Insights from the classical MD simulations

Salt bridges and ionic interactions play an important role in protein stability, protein-protein interactions, and protein folding. Here, we provide the classical MD simulations of the structure and IR signatures of the arginine (Arg)–glutamate (Glu) salt bridge. The Arg-Glu model is based on the infinite polyalanine antiparallel two-stranded β-sheet structure. The 1 μs NPT simulations show that it preferably exists as a salt bridge (a contact ion pair). Bidentate (the end-on and side-on structures) and monodentate (the backside structure) configurations are localized [Donald et al., Proteins 79, 898–915 (2011)]. These structures are stabilized by the short +N–H⋯O− bonds. Their relative stability depends on a force field used in the MD simulations. The side-on structure is the most stable in terms of the OPLS-AA force field. If AMBER ff99SB-ILDN is used, the backside structure is the most stable. Compared with experimental data, simulations using the OPLS all-atom (OPLS-AA) force field describe the stability of the salt bridge structures quite realistically. It decreases in the following order: side-on > end-on > backside. The most stable side-on structure lives several nanoseconds. The less stable backside structure exists a few tenth of a nanosecond. Several short-living species (solvent shared, completely separately solvated ionic groups ion pairs, etc.) are also localized. Their lifetime is a few tens of picoseconds or less. Conformational flexibility of amino acids forming the salt bridge is investigated. The spectral signature of the Arg-Glu salt bridge is the IR-intensive band around 2200 cm−1. It is caused by the asymmetric stretching vibrations of the +N–H⋯O− fragment. Result of the present paper suggests that infrared spectroscopy in the 2000–2800 frequency region may be a rapid and quantitative method for the study of salt bridges in peptides and ionic interactions between proteins. This region is usually not considered in spectroscopic studies of peptides and proteins

Synthetic Biology: Risks and Prospects

New area of biological studies that goes under general name of “synthetic biology” is a next step in the development of gene engineering associated with design and construction of unique biological systems with “preset” functions and properties, having no natural analogues. Nowadays synthetic biology is a source of innovations that offer solution to a number of global problems facing the humanity, including production of artificial genome-based multi-diagnostic panels, medicinal preparations, synthetic vaccine, etc. The process of unnatural life form creation requires conduction of monitoring both on the international and national scales using advanced system of biological risk assessment

Development of the PCR Assay with Universal Primers for the Detection of Different <i>tcpA</i> Gene Variants

Developed is the PCR assay for the detection of the structural genes of toxin co-regulated adhesion piluses - tcpA of different types. Determined are the universal primers, the usage of which provides for the detection of the stated above genes in V. cholerae of various serogroups. With the help of this PCR assay identified is a new variant of tcpA gene in toxigenic cholera vibrio of non-O1/non-O139 serogroup

Comparative Analysis of the Nutrient Requirements among <I>Yersinia pestis</I> Strains of the Main and Non-Main Subspecies as well as Genetic Causes of Their Auxotrophy

Studied is the nutrient demand for growth factors among 185 Yersinia pestis strains of the main and non-main subspecies ( altaica, caucasica, hissarica , and ulegeica ) isolated in 38 natural plague foci of Russia and neighboring states. Revealed is the fact that all the strains of Yersinia pestis main subspecies manifest equal dependence in growth on three amino-acids such as methionine, phenylalanine, and threonine, while strains isolated from certain natural foci have an additional demand for cysteine, leucine, and arginine. Strains of non-main subspecies differ in their nutrient requirements both from the strains of the main subspecies and among themselves. Strains of subspecies caucasica stand in need of thiamine and such amino-acids as phenylalanine, tyrosine and arginine; subspecies altaica – phenylalanine, arginine and leucine; subspecies hissarica – phenylalanine, leucine and methionine; ulegeica – phenylalanine. Detected is a number of mutations that lead to auxotrophy in Yersinia pestis strains of different subspecies

Development of a Set of Primers for Drug-Resistance Genes Detection in the Agents of Dangerous Bacterial Infections as Exemplified by <I>Yersinia pestis</I>, <I>Vibrio cholerae</I>, <I>Escherichia coli</I> Strains

A set of primers for detection of genes encoding resistance to streptomycin ( strA, strB ), tetracyclin ( tetA, tetR ), chloramphenicol ( catА ), kanamycin ( npt , aphA ), vankomycin ( sanA ), polymyxin ( pmrD ) has been developed with the aim of rapid and effective detection of drug-resistant strains of dangerous bacterial infections agents. Efficacy of constructed primers has been confirmed against a panel of 40 Yersinia pestis, 49 Vibrio cholerae, and 2 Escherichia coli strains from the State collection of pathogenic bacteria of the RAPI “Microbe”. Drug-resistance genes ntp and catA have been detected in plague agent strains , strA, strB , npt , aphA , tetA and tetR - in cholera agent; strA , tetR , ntp and aphA - in pathogenic strain E. coli О157:H7. Determined is universal character of the designed primers for drug-resistance genes detection in these pathogenic bacteria species

