Loss of mutant p53 in HaCaT keratinocytes promotes cadmium-induced keratin 17 expression and cell death.

BACKGROUND: Cadmium exposure induces dermatotoxicity and epidermal barrier disruption and leads to the development of various pathologies. HaCaT cells are immortalized human keratinocytes that are widely used as alternatives to primary human keratinocytes, particularly for evaluating cadmium toxicity. HaCaT cells bear two gain-of-function (GOF) mutations in the TP53 gene, which strongly affect p53 function. Mutant forms of p53 are known to correlate with increased resistance to various stimuli, including exposure to cytotoxic substances. In addition, keratin 17 (KRT17) was recently shown to be highly expressed in HaCaT cells in response to genotoxic stress. Moreover, p53 is a direct transcriptional repressor of KRT17. However, the impact of TP53 mutations in HaCaT cells on the regulation of cell death and keratin 17 expression is unclear. In this study, we aimed to evaluate the impact of p53 on the response to Cd-induced cytotoxicity. METHODS AND RESULTS: Employing the MTT assay and Annexin V/propidium iodide staining, we demonstrated that knockout of TP53 leads to a decrease in the sensitivity of HaCaT cells to the cytotoxic effects of cadmium. Specifically, HaCaT cells with TP53 knockout (TP53 KO HaCaT) exhibited cell death at a cadmium concentration of 10 µM or higher, whereas wild-type cells displayed cell death at a concentration of 30 µM. Furthermore, apoptotic cells were consistently detected in TP53 KO HaCaT cells upon exposure to low concentrations of cadmium (10 and 20 µM) but not in wild-type cells. Our findings also indicate that cadmium cytotoxicity is mediated by reactive oxygen species (ROS), which were significantly increased only in TP53 knockout cells treated with 30 µM cadmium. An examination of proteomic data revealed that TP53 knockout in HaCaT cells resulted in the upregulation of proteins involved in the regulation of apoptosis, redox systems, and DNA repair. Moreover, RT‒qPCR and immunoblotting showed that cadmium toxicity leads to dose-dependent induction of keratin 17 in p53-deficient cells but not in wild-type cells. CONCLUSIONS: The connection between mutant p53 in HaCaT keratinocytes and increased resistance to cadmium toxicity was demonstrated for the first time. Proteomic profiling revealed that TP53 knockout in HaCaT cells led to the activation of apoptosis regulatory circuits, redox systems, and DNA repair. In addition, our data support the involvement of keratin 17 in the regulation of DNA repair and cell death. Apparently, the induction of keratin 17 is p53-independent but may be inhibited by mutant p53.

Exploring the Functions of Mutant p53 through TP53 Knockout in HaCaT Keratinocytes.

Approximately 50% of tumors carry mutations in TP53; thus, evaluation of the features of mutant p53 is crucial to understanding the mechanisms underlying cell transformation and tumor progression. HaCaT keratinocytes represent a valuable model for research in this area since they are considered normal, although they bear two gain-of-function mutations in TP53. In the present study, transcriptomic and proteomic profiling were employed to examine the functions of mutant p53 and to investigate the impact of its complete abolishment. Our findings indicate that CRISPR-mediated TP53 knockout results in significant changes at the transcriptomic and proteomic levels. The knockout of TP53 significantly increased the migration rate and altered the expression of genes associated with invasion, migration, and EMT but suppressed the epidermal differentiation program. These outcomes suggest that, despite being dysfunctional, p53 may still possess oncosuppressive functions. However, despite being considered normal keratinocytes, HaCaT cells exhibit oncogenic properties.

Exopolysaccharides Producing Bacteria: A Review.

