The Effects of a High Concentration of Dissolved Oxygen on Actinobacteria from Lake Baikal.

Among the diversity of microorganisms, the rarest and least explored are microorganisms that live in conditions of high oxygen in the environment and can experience the effects of natural oxidative stress. Here we suggest that the actinobacteria of Lake Baikal, sampled in the littoral zone, may produce natural products with antioxidant activity. The current study aimed to assess the effects of experimentally increased amounts of oxygen and ozone on the morphology of actinobacteria, DNA mutations, and antioxidant potential. In this experiment, we cultivated actinobacteria in liquid culture under conditions of natural aeration and increased concentrations of dissolved oxygen and ozone. Over a period of three months, bacterial samples were collected every week for further analysis. Morphological changes were assessed using the Gram method. A search for DNA mutations was conducted for the highly conserved 16S rRNA gene. The evaluation of antioxidant activity was performed using the DPPH test. The biotechnological potential was evaluated using high-resolution liquid chromatography-mass spectrometry approaches supplemented with the dereplication of natural products. We demonstrated the synthesis of at least five natural products by the Streptomyces sp. strain only under conditions of increased oxygen and ozone levels. Additionally, we showed morphological changes in Streptomyces sp. and nucleotide mutations in Rhodococcus sp. exposed to increased concentrations of dissolved oxygen and oxidative stress. Consequently, we demonstrated that an increased concentration of oxygen can influence Lake Baikal actinobacteria.

Composition of nutrient media and temperature of cultivation imposes effect on the content of secondary metabolites of Nocardiopsis sp. isolated from a Siberian Cave.

Growth of human population leads to many global and medical problems. The problems include the crisis of health, antibiotic resistance, drug discovery, etc. Increasing antimicrobial resistance of microorganisms results in the need to screen natural products (incl. antibiotics and antimicrobial peptides) and their producers in different ecological niches. The purpose of this study was to estimate antibiotic activity and biotechnological potential of rare actinobacteria Nocardiopsis sp. The strain was isolated from Okhotnichya cave located in Siberia. Here, we cultivated the strain at 3 temperature modes (13 °C, 28 °C, 37 °C) in 11 liquid nutrient (rich and poor) media. Using modern assays of liquid chromatography and high-resolution mass spectrometry, we estimated the content and number of produced natural products, distribution of their masses, and potential rate of novel secondary metabolites. We demonstrated that minimal nutrient media with l-asparagine and SM25 media with malt extract were less productive at current experimental parameters. As it was shown, this strain was characterized by antibiotic properties against Bacillus subtilis when cultivated at 28 °C. Also, weak antibiotic activity of crude extracts was found in strain cultivation at 13 °C. Also, we detected a high number of novel amphiphilic and hydrophobic NPs produced by this strain. We demonstrated both the influence of the nutrient media composition and cultivation temperature on biosynthetic capabilities of rare strain Nocardiopsis sp. Finally, high level of natural products that were predicted as novel confirms high biotechnological value of rare genera of Actinobacteria that could be explained by the evolution of microorganisms in the isolated environment of cave ecosystem.

Thermal reaction norms of key metabolic enzymes reflect divergent physiological and behavioral adaptations of closely related amphipod species.

Lake Baikal is inhabited by more than 300 endemic amphipod species, which are narrowly adapted to certain thermal niches due to the high interspecific competition. In contrast, the surrounding freshwater fauna is commonly represented by species with large-scale distribution and high phenotypic thermal plasticity. Here, we investigated the thermal plasticity of the energy metabolism in two closely-related endemic amphipod species from Lake Baikal (Eulimnogammarus verrucosus; stenothermal and Eulimnogammarus cyaneus; eurythermal) and the ubiquitous Holarctic amphipod Gammarus lacustris (eurythermal) by exposure to a summer warming scenario (6-23.6 °C; 0.8 °C d-1). In concert with routine metabolic rates, activities of key metabolic enzymes increased strongly with temperature up to 15 °C in E. verrucosus, whereupon they leveled off (except for lactate dehydrogenase). In contrast, exponential increases were seen in E. cyaneus and G. lacustris throughout the thermal trial (Q10-values: 1.6-3.7). Cytochrome-c-oxidase, lactate dehydrogenase, and 3-hydroxyacyl-CoA dehydrogenase activities were found to be higher in G. lacustris than in E. cyaneus, especially at the highest experimental temperature (23.6 °C). Decreasing gene expression levels revealed some thermal compensation in E. cyaneus but not in G. lacustris. In all species, shifts in enzyme activities favored glycolytic energy generation in the warmth. The congruent temperature-dependencies of enzyme activities and routine metabolism in E. verrucosus indicate a strong feedback-regulation of enzymatic activities by whole organism responses. The species-specific thermal reaction norms reflect the different ecological niches, including the spatial distribution, distinct thermal behavior such as temperature-dependent migration, movement activity, and mating season.

