M. A. Grachev

Conserved motif CMLD in silicic acid transport proteins of diatoms

Sequenced fragments of genes coding for silicon transporters (SITs) were analyzed for diatoms of evolutionarily distant classes (centric Chaetoceros muelleri Lemmermann, pennate araphid Synedra acus Kützing, pennate raphid Phaeodactylum tricornutum Bohlin, and pennate Cylindrotheca fusiformis Reimann et Lewin with a keeled raphe system). SITs were found to contain a conserved motif, CMLD. Hydropathy profiles showed that the motif CMLD is between two transmembrane domains lacking Lys and Arg, and the domains were consequently assumed to play a role in the formation of a channel mediating silicic acid transport. The motif CMLD proved to be rare. Since Zn2+ is necessary for silica incorporation into diatom cells, a hypothesis was advanced that the motif CMLD acts as a Zn-binding site. Diatom growth suppression was observed in the presence of the alkylating agent N-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonic acid (AEDANS), which does not penetrate into the cell. Cys of the motif CMLD was assumed to act as a target for AEDANS. Zinc ions inhibited Cys alkylation in the synthetic peptide NCMLDY, testifying to the above hypothesis.

Novel fluorescent dyes based on oligopropylamines for the in vivo staining of eukaryotic unicellular algae

Weakly basic fluorescent dyes are used to visualize organelles within live cells due to their affinity to acidic subcellular organelles. In particular, they are used to stain the silica deposited in the silica deposition vesicles (SDVs) of diatoms during the course of their frustule synthesis. This study involved the synthesis of fluorescent dyes derived from oligopropylamines, compounds similar to those found in diatoms. The dyes were obtained by reacting oligopropylamines with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. The reaction was realized using methylated oligopropylamines with two or three nitrogen atoms and yielded two novel fluorescent dyes: NBD-N2 and NBD-N3. The dyes appeared to be highly efficient in the in vivo staining of growing siliceous frustules of diatoms at concentrations at least 10 times lower than those required for staining with HCK-123. NBD-N3 also efficiently stained other subcellular vesicles of eukaryotic unicellular algae. NBD-N2 stained only growing diatom frustules, whereas NBD-N3 also stained various subcellular organelles of different eukaryotic unicellular algae. NBD-N2 and NBD-N3 were not removed from stained diatom frustules by drastic treatments with H(2)SO(4) and H(2)O(2). Fluorescent silica can also be obtained by its chemical precipitation in the presence of NBD-N2 and NBD-N3.

A 1‐Myr record of paleoclimates from Lake Khubsugul, Mongolia

The ancient Lake Khubsugul in North Mongolia is situated at the far reach of moisture from the North Atlantic into continental Asia [Kuznetsova, 1978]. It is remote from the Pacific Ocean and shielded from the China Sea and the Indian Ocean by high mountains. Information on the paleoclimates in this territory is scarce and sometimes controversial. In winter of 2003, due to a joint project (Khubsugul Drilling Project or KDP) of the Siberian Branch of the Russian Academy of Sciences and the Academy of Sciences of Mongolia, a 53-m drilling core (KDP-1) of sediments of Lake Khubsugul was obtained. This article describes the first results of paleomagnetic and geochemical studies of this core, which was found to span the time interval of the last 1.05 Myr. The data suggest that global climate oscillations manifested themselves in repeated desiccation and filling of the lake due to changes in humidity.

DO DIATOM ALGAE FRUSTULES ACCUMULATE URANIUM

Abstract Neutron Activation Analysis (NAA) and Synchrotron Radiation X-ray Fluorescent Analysis (SRXRFA) were used to measure the content of uranium and a few other trace elements in samples of bottom sediments of Lake Baikel separated into biogenic (diatom algae frustules) and clastic components by an aerodynamic method. Uranium is rejected, rather than accumulated by diatom algae frustules.

Aberration of morphogenesis of siliceous frustule elements of the diatom Synedra acus in the presence of germanic acid.

