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Актуальные аспекты современной микробиологии

Журнальные статьи

1.U45336
Barbau-Piednoir E. и др. Development and validation of qualitative SYBR (R) Green Real-Time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes // Applied Microbiology and Biotechnology. 2013. Т. 97. № 9. С. 4021–4037.

A combination of four qualitative SYBRA (R) Green qPCR screening assays targeting two levels of discrimination: Listeria genus (except Listeria grayi) and Listeria monocytogenes, is presented. These assays have been developed to be run simultaneously using the same polymerase chain reaction (PCR) programme. The paper also proposes a new validation procedure to specifically validate qPCR assays applied to food microbiology according to two guidelines: the ISO 22118 norm and the "Definition of minimum performance requirements for analytical methods of GMO testing". The developed assays target the iap, prs and hlyA genes that belong to or neighbour the virulence cluster of Listeria spp. The selected primers were designed to amplify short fragments (60 to 103 bp) in order to obtain optimal PCR efficiency (between 97 and 107 % efficiency). The limit of detection of the SYBRA (R) Green qPCR assays is two to five copies of target genes per qPCR reaction. These assays are highly accurate (98.08 and 100 % accuracy for the Listeria spp. and L. monocytogenes assays, respectively).

2. U62100
Matantseva O.V., Skarlato S.O. Mixotrophy in microorganisms: Ecological and cytophysiological aspects // Journal of Evolutionary Biochemistry and Physiology. 2013. Т. 49. № 4. С. 377–388.

Mixotrophy is the ability to combine autotrophic and heterotrophic modes of nutrition. It is widely spread in a variety of microorganisms including such important plankton groups as dinoflagellates and cyanobacteria. In marine ecosystems, mixotrophy complicates our concept of the flow of materials and energy and therefore has been thoroughly studied for recent decades. Nevertheless, the exact data on the auto/heterotrophy balance during mixotrophic growth are still lacking, mainly due to insufficient knowledge of physiological and molecular grounds of this phenomenon. In this review, we address the ecological and cytophysiological aspects of the problem of mixotrophy in microorganisms as well as discuss possible causes of the relatively slow progress in this field.

3.U45586
Frontasyeva M., Kirkesali E. Epithermal neutron activation analysis in applied microbiology // Journal of Radioanalytical and Nuclear Chemistry. 2012. Т. 291. № 2. С. 421–426.

Some results from applying epithermal neutron activation analysis at FLNP JINR, Dubna, Russia, in medical biotechnology, environmental biotechnology and industrial biotechnology are reviewed. In the biomedical experiments biomass from the blue-green alga Spirulina platensis (S. platensis) has been used as a matrix for the development of pharmaceutical substances containing such essential trace elements as selenium, chromium and iodine. The feasibility of target-oriented introduction of these elements into S. platensis biocomplexes retaining its protein composition and natural beneficial properties was shown. The absorption of mercury on growth dynamics of S. platensis and other bacterial strains was observed. Detoxification of Cr and Hg by Arthrobacter globiformis 151B was demonstrated. Microbial synthesis of technologically important silver nanoparticles by the novel actinomycete strain Streptomyces glaucus 71 MD and blue-green alga S. platensis were characterized by a combined use of transmission electron microscopy, scanning electron microscopy and energy-dispersive analysis of X-rays. It was established that the tested actinomycete S. glaucus 71 MD produces silver nanoparticles extracellularly when acted upon by the silver nitrate solution, which offers a great advantage over an intracellular process of synthesis from the point of view of applications. The synthesis of silver nanoparticles by S. platensis proceeded differently under the short-term and long-term silver action.

4.U03047
Harrison J.P., Hallsworth J.E., Cockell C.S. Reduction of the Temperature Sensitivity of Halomonas hydrothermalis by Iron Starvation Combined with Microaerobic Conditions // Applied and Environmental Microbiology. 2015. Т. 81. № 6. С. 2156–2162.

