[The beta-helical domain of bacteriophage T4 controls the folding of the fragment of long tail fibers in a chimeric protein].

The key stage of the infection of the Escherichia coli cell with bacteriophage T4, the binding to the surface of the host cell, is determined by the specificity of the long tail fiber proteins of the phage, in particular, gp37. The assembly and oligomerization of this protein under natural conditions requires the participation of at least two additional protein factors, gp57A and gp38, which strongly hinders the production of the recombinant form of gp37. To overcome this problem, a modern protein engineering strategy was used, which involves the construction of a chimeric protein containing a carrier protein that drives the correct folding of the target protein. For this purpose, the trimeric beta-helical domain of another protein of phage T4, gp5, was used. It was shown that this domain, represented as a rigid trimeric polypeptide prism, has properties favorable for use as a protein carrier. A fragment of protein gp37 containing five pentapeptides repeats, Gly-X-His-X-His, which determine the binding to the receptors on the bacterial cell surface, was fused in a continuous reading frame to the C-terminus of the domain of gp5. The resulting chimeric protein forms a trimer that has the native conformation of gp37 and exhibits biological activity.

[Search for destruction factors of bacterial biofilms: comparison of phage properties in a group of Pseudomonas putida bacteriophages and specificity of their halo-formation products].

Comparison of Pseudomonas putida group of phages attributed to five species (af, phi15, phi27, phi2F, and pf16) with their common property of halo-formation (formation of lightening zones) around phage plaques was conducted. The halo around phage plaques appears as a result of reduction or disappearance of bacterial polysaccharide capsules. The concentration of viable bacteria remains unchanged within the halo. A comparison of specificities of halo-formation products from various phages was conducted by a simple method. These products were shown to be highly specific and inactive on other species of pseudomonads. Phage-resistant P. putida mutants scored with respect to various phages, which lost phage adsorption ability, were tolerant to the effect of halo-formation products in most cases. Apparently, the capsular polysaccharides, which serve as a substrate for depolymerases and are the primary phage receptors, may be often lost. Results of partial sequencing of the af phage genome revealed an open reading frame that encodes the enzyme transglycosylase similar rather to transglycosylases of oligotrophic bacteria belonging to different species than to lysozymes of other phages. Possibly, it is a polyfunctional enzyme combining functions of lysozyme and an enzyme that executes the penetration of phage particle across extracellular slime and capsule.

[Pseudomonas aeruginosa bacteriophage SN: 3D-reconstruction of the capsid and identification of surface proteins by electron microscopy].

The virulent P. aeruginosa bacteriophage SN belongs to the PB1-like species of the Myoviridae family. The comparatively small (66391 bp) DNA genome of this phage encodes 89 predicted open reading frames and the proteome involves more than 20 structural proteins. A 3D model of the phage capsid to approximately 18 A resolution reveals certain peculiarities of capsomer structure typical of only this bacteriophage species. In the present work recombinant structural proteins SN gp22 and gp29 were expressed and purified; and specific polyclonal antibodies were obtained. Immune-electron microscopy of purified phage SN using secondary gold-conjugated antibodies has revealed that gp29 forms a phage sheath, and gp22 decorates the capsid. Precise identification of multicopy major capsid proteins is essential for subsequent construction of gene-engineered phages bearing non-native peptides on their surfaces (phage display).

[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.

[Comparison of the genome for phylogenetically related bacteriophages phiKZ and EL of Pseudomonas aeruginosa: evolutionary aspects and minimal genome size].

Bacteriophages of the family Myoviridae represent one of the most widespread domains of the biosphere substantially affecting the ecological balance of microorganisms. Interestingly, sequence analysis of genomic DNAs of large bacteriophages revealed many genes coding for proteins with unknown functions. A new approach is proposed to improve the functional identification of genes. This approach is based on comparing the genome sequence for phylogenetically and morphologically related phages showing no considerable homology at the level of genomic DNA. It is assumed that gene functions essential for the development of phages of a given family are conserved and that the corresponding genes code for similar orthologous proteins even when lacking sequence homology. The genome was sequenced and compared for two Pseudomonas aeruginosa giant bacteriophages, phiKZ and EL, which belong to a group of (phiKZ-related phages. A substantial difference in genome organization was observed, suggesting specific features of phage evolution. In addition, the problem of the minimal genome of the superfamily is discussed on the basis of the difference in size and structure between the phiKZ and EL genomes.

