Proteomic and transcriptomic analyses of Cutibacterium acnes biofilms and planktonic cultures in presence of epinephrine.

Transcriptomic and proteomic analysis were performed on 72 h biofilms of the acneic strain Cutibacterium acnes and planktonic cultures in the presence of epinephrine. Epinephrine predominantly downregulated genes associated with various transporter proteins. No correlation was found between proteomic and transcriptomic profiles. In control samples, the expression of 51 proteins differed between planktonic cultures and biofilms. Addition of 5 nM epinephrine reduced this number, and in the presence of 5 µM epinephrine, the difference in proteomic profiles between planktonic cultures and biofilms disappeared. According to the proteomic profiling, epinephrine itself was more effective in the case of C. acnes biofilms and potentially affected the tricarboxylic acid cycle (as well as alpha-ketoglutarate decarboxylase Kgd), biotin synthesis, cell division, and transport of different compounds in C. acnes cells. These findings are consistent with recent research on Micrococcus luteus, suggesting that the effects of epinephrine on actinobacteria may be universal.

Epinephrine affects gene expression levels and has a complex effect on biofilm formation in M icrococcus luteus strain C01 isolated from human skin.

In this study, the effect of epinephrine on the biofilm formation of Micrococcus luteus C01 isolated from human skin was investigated in depth for the first time. This hormone has a complex effect on biofilms in various systems. In a system with polytetrafluoroethylene (PTFE) cubes, treatment with epinephrine at a physiological concentration of 4.9 × 10-9 M increased the total amount of 72-h biofilm biomass stained with crystal violet and increased the metabolic activity of biofilms, but at higher and lower concentrations, the treatment had no significant effect. On glass fiber filters, treatment with the hormone decreased the number of colony forming units (CFUs) and changed the aggregation but did not affect the metabolic activity of biofilm cells. In glass bottom plates examined by confocal microscopy, epinephrine notably inhibited the growth of biofilms. RNA-seq analysis and RT-PCR demonstrated reproducible upregulation of genes encoding Fe-S cluster assembly factors and cyanide detoxification sulfurtransferase, whereas genes encoding the co-chaperone GroES, the LysE superfamily of lysine exporters, short-chain alcohol dehydrogenase and the potential c-di-GMP phosphotransferase were downregulated. Our results suggest that epinephrine may stimulate matrix synthesis in M. luteus biofilms, thereby increasing the activity of NAD(H) oxidoreductases. Potential c-di-GMP pathway proteins are essential in these processes.

[Multiplex polymerase chain reaction for genetically modified potato event AV43-6-G7 quantification. Proof of efficiency].

One of the ways to improve the laboratory control methodology of genetically modified organisms of plant origin (GMO) is to use multiplexing - an approach that allows you to increase the number of targets and enlarge the number of simultaneously processed samples, maximizing the potential of polymerase chain reaction in real time (PCR-RT). The aim of the study is to develop a quantitative identification protocol for genetically engineered (GE) potato event AV43-6-G7 in the format of duplex PCR-RT with the use of TaqMan® PCR technology. Material and methods. The duplex system included 2 types of specific DNA-primers and fluorescence-labeled probes: the first one is for detection of transformation event AV43- 6-G7 DNA sequence, the second is for detection of Stp23 taxon-specific potato gene. PCR parameters were chosen by empirical selection of concentrations of primers and probes, Mg2+ ions, deoxyribonucleotides, stabilizing agent for polymerase, as well as primer annealing temperature and incubation duration for each stage of the cycle. Results. As a result of these studies, the composition of the reaction mixture was optimized for the detection and quantification of GE potato event AV43-6-G7 in food. Oligonucleotide primers and fluorescent probes were selected. The compositions of reaction mixtures and temperature-time parameters of PCR were tested: 2.5-fold reaction buffer for PCR-RT in the presence of ROX (carboxy-X-rhodamine), specific to the GE component primers (AV43-6-G7-f/AV43-6-G7-r) and target taxon (GRF3/ GRR3) at 300 nM/300 nM and 100 nM/100 nM, probes at 200 nM and 200 nM, respectively; bovine serum albumin - 0.04%; MgCl2 - 3.5 mM, deoxynucleoside triphosphates - 0.3 mM, as well as the temperature-time profile of the reaction (initial denaturation of 95 °C - 5 min, followed by 45 cycles: 95 °Ð¡ - 20 sec, 58 °Ð¡ - 20 sec, 62 °Ð¡ - 40 sec). Conclusion. The validity of the developed method is confirmed by laboratory studies and testifies to its reliability.