Addition of germanic acid into the culture medium of the diatom Synedra acus subsp. radians (Kutz.) Skabitsch. had nearly no influence on the culture growth at the Ge/Si molar ratio 0.01, but stopped it at ratios 0.05 and higher. It was shown by mass-spectrometry that at the Ge/Si ratio 0.01 germanium was incorporated in both the cytoplasm and siliceous valves, whereas at Ge/Si 0.05 it was incorporated into the cytoplasm but almost failed to accumulate in the valves. At Ge/Si 0.1 germanium was accumulated in the cytoplasm, but its incorporation into the valves terminated. Studies on the cell morphology by light, epifluorescence, and transmission electron microscopy showed that high concentrations of germanic acid induced disorders in morphogenesis of the siliceous frustule and accumulation of large rhodamine-stainable electron-dense inclusions. Model chemical experiments with over-saturated solutions of silicic acid in the presence of polyallylamine revealed that addition of 5% germanic acid considerably accelerated coagulation of silica. Hence, the toxic effect of germanic acid on diatoms could be caused by changes in coagulation of silica.

SR-XFA as a method of choice in the search of signals of changing palaeoclimates in the sediments of Lake Baikal, compared to INAA and ICP-MS

Abstract Sediments of Lake Baikal obtained on top of the underwater Akademichesky Ridge for reconstruction of the palaeoclimates of Holocene and Upper Pleistocene were subjected to elemental analysis with three methods: (i) synchrotron radiation X-ray fluorescent analysis (SR-XFA); (ii) instrumental neutron activation analysis (INAA); (iii) induction-coupled plasma mass-spectrometry (ICP-MS). Comparison of the results obtained is accompanied by statistical tests and shows that, due to its high sensitivity, simplicity, and non-destructive nature, SR-XFA can be recommended as a method of choice in the search of geochemical signals of changing palaeoclimates.

CULTIVATION AND AUTOMATIC COUNTING OF DIATOM ALGAE CELLS IN MULTI-WELL PLASTIC PLATES

The planktonie diatoms, Asterionella formosa Kütz. and Synedra acus Hass., were cultivated in 96-well flat-bottom plastic plates with 100 μL of medium per well. At regular time intervals, cells were counted using an inverted microscope. Typically, the number of cells per well increased over 10 days from 50-200 to >1000. The first growth inhibition experiments were successfully run at room temperature using natural illumination. However, for better control of the growth conditions, a special micro-incubator was manufactured. This device hosted two incubation plates and was equipped with Peltier elements for cooling below room temperature, and a small luminescent lamp. For automatic counting, two digital micro-photographs of each well were taken, first at a 0.2 msec exposure and the second at a 5 msec exposure. The images were treated by means of custom software based on ImagePro to identify and count the diatom cells. Cultivation of diatoms on the micro-scale proved to be a convenient technique. Using this technique, we were able to study the effects of mercaptoethanol, Cu2+ on A. formosa and Cu2+, Hg2+, Cd2+ and phenanthroline upon the growth of S. acus.

Similarity of structure of taxonomic bacterial communities in the photic layer of Lake Baikal’s three basins differing in spring phytoplankton composition and abundance

Pyrosequencing of amplicons V3-V4 of the 16S rRNA gene of 30 samples from the photic layer across the three basins of Lake Baikal has revealed 44 958 sequences of the domain Bacteria. The most representative phylums among all classified sequences in the samples were Actinobacteria, Bacteroidetes, Verrucomicrobia, Proteobacteria, Acidobacteria, and Cyanobacteria. As much as 38.5% of the total number of sequences has been determined to the level of genus. The taxonomic composition of bacterial communities was similar regardless of some differences in the composition and abundance of spring phytoplankton. Closely related or identical sequences of bacteria inhabiting the epilimnion of other lakes worldwide have been discovered for most sequences. This confirms the similarity of microbial communities in freshwater ecosystems.

Sequencing of the complete genome of an araphid pennate diatom Synedra acus subsp. radians from Lake Baikal

High-throughput method of sequencing was applied to determine the complete nucleotide sequence of an araphid pennate diatom Synedra acus subsp. radians from Lake Baikal (East Siberia). The assembled genome has a total length of 98 Mbp, the mean coverage is 33x. Structure-functional annotation of the genome was performed.

Detection of the silicic acid transport protein in the freshwater diatom Synedra acus by immunoblotting and immunoelectron microscopy.

295 Silicic acid transport proteins, or silicon transporters (SITs), ensure the key function of Bacillariophyta—the ability to capture silicic acid from the environment and transport it into cells for forming new silica frustules [1]. Silica cycle is one of the most large-scale processes in the biosphere [2]. Progress in understanding the mechanism of silica transport is restrained by the fact that individual SIT proteins have not been isolated thus far, and their location in the cell remains obscure.