Аннотация The limits to biological processes on Earth are determined by physicochemical parameters, such as extremes of temperature and low water availability. Research into microbial extremophiles has enhanced our understanding of the biophysical boundaries which define the biosphere. However, there remains a paucity of information on the degree to which rates of microbial multiplication within extreme environments are determined by the availability of specific chemical elements. Here, we show that iron availability and the composition of the gaseous phase (aerobic versus microaerobic) determine the susceptibility of a marine bacterium, Halomonas hydrothermalis, to suboptimal and elevated temperature and salinity by impacting rates of cell division (but not viability). In particular, iron starvation combined with microaerobic conditions (5% [vol/vol]O-2, 10% [vol/vol]CO2, reduced pH) reduced sensitivity to temperature across the 13 degrees C range tested. These data demonstrate that nutrient limitation interacts with physicochemical parameters to determine biological permissiveness for extreme environments. The interplay between resource availability and stress tolerance, therefore, may shape the distribution and ecology of microorganisms within Earth's biosphere. The ecological interaction between microorganisms and seaweeds depends on the production of secondary compounds that can influence microbial diversity in the water column and the composition of reef environments. We adapted the H-3-leucine incorporation technique to measure bacterial activity in biofilms associated with the blades of the macroalgae Sargassum spp. We evaluated (1) if the epiphytic bacteria on the blades were more active in detritus or in the biofilm, (2) substrate saturation and linearity of H-3-leucine incorporation, (3) the influence of specific metabolic inhibitors during H-3-leucine incorporation under the presence or absence of natural and artificial light, and (4) the efficiency of radiolabeled protein extraction. Scanning electron microscopy showed heterogeneous distribution of bacteria, diatoms, and polymeric extracellular secretions. Active bacteria were present in both biofilm and detritus on the blades. The highest H-3-leucine incorporation was obtained when incubating blades not colonized by macroepibionts. Incubations done under field conditions reported higher H-3-leucine incorporation than in the laboratory. Light quality and sampling manipulation seemed to be the main factors behind this difference. The use of specific metabolic inhibitors confirmed that bacteria are the main group incorporating H-3-leucine but their association with primary production suggested a symbiotic relationship between bacteria, diatoms, and the seaweed. Despite tremendous advances in microbial ecology over the past two decades, traditional cultivation methods have failed to grow ecologically more relevant microorganisms in the laboratory, leading to a predominance of weed-like species in the world's culture collections. In this review, we highlight the gap between culture-based and culture-independent methods of microbial diversity analysis, especially in investigations of slow growers, oligotrophs, and fastidious and recalcitrant microorganisms. Furthermore, we emphasize the importance of microbial cultivation and the acquisition of the cultivation-based phenotypic data for the testing of hypotheses arising from genomics and proteomics approaches. Technical difficulties in cultivating novel microorganisms and how modern approaches have helped to overcome these limitations are highlighted. After cultivation, adequate preservation without changes in genotypic and phenotypic features of these microorganisms is necessary for future research and training. Hence, the contribution of microbial resource centers in the handling, preservation, and distribution of this novel diversity is discussed. Finally, we explore the concept of microbial patenting and re

5.U03047
Portillo M.C. и др. Cell Size Distributions of Soil Bacterial and Archaeal Taxa // Applied and Environmental Microbiology. 2013. Т. 79. № 24. С. 7610–7617.

Cell size is a key ecological trait of soil microorganisms that determines a wide range of life history attributes, including the efficiency of nutrient acquisition. However, because of the methodological issues associated with determining cell sizes in situ, we have a limited understanding of how cell abundances vary across cell size fractions and whether certain microbial taxa have consistently smaller cells than other taxa. In this study, we extracted cells from three distinct soils and fractionated them into seven size ranges (5 mu m to 0.2 mu m) by filtration. Cell abundances in each size fraction were determined by direct microscopy, with the taxonomic composition of each size fraction determined by high-throughput sequencing of the 16S rRNA gene. Most of the cells were smaller than cells typically grown in culture, with 59 to 67% of cells <1.2 mu m in diameter. Furthermore, each size fraction harbored distinct bacterial and archaeal communities in each of the three soils, and many of the taxa exhibited distinct size distribution patterns, with the smaller size fractions having higher relative abundances of taxa that are rare or poorly characterized (including Acidobacteria, Gemmatimonadetes, Crenarchaeota, Verrucomicrobia, and Elusimicrobia). In general, there was a direct relationship between average cell size and culturability, with those soil taxa that are poorly represented in culture collections tending to be smaller. Size fractionation not only provides important insight into the life history strategies of soil microbial taxa but also is a useful tool to enable more focused investigations into those taxa that remain poorly characterized.