[Multiplex PCR for detection and quantification of GM potato event EH92-527-1 in food].

Aim - the elaboration of the protocol for the quantitative detection of genetically modified (GM) potato event EH92-527-1 in the format of duplex real-time polimerase chain reaction (RT-PCR) with TaqMan® PCR technology. Material and methods. The duplex system included two types of specific DNA primers and fluorescent probes: the 1st was for identifying of the event-specific EH92-527-1 DNA, the 2nd was for identifying of the taxon-specific potato gene Stp23. The selection of PCR parameters was carried out by empirical assortment of primers and probes, Mg2+-ions, deoxyribonucleotides and polymerase stabilizing agent, primers annealing temperature and incubation time for the each cycle stage. The results of this research was the optimization of the reaction mixture composition for EH92-527-1 event and Stp23 gene fragment identification in the duplex system: 2.5× RT-PCR buffer in the presence of ROX, primers specific for the GM potato (EH92- f/EH92-r) and target taxon (GРF3/GРR3) in the amount of 250/250 and 100/100 nM, probes - 200 and 200 nM, respectively; bovine serum albumin - 0.04%; MgCl2 - 3.5 mM, deoxynucleoside triphosphates - 0.3 mM, as well as the temperature-time reaction profile (initial denaturation at 95°C - 5 minutes, the next 45 cycles: 95 °C - 20 seconds, 58 °C - 20 seconds, 62 °C - 40 seconds). Conclusion. The method for the quantitative detection of genetically modified (GM) potato event EH92-527-1 in the format of duplex RT-PCR has been elaborated. The linearity, precision, accuracy and limit of the method definition are confirmed with in vitro studies, that results testify to the method's reliability.

[Molecular and genetic studies of genetically engineered potato: transformation event PH05-026-0048].

Expert evaluation of genetically engineered organisms (GMO) identification methods is aimed at confirmation their adequacy with the tool and methodological base used in the institutions of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing to control market turnover and labelling of genetically engineered food. The primer system's specificity was experimentally confirmed in studies with other GM potato lines, as well as with the results of the BLAST-analysis. The efficiency, linearity and correctness of the method meet the requirements of the European Union Reference Laboratory for GM Food and Feed. Limit of detection and limit of quantification of GM potato line PH05-026-0048 genomic DNA were 0.019% (11 copies of the GM potato genomic DNA) and 0.06% (36 copies of the GM genomic DNA of the potato) per 100 ng of total DNA, respectively.

[Characterization of a new strain of a purple nonsulfur bacterium from a thermal spring].

A new budding nonsulfur purple bacterium of the genus Rhodobacter (strain Ku-2) was isolated from a mat of a moderately thermal spring (Baikal rift zone, Buryat Republic, Russia). The bacterium had lamellar photosynthetic membranes, which are typica of only one Rhodobacter species, Rba. blasticus. The cells contined spheroidene carotenoids and bacteriochlorophyll a (Bchl a). In vivo absorption spectrum of the cells had the major maximum at 863 nm and an additional peak at 887 nm, which is characteristic of the pigment-protein complexes of Bchl a-containing membranes. The previously described Rba. blasticus strains did not exhibit a 887-nm maximum. The new isolate was photoheterotrophic, with optimal growth occurring at 35 degrees C, 3 g/L NaCl, and pH 7-8. The DNA G+C content was 64.4 mol %. The similarity between the 16S rRNA gene sequences of strain Ku-2 and the Rba. blasticus type strain was 98.7%. The similarity between the PufM amino acid sequences of strain Ku-2 and the previously studied Rba. blasticus strain was 89.0%. Thus, the bacterial strain Ku-2 belonged to the genus Rhodobacter and was phylogenetically related to Rba. blasticus.

