Ekaterina Krumova

Temperature downshift induces antioxidant response in fungi isolated from Antarctica

Although investigators have been studying the cold-shock response in a variety of organisms for the last two decades or more, comparatively little is known about the difference between antioxidant cell response to cold stress in Antarctic and temperate microorganisms. The change of environmental temperature, which is one of the most common stresses, could be crucial for their use in the biotechnological industry and in ecological research. We compared the effect of short-term temperature downshift on antioxidant cell response in Antarctic and temperate fungi belonging to the genus Penicillium. Our study showed that downshift from an optimal temperature to 15° or 6°C led to a cell response typical of oxidative stress: significant reduction of biomass production; increase in the levels of oxidative damaged proteins and accumulation of storage carbohydrates (glycogen and trehalose) in comparison to growth at optimal temperature. Cell response against cold stress includes also increase in the activities of SOD and CAT, which are key enzymes for directly scavenging reactive oxygen species. This response is more species-dependent than dependent on the degree of cold-shock. Antarctic psychrotolerant strain Penicilliumolsonii p14 that is adapted to life in extremely cold conditions demonstrated enhanced tolerance to temperature downshift in comparison with both mesophilic strains (Antarctic Penicilliumwaksmanii m12 and temperate Penicillium sp. t35).

Antioxidant enzyme activity of filamentous fungi isolated from Livingston Island, Maritime Antarctica

From 18 soil samples taken in the vicinity of the permanent Bulgarian Antarctic base “St. Kliment Ohridski” (62°38′29″S, 60°21′53″W) on Livingston Island, 109 filamentous fungi were isolated on selective media. The most widespread fungal species were members of the genera Cladosporium, Geomyces, Penicillium and Aspergillus. Other species, already recorded in Antarctic environment, were also isolated: Lecanicillium muscarium, Epicoccum nigrum and Alternaria alternata. Thirty strains demonstrating good growth were screened for antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) that play an important role in the defense of aerobic organisms against oxidative stress, by converting reactive oxygen species into nontoxic molecules. Six of them showed high enzyme activity. The tested strains produced SOD with statistically significant higher activity at 15°C than at 30°C suggesting that this enzyme is cold-active. Such SOD could be useful in medicine and cosmetics. The best producer of cold-active SOD, Aspergillus glaucus 363, cultivated in bioreactors, demonstrated optimal growth temperature at 25°C and maximum enzyme activities at 25 and 30°C for SOD and CAT, respectively. The electrophoretical analysis showed that the fungus possesses Cu/Zn-SOD.

Isolation and Identification of Filamentous Fungi from Island Livingston, Antarctica

ABSTRACT Over the last decades, the Antarctic regions have been investigated mainly for the presence and exploitation of psychrophilic bacteria and archea, occasionally for algae and more rarely for fungi. The present study reports results concerning the isolation and identification of filamentous fungi from samples of soil taken from Livingston Island, South Shetland Archipelago, West Antarctica. Using conventional media and techniques, all collection sites yielded populations of filamentous fungi, belonging to the phylum Ascomycota (7 genera), Deuteromycota (2), Zygomycota (2) and Basidiomycota (1). Mucor, Cladosporium, Alternaria, Aspergillus and Penicillium were predominant genera. Lecanicillium, Botrytis, Geomyces, Monodictys and Rhizopus were the most frequently isolated genera. Most of the fungal isolates proved to be cold-tolerant

Physiological aspects of immobilised Aspergillus niger cells producing polymethylgalacturonase

Abstract Conidia of Aspergillus niger 26 spores were immobilised in 3% Ca-alginate beads and some intrinsic kinetic characteristics including growth, polymethylgalacturonase (PMG) production and specific oxygen uptake rates determined. A negative correlation between the specific growth rate and specific PMG production indicated a non-growth-associated enzyme formation. Immobilisation did not change the model of PMG synthesis even in repeated replacement cultures and leads under certain conditions to an enhanced PMG production. The immobilised mycelium was concentrated at the surface layer of the alginate beads and the fungal hyphae could not be found in the central part of the gel. The dense growth of the mycelia on the outer bead surface allowed the formation of a fur-like mycelium coat, which had a large surface for oxygen uptake. This growth behaviour is a reason why the effectiveness factor increased during incubation from 0.54 to 0.93.