Cultivable Actinobacteria First Found in Baikal Endemic Algae Is a New Source of Natural Products with Antibiotic Activity.

Inadequate use of antibiotics has led to spread of microorganisms resistant to effective antimicrobial compounds for humans and animals. This study was aimed to isolate cultivable strains of actinobacteria associated with Baikal endemic alga Draparnaldioides baicalensis and estimate their antibiotic properties. During this study, we isolated both widespread and dominant strains related to the genus Streptomyces and representatives of the genera Saccharopolyspora, Nonomuraea, Rhodococcus, and Micromonospora. For the first time, actinobacteria belonging to the genera Nonomuraea and Saccharopolyspora were isolated from Baikal ecosystem. Also, it was the first time when actinobacteria of the genus Nonomuraea were isolated from freshwater algae. Some rare strains demonstrated activity inhibiting growth of bacteria and yeasts. Also, it has been shown that the strains associated with Baikal alga D. baicalensis are active against both Gram-positive and Gram-negative bacteria. According to this study and previously published materials, diversity of cultivable actinobacteria and rare strains isolated from D. baicalensis is comparable to that of cultivable actinobacteria previously isolated from plant sources of Lake Baikal. Also, it exceeds the cultivable actinobacteria diversity previously described for macroinvertebrates, water, or sediments of Lake Baikal. The large number of rare and active strains associated with the endemic alga D. baicalensis could be the promising sources for biopharmaceutical and biotechnological developments and discovery of new natural compounds.

Estimation of antimicrobial activities and fatty acid composition of actinobacteria isolated from water surface of underground lakes from Badzheyskaya and Okhotnichya caves in Siberia.

Extreme and unusual ecosystems such as isolated ancient caves are considered as potential tools for the discovery of novel natural products with biological activities. Actinobacteria that inhabit these unusual ecosystems are examined as a promising source for the development of new drugs. In this study we focused on the preliminary estimation of fatty acid composition and antibacterial properties of culturable actinobacteria isolated from water surface of underground lakes located in Badzheyskaya and Okhotnichya caves in Siberia. Here we present isolation of 17 strains of actinobacteria that belong to the Streptomyces, Nocardia and Nocardiopsis genera. Using assays for antibacterial and antifungal activities, we found that a number of strains belonging to the genus Streptomyces isolated from Badzheyskaya cave demonstrated inhibition activity against bacteria and fungi. It was shown that representatives of the genera Nocardia and Nocardiopsis isolated from Okhotnichya cave did not demonstrate any tested antibiotic properties. However, despite the lack of antimicrobial and fungicidal activity of Nocardia extracts, those strains are specific in terms of their fatty acid spectrum. When assessing fatty acid profile, we found that polyunsaturated fatty acids were quantitatively dominant in extracts of Nocardia sp. and Streptomyces sp. grown in different media. Saturated fatty acids were the second most abundant type in the fatty acid profile. It was due to palmitic acid. Also, a few monounsaturated fatty acids were detected. The obtained materials can become a basis for development of approaches to use bacteria isolated from caves as a biological sources of bioactive compounds to create medical and veterinary drugs.

The diversity and antibiotic properties of actinobacteria associated with endemic deepwater amphipods of Lake Baikal.

The emergence of pathogenic bacteria resistant to antibiotics increases the need for discovery of new effective antimicrobials. Unique habitats such as marine deposits, wetlands and caves or unexplored biological communities are promising sources for the isolation of actinobacteria, which are among the major antibiotic producers. The present study aimed at examining cultivated actinobacteria strains associated with endemic Lake Baikal deepwater amphipods and estimating their antibiotic activity. We isolated 42 actinobacterial strains from crustaceans belonging to Ommatogammarus albinus and Ommatogammarus flavus. To our knowledge, this is the first report describing the isolation and initial characterization of representatives of Micromonospora and Pseudonocardia genera from Baikal deepwater invertebrates. Also, as expected, representatives of the genus Streptomyces were the dominant group among the isolated species. Some correlations could be observed between the number of actinobacterial isolates, the depth of sampling and the source of the strains. Nevertheless, >70% of isolated strains demonstrated antifungal activity. The dereplication analysis of extract of one of the isolated strains resulted in annotation of several known compounds that can help to explain the observed biological activities. The characteristics of ecological niche and lifestyle of deepwater amphipods suggests that the observed associations between crustaceans and isolated actinobacteria are not random and might represent long-term symbiotic interactions.