Methane oxidation activity and diversity of aerobic methanotrophs in pH-neutral and semi-neutral thermal springs of the Kunashir Island, Russian Far East.

Aerobic methane oxidation has been mostly studied in environments with moderate to low temperatures. However, the process also occurs in terrestrial thermal springs, where little research on the subject has been done to date. The potential activity of methane oxidation and diversity of aerobic methanotrophic bacteria were studied in sediments of thermal springs with various chemical and physical properties, sampled across the Kunashir Island, the Kuriles archipelago. Activity was measured by means of the radioisotope tracer technique utilizing (14)C-labeled methane. Biodiversity assessments were based on the particulate methane monooxygenase (pmoA) gene, which is found in all known thermophilic and thermotolerant methanotrophs. We demonstrated the possibility of methane oxidation in springs with temperature exceeding 74 °C, and the most intensive methane uptake was shown in springs with temperatures about 46 °C. PmoA was detected in 19 out of 30 springs investigated and the number of pmoA gene copies varied between 10(4) and 10(6) copies per ml of sediment. Phylogenetic analysis of PmoA sequences revealed the presence of methanotrophs from both the Alpha- and Gammaproteobacteria. Our results suggest that methanotrophs inhabiting thermal springs with temperature exceeding 50 °C may represent novel thermophilic and thermotolerant species of the genera Methylocystis and Methylothermus, as well as previously undescribed Gammaproteobacteria.

[Anoxygenic phototrophic bacteria from microbial communities of Goryachinsk Thermal Spring (Baikal Area, Russia)].

Species composition of anoxygenic phototrophic bacteria in microbial mats of the Goryachinsk thermal spring was investigated along the temperature gradient. The spring belonging to nitrogenous alkaline hydrotherms is located at the shore of Lake Baikal 188 km north-east from Ulan-Ude. The water is of the sulfate-sodium type, contains trace amounts of sulfide, salinity does not exceed 0.64 g/L, pH 9.5. The temperature at the outlet of the spring may reach 54 degrees C. The cultures of filamentous anoxygenic phototrophic bacteria, nonsulfur and sulfur purple bacteria, and aerobic anoxygenic phototrophic bacteria were identified using the pufLM molecular marker. The fmoA marker was used for identification of green sulfur bacteria. Filamentous cyanobacteria predominated in the mats, with anoxygenic phototrophs comprising a minor component of the phototrophic communities. Thermophilic bacteria Chloroflexus aurantiacus were detected irn the samples from both the thermophilic and mesophilic mats. Cultures ofnonsulfur purple bacteria similar to Blastochloris sulfoviridis and Rhodomicrobium vannielii were isolatd from the mats developing at high (50.6-49.4 degrees C) and low temperatures (45-20 degrees C). Purple sulfur bacteria Allochromatium sp. and Thiocapsa sp., as well as green sulfur bacteria Chlorobium sp., were revealedin low-temperature mats. Truly thermophilic purple and gree sulfur bacteria were not found in the spring. Anoxygenic phototrophic bacteria found in the spring were typical of the sulfuret communities, for which the sulfur cycle is mandatory. The presence of aerobic bacteriochlorophylla-containing bacteria identified as Agrobacterium (Rhizobium) tumifaciens in the mesophilic (20 degrees C) mat is of interest.

[Diversity of diazotrophs in the sediments of hypersaline salt and soda lakes analyzed with the use of the nifH gene as a molecular marker].

