Bioaccumulation of molybdate ions by alkanotrophic Rhodococcus leads to significant alterations in cellular ultrastructure and physiology.

Alkanotrophic Rhodococcus strains from the Regional Specialised Collection of Alkanotrophic Microorganisms (acronym IEGM, www.iegmcol.ru) were screened for accumulation and sorption of MoO42- ions. Morphological and ultrastructural changes observed in bacterial cells during their cultivation in the molybdenum-containing medium are described. The species peculiarities, growth substrate preferences, and other physiological features allowing for the efficient removal of molybdate ions from the culture medium are discussed. Bioinformatics analysis of genes and proteins responsible for resistance to and accumulation of molybdenum was carried out using the sequenced R. ruber IEGM 231 and other published Rhodococcus genomes. n-Hexadecane growing strains with high (up to 85 %) accumulative activity and resistance to elevated (up to 20.0 mM) molybdenum concentrations were selected, which can be used for bioremediation of environments co-contaminated with heavy metals and hydrocarbons. Transmission electron microscopy and energy dispersive X-ray spectroscopy (TEM-EDX) revealed the ability of Rhodococcus not only to accumulate, but also to chemically convert soluble toxic molybdenum into insoluble compounds detected in the form of electron-dense nanoparticles.

Distinct Effects of Moxifloxacin and Bedaquiline on Growing and 'Non-Culturable' Mycobacterium abscessus.

Mycobacterium abscessus has recently emerged as the cause of an increasing number of human infections worldwide. Unfortunately, it is highly resistant to existing drugs, and new specific agents to combat M. abscessus have not yet been found. The discovery of antibiotics that are effective not only against replicating but also against dormant and often recalcitrant cells is a daunting challenge. In this study, we developed a model of non-replicating M. abscessus, which represents a valuable screening tool for antibacterial agents. Thus, we demonstrated that, under a deficiency of potassium ions in the growth media and prolonged incubation, M. abscessus entered a 'non-culturable' state with a significant loss of colony-forming ability, but it retained viability, as confirmed using the most-probable-number (MPN) assay. The 'non-culturable' mycobacteria possessed decelerated cellular metabolism and noticeable differences in cell morphology from actively growing mycobacteria. 'Non-culturable' cells were used in a comprehensive screening of the efficacy of antibiotics, along with actively growing cells. Both CFU and MPN tests confirmed the prominent bactericidal effect of moxifloxacin on actively growing and 'non-culturable' M. abscessus, as proven by less than 0.01% of cells surviving after antibiotic treatment and prolonged storage. Bedaquiline exhibited a comparable bactericidal effect only on metabolically inactive non-culturable cells aged for 44 days. There were reductions ranging from 1000 to 10,000-fold in CFU and MPN, but it was not so efficient with respect to active cells, resulting in a bacteriostatic effect. The demonstrated specificity of bedaquiline in relation to inert non-replicating M. abscessus offers a new and unexpected result. Based on the findings of this research, moxifloxacin and bedaquiline can be regarded as potential treatments for infections caused by M. abscessus. In addition, a key outcome is the proposal to include the combination of viability assays for comprehensive testing of drug candidates. Relying on CFU-based assays alone resulted in overestimates of antibacterial efficacy, as demonstrated in our experiments.

Highly purified DNA-containing cell envelopes from fungi for direct use in PCR.

Efficient DNA sample preparation from fungi with the rigid cell walls is still critical for successful polymerase chain reaction (PCR), one of the basic platforms in molecular diagnostics of fungi, especially in medical mycology. Common methods that involve different chaotropes to yield DNA samples have found a limited application for fungi. Here we describe a novel procedure for efficient production of permeable fungal cell envelopes with DNA inside as suitable templates for PCR. This procedure is facile, relies on boiling of fungal cells in aqueous solutions of selected chaotropic agents and additives and enables to remove RNA and proteins from PCR template samples. The use of chaotropic solutions containing 7 M urea, 1% sodium dodecyl sulfate (SDS), up to100 mM ammonia and/or 25 mM sodium citrate was the best option to yield highly purified DNA-containing cell envelopes from all fungal strains under study, including clinical Candida and Cryptococcusisolates. After treatment with the selected chaotropic mixtures, the fungal cell walls had undergone loosening and were no longer a barrier to release DNA in PCR as evident from electron microscopy examinations and successful target gene amplifications. Overall, the developed simple, fast, and low-cost approach to produce PCR-suitable templates in the form of DNA encased by permeable cell walls can find application in molecular diagnostics.