Uptake Kinetics and Subcellular Compartmentalization Explain Lethal but Not Sublethal Effects of Cadmium in Two Closely Related Amphipod Species.

Eulimnogammarus cyaneus and Eulimnogammarus verrucosus, closely related amphipod species endemic to Lake Baikal, differ with respect to body size (10- to 50-fold lower fresh weights of E. cyaneus) and cellular stress response (CSR) capacity, potentially causing species-related differences in uptake, internal sequestration, and toxic sensitivity to waterborne cadmium (Cd). We found that, compared to E. verrucosus, Cd uptake rates, related to a given exposure concentration, were higher, and lethal concentrations (50%; LC50) were 2.3-fold lower in E. cyaneus (4 weeks exposure; 6 °C). Upon exposures to species-specific subacutely toxic Cd concentrations (nominal LC1; E. cyaneus: 18 nM (2.0 µg L-1); E. verrucosus: 115 nM (12.9 µg L-1); 4 weeks exposure; 6 °C), Cd amounts in metal sensitive tissue fractions (MSF), in relation to fresh weight, were similar in both species (E. cyaneus: 0.25 ± 0.06 µg g-1; E. verrucosus: 0.26 ± 0.07 µg g-1), whereas relative Cd amounts in the biologically detoxified heat stable protein fraction were 35% higher in E. cyaneus. Despite different potencies in detoxifying Cd, body size appears to mainly explain species-related differences in Cd uptake and sensitivities. When exposed to Cd at LC1 over 4 weeks, only E. verrucosus continuously showed 15-36% reduced oxygen consumption rates indicating metabolic depression and pointing to particular sensitivity of E. verrucosus to persisting low-level toxicant pressure.

Draft Genome Sequence of Streptomyces sp. Strain IB2014011-1, Isolated from Trichoptera sp. Larvae of Lake Baikal.

Unique ecosystems with specific environmental conditions have been proven to be a promising source for isolation of new actinobacterial strains. Ancient Lake Baikal is one of the greatest examples of an ecosystem with high species biodiversity and endemicity caused by long-lasting isolated evolution and stable environmental conditions. Herein we report the draft genome sequence of Streptomyces sp. strain IB2014011-1, which was isolated from insect Trichoptera sp. larvae collected at the bottom of Lake Baikal.

Salinity modulates thermotolerance, energy metabolism and stress response in amphipods Gammarus lacustris.

Temperature and salinity are important abiotic factors for aquatic invertebrates. We investigated the influence of different salinity regimes on thermotolerance, energy metabolism and cellular stress defense mechanisms in amphipods Gammarus lacustris Sars from two populations. We exposed amphipods to different thermal scenarios and determined their survival as well as activity of major antioxidant enzymes (peroxidase, catalase, glutathione S-transferase) and parameters of energy metabolism (content of glucose, glycogen, ATP, ADP, AMP and lactate). Amphipods from a freshwater population were more sensitive to the thermal challenge, showing higher mortality during acute and gradual temperature change compared to their counterparts from a saline lake. A more thermotolerant population from a saline lake had high activity of antioxidant enzymes. The energy limitations of the freshwater population (indicated by low baseline glucose levels, downward shift of the critical temperature of aerobic metabolism and inability to maintain steady-state ATP levels during warming) was observed, possibly reflecting a trade-off between the energy demands for osmoregulation under the hypo-osmotic condition of a freshwater environment and protection against temperature stress.

Actinobacteria possessing antimicrobial and antioxidant activities isolated from the pollen of scots pine (Pinus sylvestris) grown on the Baikal shore.

Isolated ecosystems existing under specific environmental conditions have been shown to be promising sources of new strains of actinobacteria. The taiga forest of Baikal Siberia has not been well studied, and its actinobacterial population remains uncharacterized. The proximity between the huge water mass of Lake Baikal and high mountain ranges influences the structure and diversity of the plant world in Siberia. Here, we report the isolation of eighteen actinobacterial strains from male cones of Scots pine trees (Pinus sylvestris) growing on the shore of the ancient Lake Baikal in Siberia. In addition to more common representative strains of Streptomyces, several species belonging to the genera Rhodococcus, Amycolatopsis, and Micromonospora were isolated. All isolated strains exhibited antibacterial and antifungal activities. We identified several strains that inhibited the growth of the pathogen Candida albicans but did not hinder the growth of Saccharomyces cerevisiae. Several isolates were active against Gram-positive and Gram-negative bacteria. The high proportion of biologically active strains producing antibacterial and specific antifungal compounds may reflect their role in protecting pollen against phytopathogens.