ROSMARINIC ACID AND ANTIOXIDANT ENZYME ACTIVITIES IN LAVANDULA VERA MM CELL SUSPENSION CULTURE: A COMPARATIVE STUDY

The growth and intracellular protein content of lavender (Lavandula vera MM) cell suspension culture was followed along with some antioxidant defense system members—non-enzymatic (rosmarinic acid) and enzymatic [superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6)]. It was found that the media content and the cultivation mode strongly influenced the production of plant defense compounds as well as the ratio between non-enzymatic and enzymatic ones. The bioreactor culture contains about two times more rosmarinic acid, superoxide dismutase, and catalase compared to the shake-flask cultivation. These findings are discussed with respect to the relative stress levels and plant antioxidant orchestra system. It was concluded that investigated defense system components (enzymatic and non-enzymatic) were closely associated in a complex balance. The three isoenzyme forms of SOD (Cu/ZnSOD, FeSOD, and MnSOD) in the cells of Lavandula vera were revealed by polyacrylamide gel electrophoresis analysis, and the FeSOD isoform exhibited highest activity.

Unusual location and characterization of Cu/Zn-containing superoxide dismutase from filamentous fungus Humicola lutea

The present study aims to provide new information about the unusual location of Cu/Zn-superoxide dismutase (Cu/Zn-SOD) in lower eukaryotes such as filamentous fungi. Humicola lutea, a high producer of SOD was used as a model system. Subcellular fractions [cytosol, mitochondrial matrix, and intermembrane space (IMS)] were isolated and tested for purity using activity measurements of typical marker enzymes. Evidence, based on electrophoretic mobility, sensitivity to KCN and H2O2 and immunoblot analysis supports the existence of Cu/Zn-SOD in mitochondrial IMS, and the Mn-SOD in the matrix. Enzyme activity is almost equally partitioned between both the compartments, thus suggesting that the intermembrane space could be one of the major sites of exposure to superoxide anion radicals. The mitochondrial Cu/Zn-SOD was purified and compared with the previously published cytosolic enzyme. They have identical molecular mass, cyanide- and H2O2-sensitivity, N-terminal amino acid sequence, glycosylation sites and carbohydrate composition. The H. lutea mitochondrial Cu/Zn-SOD is the first identified naturally glycosylated enzyme, isolated from IMS. These findings suggest that the same Cu/Zn-SOD exists in both the mitochondrial IMS and cytosol.

Induction of polymethylgalacturonase biosynthesis by immobilized cells of Aspergillus niger 26

The involvement of the regulatory mechanism induction in the biosynthesis of polymethylgalacturonase (PMG) by immobilized cells of Aspergillus niger 26 was studied. The immobilization altered the spectrum of pectin-degrading enzymes. The free cell cultures produced four pectinolytic enzyme activities, namely PMG, polygalacturonase (PG), pectinesterase (PE) and pectinylase (PL), while the entrapped mycelium synthesized PMG and PG only. The best inducer for PMG synthesis was highly esterified apple pectin. The effects of various pectin concentrations and time of incubation with inducer showed the induction model of PMG biosynthesis. The deinduction experiments provided evidence that the presence of the inducer in every cycle was obligatory for the PMG biosynthesis during repeated replacement cultivation.