Phylogenetic analysis of the nifH genes, encoding the Fe protein of the nitrogenas enzymatic complex, was carried out for pure cultures of anoxygenic phototrophic bacteria of diverse origin, as well as for heterotrophic alkaliphilic sulfate reducers isolated from saline and soda lakes. Topology of the nitrogenase tree correlated with that of the 16S rRNAgene tree to a considerable degree; which niade it possible to use the nifH gene as a molecular marker for investigation of diazotrophic bacterialcommunities in silty sediments of saline and sodalakes. Although diazotrophs were revealed in all environmentalsamples, their phylogenetic diversity was relatively low. Sulfate-reducing deltaproteobacteria and photo- and chemotrophicgammaproteobacteria were predominant in samples integrated over sediment thickness. Analysis of samples fromthe upper sediment layers revealed predominance of phototrophic diazotrophs of various phyla, including purple sulfur and nonsulfur proteobacteria, green nonsulfur bacteria, heliobacteria; and cyanobacteria. Some phylotypes could not be identified, probably indicating the presence of bacterial groups which have not yet been studied by conventional microbiological techniques.

[Hydrocarbon-Oxidizing potential and the genes for n-alkane biodegradation in a new acidophilic mycobacterial association from sulfur blocks].

Capacity of AG(S10), a new aerobic acidophilic (growing within the pH range from 1.3 to 4.5 with the optimum at 2.0-2.5) bacterial association from sulfur blocks of the Astrakhan gas-processing complex (AGC), for oxidation of hydrocarbons of various chemical structure was investigated. A broad spectrum of normal (C10-C21) and iso-alkanes, toluene, naphthalene, andphenanthrene, as well as isoprenoids resistant to microbial degradation, pristane and phytane (components of paraffin oil), and 2,2,4,4,6,8,8,-heptamethylnonane, a branched hydrocarbon, were biodegraded under acidic conditions. Microbiological investigation revealed the dominance of mycobacteria in the AGS10 association, which was confirmed by analysis of the 16S rRNA gene clone library. In the phylogenetic tree, the 16S rRNA sequences formed a branch within the cluster of slow-growing mycobacteria, with 98% homology to the closest species Mycobacterium florentinum. Genomic DNA of AG(S10) culture grown on C14-C17 n-alkanes at pH 2.5 was found to contain the genes of two hydroxylase families, alkB and Cyp 153, indicating their combined involvement in hydrocarbon biodegradation. The high hydrocarbon-oxidizing potential of the AGS10 bacterial association, indicated that further search for the genes responsible for degradation of various hydrocarbons in acidophilic mycobacteria could be promising.

Bacterial chitin utilisation at extremely haloalkaline conditions.

Chitin is produced in large amounts in hypersaline habitats with neutral pH due to the high biomass production of brine shrimp Artemia. Recently, a high abundance of Artemia was also noticed in hypersaline soda lakes in the Kulunda Steppe (Altai, Russia), which prompted us to survey the possibility of microbial chitin utilization at extremely haloalkaline conditions in soda brines. Most active chitin utilisation-supporting microbial growth was found at anaerobic conditions at pH 10 and up to 3.5 M total Na(+). At aerobic conditions, the degradation of chitin was slower, mostly incomplete and active at <2 M total Na(+), although very slow partial degradation was possible up to 4 M Na(+). Anaerobic enrichments at pH 10 yielded two different groups of obligately haloalkaliphilic fermentative anaerobes, exclusively specialized to utilise insoluble chitin as the only growth substrate. One group was represented by a single strain growing at moderate salinity, and another comprised multiple isolates growing up to 3.5 M Na(+). These groups represent two novel bacterial phyla not closely related to any other cultured bacteria. Aerobic enrichments from the lake sediments were dominated by several obligately haloalkaliphilic members of the genus Marinimicrobium in the Gammaproteobacteria. They were less specialised than the anaerobes and grew with chitin and its monomer and oligomers at a pH of 10 up to 2.5 M Na(+). Furthermore, several strains of haloalkaliphilic Gram-positive chitinolytics belonging to bacilli and actinobacteria were isolated from soda lake sediments and surrounding soda soils. In general, the results indicate the presence of an active and diverse haloalkaliphilic chitinolytic microbial community in hypersaline soda habitats.