Bacterial exopolysaccharides (EPS) are essential natural biopolymers used in different areas including biomedicine, food, cosmetic, petroleum, and pharmaceuticals and also in environmental remediation. The interest in them is primarily due to their unique structure and properties such as biocompatibility, biodegradability, higher purity, hydrophilic nature, anti-inflammatory, antioxidant, anti-cancer, antibacterial, and immune-modulating and prebiotic activities. The present review summarizes the current research progress on bacterial EPSs including their properties, biological functions, and promising applications in the various fields of science, industry, medicine, and technology, as well as characteristics and the isolation sources of EPSs-producing bacterial strains. This review provides an overview of the latest advances in the study of such important industrial exopolysaccharides as xanthan, bacterial cellulose, and levan. Finally, current study limitations and future directions are discussed.

A study on L-threonine and L-serine uptake in Escherichia coli K-12.

In the current study, we report the identification and characterization of the yifK gene product as a novel amino acid carrier in E. coli K-12 cells. Both phenotypic and biochemical analyses showed that YifK acts as a permease specific to L-threonine and, to a lesser extent, L-serine. An assay of the effect of uncouplers and composition of the reaction medium on the transport activity indicates that YifK utilizes a proton motive force to energize substrate uptake. To identify the remaining threonine carriers, we screened a genomic library prepared from the yifK-mutant strain and found that brnQ acts as a multicopy suppressor of the threonine transport defect caused by yifK disruption. Our results indicate that BrnQ is directly involved in threonine uptake as a low-affinity but high-flux transporter, which forms the main entry point when the threonine concentration in the external environment reaches a toxic level. By abolishing YifK and BrnQ activity, we unmasked and quantified the threonine transport activity of the LIV-I branched chain amino acid transport system and demonstrated that LIV-I contributes significantly to total threonine uptake. However, this contribution is likely smaller than that of YifK. We also observed the serine transport activity of LIV-I, which was much lower compared with that of the dedicated SdaC carrier, indicating that LIV-I plays a minor role in the serine uptake. Overall, these findings allow us to propose a comprehensive model of the threonine/serine uptake subsystem in E. coli cells.

Adaptation of Lacticaseibacillus rhamnosus CM MSU 529 to Aerobic Growth: A Proteomic Approach.

The study describes the effect of aerobic conditions on the proteome of homofermentative lactic acid bacterium Lacticaseibacillus rhamnosus CM MSU 529 grown in a batch culture. Aeration caused the induction of the biosynthesis of 43 proteins, while 14 proteins were downregulated as detected by label-free LC-MS/MS. Upregulated proteins are involved in oxygen consumption (Pox, LctO, pyridoxine 5'-phosphate oxidase), xylulose 5-phosphate conversion (Xfp), pyruvate metabolism (PdhD, AlsS, AlsD), reactive oxygen species (ROS) elimination (Tpx, TrxA, Npr), general stress response (GroES, PfpI, universal stress protein, YqiG), antioxidant production (CysK, DkgA), pyrimidine metabolism (CarA, CarB, PyrE, PyrC, PyrB, PyrR), oligopeptide transport and metabolism (OppA, PepO), and maturation and stability of ribosomal subunits (RbfA, VicX). Downregulated proteins participate in ROS defense (AhpC), citrate and pyruvate consumption (CitE, PflB), oxaloacetate production (AvtA), arginine synthesis (ArgG), amino acid transport (GlnQ), and deoxynucleoside biosynthesis (RtpR). The data obtained shed light on mechanisms providing O2-tolerance and adaptation to aerobic conditions in strain CM MSU 529. The biosynthesis of 39 from 57 differentially abundant proteins was shown to be O2-sensitive in lactic acid bacteria for the first time. To our knowledge this is the first study on the impact of aerobic cultivation on the proteome of L. rhamnosus.

Cyanobacterial Root Associations of Leafless Epiphytic Orchids.

