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The strain Gordonia polyisoprenivorans 135 is able to utilize a wide range of aromatic compounds. The aim of this work was to study the features of genetic organization and biotechnological potential of the strain G. polyisoprenivorans 135 as a degrader of aromatic compounds. The study of the genome of the strain 135 and the pangenome of the G. polyisoprenivorans species revealed that some genes, presumably involved in PAH catabolism, are atypical for Gordonia and belong to the pangenome of Actinobacteria. Analyzing the intergenic regions of strain 135 alongside the "panIGRome" of G. polyisoprenivorans showed that some intergenic regions in strain 135 also differ from those located between the same pairs of genes in related strains. The strain G. polyisoprenivorans 135 in our work utilized naphthalene (degradation degree 39.43%) and grew actively on salicylate. At present, this is the only known strain of G. polyisoprenivorans with experimentally confirmed ability to utilize these compounds.
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Biodegradation of phenol is an effective method for removing this toxicant from contaminated sites. Phenol is a toxic compound for living cells, so many bacteria degrade phenol in relatively low concentrations, up to 0.75 g L-1. The Rhodococcus opacus strain 1CP is an effective destructor of a wide range of pollutants. In the absence of a carbon source in the medium, cells of the R. opacus 1CP strain easily form cyst-like resting cells (CLC). The purpose of this work was to evaluate the viability of cells during long-term storage and the efficiency of the process of phenol destruction by R. opacus 1CP cells germinating after dormancy. Resting cells were obtained by simple cultivation in a rich medium followed by storage under static conditions. This is a simple approach to obtain a large amount of biomass. Decomposition of phenol proceeded via catechol followed by ortho-cleavage of aromatic ring. The induction of three phenol hydroxylases was detected by RT-PCR in cells germinated in a mineral medium with phenol as the carbon source. The stability of the genome of cells germinating after dormancy is shown by box-PCR. Dormant R. opacus 1CP cells, both suspended and immobilized, can be directly used for the decomposition of phenol after 4-12 months storage. In addition to phenol, after 9 months of storage, immobilized germinating cells easily metabolized 4-chlorophenol and 2,4,6-trichlorophenol. The results demonstrate a potential and simple approach toward achieving long-term storage of cells for further use in bioremediation.
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Bacteria, designated as A1.1 and A1.2, were isolated from poultry waste based on the ability to form ammonia on LB nutrient medium. Whole genome sequencing identified the studied strains as Peribacillus frigoritolerans VKM B-3700D (A1.1) and Bacillus subtilis VKM B-3701D (A1.2) with genome sizes of 5462638 and 4158287 bp, respectively. In the genome of B. subtilis VKM B-3701D, gene clusters of secondary metabolites of bacillin, subtilisin, bacilisin, surfactin, bacilliacin, fengycin, sactipeptide, and ratipeptide (spore killing factor) with potential antimicrobial activity were identified. Clusters of coronimine and peninodin production genes were found in P. frigoritolerans VKM B-3700D. Information on coronimine in bacteria is extremely limited. The study of the individual properties of the strains showed that the cultures are capable of biosynthesis of a number of enzymes, including amylases. The B. subtilis VKM V-3701D inhibited the growth of bacterial test cultures and reduced the growth rate of the mold fungus Aspergillus unguis VKM F-1754 by 70% relative to the control. The antimicrobial activity of P. frigoritolerans VKM V-3700D was insignificant. At the same time, a mixture of cultures P. frigoritolerans VKM B-3700D/B. subtilis VKM B-3701D reduced the growth rate of A. unguis VKM F-1754 by 24.5%. It has been shown that strain A1.1 is able to use nitrogen compounds for assimilation processes. It can be assumed that P. frigoritolerans VKM V-3700D belongs to the group of nitrifying or denitrifying microorganisms, which may be important in developing methods for reducing nitrogen load and eutrophication.