Analysis of the Genome Wide Sequence of Yersinia pestis strains Based on the Consecutive 680-SNP Algorithm

Designed is an optimized scheme for SNP analysis of the genome wide sequences of plague agent strains, based on the consecutive algorithm of clusterization of individual intraspesific Y. pestis groups. The scheme comprises 680 singular polymorphic nucleotides (SNPs), the usage of which provides not only for the clustering of closely related strains, but for the determination of phylogenetic bonds between groups of strains and order of their divergence from the common tree-trunk of the plague agent evolution. Based on the consecutive 680-SNP algorithm, carried out is the analysis of genome wide sequences of C-627 Y. pestis strain isolated in Central-Caucasian high-mountain focus (Russia), strain 1454 isolated in Altay mountain focus (Russia), strain 231 (708) – Aksay high mountain focus (Kirgizia), and vaccine EV NIIEG (Research Institute of Epidemiology and Hygiene) strain, applied for immunization in the Russian Federation and in a number of other countries. Identified are the genome variants of the strains: 1. ORI3 (EV NIIEG), 2. MED0 (C-627), 0.ANT3 (231(708)), 0.ANT4 (1454), and their place in the scheme of Y. pestis global variation. Evidenced is the absence of the chromosomal region responsible for pigmentation in the genome wide sequence of the vaccine strain EV NIIEG, which testifies to its safe use for specific plague prophylaxis

Structural-Functional Analysis of Cryptic Plasmids in <i>Yersinia pestis</i> Strains from Two Natural Plague Foci of Russia

Identified are the complete nucleotide sequences of two cryptic plasmids - pCKF from the Central-Caucasian high-mountain plague focus and pTP33 - from Tuva mountain plague focus in Russia. It is established that the size of the pCKF is 5.4 kbp, and its G-C pair composition accounts for 38.4 %. The plasmid contains 8 open reading frames, encoding transport and secretion functions, in particular, secretion systems of the VI type. The size of another cryptic plasmid, pTP33, is 33.8 kbp, and G-C pair composition accounts for 50.3 %. It contains 52 open reading frames, the majority of which have been classified as phage proteins. Hence, it is inferred that pTP33 is a ring genome of phage. It also has two genes of two-component protein system, toxin-antitoxin, YoeB/YefM, that affects replication apparatus of bacteria

Analysis of Genetic Variability of Yersinia pestis Strains (Medieval Biovar) Isolated in Natural Plague Foci of the Russian Federation and Mongolia

Carried out is the analysis of genetic peculiarities of Yersinia pestis strains (main ssp.), isolated in natural foci of the Russian Federation and Mongolia. Determined is the fact that strains of medieval biovar characterized by typical glpD1 napA2 rhaS1 genotype circulate in 7 out of 9 marmot and sandy-type foci. Strains of antique biovar characterized by glpD1 napA1 rhaS1 genotype prevail in the Trans-Baikal steppe foci. Y. pestis strains from Tuva mountain focus have similar genotype, which correlates with denitrification activity and absence of marker mutation of medieval biovar – single nucleotide substitution G→T in the 613 position of napA gene, and testifies to affiliation of these strains to antique biovar. Among Y. pestis strains isolated in Mongolia, identified are two atypical strains incapable of reducing nitrates but pertinent to antique biovar

Variability of <i>phoP/phoQ</i> and <i>rovA</i> Genes Sequences - Global Regulators of the Plague Agent Life Span

Compared are the gene sequences of the global phoP/phoQ and rovA transcription regulators in original Yersinia pestis strains of different subtypes. Detected is overall identity of the sequenced phoQ fragment and high conservatism of the rovA gene. All the plague agent strains belonging to the main subspecies have a missense mutation in phoP gene. It is a substitution of a single nucleotide G→A in the position 643 from the beginning of the gene, which causes amino-acid residue shift Gly→Ser in the position 215 in polypeptide chain of the PhopP protein, and, is a possible cause of alteration of the PhoP transcription activity in Yersinia pestis strains belonging to the main subspecies

Studies of Biofilm Formation in Non-Pigmented and Plasmid-Deprived Mutants of <I>Yersinia pestis</I> on Biotic Surfaces, <I>in vivo</I> and <I>in vitro</I> Conditions

In non-pigmented and plasmid-deprived mutants – isogenic variants of highly virulent Yersinia pestis 231 strain – studied is the mechanism of biofilm formation on biotic surfaces, both in vitro (on the laboratory model of nematode Caenorhabdiitis elegans) and in vivo (inside the alimentary tract of Nosopsyllus laeviceps flea). It is determined that spontaneous loss of ability to form biofilms and generate pigmented colonies in the mutants is probably caused not only by the deletion of the whole chromosome pigmentation fragment, but also by a point(single base) mutation in structural hms operon. It is demonstrated that the absence of pCad, pFra or pPst plasmids does not have an impact on the ability of plasmid-deprived mutants to form biofilm on the cuticle of nematode C. elegans