The oxidative stress response of the filamentous yeast Trichosporon cutaneum R57 to copper, cadmium and chromium exposure

Despite the intensive research in the past decade on the microbial bioaccumulation of heavy metals, the significance of redox state for oxidative stress induction is not completely clarified. In the present study, we examined the effect of redox-active (copper and chromium) and redox-inactive (cadmium) metals on the changes in levels of oxidative stress biomarkers and antioxidant enzyme defence in Trichosporon cutaneum R57 cells. This filamentous yeast strain showed significant tolerance and bioaccumulation capability of heavy metals. Our findings indicated that the treatment by both redox-active and redox-inactive heavy metal induced oxidative stress events. Enhanced concentrations of Cu2+, Cr6+ and Cd2+ caused acceleration in the production of reactive oxygen species (ROS), increase in the level of oxidatively damaged proteins and accumulation of reserve carbohydrates (glycogen and trehalose). Cell response against heavy metal exposure also includes elevation in the activities of antioxidant enzymes, superoxide dismutase and catalase, which are key enzymes for directly scavenging of ROS. Despite the mentioned changes in the stress biomarkers, T. cutaneum did not show a significant growth diminution. Probably, activated antioxidant defence contributes to the yeast survival under conditions of heavy metal stress.

Differential Effect of Paraquat and Hydrogen Peroxide on the Oxidative Stress Response in Vibrio Cholerae Non O1 26/06

ABSTRACT Vibrio cholerae non O1 26/06, a non-pathogenic strain, was subjected to treatment by different concentration of paraquat (PQ) and H2O2. Exposure to PQ for 1 h caused induction of reactive oxygen species (ROS) such as superoxide anion radical (•O2−) and H2O2. At the same time, second stress factor significantly inhibited •O2− production and enhanced the intracellular H2O2 content. The enhanced ROS generation resulted in a significant increase in the levels of oxidatively damaged proteins in comparison to the control variant. Thus, the exposure of V. cholerae cells to PQ and H2O2 promoted oxidative stress. Cell response against this stress includes activation of antioxidant enzyme defence. The treatment with PQ concentrations in the range of 0 - 5 mM resulted mainly in activation of SOD, but not noticeably changed CAT activity in V. cholerae non-O1 26/06. In contrast, effect of H2O2treatment on antioxidant enzyme synthesis in our Vibrio strain was still much more pronounced for CAT than for SOD. Therefore, oxidative stress responses induced by •O2− (generated intracellularly by PQ) and H2O2 demonstrated differential adaptation of Vibrio cells to different toxic agents.

Transient Cold Shock Induces Oxidative Stress Events in Antarctic Fungi

The Antarctic biota has evolved under the influence of a suite of geological and climatic factors, including the geographic isolation of the landmass and the continental shelves, extremely low temperatures and intense seasonality (Russo et al., 2010). The isolation and environmental history of Antarctica have led to a unique biota. Many groups of organisms became extinct in Antarctica as a result of the extremely cold conditions. Although this continent is the coldest, highest, windiest, driest, wildest and most pristine of all of the continents, it is full of life. In addition to its well-known inhabitants, such as penguins and seals, it also has a diverse and unique range of microbial diversity (Nichols et al., 1999; Vincent, 2000). Microorganisms successfully colonise cold habitats and play a major role in the processes of nutrient turnover at low temperatures. In recent years, a growing attention in research has been devoted to cold-adapted microorganisms. This interest in Antarctic microorganisms stems from several reasons. Antarctica’s environmental extremes present conditions in which microorganisms have evolved unique characteristics for survival, which are of great scientific interest. Moreover, the availability of novel Antarctic species, which are generally isolated from extreme environments, opens the door for biotechnological exploration. Investigations of psychrotolerant and psychrophilic microorganisms are also important for human health because microorganisms can cause food spoilage and food-borne diseases. Research on cold shock raises a number of questions: which cellular function is affected most upon cold shock, what makes cell growth stop, and are there well-conserved or common cold shock proteins as in the case of heat-shock proteins? These questions are no less important than those in the case of heat shock (Inouye, 1999).