V. N. Danilevich

Changes in the Fine Structure of Microbial Cells Induced by Chaotropic Salts

The electron microscopic examination of thin sections of cells of the yeasts Saccharomyces cerevisiae and Pichia pastoris and the gram-positive bacteria Micrococcus luteus and Bacillus subtilis showed that cell treatment with the chaotropic salts guanidine hydrochloride (6 M) and guanidine thiocyanate (4 M) at 37°C for 3–5 h or at 100°C for 5–6 min induced degradative processes, which affected almost all cellular structures. The cell wall, however, retained its ultrastructure, integrity, and rigidity, due to which the morphology of cells treated with the chaotropic salts did not change. High-molecular-weight DNA was localized in a new cell compartment, the ectoplasm (a peripheral hydrophilic zone). The chaotropic salts destroyed the outer and inner membranes and partially degraded the outer and inner protein coats of Bacillus subtilis spores, leaving their cortex (the murein layer) unchanged. The spore core became accessible to stains and showed the presence of regions with high and low electron densities. The conditions of cell treatment with the chaotropic salts were chosen to provide for efficient in situ PCR analysis of the 16S and 18S rRNA genes with the use of oligonucleotide primers.

Intraspecies diversity of dormant forms of Mycobacterium smegmatis

The non-spore-forming gram-positive bacterium Mycobacterium smegmatis mc2 155, related to M. tuberculosis, was revealed to be capable of forming different types of dormant forms (DFs) during the life cycle of its cultures. The relationship between the intraspecies diversity of DFs and the cultivation conditions of the mycobacterium was established. The DFs possessed the following common properties: (i) maintenance of viability for a long period of time (5 months), (ii) resistance to deleterious factors such as heat treatment, and (iii) morphological and ultrastructural peculiarities that distinguish DFs from vegetative cells. The diversity of M. smegmatis DFs manifested itself in differences in terms of structural organization, conditions required for growth renewal, and capacity to produce antibiotic-resistant variants upon germination on selective media. Well-differentiated cystlike dormant cells (CDCs) were formed in the cultures grown in synthetic SR1 medium with fivefold-decreased nitrogen content. The structural organization of CDCs differed from that of other DF types mainly in the presence of club-shaped cells, thickened lamellar cell walls, coarse cytoplasm texture, and large electron-transparent triacylglyceride inclusion bodies. It was possible to use mycobacterial CDCs as a source of PCR-competent DNA. CDC populations were heterogeneous in cell buoyant density, and the individual fractions, which we isolated, were found to differ in thermal stability and the ability to revert to growth under standard conditions. Coccoid DFs, which retained their colony-forming capacity for a long time but were less heat-resistant than the CDCs, were formed by mycobacteria grown in standard Sauton’s medium with initial pH value decreased to 6.2. Poorly differentiated DFs resulted from growing mycobacterial cultures in Sauton’s medium with a fivefold-decreased phosphorus content. Upon germination of various DF types, the variants resistant to kanamycin (200 μg/ml) and tetracycline (20 μg/ml) were obtained. CDC suspensions incubated for 5 months demonstrated the highest percentage (1.5%) of antibiotic-resistant clones. The data obtained on the DF diversity of M. smegmatis, a fast-growing relative of M. tuberculosis, contribute to our understanding of the flexibility of the survival strategy of this bacterium in nature and in the host organism.

Interaction of Chromosomal and Plasmid DNA in Acidithiobacillus ferrooxidans Strains Adapted to Different Oxidation Substrates

Restriction analysis of plasmids pTFK1 and pTFK2 of theAcidithiobacillus ferrooxidans strain TFBk was carried out, and the sizes of these plasmids were determined (13.5 and 30 kb, respectively). A macrorestriction map was built for plasmid pTFK1. DNA–DNA hybridization revealed that the plasmids contained homologous nucleotide sequences. Plasmid pTFK2 labeled with 32P was used as a probe for Southern hybridization with blots of XbaI-generated fragments of the chromosomal DNA of A. ferrooxidans strains grown on a medium containing Fe2+ or adapted to different oxidation substrates. Low-intensity hybridization signals were observed for many fragments of the chromosomal DNA of the strains studied. In the process of adaptation to new oxidation substrates, the localization of bands producing the low-intensity hybridization signals changed in a number of cases. Certain fragments of the chromosomal DNA of the strains adapted to different oxidation substrates produced strong hybridization signals with pTFK2. The data obtained are discussed in terms of the possible role of IST elements and plasmids in the adaptation of A. ferrooxidans to new energy substrates, microevolution, and strain polymorphism.