Desulfuribacillus alkaliarsenatis gen. nov. sp. nov., a deep-lineage, obligately anaerobic, dissimilatory sulfur and arsenate-reducing, haloalkaliphilic representative of the order Bacillales from soda lakes.

An anaerobic enrichment culture inoculated with a sample of sediments from soda lakes of the Kulunda Steppe with elemental sulfur as electron acceptor and formate as electron donor at pH 10 and moderate salinity inoculated with sediments from soda lakes in Kulunda Steppe (Altai, Russia) resulted in the domination of a Gram-positive, spore-forming bacterium strain AHT28. The isolate is an obligate anaerobe capable of respiratory growth using elemental sulfur, thiosulfate (incomplete reduction) and arsenate as electron acceptor with H2, formate, pyruvate and lactate as electron donor. Growth was possible within a pH range from 9 to 10.5 (optimum at pH 10) and a salt concentration at pH 10 from 0.2 to 2 M total Na+ (optimum at 0.6 M). According to the phylogenetic analysis, strain AHT28 represents a deep independent lineage within the order Bacillales with a maximum of 90 % 16S rRNA gene similarity to its closest cultured representatives. On the basis of its distinct phenotype and phylogeny, the novel haloalkaliphilic anaerobe is suggested as a new genus and species, Desulfuribacillus alkaliarsenatis (type strain AHT28(T) = DSM24608(T) = UNIQEM U855(T)).

[Search for lactate oxidase producer microorganisms].

Using the method of enrichment cultures, eight lactate oxidase producer strains of the fungus Geotrichum candidum were identified. The microorganisms were isolated from diverse specimens of fermented vegetables and manure. Variation in the content of glucose and lactate and the degree of aeration made it possible to attain lactate oxidase activities of up to 130-140 U per 11 grown medium containing microbial cells.

[Properties and prospects of practical use of extracellular L-glutamate oxidase from Streptomyces sp. Z-11-6]. / Svoistva i perspektivy prakticheskogo primeneniia vnekletochnoi L-glutamatoksidazy Streptomyces sp. Z-11-6.

The properties of extracellular L-glutamate oxidase, isolated and purified from Streptomyces sp. Z-11-6 (specific activity, 50.8 U/mg protein; yield, 40%), were studied. A photometrical method of determination of activities of alanine- and aspartate aminotransferases, based on the use of the L-glutamate oxidase and peroxidase, has been developed. This method is sufficiently sensitive to be used for the determination of aminotransferase activities in biological fluids. The presence of other amino acids did not interfere with the analysis and had no effect on the results of determination.

[Isolation and properties of glucose oxidase from Penicillum funiculosum 433]. / Poluchenie i svoistva gliukozooksidazy iz Penicillium funiculosum 433.

A method for isolation of extracellular glucose oxidase from Penicillium funiculosum 433 and its purification is proposed. The enzymatic preparation was produced with a yield of 56% and a specific activity of 3730 AU per 1 mg protein. The enzyme studied displayed a high thermostability, resistance to metal ions, and performance in a wide pH range and was equal in its properties to foreign analogues.

[Photometric assay for methanol in the presence of ethanol]. / Fotometricheskii metod opredeleniia metanola v prisutstvii étanola.

A new enzymatic photometric assay for determination of methanol and ethanol in solutions containing both alcohols is described. The assay allows detecting methanol in the concentration range of tens ppm of in the presence of tens per cent of ethanol. The lower determination threshold for methanol is at least 0.002% in the presence of 45% ethanol, with a coefficient of variation of 0.02-0.05. General-purpose spectrophotometers and photoelectric colorimeters can be used in the measurements.

[Photometric method for determining ethanol]. / Fotometricheskii metod opredeleniia étanola.

A novel enzymatic photometric assay for ethanol determination using alcohol oxidase and peroxidase is described. The sensitivity of the method allows detecting ethanol in biological fluids (saliva and blood serum). Secondary alcohols and other organic compounds do not interfere with the assay. General-purpose spectrophotometers and photoelectric colorimeters can be used in the measurements. Methanol and propanol can also be determined by this technique.