6.U45336
Prakash O. и др. Microbial cultivation and the role of microbial resource centers in the omics era // Applied Microbiology and Biotechnology. 2013. Т. 97. № 1. С. 51–62.

Despite tremendous advances in microbial ecology over the past two decades, traditional cultivation methods have failed to grow ecologically more relevant microorganisms in the laboratory, leading to a predominance of weed-like species in the world's culture collections. In this review, we highlight the gap between culture-based and culture-independent methods of microbial diversity analysis, especially in investigations of slow growers, oligotrophs, and fastidious and recalcitrant microorganisms. Furthermore, we emphasize the importance of microbial cultivation and the acquisition of the cultivation-based phenotypic data for the testing of hypotheses arising from genomics and proteomics approaches. Technical difficulties in cultivating novel microorganisms and how modern approaches have helped to overcome these limitations are highlighted. After cultivation, adequate preservation without changes in genotypic and phenotypic features of these microorganisms is necessary for future research and training. Hence, the contribution of microbial resource centers in the handling, preservation, and distribution of this novel diversity is discussed. Finally, we explore the concept of microbial patenting and requisite guidelines of the "Budapest Treaty" for establishment of an International Depositary Authority.

7.U03047
Turroni F. и др. Global Genome Transcription Profiling of Bifidobacterium bifidum PRL2010 under In Vitro Conditions and Identification of Reference Genes for Quantitative Real-Time PCR // Applied and Environmental Microbiology. 2011. Т. 77. № 24. С. 8578–8587.

Bifidobacteria have attracted significant scientific attention due to their perceived role as health-promoting microorganisms, although the genetics of the bacterial group is still underexplored. In this study, we investigated the transcriptome of Bifidobacterium bifidum PRL2010 during in vitro growth by microarray technology. When B. bifidum PRL2010 was grown in liquid broth, 425 of the 1,644 PRL2010 genes represented on the array were expressed in at least one of the three investigated growth phases, i.e., the lag, exponential, and stationary phases. These transcriptional analyses identified a core in vitro transcriptome encompassing 150 genes that are expressed in all phases. A proportion of these genes were further investigated as potential reference genes by quantitative real-time reverse transcription-PCR (qRT-PCR) assays. Their expression stability was evaluated under different growth conditions, which included cultivation on different carbon sources, exposure to environmental stresses (thermal, acidic, and osmotic), and growth phases. Our analyses validated six reference genes suitable for normalizing mRNA expression levels in qRT-PCR experiments applied to bifidobacteria.

8.U45336
Zheng D.-Q. и др. Comparative functional genomics to reveal the molecular basis of phenotypic diversities and guide the genetic breeding of industrial yeast strains // Applied Microbiology and Biotechnology. 2013. Т. 97. № 5. С. 2067–2076.

An understanding of the genetic basis underlying the phenotypic variations of yeast strains would guide the breeding of this useful microorganism. Here, comparative functional genomics (CFG) of two bioethanol Saccharomyces cerevisiae strains (YJS329 and ZK2) with different stress tolerances and ethanol fermentation performances were performed. Our analysis indicated that different patterns of gene expression in the central carbon metabolism, antioxidative factors, and membrane compositions of these two strains are the main contributors to their various traits. Some of the differently expressed genes were directly caused by the genomic structural variations between YJS329 and ZK2. Moreover, CFG of these two strains also led to novel insights into the mechanism of stress tolerance in yeast. For example, it was found that more oleic acid in the plasma membrane contributes to the acetic acid tolerance of yeast. Based on the genetic information particular to each strain, strategies to improve their adaptability and ethanol fermentation performances were designed and confirmed. Thus, CFG could not only help reveal basis of phenotypic diversities but also guide the genetic breeding of industrial microorganisms.

