DNA-Binding Protein Dps Protects Escherichia coli Cells against Multiple Stresses during Desiccation.

Gradual dehydration is one of the frequent lethal yet poorly understood stresses that bacterial cells constantly face in the environment when their micro ecotopes dry out, as well as in industrial processes. Bacteria successfully survive extreme desiccation through complex rearrangements at the structural, physiological, and molecular levels, in which proteins are involved. The DNA-binding protein Dps has previously been shown to protect bacterial cells from many adverse effects. In our work, using engineered genetic models of E. coli to produce bacterial cells with overproduction of Dps protein, the protective function of Dps protein under multiple desiccation stresses was demonstrated for the first time. It was shown that the titer of viable cells after rehydration in the experimental variants with Dps protein overexpression was 1.5-8.5 times higher. Scanning electron microscopy was used to show a change in cell morphology upon rehydration. It was also proved that immobilization in the extracellular matrix, which is greater when the Dps protein is overexpressed, helps the cells survive. Transmission electron microscopy revealed disruption of the crystal structure of DNA-Dps crystals in E. coli cells that underwent desiccation stress and subsequent watering. Coarse-grained molecular dynamics simulations showed the protective function of Dps in DNA-Dps co-crystals during desiccation. The data obtained are important for improving biotechnological processes in which bacterial cells undergo desiccation.

Building a Cell House from Cellulose: The Case of the Soil Acidobacterium Acidisarcina polymorpha SBC82T.

Acidisarcina polymorpha SBC82T is a recently described representative of the phylum Acidobacteriota from lichen-covered tundra soil. Cells of this bacterium occur within unusual saccular chambers, with the chamber envelope formed by tightly packed fibrils. These extracellular structures were most pronounced in old cultures of strain SBC82T and were organized in cluster-like aggregates. The latter were efficiently destroyed by incubating cell suspensions with cellulase, thus suggesting that they were composed of cellulose. The diffraction pattern obtained for 45-day-old cultures of strain SBC82T by using small angle X-ray scattering was similar to those reported earlier for mature wood samples. The genome analysis revealed the presence of a cellulose biosynthesis locus bcs. Cellulose synthase key subunits A and B were encoded by the bcsAB gene whose close homologs are found in genomes of many members of the order Acidobacteriales. More distant homologs of the acidobacterial bcsAB occurred in representatives of the Proteobacteria. A unique feature of bcs locus in strain SBC82T was the non-orthologous displacement of the bcsZ gene, which encodes the GH8 family glycosidase with a GH5 family gene. Presumably, these cellulose-made extracellular structures produced by A. polymorpha have a protective function and ensure the survival of this acidobacterium in habitats with harsh environmental conditions.

Brain Natriuretic Peptide (BNP) Affects Growth and Stress Tolerance of Representatives of the Human Microbiome, Micrococcus luteus C01 and Alcaligenes faecalis DOS7.

Brain natriuretic peptide (BNP) is secreted by the ventricles of the heart during overload to signal heart failure. Slight bilateral skin itching induced by BNP has been associated with response activity of the skin microbiota. In this work, we studied the effect of 25-250,000 pg BNP/mL on the growth, long-term survival, and stress (H2O2, antibiotics, salinity, heat and pH shock) resistance of human symbiont bacteria: Gram-positive Micrococcus luteus C01 and Gram-negative Alcaligenes faecalis DOS7. The effect of BNP turned out to be dose-dependent. Up to 250 pg BNP/mL made bacteria more stress resistant. At 2500 pg BNP/mL (heart failure) the thermosensitivity of the bacteria increased. Almost all considered BNP concentrations increased the resistance of bacteria to the action of tetracycline and ciprofloxacin. Both bacteria survived 1.3-1.7 times better during long-term (up to 4 months) storage. Our findings are important both for clinical medical practice and for practical application in other areas. For example, BNP can be used to obtain stress-resistant bacteria, which is important in the collection of microorganisms, as well as for the production of bacterial preparations and probiotics for cosmetology, agriculture, and waste management.

Multi-crystal data collection using synchrotron radiation as exemplified with low-symmetry crystals of Dps.

Multi-crystal data collection using synchrotron radiation was successfully applied to determine the three-dimensional structure of a triclinic crystal form of Dps from Escherichia coli at 2.0 Šresolution. The final data set was obtained by combining 261 partial diffraction data sets measured from crystals with an average size of approximately 5 µm. The most important features of diffraction data measurement and processing for low-symmetry crystals are discussed.

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.

Projection structures reveal the position of the DNA within DNA-Dps Co-crystals.

One of the universal mechanisms for the response of Escherichia coli to stress is the increase of the synthesis of specific histone-like proteins that bind the DNA, Dps. As a result, two-and three-dimensional crystalline arrays may be observed in the cytoplasm of starving cells. Here, we determined the conditions to obtain very thin two-dimensional DNA-Dps co-crystals in vitro, and studied their projection structures, using electron microscopy. Analysis of the projection maps of the free Dps crystals revealed two lattice types: hexagonal and rectangular. We used the fluorescently labeled DNA to prove that the DNA is present within the co-crystals with Dps in vitro, and visualized its position using transmission electron microscopy. Molecular modeling confirmed the DNA position within the crystal. We have also suggested a structural model for the DNA-Dps co-crystal dissolving in the presence of Mg2+ ions.

Interaction of deoxyribonucleic acid with deoxyribonucleic acid-binding protein from starved cells: cluster formation and crystal growing as a model of initial stages of nucleoid biocrystallization.

The paper represents the study of interaction between deoxyribonucleic acid (DNA) and deoxyribonucleic acid-binding protein from starved cells (DPS) cluster formation and crystal growing within a cell. This study is a part of the project that includes European Synchrotron Radiation Facility (ESRF) investigations of in vivo and in vitro nanocrystallization processes of Escherichia coli (E. coli) nucleoid under stress condition combined with theoretical molecular dynamics approaches. Nucleoid biocrystallization is an adaptive mechanism of bacterial cells under stress. It is poorly understood at the present time. Understanding crystal formation process is a very important for molecular biology, pharmacology and other areas. In the simulation part the coarse-grained modeling of various combinations of the following molecules was used: DPS proteins (from 1 to 108 DPS dodecamers in simulation box), short DNA fragments with a length of 24 base pairs (b.p., from 1 to 100 DNA fragments in simulation box) and a part of pBluescript SK(+) plasmide with a length of 161 b.p., in the presence of ions. We defined structural, energetic and dynamic properties of DPS-DPS and DPS-DNA complexes in clusters and crystals that allow us to predict crystal formation and the structure of these crystals in experimental systems.  Communicated by Ramaswamy H. Sarma.