Ways of Long-Term Survival of Hydrocarbon-Oxidizing Bacteria in a New Biocomposite Material-Silanol-Humate Gel.

Immobilized bacterial cells are presently widely used in the development of bacterial preparations for the bioremediation of contaminated environmental objects. Oil hydrocarbons are among the most abundant pollutants. We have previously described a new biocomposite material containing hydrocarbon-oxidizing bacteria (HOB) embedded in silanol-humate gels (SHG) based on humates and aminopropyltriethoxysilane (APTES); high viable cell titer was maintained in this material for at least 12 months. The goal of the work was to describe the ways of long-term HOB survival in SHG and the relevant morphotypes using the techniques of microbiology, instrumental analytical chemistry and biochemistry, and electron microscopy. Bacteria surviving in SHG were characterized by: (1) capacity for rapid reactivation (growth and hydrocarbon oxidation) in fresh medium; (2) ability to synthesize surface-active compounds, which was not observed in the cultures stored without SHG); (3) elevated stress resistance (ability to grow at high Cu2+ and NaCl concentrations); (4) physiological heterogeneity of the populations, which contained the stationary hypometabolic cells, cystlike anabiotic dormant forms (DF), and ultrasmall cells; (5) occurrence of piles in many cells, which were probably used to exchange genetic material; (6) modification of the phase variants spectrum in the population growing after long-term storage in SHG; and (7) oxidation of ethanol and acetate by HOB populations stored in SHG. The combination of the physiological and cytomorphological properties of the cells surviving in SHG for long periods may indicate a new type of long-term bacterial survival, i.e., in a hypometabolic state.

Microbial Biofilms at Meat-Processing Plant as Possible Places of Bacteria Survival.

Biofilm contamination in food production threatens food quality and safety, and causes bacterial infections. Study of food biofilms (BF) is of great importance. The taxonomic composition and structural organization of five foods BF taken in different workshops of a meat-processing plant (Moscow, RF) were studied. Samples were taken from the surface of technological equipment and premises. Metagenomic analysis showed both similarities in the presented microorganisms dominating in different samples, and unique families prevailing on certain objects were noted. The bacteria found belonged to 11 phyla (no archaea). The dominant ones were Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. The greatest diversity was in BFs taken from the cutting table of raw material. Biofilms' bacteria may be the cause of meat, fish and dairy products spoilage possible representatives include Pseudomonas, Flavobacterium, Arcobacter, Vagococcus, Chryseobacterium, Carnobacterium, etc.). Opportunistic human and animal pathogens (possible representatives include Arcobacter, Corynebacterium, Kocuria, etc.) were also found. Electron-microscopic studies of BF thin sections revealed the following: (1) the diversity of cell morphotypes specific to multispecies BFs; (2) morphological similarity of cells in BFs from different samples, micro-colonial growth; (3) age heterogeneity of cells within the same microcolony (vegetative and autolyzed cells, resting forms); (4) heterogeneity of the polymer matrix chemical nature according to ruthenium red staining.

Eurotium Cristatum Postfermentation of Fireweed and Apple Tree Leaf Herbal Teas.

Fungi Eurotium spp. are the main biological agents that ferment the leaves of the Camellia sinensis tea bush to form a popular food product, postfermented tea. The fungus E. cristatum, stored in the collection of the Gause Institute of New Antibiotics under the number INA 01267, was isolated and identified from a briquette of Fujian Chinese tea. The species identification was carried out based on morphocultural characteristics and DNA sequencing. This study is aimed at determining the feasibility of making postfermented herbal teas using E. cristatum and to evaluate their quality. Autofermented herbal teas from Chamaenerion angustifolium (fireweed) and Malus domestica (apple tree) served as the starting material for this study. The change in the concentration of phenolic compounds, organic acids, sugars, and free amino acids was observed for herbal teas subjected to postfermentation with E. cristatum INA 01267. It was found that the E. cristatum INA 01267 strain does not have antimicrobial activity and does not form mycotoxins, which is an indicator of food safety.

Morphological peculiarities of the DNA-protein complexes in starved Escherichia coli cells.