[Phenogenetic characterization of a group of giant Phi KZ-like bacteriophages of Pseudomonas aeruginosa]. / Fenogeneticheskaia kharakteristika gruppy gigantskikh Phi KZ-podobnykh bakteriofagov Pseudomonas aeruginosa.

A comparative study was made of a group of Pseudomonas aeruginosa virulent giant DNA bacteriophages similar to phage phi KZ in several genetic and phenotypic properties (particle size, particle morphology, genome size, appearance of negative colonies, high productivity, broad spectrum of lytic activity, ability to overcome the suppressing effect of plasmids, absence of several DNA restriction sites, capability of general transduction, pseudolysogeny). We have recently sequenced the phage phi KZ genome (288,334 bp) [J. Mol. Biol., 2002, vol. 317, pp. 1-19]. By DNA homology, the phages were assigned to three species (represented by phage phi KZ, Lin68, and EL, respectively) and two new genera (phi KZ and EL). Restriction enzyme analysis revealed the mosaic genome structure in four phages of the phi KZ species (phi KZ, Lin21, NN, and PTB80) and two phages of the EL species (EL and RU). Comparisons with respect to phage particle size, number of structural proteins, and the N-terminal sequences of the major capsid protein confirmed the phylogenetic relatedness of the phages belonging to the phi KZ genus. The origin and evolution of the phi KZ-like phages are discussed. Analysis of protein sequences encoded by the phage phi KZ genome made it possible to assume wide migration of the phi KZ-like phages (wandering phages) among various prokaryotes and possibly eukaryotes. Since the phage phi KZ genome codes for potentially toxic proteins, caution must be exercised in the employment of large bacteriophages in phage therapy.

[Thermostability of bacteriophage T4 fibritin and its deletion mutants]. / Termostabil'nost' fibritina bakteriofaga T4 i ego deletsionnykh mutantov.

The thermal stability of a series of recently obtained mutants of fibritin from bacteriophage T4 (a superhelical fibrous homotrimer with parallel-packed subunits each containing 486 amino acid residues) progressively truncated from the subunit N-end was studied during incubation at 40-90 degrees C in the presence of a surfactant (2% SDS). The mutant fibritins, G, B, C, and E, contained 443, 276, 231, and 120 amino acid residues, respectively. One more truncated mutant (fibritin S1, 108 amino acid residues) was obtained. The 2% SDS-PAGE showed that the migration mobilities of all these proteins corresponded to apparent molecular masses substantially greater than those of the preliminarily heated samples (3 min at 100 degrees C). The heating of the intact fibritin and the mutant G at 50-70 degrees C for 10 min resulted in the formation of a form with an apparent molecular mass higher than 200 kDa. This form probably represented a trimeric protein with a partly denatured N-terminal part. Fibritins B and C were more stable and were only partly denatured into monomers even at 70-90 degrees C. The short mutants E and S1 dissociated into monomers at temperatures from 45 to 50 degrees C. The denaturation of mutants B, C, E, and S1 proceeded in one stage without formation of any intermediate form. The stability of the trimeric molecules of native fibritin under PAGE denaturing conditions and the behavior of the intact protein during heating in the temperature range of 50-70 degrees C might be used for the identification of fibritin intermediate forms upon folding in vivo. The refolding capability was found for fibritin and its mutants denatured by heating at low temperatures in the presence of 2% SDS.

[Detection of genomic RNA of the hepatitis A virus in clinical samples using the blot hybridization method]. / Detektsiia genomnoi RNK virusa gepatita A v klinicheskikh obraztsakh s pomoshch'iu metoda tochechno gibridizatsii.