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The application of Gordonia strains in biotechnologies of environmental purification as degraders of pollutants of different chemical structures is an interesting research topic. The strain Gordonia rubripertincta 112 (IEGM112) is capable of utilizing diesel fuel, alkanes, and aromatic compounds. The aim of this work was to study the potential of G. rubripertincta 112 as a degrader of aromatic and aliphatic compounds and analyze its complete genome in comparison with other known G. rubripertincta strains. The genome had a total length of 5.28 Mb and contained 4861 genes in total, of which 4799 were coding sequences (CDS). The genome contained 62 RNA genes in total, of which 50 were tRNAs, three were ncRNAs, and nine were rRNAs. The strain bears plasmid elements with a total length of 189,570 nucleotides (plasmid p1517). The strain can utilize 10.79 ± 1.17% of hexadecane and 16.14 ± 0.16% of decane over 3 days of cultivation. In the genome of the strain, we have found metabolic pathways of alkane (cytochrome P450 hydroxylases) and catechol (ortho- and meta-pathways) degradation. These results will help us to further approach the fundamental study of the processes occurring in the strain cells and to enrich our knowledge of the catabolic capabilities of G. rubripertincta.
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Gordonia polyisoprenivorans 135 is a promising degrader of aromatic hydrocarbons. It can utilize phenanthrene, anthracene, benzoate, chlorobenzoates, and phenol. The genome of strain 135 was completely sequenced; it consists of a single 5,988,360-bp circular chromosome (GC content of 67.01%).
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In this work, a new Ch2 strain was isolated from soils polluted by agrochemical production wastes. This strain has a unique ability to utilize toxic synthetic compounds such as epsilon-caprolactam (CAP) as a sole carbon and energy source and the herbicide glyphosate (GP) as a sole source of phosphorus. Analysis of the nucleotide sequence of the 16S rRNA gene of Ch2 revealed that the strain belongs to the species Pseudomonas putida. This strain grew in the mineral medium containing CAP in a concentration range of 0.5 to 5.0 g/L and utilized 6-aminohexanoic acid and adipic acid, which are the intermediate products of CAP catabolism. The ability of strain Ch2 to degrade CAP is determined by a conjugative megaplasmid that is 550 kb in size. When strain Ch2 is cultured in a mineral medium containing GP (500 mg/L), more intensive utilization of the herbicide occurs in the phase of active growth. In the phase of declining growth, there is an accumulation of aminomethylphosphonic acid, which indicates that the C-N bond is the first site cleaved during GP degradation (glyphosate oxidoreductase pathway). Culture growth in the presence of GP during the early step of its degradation is accompanied by unique substrate-dependent changes in the cytoplasm, including the formation of vesicles of cytoplasmic membrane consisting of specific electron-dense content. There is a debate about whether these membrane formations are analogous to metabolosomes, where the primary degradation of the herbicide can take place. The studied strain is notable for its ability to produce polyhydroxyalkanoates (PHAs) when grown in mineral medium containing GP. At the beginning of the stationary growth phase, it was shown that, the amount and size of PHA inclusions in the cells drastically increased; they filled almost the entire volume of cell cytoplasm. The obtained results show that the strain P. putida Ch2 can be successfully used for the PHAs' production. Moreover, the ability of P. putida Ch2 to degrade CAP and GP determines the prospects of its application for the biological cleanup of CAP production wastes and in situ bioremediation of soil polluted with GP.
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epsilon-Caprolactam (Caprolactam, CAP), a monomer of the synthetic non-degradable polymer nylon-6, is the major wastewater component in the production of caprolactam and nylon-6. Biological treatment of CAP, using microbes could be a potent alternative to the current waste utilization techniques. This work focuses on the characterization and potential use of caprolactam-degrading bacterial strain BS3 isolated from soils polluted by CAP production wastes. The strain was identified as Brevibacterium epidermidis based on the studies of its morphological, physiological, and biochemical properties and 16S rRNA gene sequence analysis. This study is the first to report the ability of Brevibacterium to utilize CAP. Strain BS3 is an alcalo- and halotolerant organism, that grows within a broad range of CAP concentrations, from 0.5 up to 22.0 g/L, optimally at 1.0-2.0 g/L. A caprolactam biodegradation experiment using gas chromatography showed BS3 to degrade 1.0 g/L CAP over 160 h. In contrast to earlier characterized narrow-specific CAP-degrading bacteria, strain BS3 is also capable of utilizing linear nylon oligomers (oligomers of 6-aminohexanoic acid), CAP polymerization by-products, as sole sources of carbon and energy. The broad range of utilized toxic pollutants, the tolerance for high CAP concentrations, as well as the physiological properties of B. epidermidis BS3, determine the prospects of its use for the biological cleanup of CAP and nylon-6 production wastes that contain CAP, 6-aminohexanoic acid, and low molecular weight oligomer fractions.