9. 011522
Арсенюк А.Ю., Павлова И.Б., Игнатьев П.С. ИССЛЕДОВАНИЕ ПРОЦЕССА L-ТРАНСФОРМАЦИИ В ПОПУЛЯЦИИ САЛЬМОНЕЛЛ МЕТОДАМИ ЭЛЕКТРОННОЙ И ЛАЗЕРНОЙ ИНТЕРФЕРЕНЦИОННОЙ МИКРОСКОПИИ//Сельскохозяйственная биология. 2013. № 6. С. 55-60.

"Существование бактерий в отсутствие клеточной стенки — весьма своеобразное и широко распространенное явление, к которому неприменимы принципы классической микробиологии. L-трансформация бактерий с образованием L-форм затрудняет обнаружение патогенов и служит причиной многочисленных ложноотрицательных заключений по результатам биопроб, так как L-формы находятся в некультивируемом состоянии. Процесс L-трансформации при воздействии абиотического (тетрациклин) и биотического (биологически активные вещества Bacillus subtilis ) факторов, а также реверсию нестабильных L-форм исследовали в популяции Salmonella enterica serovar typhimurium методами просвечивающей и сканирующей электронной (ПЭМ и СЭМ), а также лазерной модуляционной интерференционной (МИМ) микроскопии. Выявлены закономерные морфологические признаки L-форм, способных реверсировать в исходное состояние. С появлением методов оптической микроскопии, обладающих сверхразрешением, стало доступно изучение процесса L-трансформации в режиме реального времени на живых бактериях (без фиксации и окрашивания). Полученные на лазерном интерференционном микроскопе МИМ-321 (Россия) трехмерные фазовые портреты позволили не только однозначно идентифицировать L-формы бактерий, но и с высокой точностью определить их основные морфологические параметры (диаметр, периметр, высота, площадь, объем). С применением современных алгоритмов обработки этих данных внутри нестабильных L-форм зарегистрированы кольцевые структуры, идентифицированные нами как ДНК. Гетероморфизм со всеми проявлениями L-трансформации характерен для патогенных и условно патогенных бактерии (как в норме, так и при действии любых абиотических и биотических факторов), что способствует выживанию вида. Изучение биологических особенностей L-трансформации имеет важное значение для понимания процессов существования потенциальных резервуаров возбудителей в природе, а также в патогенезе рецидивов хронических заболеваний.

10. 011433
Дольник А.С., Тамазян Г.С., Першина Е.В., Вяткина К.В., Порозов Ю.Б., Пинаев А.Г., Андронов Е.Е.КОНЦЕПЦИЯ УНИВЕРСАЛЬНОЙ ТАКСОНОМИЧЕСКОЙ СИСТЕМЫ БАКТЕРИЙ: ЭВОЛЮЦИОННОЕ ПРОСТРАНСТВО ГЕНА 16S-РРНК V. 1.0//Сельскохозяйственная биология. 2012. № 5. С. 112-120.

"Проблема системности в таксономии, в основе своей связанная с вопросами эволюции, остается одной из сложнейших в современной биологии, и в частности в микробиологии. Эта проблема всегда привлекала внимание ученых, в том числе Н.И. Вавилова, закон гомологических рядов которого, несомненно, следует отнести к числу наиболее ярких попыток внесения упорядоченности в анализ биоразнообразия. В молекулярной экологии микроорганизмов востребованность универсальной таксономической системы особенно очевидна. Анализ таксономической структуры почвенных микробиомов с использованием секвенаторов нового поколения сталкивается с многочисленными трудностями, одна из которых — невозможность точной идентификации значительной части выявляемых в окружающей среде вариантов гена 16S-рРНК из-за отсутствия близкородственных последовательностей в базах данных. Для решения этой проблемы мы предлагаем концепцию «эволюционного пространства» гена 16S-рРНК, или своеобразной системы, в которой есть место для любой последовательности указанного гена вне зависимости от того, присутствует ли она в базах данных/биосфере и даже реализована ли она в ходе эволюции. В такой системе любой вариант гена 16S-рРНК получает фиксированные координаты. Эволюционное пространство открывает возможность для создания универсальной «таксономической карты» и привлечения ряда мощных алгоритмов для анализа микробного сообщества как единого целого. В настоящей публикации описана первая версия эволюционного пространства гена 16S-рРНК бактерий минимально возможной размерности (13D). "

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