Study of the diversity in a group of phages of Pseudomonas aeruginosa species PB1 (Myoviridae) and their behavior in adsorbtion-resistant bacterial mutants

A group of 12 Pseudomonas aeruginosa virulent bacteriophages of different origin scored with regard to the plaque phenotype are assigned to PB1-like species based on the similarity in respect to morphology of particles and high DNA homology. Phages differ in restriction profile and the set of capsid major proteins. For the purpose of studying adsorption properties of these phages, 20 random spontaneous mutants of P. aeruginosa PAO1 with the disturbed adsorption placed in two groups were isolated. Mutants of the first group completely lost the ability to adsorb all phages of this species. It is assumed that their adsorption receptors are functionally inactive or lost at all, because the attempt to isolate phage mutants or detect natural phages of PB1 species capable of overcoming resistance of these bacteria failed. The second group includes five bacterial mutants resistant to the majority of phages belonging to species PB1, These mutants maintain the vigorous growth of phage SN and poor growth of phage 9/3, which forms turbid plaques with low efficiency of plating. In the background of weak growth, phage 9/3 yields plaques that grew well. The examination of the progeny of phage 9/3, which can grow on these bacteria, showed that its DNA differed from DNA of the original phage 9/3 by restriction profile and is identical to DNA of phage PB1 with regard to this trait. Data supported a suggestion that this phage variant resulted from recombination of phage 9/3 DNA with the locus of P. aeruginosa PAO1 genome encoding the bacteriocinogenic factor R. However, this variant of phage 9/3 did not manifest the ability to grow on phage-resistant mutants of the first group. Possible reasons for the difference between phages 9/3 or SN and the remaining phages of PB1 species are discussed. A preliminary formal scheme of the modular structure for adsorption receptors on the surface of P. aeruginosa PAO1 bacteria was constructed based on the analysis of growth of some other phage species on adsorption mutants of the first type.     

Specific features of the zooplankton of the coastal zone of Lake Sevan

Specific features of the Lake Sevan zooplankton development during a rise in water level in July 2007 and a change in the fish density were revealed. Compared to earlier data (from the second half of the 20th century), the pelagial zooplankton quantitatively dominates over the zooplankton of shallows. It is shown that the diversity and abundance of rotifers increase in the coastal zones subject to inundation. The rotifer indicator species of eutrophic and α-mesosaprobic waters contribute to this increase. The stimulating effect of the waste products of a bird colony on the zooplankton of the lake shallows is revealed.     

Analysis of calorimetric data for the binding of monomeric bis-benzimidazole,an analog of the Hoechst 33258 dye,to poly(dA) · poly(dT)

Monomeric bis-benzimidazole (MB) is an analog of the Hoechst 33258 dye. The enthalpy and entropy of MB binding were evaluated by analyzing the calorimetric data on MB reverse titration with poly(dA) · poly(dT). A mathematical model was developed to estimate the thermodynamic parameters of binding on the basis of calorimetric data. The results agree well with spectrophotometric data on the binding of analogous compounds. The model was used to estimate the parameters of binding with poly(dA) · poly(dT) for dimeric bis-benzimidazole (DB), which consists of two bis-benzimidazole monomers linked via a flexible chain. The ligand was assumed to produce different types of complexes with the polymer.     

DNA aptamers for as analytical tools for the quantitative analysis of DNA-dealkylating enzymes

The AlkB family of oxygenases catalyze the removal of alkyl groups from nucleic acid substrates in an iron and 2-oxoglutarate-dependent manner and have roles including in DNA repair. To understand the biological functions of these DNA-dealkylating enzymes it is desirable to measure their expression levels in vitro and in vivo in complex biological matrixes. Quantitative analyses of the enzymes require affinity probes capable of binding AlkB family members selectively and with high affinity. Here we report that DNA aptamers can serve as efficient affinity probes for quantitative detection of such enzymes in vitro. Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) was applied as a general tool for: (i) selection of DNA aptamers, (ii) characterization of binding parameters for the aptamers, and (iii) quantitative detection of the target in an aptamer-based affinity analysis. The selected aptamers have a range of K_d values between 20 and 240 nM. The aptamers enabled accurate quantitative analysis of AlkB even in the presence of the Escherichia coli cell lysate. Aptamers can likely be developed for other nucleic acid repair enzymes. They may also be developed for use in in vitro and potentially in vivo studies of known nucleic acid-modifying enzymes including for functional analysis.     

