Structural,Functional, and Evolutionary Characteristics of Proteins with Repeats

Molecular Biology - The review summarizes and systematizes the data on the classification, taxonomic distribution, structural features, and functions of proteins with structural repeats. Modern...     

Ancylobacter lacus sp. nov. and Ancylobacter plantiphilus sp. nov., Novel Aerobic Facultative Methylotrophic Bacteria

Microbiology - Novel aerobic facultatively methylotrophic bacteria were isolated from the water of a freshwater lake (strain F30LT), soil sample of rhizosphere of white clover Trifolium repens L....     

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.     

Bacterial Cellulose as a Matrix for Microorganisms in Bioelectrocatalytic Systems

Applied Biochemistry and Microbiology - Bacterial cellulose (BC) produced by the Komagateibacter sucrofermentas VKPM B-11267 bacteria was used as a carrier for immobilization of acetic acid...     

Probiotic Properties of Lactobacillus crispatus 2,029: Homeostatic Interaction with Cervicovaginal Epithelial Cells and Antagonistic Activity to Genitourinary Pathogens

Lactobacillus crispatus 2029 isolated upon investigation of vaginal lactobacilli of healthy women of reproductive age was selected as a probiotic candidate. The aim of the present study was elucidation of the role of L. crispatus 2029 in resistance of the female reproductive tract to genitourinary pathogens using cervicovaginal epithelial model. Lactobacillus crispatus 2029 has surface layers (S-layers), which completely surround cells as the outermost component of their envelope. S-layers are responsible for the adhesion of lactobacilli on the surface of cervicovaginal epithelial cells. Study of interactions between L. crispatus 2029 and a type IV collagen, a major molecular component of epithelial cell extracellular matrix, showed that 125I-labeled type IV collagen binds to lactobacilli with high affinity (Kd = (8.0 ± 0.7) × 10^?10 M). Lactobacillus crispatus 2029 consistently colonized epithelial cells. There were no toxicity, epithelial damage and apoptosis after 24 h of colonization. Electronic microscope images demonstrated intimate association between L. crispatus 2029 and epithelial cells. Upon binding to epithelial cells, lactobacilli were recognized by toll-like 2/6 receptors. Lactobacillus crispatus induced NF-κB activation in epithelial cells and did not induce expression of innate immunity mediators IL-8, IL-1β, IL-1α and TNF-α. Lactobacillus crispatus 2029 inhibited IL-8 production in epithelial cells induced by MALP-2 and increased production of anti-inflammatory cytokine IL-6, maintaining the homeostasis of female reproductive tract. Lactobacillus crispatus 2029 produced H₂O₂ and provided wide spectrum of antagonistic activity increasing colonization resistance to urinary tract infections by bacterial vaginosis and vulvovaginal candidiasis associated agents.     

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.     

The S1 ribosomal protein family contains a unique conservative domain

The number of amino acid residues contained in the S1 ribosomal protein of various bacteria varies in a wide range: from 111 to 863 residues in Spiroplasma kunkelii and Treponema pallidum, respectively. The architecture of this protein is traditionally (in particular, because of unknown spatial structure) represented as repeated S1 domains, the copy number of which depends on the protein length. The data on the copy number and boundaries of these domains is available in specialized databases, such as SMART, Pfam, and PROSITE; however, these data can be rather different for the same object. In this work, we used the approach utilizing analysis of predicted secondary structure (PsiPred program). This allowed us to detect the structural domains in S1 protein sequences; their copy number varied from one to six. Alignment of the S1 proteins containing different numbers of domains with the S1 RNA-binding domain of Escherichia coli polynucleotide phosphorylase provided for discovering a domain within this family displaying the maximal homology to the E. coli domain. This conservative domain migrates along the chain, and its location in the proteins with different numbers of domains follows a certain pattern. Similar to the S1 domain of polynucleotide phosphorylase, residues Phe19, Phe22, His34, Asp64, and Arg68 in this conservative domain are clustered on the surface to form an RNA-binding site.     

Investigation of site-specific DNA binding with nicking endonuclease Nt.BspD6I at single molecule level by atomic force microscopy

Nicking endonuclease Nt.BspD6I is a heterodimeric restriction endonuclease, one subunit of which exhibits specific nicking activity. It gets bound to double-stranded DNA and makes a break (nick) in one chain at a distance of 4 nucleotides from the binding site. In this work, for visualization of the specific binding and protein landing site an atomic force microscopy was used. In five minutes after incubation of DNA solution with nicking endonuclease, the DNA molecules with associated proteins which located at the expected binding site and "shared" DNA strand into two segments (approximately, 1/3 and 2/3 of length) were observed in the images. In addition, near the binding site DNA molecule had a height corresponding to a single-stranded DNA molecule, which was in good agreement with single-stranded cleavage by nickase in the course of complex formation.     

Investigation of site-specific DNA binding with nicking endonuclease Nt.BspD6I at single molecule level by atomic force microscopy

Nicking endonuclease Nt.BspD6I is a heterodimeric restriction endonuclease, one subunit of which exhibits specific nicking activity. It gets bound to double-stranded DNA and makes a break (nick) in one chain at a distance of 4 nucleotides from the binding site. In this work, for visualization of the specific binding and protein landing site, atomic force microscopy was used. In five minutes after incubation of DNA solution with nicking endonuclease, DNA molecules with associated proteins which located at the expected binding site and “shared” the DNA strand into two segments (approximately, 1/3 and 2/3 of length) were observed in the images. In addition, near the binding site the DNA molecule had a height corresponding to a single-stranded DNA molecule, which was in good agreement with single-stranded cleavage by nickase in the course of complex formation.