One of the adaptive strategies for the constantly changing conditions of the environment utilized in bacterial cells involves the condensation of DNA in complex with the DNA-binding protein, Dps. With the use of electron microscopy and electron tomography, we observed several morphologically different types of DNA condensation in dormant Escherichia coli cells, namely: nanocrystalline, liquid crystalline, and the folded nucleosome-like. We confirmed the presence of both Dps and DNA in all of the ordered structures using EDX analysis. The comparison of EDX spectra obtained for the three different ordered structures revealed that in nanocrystalline formation the majority of the Dps protein is tightly bound to nucleoid DNA. The dps-null cells contained only one type of condensed DNA structure, liquid crystalline, thus, differing from those with Dps. The results obtained here shed some light on the phenomenon of DNA condensation in dormant prokaryotic cells and on the general problem of developing a response to stress. We demonstrated that the population of dormant cells is structurally heterogeneous, allowing them to respond flexibly to environmental changes. It increases the ability of the whole bacterial population to survive under extreme stress conditions.

The response of soil Arthrobacter agilis lush13 to changing conditions: Transition between vegetative and dormant state.

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.

Drotaverine hydrochloride degradation using cyst-like dormant cells of Rhodococcus ruber.

This work has a focus on adaptive capabilities of the actinobacterium Rhodococcus ruber IEGM 326 to cope with drotaverine hydrochloride (DH), a known pharmaceutical pollutant. Cultivation of R. ruber in a nitrogen-limited medium with incubation at the ambient temperature resulted in the formation of cyst-like dormant cells (CLDCs). They maintained viability for 2-7 months, possessed the undetectable respiratory activity and elevated resistance to heating, and had a specific morphology. CLDCs are regarded to ensure long-term survival in various habitats and may be used as storage formulations. R. ruber IEGM 326 was tolerant to DH (MIC, 200 mg/l) and displayed different abilities to degrade this compound, depending on inoculum, temperature, and the presence of glucose as co-oxidized substrate. Thus, the loss of DH (20 mg/l) over 48 h at the optimal temperature (27 ± 2 °C) was 5-8 % in the absence of glucose after inoculating with vegetative cells. The addition of glucose (5 g/l) increased DH degradation up to 46 %. Noteworthy, CLDCs as inoculum were advantageous over vegetative cells to degrade DH at the non-optimal temperature (35 ± 2 °C) at reduced bulk respiratory activity. The obtained results are promising to improve the biodegrading capabilities of other Rhodococcus strains.

Improved xenobiotic-degrading activity of Rhodococcus opacus strain 1cp after dormancy.

The goals of the present work were as follows: to obtain the dormant forms of R. opacus 1cp; to study the phenotypic variability during their germination; to compare phenotypic variants during the growth on selective and elective media; and to reveal changes in the ability of the strain to destruct xenobiotics that had not been degradable before dormancy. It was shown that Rhodococcus opacus 1cp (the strain degrading chlorinated phenols) became able to utilize a broader spectrum of xenobiotics after storage in the dormant state. Germination of the dormant forms of R. opacus 1cp on an agarized medium was followed by emergence and development of phenotypic variants that could grow on 4-chlorophenol and 2,4,6-trichlorophenol without adaptation. The cells of R. opacus 1cp phenotypic variants also utilized all of the tested chlorinated phenols: 2,3-, 2,5-, and 2,6-dichloro-, 2,3,4- and 2,4,5-trichloro-, pentachlorophenol, and 1,2,4,5-tetrachlorobenzene in concentrations up to 60 mg/L, though at the lower rates than 4-CP and 2,4,6-TCP. The improved degradation of chlorinated phenols by R. opacus strain 1cp exposed to the growth arrest conditions demonstrates the significance of dormancy for further manifestation of the adaptive potential of populations. A new principle of selection of variants with improved biodegradative properties was proposed. It embraces introduction of the dormancy stage into the cell life cycle with subsequent direct inoculation of morphologically different colonies into the media with different toxicants, including those previously not degraded by the strain.

Effect of alkyl hydroxybenzenes on the properties of dioxygenases.