The leafless orchids are rare epiphytic plants with extremely reduced leaves, and their aerial roots adopted for photosynthesis. The beneficial plant-microbial interactions contribute significantly to host nutrition, fitness, and growth. However, there are no data available on the bacterial associations, inhabiting leafless orchids. Here, we describe the diversity of cyanobacteria, which colonize the roots of greenhouse Microcoelia moreauae and Chiloschista parishii. The biodiversity and structure of the cyanobacterial community were analyzed using a complex approach, comprising traditional cultivable techniques, denaturing gradient gel electrophoresis (DGGE), and phylogenetic analysis, as well as the light and scanning electron microscopy (SEM). A wide diversity of associated bacteria colonize the root surface, forming massive biofilms on the aerial roots. The dominant populations of filamentous nitrogen-fixing cyanobacteria belonged to the orders Oscillatoriales, Synechococcales, and Nostocales. The composition of the cyanobacterial community varied, depending on the nitrogen supply. Two major groups prevailed under nitrogen-limiting conditions, belonging to Leptolyngbya sp. and Komarekiella sp. The latter was characterized by DGGE profiling and sequencing, as well as by its distinctive features of morphological plasticity. The leading role of these phototrophophic and diazotrophic cyanobacteria is discussed in terms of the epiphytic lifestyle of the leafless orchids.

Multiple Drug-Induced Stress Responses Inhibit Formation of Escherichia coli Biofilms.

In most ecosystems, bacteria exist primarily as structured surface-associated biofilms that can be highly tolerant to antibiotics and thus represent an important health issue. Here, we explored drug repurposing as a strategy to identify new antibiofilm compounds, screening over 1,000 compounds from the Prestwick Chemical Library of approved drugs for specific activities that prevent biofilm formation by Escherichia coli Most growth-inhibiting compounds, which include known antibacterial but also antiviral and other drugs, also reduced biofilm formation. However, we also identified several drugs that were biofilm inhibitory at doses where only a weak effect or no effect on planktonic growth could be observed. The activities of the most specific antibiofilm compounds were further characterized using gene expression analysis, proteomics, and microscopy. We observed that most of these drugs acted by repressing genes responsible for the production of curli, a major component of the E. coli biofilm matrix. This repression apparently occurred through the induction of several different stress responses, including DNA and cell wall damage, and homeostasis of divalent cations, demonstrating that biofilm formation can be inhibited through a variety of molecular mechanisms. One tested drug, tyloxapol, did not affect curli expression or cell growth but instead inhibited biofilm formation by suppressing bacterial attachment to the surface.IMPORTANCE The prevention of bacterial biofilm formation is one of the major current challenges in microbiology. Here, by systematically screening a large number of approved drugs for their ability to suppress biofilm formation by Escherichia coli, we identified a number of prospective antibiofilm compounds. We further demonstrated different mechanisms of action for individual compounds, from induction of replicative stress to disbalance of cation homeostasis to inhibition of bacterial attachment to the surface. Our work demonstrates the potential of drug repurposing for the prevention of bacterial biofilm formation and suggests that also for other bacteria, the activity spectrum of antibiofilm compounds is likely to be broad.

In Vitro Evaluation of Probiotic Potential of Selected Lactic Acid Bacteria Strains.

Research of human microbiome demonstrates that in order to develop next generation of probiotic agents, it is necessary to choose bacterial strains featured by special properties, such as the ability of the cells to attach to intestinal walls, resistance to bile and acids, bacteriocin synthesis, antioxidative and antipathogenic activity, and survivability in intestines. Thirty-three strains of lactic acid bacteria of Lactobacillus and Lactococcus genera from the Lomonosov Moscow State University Collection of Microorganisms (CM MSU) have been tested for important probiotic properties which assist these bacteria to settle effectively in intestines: cell adhesion, ability to form biofilms, agglutination with lectin (concanavalin A), and antimicrobial activity. The results of experiments clearly demonstrate that all these properties can be classified as strain characteristics and differ even within the same species. Besides the cultures of Lactobacillus with good agglutination ability with concanavalin A (Lact. caucasicus CM MSU 155, Lact. brevis CM MSU 521), we also discovered strains with high adhesion properties (Lact. acidophilus CM MSU 146-89% affinity for hexadecane; Lact. paracasei CM MSU 527-85%; Lact. plantarum CM MSU 508-78%; Lact. caucasicus CM MSU 155-70%; and Lact. delbrueckii CM MSU 571-57%), biofilm formation ability with a hydrophobic carrier (Lact. plantarum CM MSU 588-OD590 of crystal violet extracts = 1.336; Lact. brevis CM MSU 521-OD590 = 1.207; and Lact. brevis CM MSU 535-OD590 = 1.151), and with high antimicrobial activity specially to Staphylococcus aureus. Lact. brevis CM MSU 521 possesses the best property combination, which makes it potentially applicable as a very good lactic acid probiotic strain.