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A new biopreparation is developed to clean soils from oil pollution in the arid climate of the Republic of Kazakhstan. The biopreparation includes bacterial strains R. qingshengii F2-1, R. qingshengii F2-2, and P. alloputida BS3701. When using the biopreparation in a liquid mineral medium with 15% crude oil, laboratory studies have revealed degradation of 48% n-alkanes and 39% of PAHs after 50 days. The effectiveness of the biopreparation has been demonstrated in field experiments in the soil contaminated with 10% crude oil at the K-Kurylys landfill, Republic of Kazakhstan. During the six-month field experiment, the number of oil degraders reached 107 CFU/g soil, which degraded 70% of crude oil by the end of the experiment.
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Bacteria make a huge contribution to the purification of the environment from toxic stable pollutants of anthropogenic and natural origin due to the diversity of their enzyme systems. For example, the ability to decompose 3-chlorobenzoate (3CBA) by the four representative genera of Actinobacteria, such as Rhodococcus, Gordonia, Microbacterium, and Arthrobacter, was studied. In most cases, the formation of 4-chlorocatechol as the only key intermediate during the decomposition of 3CBA was observed. However, Rhodococcus opacus strain 1CP was an exception, whose cells decomposed 3CBA via both 3-chloro- and 4-chlorocatechol. The enzyme 3-Chlorobenzoate 1,2-dioxygenase (3CBDO) induced during the growth of these bacteria in the presence of 3CBA differed significantly in substrate specificity from the benzoate dioxygenases induced upon growth in the presence of benzoate. The R. opacus 6a strain was found to contain genes encoding chlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, and dienelactone hydrolase, whose nucleotide sequence was 100% consistent with the sequences of the corresponding genes encoding the enzymes of the modified 4-chlorocatechol ortho-cleavage pathway of the strain R. opacus 1CP. However, the gene encoding chloromuconolactone dehalogenase (clcF) was not found in the representatives of the actinomycete genera, including Gordonia and Arthrobacter. A linear mega-plasmid carrying 3-chlorocatechol degradation genes remained stable after maintaining the R. opacus 1CP strain on an agar-rich medium for 25 years. In general, a similar plasmid was absent in actinobacteria of other genera, as well as in closely related species of R. opacus 6a.
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BACKGROUND: Halogenated aromatic compounds are more resistant to microbial degradation than non-halogenated aromatic compounds. Microbial degradation of sodium benzoate in the presence of sodium 3-chlorobenzoate is of interest. The ability to degrade aromatic compounds is largely determined by the substrate specificity of the first enzyme that initiates degradation, namely, benzoate 1,2-dioxygenase for benzoate degradation, and 3-chlorobenzoate 1,2-dioxygenase for 3-chlorobenzoate degradation. In this study, the perspective of immobilized cells of Rhodococcus opacus 1CP actinobacterium for degradation of benzoate and 3-chlorobenzoate was explored. METHODS: The biosensor approach (a membrane microbial sensor based on immobilized cells of Rhodococcus opacus 1CP and the Clark-type oxygen electrode as a transducer) was applied to evaluate the actinobacterial cells' responses to benzoate and 3-chlorobenzoate in the absence of both enzymes, benzoate 1,2-dioxygenase and 3-chlorobenzoate 1,2-dioxygenase, or in the presence of one of the said enzymes. RESULTS: Data obtained show that 1CP actinobacterium possessed a constitutive system for the transport of benzoate and 3-chlorobenzoate into culture cells. The affinity of the transport system for benzoate was higher than that for 3-chlorobenzoate. Moreover, adaptation to one substrate did not preclude the use of the second substrate. Probably, porins facilitated the penetration of benzoate and 3-chlorobenzoate into 1CP cells. Analyzing V vs. S dependencies, negative cooperativity was found, when benzoate 1,2-dioxygenase bound substrate (3-chlorobenzoate), while positive cooperativity was determined at benzoate binding. The observed difference could be associated with the presence of at least two systems of 3-chlorobenzoate transport into actinobacterial cells and allosteric interaction of active sites of benzoate 1,2-dioxygenase in the presence of 3-chlorobenzoate. CONCLUSIONS: The membrane microbial sensor based on immobilized Rhodococcus opacus 1CP cells could be useful as a perspective tool for comparative evaluation of enzymes of complex structure such as benzoate- and 3-chlorobenzoate 1,2-dioxygenase.