The structural peculiarities of condensed DNA micro- and nanoparticles formed in PCR

Studies of DNA condensation have opened new perspectives in biotechnology and medicine. DNA condensation induced by polyamines or trivalent metal ions in vitro at room temperature has been investigated in detail. Our recent studies have demonstrated Mg2+-mediated formation of DNA condensates during the PCR. In this study, we report the unique morphology and fine structure of PCR-generated condensed DNA particles using electron and atomic force microscopy. The principal morphologies of studied DNA condensates are 3D particles of micrometer dimensions, oval microdisks of nanometer thickness, filaments, and compact nano-sized particles. SEM examinations have revealed a new structural type of spherical and elliptical 3D microparticles formed by numerous definitely oriented microdisks and their segments. AFM revealed a granular structure of the microdisk surface and the smallest nano-sized disks and thinnest nanofibrils – that appear to be the primary products of DNA condensation during the PCR. We suggest that the formation of DNA nanofibrils and nanodisks in PCR occurs due to Mg2+ – mediated intermolecular (lateral) and intramolecular condensation of ssDNA. Aggregation of elementary nanodisks in the course of thermal PCR cycles, occurring both by magnesium cations and via complementary interactions, give a rise to large nano-sized aggregates and more complex microparticles.

Rapid and efficient technique for the production of condensed DNA and RNA nanoparticles using thermal cycling

The phenomenon of formation of condensed forms of DNA (micro and nanoparticles) in the course of polymerase chain reaction (PCR) was described recently [7–10]. These particles are formed at late stages of PCR, under conditions of accumulation of significant amounts of the PCR product—double stranded linear amplicon DNA. The DNA micropar ticles formed as a result of PCR have a unique struc ture (ultrastructure). These are mostly large forma tions (0.5 to 10 μm), their size and morphology depend on the template used in the PCR (genomic DNA of microorganisms or plasmid DNAs) and the type of thermostable DNA polymerase (Taq or Klen Taq polymerase) [9, 10]. The microparticles are stable in water but are easily destroyed (dissociated) in the presence of chelates (1 mM EDTA); the main product of this dissociation is a double stranded amplicon DNA [7, 8]. The destruction of microparticles in the presence of EDTA indicates that Mg2+ cations con tained in the PCR buffer play the key role in the con densation of amplicon DNA. Recently, we have estab lished that, along with Mg2+ cations, single stranded DNA fragments are also required for the condensation of DNA during PCR [9].

Micro- and nanoparticles of condensed DNA Formed in PCR with Taq polymerase and plasmid DNA as a template

Formation of micro- and nanoparticles of condensed DNA during PCR with microbial genomic DNA or plasmid DNA as templates was reported previously. Initially, the microparticles were formed using a thermostable KlenTaq polymerase, which is a deletion variant of Taq polymerase. The present work shows that Taq polymerase is also capable of efficient formation of micro- and nanoparticles of condensed DNA in PCR. Electron microscopy revealed a number of morphological types (more than four) of microparticles produced in PCR with different reaction buffers in the presence of Taq polymerase and different plasmid DNAs as a template. In the case of some kinds of amplicons, an increase in the number of thermal cycles was shown to result in production of numerous nanowires and electron-dense spherical nanoparticles. The PCR conditions for preferential formation of discs (or ellipsoids) a few micrometers in diameter and several dozens of nanometers in thickness were determined. The structure of microparticles formed in the presence of Taq polymerase was found to depend on the level of synthesis of single-stranded DNA fragments in PCR. Experiments with nuclease S1 revealed that, along with double-stranded DNAs of the amplicon, micro- and nano-particles contained single-stranded DNA fragments, which were absolutely necessary for their formation. In light of these data, the molecular mechanism of micro- and nanoparticle formation in the course of PCR is discussed.

Fluorescence Microscopic Study of Microorganisms Treated with Chaotropic Agents

The yeasts Saccharomyces cerevisiae and Pichia pastoris and the bacteria Micrococcus luteus, Bacillus subtilis, and Anaerobacter polyendosporus have been treated with the chaotropic agents guanidine hydrochloride and guanidine thiocyanate and certain detergents and studied using fluorescence microscopy. Studies with the use of fluorochromes that can selectively stain nucleic acids (diamidino-2-phenylindole (DAPI), propidium iodide, and acridine orange) show that treatment of the bacterial and yeast cells at 37°C for 3–5 h induces a release of DNA from the cytoplasm and its accumulation in the cellular zone, known as ectoplasm, located between the cell wall and the remainder of the cytoplasm (called endoplasm) in the form of one or several large granules. After treating the cells with the chaotropic agents at 100°C for 5–6 min, the DNA is diffusively distributed over the ectoplasm. The fluorochromes used do not allow the detection of RNA. These findings are in agreement with previous data obtained from electron microscopic study of thin cell sections. After 33 PCR cycles, a considerable portion of DNA leaves the cells; as a result, they show a low level of diffusive fluorescence when stained with DAPI. When endospores of B. subtilis are treated with the chaotropic agents, they become highly permeable to the fluorochromes. Fluorescence microscopic study of such endospores shows that they contain DNA in the central part of their cores.