Composition of the Biofilm Matrix of Cutibacterium acnes Acneic Strain RT5.

In skin, Cutibacterium acnes (former Propionibacterium acnes) can behave as an opportunistic pathogen, depending on the strain and environmental conditions. Acneic strains of C. acnes form biofilms inside skin-gland hollows, inducing inflammation and skin disorders. The essential exogenous products of C. acnes accumulate in the extracellular matrix of the biofilm, conferring essential bacterial functions to this structure. However, little is known about the actual composition of the biofilm matrix of C. acnes. Here, we developed a new technique for the extraction of the biofilm matrix of Gram-positive bacteria without the use of chemical or enzymatic digestion, known to be a source of artifacts. Our method is based on the physical separation of the cells and matrix of sonicated biofilms by ultracentrifugation through a CsCl gradient. Biofilms were grown on the surface of cellulose acetate filters, and the biomass was collected without contamination by the growth medium. The biofilm matrix of the acneic C. acnes RT5 strain appears to consist mainly of polysaccharides. The following is the ratio of the main matrix components: 62.6% polysaccharides, 9.6% proteins, 4.0% DNA, and 23.8% other compounds (porphyrins precursors and other). The chemical structure of the major polysaccharide was determined using a nuclear magnetic resonance technique, the formula being →6)-α-D-Galp-(1→4)-ß-D-ManpNAc3NAcA-(1→6)-α-D-Glcp-(1→4)-ß-D-ManpNAc3NAcA-(1→3)-ß-GalpNAc-(1→. We detected 447 proteins in the matrix, of which the most abundant were the chaperonin GroL, the elongation factors EF-Tu and EF-G, several enzymes of glycolysis, and proteins of unknown function. The matrix also contained more than 20 hydrolases of various substrata, pathogenicity factors, and many intracellular proteins and enzymes. We also performed surface-enhanced Raman spectroscopy analysis of the C. acnes RT5 matrix for the first time, providing the surface-enhanced Raman scattering (SERS) profiles of the C. acnes RT5 biofilm matrix and biofilm biomass. The difference between the matrix and biofilm biomass spectra showed successful matrix extraction rather than simply the presence of cell debris after sonication. These data show the complexity of the biofilm matrix composition and should be essential for the development of new anti-C. acnes biofilms and potential antibiofilm drugs.

Excision of selectable markers from the Escherichia coli genome without counterselection using an optimized λRed recombineering procedure.

The introduction of chromosomal mutations into the E. coli genome using λRed-mediated recombineering includes two consecutive steps-the insertion of an antibiotic resistance gene and the subsequent excision of the marker. The second step usually requires a counterselection method, because the efficiency of recombination is not high enough to find recombinants among non-recombinant cells. Most counterselection methods require the introduction of additional mutations into the genome or the use of expensive chemicals. In this paper, we describe the development of a reliable procedure for the removal of an antibiotic resistance marker from the E. coli genome without the need for counterselection. For this purpose, we used dsDNA cassettes consisting of two regions homologous to the sequences that flank the marker on the chromosome. We optimized the length of the homologous regions, the electroporation conditions, and the duration of recovery for the electroporated cells in order to maximize the recombination efficiency. Using the optimal parameters identified, we obtained a rate of 4-6% recombinants among the transformed cells. This high efficiency allowed us to find marker-less, antibiotic-sensitive recombinants by replica plating without the need for selection.