Asunto(s)
Dioxigenasas , Rhodococcus , Benzoatos/metabolismo , Clorobenzoatos , Dioxigenasas/metabolismo , Rhodococcus/metabolismoRESUMEN
Rhodococci are typical soil inhabitants which take part in remediation of soil polluted with hydrocarbons. In this paper, we describe a new strain, Rhodococcus qingshengii 7B, which is capable of growth and hydrocarbon degradation at 45°C and in the presence of up to 10% NaCl in the medium. The genome of the 7B strain consists of a 6,278,280 bp chromosome and two plasmids. The circular plasmid is 103,992 bp in length. The linear plasmid is 416,450 bp in length. Genome analysis revealed the genes of degradation of various hydrocarbons, resistance to salt stress and plant growth promoting activity. This strain is promising for use in remediation of oil-contaminated soils, because it has a pronounced ability to utilize crude oil, oil sludge and individual hydrocarbons in a wide temperature range. Over 15 days of the experiment, the strain utilized 51% of crude oil at 28°C and 24% at 45 °Ð¡.
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Microorganisms capable of decomposing hydrophobic substrates in cold climates are of considerable interest both in terms of studying adaptive reactions to low temperatures and in terms of their application in biotechnologies for cleaning up oil spills in a crude-oil polluted soil. The aim of this work was to investigate the genome of Rhodococcus opacus S8 and explore behavior traits of this strain grown in the presence of hexadecane. The genome size of strain S8 is 8.78 Mb, of which the chromosome size is 7.75 Mb. The S8 strain contains 2 circular plasmids of 135 kb and 105 kb and a linear plasmid with a size of 788 kb. The analysis of the genome revealed the presence of genes responsible for the degradation of alkanes and synthesis of biosurfactants. The peculiarities of morphology of microbial cells when interacting with a hydrophobic substrate were revealed. An adaptive mechanism responsible in the absence of oxygen for maintaining the process of degradation of hexadecane is discussed. The data obtained show that the strain S8 has great potential to be used in biotechnologies.
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In the process of evolution, living organisms develop mechanisms for population preservation to survive in unfavorable conditions. Spores and cysts are the most obvious examples of dormant forms in microorganisms. Non-spore-forming bacteria are also capable of surviving in unfavorable conditions, but the patterns of their behavior and adaptive reactions have been studied in less detail compared to spore-forming organisms. The purpose of this work was to study the features of transition from dormancy to active vegetative growth in one of the non-spore-forming bacteria, Gordonia polisoprenivorans 135, which is known as a destructor of such aromatic compounds as benzoate, 3-chlorobenzoate, and phenol. It was shown that G. polyisoprenivorans 135 under unfavorable conditions forms cyst-like cells with increased thermal resistance. Storage for two years does not lead to complete cell death. When the cells were transferred to fresh nutrient medium, visible growth was observed after 3 h. Immobilized cells stored at 4 °C for at least 10 months regenerated their metabolic activity after only 30 min of aeration. A study of the ultrathin organization of resting cells by transmission electron microscopy combined with X-ray microanalysis revealed intracytoplasmic electron-dense spherical membrane ultrastructures with significant similarity to previously described acidocalcisomas. The ability of some resting G. polyisoprenivorans 135 cells in the population to secrete acidocalcisome-like ultrastructures into the extracellular space was also detected. These structures contain predominantly calcium (Ca) and, to a lesser extent, phosphorus (P), and are likely to serve as depots of vital macronutrients to maintain cell viability during resting and provide a quick transition to a metabolically active state under favorable conditions. The study revealed the features of transitions from active growth to dormant state and vice versa of non-spore-forming bacteria G. polyisoprenivorans 135 and the possibility to use them as the basis of biopreparations with a long shelf life.
