Super-long Anabiosis of Ancient Microorganisms in Ice and Terrestrial Models for Development of Methods to Search for Life on Mars, Europa and other Planetary Bodies

Successful missions to Mars, Europe and other bodies of the Solar system have created a prerequisite to search for extraterrestrial life. The first attempts of microbial life detection on the Martian surface by the Viking landed missions gave no biological results. Microbiological investigations of the Martian subsurface ground ice layers seem to be more promising. It is well substantiated to consider the Antarctic ice sheet and the Antarctic and Arctic permafrost as terrestrial analogues of Martian habitats. The results of our long-standing microbiological studies of the Antarctic ice would provide the basis for detection of viable microbial cells on Mars. Our microbiological investigations of the deepest and thus most ancient strata of the Antarctic ice sheet for the first time gave evidence for the natural phenomenon of long-term anabiosis (preservation of viability and vitality for millennia years). A combination of classical microbiological methods, epifluorescence microscopy, SEM, TEM, molecular diagnostics, radioisotope labeling and other techniques made it possible for us to obtain convincing proof of the presence of pro- and eukaryotes in the Antarctic ice sheet. In this communication, we will review and discuss some critical issues related to the detection of viable microorganisms in cold terrestrial environments with regard to future searches for microbial life and/or its biological signatures on extraterrestrial objects

Biotransformation of Oleanolic Acid Using <i>Rhodococcus rhodochrous</i> IEGM 757

Using the bioresources of the Regional Specialised Collection of Alkanotrophic Microorganisms (acronym IEGM, Perm, Russia; WFCC # 285), R. rhodochrous IEGM 757 was selected, which catalyzed the C5, C22, and C23 functionalization of pentacyclic triterpenoid oleanolic acid (OA, 3β-hydroxyolean-12-en-28-oic acid, 1.0 g/L) to form a new 5α,22α-dihydroxy derivative of gypsogenic acid (3β,5α,22α-trihydroxyolean-12-ene-23,28-dioic acid) for 5 days. In silico analysis showed that, compared to the native triterpenoid, the OA metabolite may be more soluble in water and less ecotoxic, act as an apoptosis agonist and insulin promoter, and have chemopreventive and analgesic effects. Phase-contrast, fluorescent, scanning, and transmission electron microscopy and X-ray spectroscopy demonstrated the high resistance of R. rhodochrous IEGM 757 to OA. This creates opportunities for further research and development of a method for the production of the OA metabolite. New-generation sequencing of the R. rhodochrous IEGM 757 whole genome, annotation and bioinformatics analysis of the obtained sequences, and real-time PCR were applied. As a result, 24 genes encoding CYP450 enzymes were found, which are highly likely to be involved in the process of OA oxidation

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’ <i>Mycobacterium abscessus</i>

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

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

<p>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 (<i>KlenTaq</i> or <i>Taq</i>). 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.</p