New insight into formation of DNA-containing microparticles during PCR: the scaffolding role of magnesium pyrophosphate crystals

This work aims to study molecular mechanisms involved in the formation of DNA-containing microparticles and nanoparticles during PCR. Both pyrophosphate and Mg2+ ions proved to play an important role in the generation of DNA microparticles (MPs) with a unique and sophisticated structure in PCR with Taq polymerase. Thus, the addition of Tli thermostable pyrophosphatase to a PCR mixture inhibited this process and caused the destruction of synthesized DNA MPs. Thermal cycling of Na-pyrophosphate (Na-PPi)- and Mg2+-containing mixtures (without DNA polymerase and dNTPs) under the standard PCR regime yielded crystalline oval or lenticular microdisks and 3D MPs composed from magnesium pyrophosphate (Mg-PPi). As shown by scanning electron microscopy (SEM), the produced Mg-PPi microparticles consisted of intersecting disks or their segments. They were morphologically similar but simpler than DNA-containing MPs generated in PCR. The incorporation of dNTPs, primers, or dsDNA into Mg-pyrophosphate particles resulted in the structural diversification of 3D microparticles. Thus, the unusual and complex structure of DNA MPs generated in PCR is governed by the unique feature of Mg-pyrophosphate to form supramolecular particles during thermal cycling. We hypothesize the Mg-pyrophosphate particles that are produced during thermal cycling serve as scaffolds for amplicon DNA condensation.

Effective PCR detection of vegetative and dormant bacterial cells due to a unified method for preparation of template DNA encased within cell envelopes

The unified method of template preparation for PCR in the form of DNA covered by permeabilized cell envelopes was used for the cells of different physiological status (vegetative, dormant forms of different types, and nonviable micromummies). The procedure for the preparation of template DNA included one-stage (boiling in a buffer with chaotropic salts) or two-stage (boiling in a buffer with chaotropic salts followed by treatment with proteinase K) sample preparation. The proposed method proved effective for detection of not only vegetative cells but also of the bacillary spores and the cystlike dormant cells (CLC) of non-spore-forming bacteria. For example, the two-stage sample preparation of Bacillus cereus spores resulted in the PCR sensitivity increase up to the detection level of 3–30 spores per sample; the one-stage sample preparation was three orders of magnitude less efficient (104 spores per sample). An increase in the sensitivity of PCR detection (4–10-fold) owing to the use of the two-stage sample preparation was shown for bacillary, staphylococcal, and mycobacterial CLC. The possibility of PCR detection of staphylococcal micromummies with irreversibly lost viability, which were therefore undetectable by plating techniques, was also demonstrated. The application of the unified sample preparation method ensuring efficacious PCR detection of bacterial cells, irrespective of their physiological state, may be a promising approach to more complete detection of microbial diversity and the overall insemination of natural substrates.

Obtaining and characterization of DNA-containing micromummies of yeasts and gram-positive bacteria with enhanced cell wall permeability: Application in PCR

The procedure of obtaining DNA-containing cell envelopes (“micromummies”) of bacteria, yeasts, and fungi using chaotropic salts has been developed previously and the possibility of their direct application in PCR has been demonstrated. The fine structure of micromummies has been studied by electron microscopic methods. This work has demonstrated that additional treatment of micromummies of yeasts and gram-positive bacteria with proteinase K results in hydrolytic degradation of cell proteins and drastic enhancement of cell wall permeability for macromolecules (DNA). Thus, the efficiency of PCR ex situ using resultant micromummies after washing off the products of protein hydrolysis and proteinase K can be increased. The results of electron microscopic study of ultrathin sections of yeasts (Pichia pastoris, Saccharomyces cerevisiae) and gram-positive bacteria (Micrococcus luteus, Arthrobacter globiformis, Bacillus subtilis) support the biochemical data that treatment with chaotropic salts and proteinase K results in the loosening of microbial cell walls and in a decrease in the intracellular protein content. At the same time, cell walls generally maintain their integrity (continuity) and initial spherical or rodlike shape. The optimal modes of treatment of the cells of different microbial species with chaotropic salts and proteinase K have been selected to obtain permeabilized cell envelopes containing denatured or native DNA.