Effect of two cosmetic compounds on the growth, biofilm formation activity, and surface properties of acneic strains of Cutibacterium acnes and Staphylococcus aureus.

Increasing popularity of preservative-free cosmetics necessitates in-depth research, specifically as bacteria can react to local factors by important metabolic changes. In this respect, investigating the effect of cosmetic preparations on pathogenic strains of commensal species such as acneic forms of Cutibacterium acnes (former Propionibacterium acnes) and bacteria behaving both as commensals and opportunistic pathogens such as Staphylococcus aureus is of major interest. In this study, we studied the effect of commonly used cosmetics, Uriage™ thermal water (UTW) and a rhamnose-rich polysaccharide (PS291® ) on RT4 and RT5 acneic strains of C. acnes and a cutaneous strain of S. aureus. UTW affected the growth kinetic of acneic C. acnes essentially by increasing its generation time and reducing its biomass, whereas only the S. aureus final biomass was decreased. PS291 had more marginal effects. Both compounds showed a marked antibiofilm activity on C. acnes and S. aureus. For S. aureus that appeared essentially due to inhibition of initial adhesion. Cosmetics did not modify the metabolic activity of bacteria. Both C. acnes and S. aureus showed marked hydrophobic surface properties. UTW and PS291 had limited effect on C. acnes but increased the hydrophobic character of S. aureus. This work underlines the effect of cosmetics on cutaneous bacteria and the potential limitations of preservative-free products.

Development of new versatile plasmid-based systems for λRed-mediated Escherichia coli genome engineering.

Plasmid-based systems are the most appropriate for multistep lambda Red (λRed)-mediated recombineering, such as the assembly of strains for biotechnological applications. Currently, the widely used λRed-expressing plasmids use a temperature-sensitive origin of replication or temperature shift control of λRed expression. In this work, we have constructed a new, conditionally replicating vector that can be efficiently eliminated from the host strain through passaging in medium containing isopropyl-ß-d-thiogalactopyranoside. Using the new vector, we have developed two improved helper plasmids (viz., pDL17 and pDL14) for dsDNA and oligonucleotide-mediated recombineering, respectively. The plasmid pDL14 contains a dominant negative mutSK622A allele that suppresses methyl-directed mismatch repair (MMR). The coexpression of λRed and mutSK622A provides efficient oligonucleotide-mediated recombineering in the presence of active host MMR. The expression of λRed was placed under the control of the tightly regulated PrhaB promoter. Because of their low expression level under uninduced conditions, both plasmids could be maintained without elimination for multiple recombineering steps. The temperature-independent replication of the plasmids and control of λRed expression by l-rhamnose allow for all procedures to be performed at 37 °C. Thus, the new plasmids are robust, convenient, and versatile tools for Escherichia coli genome editing.

Regulation of Monospecies and Mixed Biofilms Formation of Skin Staphylococcus aureus and Cutibacterium acnes by Human Natriuretic Peptides.

Staphylococcus aureus and Cutibacterium acnes are common representatives of the human skin microbiome. However, when these bacteria are organized in biofilm, they could be involved in several skin disorders such as acne or psoriasis. They inhabit in hollows of hair follicles and skin glands, where they form biofilms. There, they are continuously exposed to human hormones, including human natriuretic peptides (NUPs). We first observed that the atrial natriuretic peptide (ANP) and the C-type natriuretic peptide (CNP) have a strong effect S. aureus and C. acnes biofilm formation on the skin. These effects are significantly dependent on the aero-anaerobic conditions and temperature. We also show that both ANP and CNP increased competitive advantages of C. acnes toward S. aureus in mixed biofilm. Because of their temperature-dependent effects, NUPs appear to act as a thermostat, allowing the skin to modulate bacterial development in normal and inflammatory conditions. This is an important step toward understanding how human neuroendocrine systems can regulate the cutaneous microbial community and should be important for applications in fundamental sciences, medicine, dermatology, and cosmetology.

