Differential Expression of Stress Adaptation Genes in a Diatom Ulnaria acus under Different Culture Conditions.

Diatoms are a group of unicellular eukaryotes that are essential primary producers in aquatic ecosystems. The dynamic nature of their habitat necessitates a quick and specific response to various stresses. However, the molecular mechanisms of their physiological adaptations are still underexplored. In this work, we study the response of the cosmopolitan freshwater diatom Ulnaria acus (Bacillariophyceae, Fragilariophycidae, Licmophorales, Ulnariaceae, Ulnaria) in relation to a range of stress factors, namely silica deficiency, prolonged cultivation, and interaction with an algicidal bacterium. Fluorescent staining and light microscopy were used to determine the physiological state of cells under these stresses. To explore molecular reactions, we studied the genes involved in the stress response-type III metacaspase (MC), metacaspase-like proteases (MCP), death-specific protein (DSP), delta-1-pyrroline-5-carboxylate dehydrogenase (ALDH12), and glutathione synthetase (GSHS). We have described the structure of these genes, analyzed the predicted amino acid sequences, and measured their expression dynamics in vitro using qRT-PCR. We demonstrated that the expression of UaMC1, UaMC3, and UaDSP increased during the first five days of silicon starvation. On the seventh day, it was replaced with the expression of UaMC2, UaGSHS, and UaALDH. After 45 days of culture, cells stopped growing, and the expression of UaMC1, UaMC2, UaGSHS, and UaDSP increased. Exposure to an algicidal bacterial filtrate induced a higher expression of UaMC1 and UaGSHS. Thus, we can conclude that these proteins are involved in diatoms' adaptions to environmental changes. Further, these data show that the molecular adaptation mechanisms in diatoms depend on the nature and exposure duration of a stress factor.

The effect of metabarcoding 18S rRNA region choice on diversity of microeukaryotes including phytoplankton.

Metabarcoding using high throughput sequencing of amplicons of the 18S rRNA gene is one of the widely used methods for assessing the diversity of microeukaryotes in various ecosystems. We investigated the effectiveness of the V4 and V8-V9 regions of the 18S rRNA gene by comparing the results of metabarcoding microeukaryotic communities using the DADA2 (ASV), USEARCH-UNOISE3 (ZOTU), and USEARCH-UPARSE (OTU with 97% similarity) algorithms. Both regions showed similar levels of genetic variability and taxa identification accuracy. Richness for DADA2 datasets of both regions was lower than for UNOISE3 and UPARSE datasets, which is due to more accurate error correction in amplicons. Microeukaryotic communities (autotrophs and heterotrophs) structure identified using both regions showed a significant relationship with phytoplankton (autotrophs) communities structure based on microscopy in a seasonal freshwater sample series. The strongest relationship was found between the phytoplankton species and V8-V9 ASVs produced by DADA2.

Variability of Microbial Communities in Two Long-Term Ice-Covered Freshwater Lakes in the Subarctic Region of Yakutia, Russia.

Although under-ice microbial communities are subject to a cold environment, low concentrations of nutrients, and a lack of light, they nevertheless take an active part in biogeochemical cycles. However, we still lack an understanding of how high their diversity is and how these communities are distributed during the long-term ice-cover period. Here, we assessed for the first time the composition and distribution of microbial communities during the ice-cover period in two subarctic lakes (Labynkyr and Vorota) located in the area of the lowest temperature in the Northern Hemisphere. The diversity distribution and abundance of main bacterial taxa as well as the composition of microalgae varied by time and habitat. The 16S rRNA gene sequencing method revealed, in general, a high diversity of bacterial communities where Proteobacteria (~ 45%) and Actinobacteria (~ 21%) prevailed. There were significant differences between the communities of the lakes: Chthoniobacteraceae, Moraxellaceae, and Pirellulaceae were abundant in Lake Labynkyr, while Cyanobiaceae, Oligoflexales, Ilumatobacteraceae, and Methylacidiphilaceae were more abundant in Lake Vorota. The most abundant families were evenly distributed in April, May, and June their contribution was different in different habitats. In April, Moraxellaceae and Ilumatobacteraceae were the most abundant in the water column, while Sphingomonadaceae was abundant both in water column and on the ice bottom. In May, the abundance of Comamonadaceae increased and reached the maximum in June, while Cyanobiaceae, Oxalobacteraceae, and Pirellulaceae followed. We found a correlation of the structure of bacterial communities with snow thickness, pH, Nmin concentration, and conductivity. We isolated psychrophilic heterotrophic bacteria both from dominating and minor taxa of the communities studied. This allowed for specifying their ecological function in the under-ice communities. These findings will advance our knowledge of the under-ice microbial life.

Seasonal Succession and Coherence Among Bacteria and Microeukaryotes in Lake Baikal.