Biotransformation of (-)-Isopulegol by Rhodococcus rhodochrous.

The ability of actinobacteria of the genus Rhodococcus to biotransform the monoterpenoid (-)-isopulegol has been established for the first time. R. rhodochrous strain IEGM 1362 was selected as a bacterium capable of metabolizing (-)-isopulegol to form new, previously unknown, 10-hydroxy (2) and 10-carboxy (3) derivatives, which may presumably have antitumor activity and act as respiratory stimulants and cancer prevention agents. In the experiments, optimal conditions were selected to provide the maximum target catalytic activity of rhodococci. Using up-to-date (TEM, AFM-CLSM, and EDX) and traditional (cell size, roughness, and zeta potential measurements) biophysical and microbiological methods, it was shown that (-)-isopulegol and halloysite nanotubes did not negatively affect the bacterial cells. The data obtained expand our knowledge of the biocatalytic potential of rhodococci and their possible involvement in the synthesis of pharmacologically active compounds from plant derivatives.

Ribonucleic acid (RNA) condensation by thermal cycling with metal cations: yield of nanoparticles and their applicability for transfection.

To the present, different efficient but expensive, multistage, and time-consuming technologies have been developed to deliver ribonucleic acids (RNA) into eukaryotic cells. Here, we report a simple and feasible solution to design RNA nanocarriers based on nucleic acid condensation by bi- and trivalent metal ions during thermal cycling. Efficient RNA conversion to nanoparticles with small size (10-50 nm) suitable for transfection was achieved using cations Ni2+, Co2+ or Cu2+ alone or in combination with Ca2+ at the specially selected concentrations (2.0 mM-3.5 mM), low ionic strength, and narrow pH range (8.0-8.5). Other ions - Mn2+, Zn2+, Tb3+, or Gd3+ - caused RNA-cleaving effect that was abolished in the presence of Ni2+, Co2+, Zn2+, or Cu2+. Naked RNA-metal ion nanoparticles were extremely unstable in phosphate buffer and sensitive to serum ribonucleases (RNases), and this problem was solved by treatment with polyarginines-16 and 8. Polyarginine-stabilized nanoparticles, containing malachite green (MG) aptamer RNA and metal cations, crossed the cell membrane, dissociated in the cytoplasm, and preserved the functionality of transported RNA, as judged from efficient transfection of human embryonic kidney 293 cells. The technology, involving RNA condensation by metal cations, can be used as a cheap alternative to produce nanoscale carriers to deliver various RNAs into cells in vitro and in vivo.Communicated by Ramaswamy H. Sarma.

Serinibacter arcticus sp. nov., isolated from a thawing ancient ice wedge.

A novel actinobacterium, strain K3-2T, was isolated in pure culture from a thawing ancient ice wedge at Mammoth Mountain (Eastern Siberia, Russia). Colonies of strain K3-2T were yellowish orange; cells had the fine structure typical of Gram-positive bacteria, were non-motile short rods and were non-spore-forming. Strain K3-2T was mesophilic (optimum growth at 28 °Ð¡), but capable of growing at 4 °Ð¡. The cell-wall peptidoglycan of strain K3-2T contained lysine (the diagnostic diamino acid), glutamic acid, alanine, ornithine, glycine and serine. The polar lipids were phosphatidylglycerol, lysophosphatidylserine, three unidentified phospholipids and glycolipids. The major fatty acids were anteiso-C15 : 0 and C16 : 0. The only menaquinone detected was MK-8(H4). 16S rRNA gene analysis indicated that strain K3-2T belongs to the genus Serinibacter. The closest taxonomically described relatives were Serinibacter salmoneus Kis4-28T and Serinibacter tropicus PS-14-7T, with 97.20 and 97.20 % 16 s rRNA gene sequence similarity, respectively. The average nucleotide identity value of the whole genome sequence between strain K3-2T and S. salmoneus Kis4-28T was 78.9 %. DNA-DNA relatedness values between strain K3-2T and S. salmoneus DSM 21801T (=Kis4-28T) and S. tropicus VKPM Ac 2044T (=PS-14-7T) were 41 and 47 %. Thus, strain K3-2T represents a novel species of the genus Serinibacter for which the name Serinibacter arcticus sp. nov. is proposed. The type strain is K3-2T (DSM 103859T=VKM Ас-2719T).