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The strain Rhodococcus qingshengii VT6 is a promising degrader of persistent pollutants and a putative biosurfactant producer. The genome of the strain was sequenced completely. It consists of a 6,457,868-bp chromosome and 4 plasmids (pLP1, 501,672 bp; pLP2, 188,969 bp; pCP1, 100,387 bp; and pCP2, 132,858 bp).
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The aim of the work was to carry out the physiological, biochemical and genetic characterization of the Exiguobacterium alkaliphilum B-3531D strain. This strain is promising for use in the field of environmental biotechnology, since it has a pronounced ability to utilize crude oil and individual hydrocarbons in a wide temperature range. The genome of the strain was sequenced and completely assembled, it consists of a 2,903,369 bp circular chromosome and two circular plasmids, namely, pE73 (73,590 bp) and pE52 (52,125 bp). When cultivated in a mineral medium containing 2% of crude oil, the strain utilized 50% within 30 days of the experiment. In simulated seawater with the same oil content, the loss of hydrocarbons was 45% over the same period. For the first time we observed in an E. alkaliphilum strain the ability to efficiently utilize crude oil, including with an increased content of sodium chloride in the cultivation medium.
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The intensive development of agriculture leads to the depletion of land and a decrease in crop yields and in plant resistances to diseases. A large number of fertilizers and pesticides are currently used to solve these problems. Chemicals can enter the soil and penetrate into the groundwater and agricultural plants. Therefore, the primary task is to intensify agricultural production without causing additional damage to the environment. This problem can be partially solved using microorganisms with target properties. Microorganisms that combine several useful traits are especially valuable. The aim of this work was to search for new microbial strains, which are characterized by the ability to increase the bioavailability of nutrients, phytostimulation, the antifungal effect and the decomposition of some xenobiotics. A few isolated strains of the genera Bacillus and Pseudomonas were characterized by high activity against fungal phytopathogens. One of the bacterial strains identified as Priestiaaryabhattai on the basis of the 16S rRNA gene sequence was characterized by an unusual cellular morphology and development cycle, significantly different from all previously described bacteria of this genus. All isolated bacteria are capable of benzoate degradation as a sign of the ability to degrade aromatic compounds. Isolated strains were shown to be prospective agents in biotechnologies.
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Microbial reactor sensors (based on freshly harvested intact microbial cells) or microbial membrane sensors (based on immobilized microbial cells) can be used as convenient instruments for studying processes that cause the response of a biosensor, such as the properties of enzymes or the characteristics of metabolism. However, the mechanisms of the formation of biosensors responses have not yet been fully understood to study only one of these processes. In this work, the results of studies on the formation of a response to juglone for intact and immobilized bacterial cells used as receptors are presented. It was shown that the contribution of reactive oxygen species (ROS) to the formation of the biosensor response depends on the culture receptor and the form of juglone, quinone, or phenolate used. The response to the quinone form of juglone both for intact and immobilized cells of catalase-positive actinobacterium is formed regardless of the presence of ROS. The response of freshly harvested intact actinobacterial cells was caused by the rate of the enzymatic conversion of juglone. The rate of the response of immobilized actinobacterial cells was influenced by the activity of transport systems and metabolism. The response of immobilized pseudomonad cells was caused by the transport of juglone into cells, the inhibitory effect of juglone-induced ROS, and juglone metabolism.