The Isolation, Identification and Analyses of Lactobacillus Genus Bacteria with Probiotic Potential.

103 strains of lactic acid bacteria of Lactobacillus genus were isolated from natural sources and identified for genus and species level with API tests and 16S rRNA sequencing. However, only 27 strains from isolated cultures demonstrated a high stability to gastric stress and from that - only 15 strains were highly resistant to intestinal stress. Results indicated that only some isolated cultures of lactobacilli possessed potential probiotical properties and could serve as new probiotics for dairy industry with high resistance to gastro-intestinal stresses.

The biotransformation of brewer's spent grain into biogas by anaerobic microbial communities.

The present study reports on the biotransformation of the brewer's spent grain (BSG) in co-digestion with Jerusalem artichoke (JA, Helianthus tuberosus L.) phytomass by thermophilic (+55 °C) and mesophilic (+30 °C) anaerobic methanogenic communities. BSG is a by-product of the beer-brewing process generated in large amounts, in which utilization provokes a negative effect on the environment. In this study, we will show an effective conversion of BSG into biogas by selected microbial communities, obtained from different sources (animal manure and previously isolated microbial consortia). The stimulation of methanogenesis was reached by the co-digestion of JA's phytomass (stem and leaves). The optimized conditions for microbial stable cultivation included the use of nutrient medium, containing yeast extract and trace element solution. The optimal BSG concentration in biogas production was 50 and 100 g L(-1). Under thermophilic conditions, the maximum total methane production reached 64%, and it comprised around 6-8 and 9-11 of L CH4 per 100 g of fermented BSG without and with co-digested JA, respectively, when the fresh inoculum was added. Although, after a year of re-cultivation, the values reduced to around 6-7, and 6-10 L CH4/100 g BSG, correspondingly, the selected microbial communities showed effective biotransformation of BSG. The supplementation of soil with the residual fermented BSG (10%, w/w) resulted in the promotion of lettuce (Lepidium sativum L.) growth. The results obtained demonstrate a potential for complete BSG utilization via biogas production and application as a soil additive.

The Probiotical Potential of Lactobacilli from Therapeutic Preventive Beverage Kurunga.

INTRODUCTION: Kurunga is a dairy drink made of a mix of lactic acid and alcoholic fermentation, characterized by high biological value based on protein composition, amino acid spectrum, fatty acid composition of lipids, vitamin and mineral substances, and physiological activity of microbiota containing lactobacilli, lactococci, bifidobacteria, and yeast. Among the probiotic correctors of normal microbiota isolated from national products, lactobacilli was of particular interest, with regards to a therapeutic - preventive effect. The aim of the study was to examine the probiotic properties of lactobacilli from kurunga. METHODS: We isolated lactic acid bacteria strains from kurunga. The isolated cultures were identified using common microbiological methods and phylogenetic analysis. The antibiotic activities of these strains were determined by measuring the growth inhibition zone of test cultures. The probiotic properties were measured as levels of resistance to bile and hydrochloric acids, in addition to the presence of superoxide dismutase (SOD) activity using the xanthine oxidase-cytochrome method. Proteolitic activity was determined at the various levels of pH (3.0, 4.2, 5.3, and 7.0). RESULTS: According to the morphological, cultural, physiological, biochemical properties and the genotypic analysis of the oligonucleotides sequence of specific genes, the most effective strain was identified as Lactobacillus diolivorans KL-2 (GenBank database KC438372). The isolated strain suppressed the growth of Gram-positive bacteria, such as Bacillus, Staphylococcus, and Listeria sp., as well as Gram-negative bacteria, such as E.coli, Proteus, Salmonella sp. They also possessed fungicidal action (based on Penicillum, Aspergillus sp, and Candida sp.). The strain was resistant to the action of the bile acids at concentrations of 0.8% to 1.0% and hydrochloric acid. The strain KL-2 possessed a relatively high SOD activity (25.74 U/mg of protein), a low proteolytic activity at a pH 3.0 (4.74·10-3 PU/ml), and high proteolytic activity at pH 4.2 (294.74·10-3 PU/ml), pH 5.3 (330.52·10-3 PU/ml) and pH 7.0 (713.68·10-3 PU/ml). CONCLUSION: The unique properties of this strain, such as stability in the gastrointestinal tract, the wide spectrum of bactericidal and fungicidal action to the pathogenic species, the relatively high superoxide dismutase and proteolytic activities, and the absence of toxicity, make it a prime candidate for probiotic culturing.