Microorganisms exhibit seasonal succession governed by physicochemical factors and interspecies interactions, yet drivers of this process in different environments remain to be determined. We used high-throughput sequencing of 16S rRNA and 18S rRNA genes to study seasonal dynamics of bacterial and microeukaryotic communities at pelagic site of Lake Baikal from spring (under-ice, mixing) to autumn (direct stratification). The microbial community was subdivided into distinctive coherent clusters of operational taxonomic units (OTUs). Individual OTUs were consistently replaced during different seasonal events. The coherent clusters change their contribution to the microbial community depending on season. Changes of temperature, concentrations of silicon, and nitrates are the key factors affected the structure of microbial communities. Functional prediction revealed that some bacterial or eukaryotic taxa that switched with seasons had similar functional properties, which demonstrate their functional redundancy. We have also detected specific functional properties in different coherent clusters of bacteria or microeukaryotes, which can indicate their ability to adapt to seasonal changes of environment. Our results revealed a relationship between seasonal succession, coherency, and functional features of freshwater bacteria and microeukaryotes.

Co-occurrence Networks Among Bacteria and Microbial Eukaryotes of Lake Baikal During a Spring Phytoplankton Bloom.

The pelagic zone of Lake Baikal is an ecological niche where phytoplankton bloom causes increasing microbial abundance in spring which plays a key role in carbon turnover in the freshwater lake. Co-occurrence patterns revealed among different microbes can be applied to predict interactions between the microbes and environmental conditions in the ecosystem. We used 454 pyrosequencing of 16S rRNA and 18S rRNA genes to study bacterial and microbial eukaryotic communities and their co-occurrence patterns at the pelagic zone of Lake Baikal during a spring phytoplankton bloom. We found that microbes within one domain mostly correlated positively with each other and are highly interconnected. The highly connected taxa in co-occurrence networks were operational taxonomic units (OTUs) of Actinobacteria, Bacteroidetes, Alphaproteobacteria, and autotrophic and unclassified Eukaryota which might be analogous to microbial keystone taxa. Constrained correspondence analysis revealed the relationships of bacterial and microbial eukaryotic communities with geographical location.

Correction to: Co-occurrence Networks Among Bacteria and Microbial Eukaryotes of Lake Baikal During a Spring Phytoplankton Bloom.

The original version of this article unfortunately contained mistakes in the legends of figures.

Sub-Ice Microalgal and Bacterial Communities in Freshwater Lake Baikal, Russia.

The sub-ice environment of Lake Baikal represents a special ecotope where strongly increasing microbial biomass causes an "ice-bloom" contributing therefore to the ecosystem functioning and global element turnover under low temperature in the world's largest freshwater lake. In this work, we analyzed bacterial and microalgal communities and their succession in the sub-ice environment in March-April 2010-2012. It was found out that two dinoflagellate species (Gymnodinium baicalense var. minor and Peridinium baicalense Kisselew et Zwetkow) and four diatom species (Aulacoseira islandica, A. baicalensis, Synedra acus subsp. radians, and Synedra ulna) predominated in the microalgal communities. Interestingly, among all microalgae, the diatom A. islandica showed the highest number of physically attached bacterial cells (up to 67 ± 16 bacteria per alga). Bacterial communities analyzed with pyrosequencing of 16S rRNA gene fragments were diverse and represented by 161 genera. Phyla Proteobacteria, Verrucomicrobia, Actinobacteria, Acidobacteria, Bacteroidetes, and Cyanobacteria represented a core community independently on microalgal composition, although the relative abundance of these bacterial phyla strongly varied across sampling sites and time points; unique OTUs from other groups were rare.

Changes in Diversity of Silica-Scaled Chrysophytes during Lake-River-Reservoir Transition (Baikal-Angara-Irkutsk Reservoir).

Hydroelectric dams create new ecosystems such as reservoirs. Several hydroelectric dams forming shallow reservoirs were built on the Angara River flowing out of Lake Baikal. The first of them in downstream Angara is Irkutsk Reservoir, with several shallow bays. Since silica-scaled chrysophytes are effective bioindicators for aquatic ecosystems, this paper aimed to determine their distribution, taxonomic structure and species richness in South Baikal and Irkutsk Reservoir, which have different environmental parameters. Thirty-one species were found using scanning and transmission electron microscopy. Only seven of them inhabited South Baikal in June 2023 at 3.66-4.51 °C and pH 7.80-8.24, with Chrysosphaerella baicalensis, Spiniferomonas trioralis f. cuspidata and Mallomonas alpina being prevalent. Only one species (M. alpina) was dominant in Irkutsk Reservoir at all stations at a water temperature of 5.33-11.55 °C and pH 8.10-8.52, alongside three other abundant species, Synura cf. glabra, Mallomonas acaroides and M. crassisquama. The maximum number of species (23) was found in a shallow bay of the reservoir at maximal values of temperature (11.5 °C) and pH (8.57) and minimal values of phosphate and nitrate concentrations during the study. The enrichment of Irkutsk Reservoir in species of silica-scaled chrysophytes was due both to cosmopolitan widespread and polyzonal species as well as to rare boreal, arctic-boreal, and unknown, possibly new species.