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Técnicas Biosensibles , Citotoxinas/farmacología , Naftoquinonas/farmacología , Bacterias , Células Inmovilizadas , Especies Reactivas de OxígenoRESUMEN
Violacein is a biotechnologically significant secondary metabolite due to its antibacterial, antifungal, and other properties. Isolation, research, and identification of violacein producing strains are of interest for the development of biotechnological processes, in order to enhance the biosynthesis of this compound. The purpose of the present work was to study the properties of a newly isolated bacterium capable of synthesizing blue-purple pigment. An aboriginal bacterium was isolated from the coastal zone of the Vezelka River in the city of Belgorod. Based on chemical and spectrophotometric studies of the crude ethanol extract, the pigment was identified as violacein, and the isolate was assigned to the group of violacein-forming bacteria, which includes bacteria of the genera Chromobacterium, Iodobacter, Janthinobacterium, Duganella, Collimonas, and Massilia. Based on cultural, morphological, tinctorial, physiological, and biochemical properties, as well as analysis of the 16S rRNA gene sequence, the new isolated strain was assigned to the genus Janthinobacterium. The isolated strain is capable of suppressing the growth of a number of fungal and bacterial phytopathogens. For representatives of the genus Janthinobacterium, their inhibitory influence on cyanobacteria was shown for the first time.
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The electrochemical reactor microbial sensor with the Clark oxygen electrode as the transducer was used for investigation of the competition between 3-chlorobenzoate (3-CBA) and its analogues, 2- and 4-chlorobenzoate (2-CBA and 4-CBA), for 3-chlorobenzoate-1,2-dioxygenase (3-CBDO) of Rhodococcus opacus 1CP cells. The change in respiration of freshly harvested R. opacus 1CP cells in response to 3-CBA served as an indicator of 3-CBDO activity. The results obtained confirmed inducibility of 3-CBDO. Sigmoidal dependency of the rate of the enzymatic reaction on the concentration of 3-CBA was obtained and positive kinetic cooperativity by a substrate was shown for 3-CBDO. The Hill concentration constant, S0.5, and the constant of catalytic activity, Vmax, were determined. Inhibition of the rate of enzymatic reaction by excess substrate, 3-CBA, was observed. Associative (competitive inhibition according to classic classification) and transient types of the 3-CBA-1,2-DO inhibition by 2-CBA and 4-CBA, respectively, were found. The kinetic parameters such as S0.5i and Vmaxi were also estimated for 2-CBA and 4-CBA. The disappearance of the S-shape of the curve of the V versus S dependence for 3-CBDO in the presence of 4-CBA was assumed to imply that 4-chlorobenzoate had no capability to be catalytically transformed by 3-chlorobenzoate-1,2-dioxygenase of Rhodococcus opacus 1CP cells.
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Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Clorobenzoatos/metabolismoRESUMEN
This work was aimed at studying the response of soil non-spore-forming actinobacterial strain Arthrobacter agilis Lush 13 to changing natural conditions, such as nutrient availability and the presence of degradable and recalcitrant aliphatic and aromatic substrates. The A. agilis strain Lush13 was able to degrade octane, nonane, hexadecane, benzoate, phenol, and 2,3-, 2,4-, 2,5-, 2,6-dichlorophenols, but not grew on 3,4-dichlorophenol, 2,3,4-, 2,4,5-, 2,4,6-trichlorophenol (TCP), pentachlorophenol (PCP), 2-chlorobenzoate, 3-chlorobenzoate, 3,5-dichlorobenzoate, 2,4-dichlorobenzoate. Under growth-arresting conditions due to nitrogen- or multiple starvation or recalcitrant (non-utilizable) carbon source, the studied strain preserved viability for prolonged periods (4-24 months) due to transition to dormancy in the form of conglomerated small and ultrasmall cyst-like dormant cells (CLC). Dormant cells were shown to germinate rapidly (30 min or later) after removal of starvation stress, and this process was followed by breakdown of conglomerates with the eliberation and further division of small multiple actively growing daughter cells. Results of this study shed some light to adaptive capabilities of soil arthrobacters in pure and polluted environments.