The aim of the present work was to investigate the influence of alkylhydroxybenzenes (AHBs) and tyrosol, which belong to cell differentiation factors d(1) group of autoregulators on properties of biodegradation enzymes, catechol 1,2-dioxygenase (Cat 1,2-DO) and methylcatechol 1,2-dioxygenase (MCat 1,2-DO) of Rhodococcus opacus 6a. AHBs were found to have a greater effect on MCat 1,2-DO than on Cat 1,2-DO. It was expressed by more pronounced changes in the activity of MCat 1,2-DO with unsubstituted catechol at different AHB concentrations and by increasing thermostability of MCat 1,2-DO compared to Cat 1,2-DO under the protective action of AHBs. The compound C(7)-AHB shifted the maximum of dioxygenase activities towards higher temperatures and increased their operation optimum. AHBs changed the specificity constant of dioxygenases by decreasing/increasing the K(m)/V(max) value. For example, the increase in the V(max) value of 3,6-dichlorocatechol oxidation by Cat 1,2-DO in the presence of C(7)-AHB was 300-fold higher compared to the same reaction without AHB. The influence of cell differentiation factors on the properties of dimeric enzymes has been shown for the first time. It gives an idea of how the specificity of enzymes can be changed in vivo when strains contact new substrates. The work has shown the possibility of modification of the properties of dimeric enzymes towards the extension of enzyme activity with difficulty converted substrates or in more extreme conditions, which may be important for biotechnological processes.

Dormant forms of Mycobacterium smegmatis with distinct morphology.

Cultivation of Mycobacterium smegmatis cells in a nitrogen-limited minimal medium (SR-1) followed by prolonged storage at room temperature without shaking resulted in the gradual accumulation of morphologically distinct ovoid forms characterized by (i) low metabolic activity; (ii) elevated resistance to antibiotics and to heat treatment; and (iii) inability to produce colonies on standard agar plates (non-platable cells). Detailed microscopic examination confirmed that ovoid cells possessed an intact cell envelope, specific fine structure and large electron-transparent bodies in the cytoplasm. Cell staining with Nile red and analysis of the lipid content by TLC revealed the presence of significant amounts of apolar lipids in these bodies. The ovoid forms could be stored for significant periods (up to 5 months) and resuscitated afterwards in a modified Sauton's medium. Importantly, resuscitation of ovoid cells was accompanied by their transformation into the typical rod-shaped cells. We suggest that the observed ovoid cells represent dormant forms, resembling morphologically distinct cells of Mycobacterium tuberculosis previously isolated from tuberculosis patients and infected animals.

Nature of DNA-bound fatty acids in Pseudomonas aurantiaca.

The existence of Pseudomonas aurantiaca DNA-bound fatty acids and lipids is presented in this work. The isolation of DNA was carried out by two different procedures, namely, phenol and detergent-based phenol isolation in order to prove the presence of DNA-bound lipids. The lipid content of DNA is expressed in terms of fatty acid profile. A high level of 16:0, 18:0 and 18:1 is characteristic for tightly bound DNA lipids. On the other hand, the fatty acids such as 14:1, iso14:0 and iso16:0 are found in trace amounts only in DNA lipid fraction, but these fatty acids are not found in the whole-cell lipids. Absolutely no 3-hydroxy fatty acids were found in DNA lipids. However, both C16 and C18 species represent the main fatty acids of whole-cell and DNA-bound lipids. The presence of DNA-bound lipids even under tough treatment of DNA allows to conclude that these lipids represent a special pool among cellular lipids.

The structure of resting bacterial populations in soil and subsoil permafrost.

The structure of individual cells in microbial populations in situ of the Arctic and Antarctic permafrost was studied by scanning and transmission electron microscopy methods and compared with that of cyst-like resting forms generated under special conditions by the non-spore-forming bacteria Arthrobacter and Micrococcus isolated from the permafrost. Electron microscopy examination of microorganisms in situ revealed several types of bacterial cells having no signs of damage, including "dwarf" curved forms similar to nanoforms. Intact bacterial cells in situ and frozen cultures of the permafrost isolates differed from vegetative cells by thickened cell walls, the altered structure of cytoplasm, and the compact nucleoid, and were similar in these features to cyst-like resting forms of non-spore-forming "permafrost" bacterial strains of Arthrobacter and Micrococcus spp. Cyst-like cells, being resistant to adverse external factors, are regarded as being responsible for survival of the non-spore-formers under prolonged exposure to subzero temperatures and can be a target to search for living microorganisms in natural environments both on the Earth and on extraterrestrial bodies.