Complete sequence of the mitochondrial genome of a diatom alga Synedra acus and comparative analysis of diatom mitochondrial genomes.

The first two mitochondrial genomes of marine diatoms were previously reported for the centric Thalassiosira pseudonana and the raphid pennate Phaeodactylum tricornutum. As part of a genomic project, we sequenced the complete mitochondrial genome of the freshwater araphid pennate diatom Synedra acus. This 46,657 bp mtDNA encodes 2 rRNAs, 24 tRNAs, and 33 proteins. The mtDNA of S. acus contains three group II introns, two inserted into the cox1 gene and containing ORFs, and one inserted into the rnl gene and lacking an ORF. The compact gene organization contrasts with the presence of a 4.9-kb-long intergenic region, which contains repeat sequences. Comparison of the three sequenced mtDNAs showed that these three genomes carry similar gene pools, but the positions of some genes are rearranged. Phylogenetic analysis performed with a fragment of the cox1 gene of diatoms and other heterokonts produced a tree that is similar to that derived from 18S RNA genes. The introns of mtDNA in the diatoms seem to be polyphyletic. This study demonstrates that pyrosequencing is an efficient method for complete sequencing of mitochondrial genomes from diatoms, and may soon give valuable information about the molecular phylogeny of this outstanding group of unicellular organisms.

Diatom genes originating from red and green algae: Implications for the secondary endosymbiosis models.

Previous phylogenomic analyses of diatoms have discovered some plastid-targeted genes apparently coming from green algae. Number of these genes varied from less than a half of EGT-compatible genes to an overwhelming majority, and their presence was treated as an evidence of cryptic green plastid. We have performed such an analysis with a novel weighted approach on an extended dataset of diatom genomes and proteomes. Approximately equal evidence was found for red and green algal origins for diatoms genes. Considering that very similar results were obtained on other secondary photosynthetic groups whose endosymbioses were independent from that of the diatom ancestors, we consider the serial plastid replacements unparsimonious. A better explanation of these data can be provided by the shopping bag model, where a future host switches numerous endosymbionts and acquires some genes from each of them. Eventually the host loses the ability to replace endosymbionts (e.g. through the loss of phagotrophy) and whatever symbiont was present at the moment gets fixed and reduced to an organelle.

De novo transcriptome assembly and analysis of the freshwater araphid diatom Fragilaria radians, Lake Baikal.

Diatoms are a group of eukaryotic microalgae populating almost all aquatic and wet environments. Their abundance and species diversity make these organisms significant contributors to biogeochemical cycles and important components of aquatic ecosystems. Although significant progress has been made in studies of Diatoms (Bacillariophyta) over the last two decades, since the spread of "omics" technologies, our current knowledge of the molecular processes and gene regulatory networks that facilitate environmental adaptation remain incomplete. Here, we present a transcriptome analysis of Fragilaria radians isolated from Lake Baikal. The resulting assembly contains 27,446 transcripts encoding 21,996 putative proteins. The transcriptome assembly and annotation were coupled with quantitative experiments to search for differentially expressed transcripts between (i) exponential growth phase and dark-acclimated cell cultures, and (ii) those changing expression level during the early response to light treatment in dark-acclimated cells. The availability of F. radians genome and transcriptome data provides the basis for future targeted studies of this species. Furthermore, our results extend taxonomic and environmental sampling of Bacillariophyta, opening new opportunities for comparative omics-driven surveys.

Co-occurrence patterns between phytoplankton and bacterioplankton across the pelagic zone of Lake Baikal during spring.

Phytoplankton and bacterioplankton play a key role in carbon cycling of aquatic ecosystems. In this study, we found that co-occurrence patterns between different types of phytoplankton, bacterioplankton, and environmental parameters in Lake Baikal during spring were different over the course of three consecutive years. The composition of phytoplankton and bacterial communities was investigated using microscopy and 16S rRNA gene pyrosequencing, respectively. Non-metric multidimensional scaling (NMDS) revealed a relationship between the structure of phytoplankton and bacterial communities and temperature, location, and sampling year. Associations of bacteria with diatoms, green microalgae, chrysophyte, and cryptophyte were identified using microscopy. Cluster analysis revealed similar correlation patterns between phytoplankton abundance, number of attached bacteria, ratio of bacteria per phytoplankton cell and environmental parameters. Positive and negative correlations between different species of phytoplankton, heterotrophic bacteria and environmental parameters may indicate mutualistic or competitive relationships between microorganisms and their preferences to the environment.

