazotrophic bacteria. Future biochemical and genetic experiments will help to understand metabolic relations between these bacterial isolates and the hydroid Dynamena pumila in the association.