TL, the new bacteriophage of Pseudomonas aeruginosa and its application for the search of halo-producing bacteriophages

The properties of new virulent bacteriophage TL of Pseudomonas aeruginosa belonging to the family Podoviridae (genome size of 46 kb) were investigated. This bacteriophage is capable of lysogenizing the bacterial lawn in halo zones around negative colonies (NC) of other bacteriophages. TL forms large NC, that are hardly distinguishable on the lawn of P. aeruginisa PAO1. At the same time, on the lawns of some phage-resistant PAO1 mutants, as well as on those produced by a number of clinical isolates, TL forms more transparent NC. It is suggested that more effective growth of the bacteriophage TL NC is associated with the differences in outer lipopolysaccharide (LPS) layer of the cell walls of different bacterial strains, as well as of the bacteria inside and outside of the halos. This TL property was used to optimize selection of bacteriophages producing halos around NC on the lawn of P. aeruginosa PAO1. As a result, a group of bacteriophages differing in the patterns of interaction between their halos and TL bacteriophage, as well as in some characters was identified. Taking into consideration the importance of cell-surfaced structures of P. aeruginosa in manifestation of virulence and pathogenicity, possible utilization of specific phage enzymes, polysacchadide depolymerases, for more effective treatment of P. aeruginosa infections is discussed.     

A genetic map of Xenopus tropicalis

We present a genetic map for Xenopus tropicalis, consisting of 2886 Simple Sequence Length Polymorphism (SSLP) markers. Using a bioinformatics-based strategy, we identified unique SSLPs within the X. tropicalis genome. Scaffolds from X. tropicalis genome assembly 2.0 (JGI) were scanned for Simple Sequence Repeats (SSRs); unique SSRs were then tested for amplification and polymorphisms using DNA from inbred Nigerian and Ivory Coast individuals. Thus identified, the SSLPs were genotyped against a mapping cross panel of DNA samples from 190 F2 individuals. Nearly 4000 SSLPs were genotyped, yielding a 2886-marker genetic map consisting of 10 major linkage groups between 73 and 132 cM in length, and 4 smaller linkage groups between 7 and 40 cM. The total effective size of the map is 1658 cM, and the average intermarker distance for each linkage group ranged from 0.27 to 0.75 cM. Fluorescence In Situ Hybridization (FISH) was carried out using probes for genes located on mapped scaffolds to assign linkage groups to chromosomes. Comparisons of this map with the X. tropicalis genome Assembly 4.1 (JGI) indicate that the map provides representation of a minimum of 66% of the X. tropicalis genome, incorporating 758 of the approximately 1300 scaffolds over 100,000 bp. The genetic map and SSLP marker database constitute an essential resource for genetic and genomic analyses in X. tropicalis.     

Potassium channel blocker crafted by α-hairpinin scaffold engineering

The α-Hairpinins are a family of plant defense peptides with a common fold presenting two short α-helices stabilized by two invariant S–S-bridges. We have shown previously that substitution of just two amino acid residues in a wheat α-hairpinin Tk-AMP-X2 leads to Tk-hefu-2 that features specific affinity to voltage-gated potassium channels K_V1.3. Here, we utilize a combined molecular modeling approach based on molecular dynamics simulations and protein surface topography technique to improve the affinity of Tk-hefu-2 to K_V1.3 while preserving its specificity. An important advance of this work compared with our previous studies is transition from the analysis of various physicochemical properties of an isolated toxin molecule to its consideration in complex with its target, a membrane-bound ion channel. As a result, a panel of computationally designed Tk-hefu-2 derivatives was synthesized and tested against K_V1.3. The most active mutant Tk-hefu-10 showed a half-maximal inhibitory concentration of ～150 nM being >10 times more active than Tk-hefu-2 and >200 times more active than the original Tk-hefu. We conclude that α-hairpinins provide an attractive disulfide-stabilized scaffold for the rational design of ion channel inhibitors. Furthermore, the success rate can be considerably increased by the proposed “target-based” iterative strategy of molecular design.     

New temperate Pseudomonas aeruginosa phage, phi297: Specific features of genome structure

The genome structure and some specific features of temperate Pseudomonas aeruginosa phage phi297 are considered. Analysis of sequencing data and genome annotation suggest that the phi297 genome displays a mosaic structure, which has formed through combining gene blocks from bacteria of taxonomically remote groups and/or their phages. The results of a comparison of the phi297 DNA homology level and pattern with the genome sequences of the currently known related P. aeruginosa bacteriophages are interpreted from the perspective of assumed active migration of these phages between different bacterial species.