Structural variability of DNA-containing Mg-pyrophosphate microparticles: optimized conditions to produce particles with desired size and morphology.

Our previous studies demonstrated the formation of structurally diverse DNA-containing microparticles (DNA MPs) in PCR with Mg-pyrophosphate (MgPPi) as the structure-forming component. These DNA MPs were referred to major structural types: microdisks (2D MPs) with nanometer thickness and 3D MPs with sophisticated morphology and constructed from intersecting disks and their segments. Little is known about factors that influence both the morphology and size of DNA MPs, and the present study was aimed at fulfilling this gap. We showed that the addition of Mn2+ cations to PCR mixtures caused the profound changes in MPs morphology, depending on DNA polymerase used (KlenTaq or Taq). Asymmetric PCR with 20-fold decrease in the concentration of one of two primers facilitated the predominant formation of microdisks with unusual structure. The addition of 1 mM Na-pyrophosphate to PCR mixtures with synthesized DNA and subsequent thermal cycling (10-15 cycles) were optimal to produce microdisks or nanometer 3D particles. Using electron microscopy, we studied also the structure of inorganic micro- and nanoparticles from MgPPi, formed during multiple heating and cooling cycles of a mixture of Mg2+ and Na-pyrophosphate in various regimes. Also, we found the conditions to yield planar (Mg·Mn)PPi nanocrystals (diameter ~100 nm and thickness ~10 nm) which efficiently adsorbed exogenous DNA. These inorganic nanoparticles are promising for DNA delivery in transfection studies. Mechanisms to be involved in structural modifications of MPs and perspectives of their practical application are discussed.

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.

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 Mg(2+) 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 Mg(2+)-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.

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.

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 Mg(2+)-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 Mg(2+) - 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.

Dormant ovoid cells of Mycobacterium tuberculosis are formed in response to gradual external acidification.

It is believed that latent tuberculosis is associated with the persistence of Mycobacterium tuberculosis (MTB) in a dormant-like state. Dormant cells of MTB with coccoid morphology were produced in some in vivo studies, but similar forms were not produced in the known in vitro models in sufficient amounts to permit their characterization. This work demonstrates the efficient formation of phase-dark ovoid cells in MTB cultures within 150 days after the onset of stationary phase. During this time the medium underwent gradual acidification (pH 8.5 → 4.7) as a result of cellular metabolism. A rapid change in the external pH resulted in cell degradation and death. In common with the dormant forms found in other organisms, the ovoid cells had thickened cell walls, a low metabolic activity and elevated resistance to antibiotics and heating. The ovoid cells had lost the ability to form colonies on solid medium and were thus regarded as operationally «non-culturable¼. At an early stage in the acidification process (about 40 days post inoculation), the ovoid cells self-resuscitated when placed in fresh liquid medium. However, ovoid cells, stored for a prolonged time, required supernatant from active MTB cells, or externally added recombinant form of resuscitation promoting factor (Rpf) for successful resuscitation. It is suggested that the adaptation of cellular metabolism leading to gradual acidification of the external medium results in the formation of morphologically distinct dormant MTB cells in vitro. The model of MTB dormancy developed here could be a useful tool for the development of new drugs against latent TB.

The role of histone-like protein, Hlp, in Mycobacterium smegmatis dormancy.

The role of histone-like protein (Hlp) in the development of a dormant state in long-incubated stationary-phase Mycobacterium smegmatis cells was studied in two models: (1) adoption of 'nonculturable' (NC) state, which is reversible due to resuscitation with proteinaceous resuscitation-promoting factor (Rpf) and (2) the formation of morphologically distinct, ovoid resting forms. In the first model, inactivation of the hlp gene resulted in prolongation of culturability of starved cells followed by irreversible nonculturability when mycobacterial cells were unresponsive to resuscitation with Rpf. In the second model, M. smegmatis strain with the inactivated hlp gene was able to form dormant ovoid cells, but they were less resistant to heating and UV radiation than those of wild-type strain. The susceptibility of ovoid cells produced by Delta hlp mutant to these damaging factors was probably due to a less condensed state of DNA, as revealed by fluorescent microscopy and DAPI staining. Evidently, Hlp is essential for cell viability at a later stage of NC dormancy or provides a greater stability of specialized dormant forms.

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.