Bacterial community composition in the water column of the deepest freshwater Lake Baikal as determined by next-generation sequencing.

The composition of bacterial communities in Lake Baikal in different hydrological periods and at different depths (down to 1515 m) has been analyzed using pyrosequencing of the 16S rRNA gene V3 variable region. Most of the resulting 34 562 reads of the Bacteria domain have clustered into 1693 operational taxonomic units (OTUs) classified with the phyla Proteobacteria, Actinobacteria, Chloroflexi, Bacteroidetes, Firmicutes, Acidobacteria and Cyanobacteria. It has been found that their composition at the family level and relative contributions to bacterial communities distributed over the water column vary depending on hydrological period. The number of OTUs and the parameters of taxonomic richness (ACE, Chao1 indices) and diversity (Shannon and inverse Simpson index) reach the highest values in water layers. The composition of bacterial communities in these layers remains relatively constant, whereas that in surface layers differs between hydrological seasons. The dynamics of physicochemical conditions over the water column and their relative constancy in deep layers are decisive factors in shaping the pattern of bacterial communities in Lake Baikal.

Inferring phylogenetic networks from gene order data.

Existing algorithms allow us to infer phylogenetic networks from sequences (DNA, protein or binary), sets of trees, and distance matrices, but there are no methods to build them using the gene order data as an input. Here we describe several methods to build split networks from the gene order data, perform simulation studies, and use our methods for analyzing and interpreting different real gene order datasets. All proposed methods are based on intermediate data, which can be generated from genome structures under study and used as an input for network construction algorithms. Three intermediates are used: set of jackknife trees, distance matrix, and binary encoding. According to simulations and case studies, the best intermediates are jackknife trees and distance matrix (when used with Neighbor-Net algorithm). Binary encoding can also be useful, but only when the methods mentioned above cannot be used.

The structure of microbial community and degradation of diatoms in the deep near-bottom layer of Lake Baikal.

Insight into the role of bacteria in degradation of diatoms is important for understanding the factors and components of silica turnover in aquatic ecosystems. Using microscopic methods, it has been shown that the degree of diatom preservation and the numbers of diatom-associated bacteria in the surface layer of bottom sediments decrease with depth; in the near-bottom water layer, the majority of bacteria are associated with diatom cells, being located either on the cell surface or within the cell. The structure of microbial community in the near-bottom water layer has been characterized by pyrosequencing of the 16S rRNA gene, which has revealed 149 208 unique sequences. According to the results of metagenomic analysis, the community is dominated by representatives of Proteobacteria (41.9%), Actinobacteria (16%); then follow Acidobacteria (6.9%), Cyanobacteria (5%), Bacteroidetes (4.7%), Firmicutes (2.8%), Nitrospira (1.6%), and Verrucomicrobia (1%); other phylotypes account for less than 1% each. For 18.7% of the sequences, taxonomic identification has been possible only to the Bacteria domain level. Many bacteria identified to the genus level have close relatives occurring in other aquatic ecosystems and soils. The metagenome of the bacterial community from the near-bottom water layer also contains 16S rRNA gene sequences found in previously isolated bacterial strains possessing hydrolytic enzyme activity. These data show that potential degraders of diatoms occur among the vast variety of microorganisms in the near-bottom water of Lake Baikal.

Immunogenicity of candidate DNA vaccine based on subtype A of human immunodeficiency virus type 1 predominant in Russia.

Human immunodeficiency virus (HIV)-1 subtype A strains circulating among the majority of HIVinfected individuals in the former Soviet Union (FSU) countries demonstrate low genetic diversity. The consensus sequence of the FSU region-specific isolate has been used for the candidate DNA vaccine development. We constructed recombinant plasmids with four viral genes: env (gp140), gag, pol (reverse transcriptase), and nef. We immunized BALB/c mice intramuscularly using equimolar mixture of four recombinant plasmids, and observed significant cytotoxic T lymphocyte response and specific CD8(+) cell production against cells presenting HIV-1 peptides. Overall, the Th1 pathway of immune response clearly dominated. Immunological properties of this candidate DNA vaccine against HIV-1 suggest the possibility of its further study in clinical trials.

Evolutionarily new sequences expressed in tumors.

BACKGROUND: Earlier we suggested the concept of the positive evolutionary role of tumors. According to this concept, tumors provide conditions for the expression of evolutionarily new and/or sleeping genes in their cells. Thus, tumors are considered as evolutionary proving ground or reservoir of expression. To support this concept we have previously characterized in silico and experimentally a new class of human tumor-related transcribed sequences. RESULTS: In this article we describe results of further studies of previously described tumor-related sequences. The results of molecular phylogeny studies, Southern hybridization experiments and computational comparison with genomes of other species are presented. CONCLUSION: These results suggest that these previously described tumor-related human transcripts are also relatively evolutionarily new.