Actinobacteria Isolated from an Underground Lake and Moonmilk Speleothem from the Biggest Conglomeratic Karstic Cave in Siberia as Sources of Novel Biologically Active Compounds.

Actinobacteria isolated from unstudied ecosystems are one of the most interesting and promising sources of novel biologically active compounds. Cave ecosystems are unusual and rarely studied. Here, we report the isolation and characterization of ten new actinobacteria strains isolated from an ancient underground lake and moonmilk speleothem from the biggest conglomeratic karstic cave in Siberia with a focus on the biological activity of the obtained strains and the metabolite dereplication of one active strain. Streptomyces genera isolates from moonmilk speleothem demonstrated antibacterial and antifungal activities. Some of the strains were able to inhibit the growth of pathogenic Candida albicans.

Microsporidian Parasites Found in the Hemolymph of Four Baikalian Endemic Amphipods.

At present, approximately 187 genera and over 1300 species of Microsporidia have been described, among which almost half infect aquatic species and approximately 50 genera potentially infect aquatic arthropods. Lake Baikal is the deepest and one of the oldest lakes in the world, and it has a rich endemic fauna with a predominance of arthropods. Among the arthropods living in this lake, amphipods (Crustacea) are the most dominant group and are represented by more than 350 endemic species. Baikalian amphipods inhabit almost all depths and all types of substrates. The age and geographical isolation of this group creates excellent opportunities for studying the diversity, evolution and genetics of host-parasite relationships. However, despite more than 150 years of study, data investigating the microsporidia of Lake Baikal remain incomplete. In this study, we used molecular genetic analyses to detect microsporidia in the hemolymph of several endemic species of amphipods from Lake Baikal. We provide the first evidence that microsporidian species belonging to three genera (Microsporidium, Dictyocoela and Nosema) are present in the hemolymph of Baikalian endemic amphipods. In the hemolymph of Eulimnogammarus verrucosus, we detected SSU rDNA of microsporidia belonging to the genus Nozema. In the hemolymph of Pallasea cancellous, we found the DNA of Microsporidium sp. similar to that in other Baikalian endemic amphipods; Dictyocoela sp. was found in the hemolymph of Eulimnogammarus marituji and Acanthogammarus lappaceus longispinus.

A cellular and metabolic assessment of the thermal stress responses in the endemic gastropod Benedictia limnaeoides ongurensis from Lake Baikal.

Our objective was to determine if the Lake Baikal endemic gastropod Benedictia limnaeoides ongurensis, which inhabits in stable cold waters expresses a thermal stress response. We hypothesized that the evolution of this species in the stable cold waters of Lake Baikal resulted in a reduction of its thermal stress-response mechanisms at the biochemical and cellular levels. Contrary to our hypothesis, our results show that exposure to a thermal challenge activates the cellular and biochemical mechanisms of thermal resistance, such as heat shock proteins and antioxidative enzymes, and alters energetic metabolism in B. limnaeoides ongurensis. Thermal stress caused the elevation of heat shock protein 70 and the products of anaerobic glycolysis together with the depletion of glucose and phosphagens in the studied species. Thus, a temperature increase activates the complex biochemical system of stress response and alters the energetic metabolism in this endemic Baikal gastropod. It is concluded that the deepwater Lake Baikal endemic gastropod B. limnaeoides ongurensis retains the ability to activate well-developed biochemical stress-response mechanisms when exposed to a thermal challenge.

Optical diagnostic test of stress conditions of aquatic organisms.

Global climate change has become a dire reality and its impact is expected to rise dramatically in the near future. Combined with the day-to-day human activities the climatic changes heavily affect the environment. In particular, a global temperature increase accompanied by a number of anthropogenic chemicals falling within the freshwater ecosystem results in a dramatic enhancement of the overall stress for most aquatic organisms. This leads to a significant shift in the species inventory and potential breakdown of the water ecosystem with severe consequences for local economies and water supply. In order to understand and predict the influence of climatic changes on the physiological and biochemical processes that take place in living aquatic organisms we explore the application of optical spectroscopy for monitoring and quantitative assessment of antioxidant enzymes activity in benthic amphipods of Lake Baikal. We demonstrate that the changes of the enzymes activity in Baikal amphipods undergoing thermal and/or hypoxia stress can be observed and documented by UV and optical spectroscopy both in vivo and in vitro.