Turning cellulose waste into electricity: hydrogen conversion by a hydrogenase electrode.

Hydrogen-producing thermophilic cellulolytic microorganisms were isolated from cow faeces. Rates of cellulose hydrolysis and hydrogen formation were 0.2 mM L(-1) h(-1) and 1 mM L(-1) h(-1), respectively. An enzymatic fuel cell (EFC) with a hydrogenase anode was used to oxidise hydrogen produced in a microbial bioreactor. The hydrogenase electrode was exposed for 38 days (912 h) to a thermophilic fermentation medium. The hydrogenase activity remaining after continuous operation under load was 73% of the initial value.

Microbial transformation of azaarenes and potential uses in pharmaceutical synthesis.

Pyridine, quinoline, acridine, indole, carbazole, and other heterocyclic nitrogen-containing compounds (azaarenes) can be transformed by cultures of bacteria and fungi to produce a variety of new derivatives, many of which have biological activity. In many cases, the microbial biotransformation processes are regio- and stereoselective so that the transformation products may be useful for the synthesis of new candidate drugs.

The positive effect of exogenous hemin on a resistance of strict anaerobic archaeon Methanobrevibacter arboriphilus to oxidative stresses.

Methanogenic archaeon Methanobrevibacter arboriphilus (strains AZ and DH1), which is a strict anaerobic microorganism not able to synthesize heme, possessed a very high catalase activity in the presence of 20-50 µM hemin in a growth medium. We investigated the effect of various oxidative stresses (hydrogen peroxide and oxygenation) on M. arboriphilus cells grown on the standard nutrient medium supplemented with 0.1 % yeast extract, and on the same medium supplemented with hemin. It was demonstrated that 30 µM hemin had a very significant positive effect on the resistance of M. arboriphilus strains to H(2)O(2) and O(2) stresses because of 30- to 40-fold increase of heme catalase activity. Thus, hydrogen peroxide (0.6-1.2 mM) or oxygen (3-5 %) had a strong negative impact on low-catalase cultures grown in the hemin-free standard medium, whereas the presence of 30 µM hemin in the medium results in a high yield of biomass even under conditions of four times stronger H(2)O(2) and two times stronger O(2) stresses. The intracellular catalase activity reached a high level in 30-60 min after hemin was added to the nutrient medium, but the activity already increased about 5-7-fold in 6 min after hemin addition. Our experimental data suggest that exogenous hemin provides an effective antioxidative defense in representatives of the genus Methanobrevibacter, specially playing an important role in the puromycin-insensitive formation of the active heme-containing catalase from presynthesized apoenzyme and heme.

Microbial transformation of antimalarial terpenoids.

The fungal and bacterial transformation of terpenoids derived from plant essential oils, especially the sesquiterpenoid artemisinin from Artemisia annua, has produced several new candidate drugs for the treatment of malaria. Obtaining new derivatives of terpenoids, including artemisinin derivatives with increased antimalarial activity, is an important goal of research in microbial biotechnology and medicinal chemistry.