The synthetic oligonucleotide sequence 3'CTCCTCGTACTTTA-5' complementing hepatitis A virus RNA was compared with cDNA probes in identification of viral genomic RNA. The clinical materials from patients in the 1-2 weeks of jaundice were screened. High specificity of the technique was demonstrated. Possibility of clinical using of the blot hybridization technique is discussed.

[Structure and regulation of the assembly of bacteriophage T4 fibrillar proteins. I. Isolation and spectral properties of long fibers]. / Struktura i reguliatsiia sborki fibrilliarnykh belkov bakteriofaga T4. I. Vydelenie i spektral'nye svoistva dlinnykh fibrill.

A preparative procedure for purification of the biological active proximal (A) and distal (BC') parts of bacteriophage T4 long-tail fibers is described. Absorption spectra of these proteins in the near ultraviolet region were measured. The absorption coefficients were determined on the basis of the nitrogen content, the absorption coefficient for the A part is epsilon 0.1% 277 nm = 0.93 +/- 0.06 and for the BC' part is epsilon 0.1%, 277,5 nm = 1.01 +/- 0.08. Calculations of the secondary structure from CD spectra show that there is a high content of beta-structure: 41% in the A part and 51% in the BC' part,--and also that alpha-helix are present in the native complex: 20% in A and 7% in BC'. A model for the spatial organisation of long fibers is proposed.

[Structure and regulation of the assembly of bacteriophage T4 fibrillar proteins. II. Isolation and initial structural characteristics of whiskers]. / Struktura i reguliatsiia sborki fibrilliarnykh belkov bakteriofaga T4. II. Vydelenie i nachal'naia strukturnaia kharakteristika vorotnichkovykh nitei.

A procedure for purification of bacteriophage T4 whiskers and it's monomeric subunits--gene product wac--has been developed. We have shown, that the whiskers are composed of two identical copies of gene product wac with molecular weight of 56 kDa each. The dimer of gene product wac is a highly ordered structure and it's length is about 70.0 +/- 10.0 nm, as revealed by electron microscopy. The amino acid composition of whiskers is very similar to that of watersoluble keratins. We have proposed a new term for the definition of the whiskers--the fibritin.

[A model of the secondary structure and distribution of antigenic determinants in the VP1 protein of hepatitis A virus]. / Model' vtorichnoi struktury i raspredeleniia antigennykh determinant v belke VP1 virusa gepatita A.

A comparative analysis of amino acid sequence of the proteins VP1 of hepatitis A virus and poliovirus of the 1 type was carried out. A model is proposed of structural organization of VP1 of hepatitis A virus providing the presence of a bilayer core formed by 8 antiparallel beta-strands. Probable candidates for surface antigenic determinants are the amino acid sequences located in unordered fragments of the polypeptide chain (residues 101-106 and 115-125), and alpha-helical region (residues 127-135).

[Structural separation of the functional loci of bacteriophage T4 short fibrillae]. / Strukturnoe razdelenie funktsional'nykh lokusov korotkikh fibrill bakteriofaga T4.

Short-tail fibers (STF) of bacteriophage T4 are a polyfunctional protein. STF appears to be a trimer of gene 12 product. The modified trimers, consisting of fragments of gene 12 product with mol mass 45 and 50 Kd, respectively, were isolated by limited proteolysis with trypsin and papain. The isolated trimers retained their bactericidal activity but were unable to complement the fiberless phage particles. The results obtained suggest that STF loci responsible for bactericidal effect are separated from the loci involved in interaction with the base plate.

[Isolation of biologically active halves of the long tail fibers and whiskers of bacteriophage T4]. / Vydelenie biologicheski aktivnykh polovinok dlinnykh khvostovykh fibrill i bakenbard bakteriofaga T4.

The following substructural elements of bacteriophage T4 have been isolated in homogeneous and biologically active state: the whole long tail fibrils, distal and proximal halves of the long tail fibrils and whiskers. It has been shown that during the infection of Escherichia coli with T4 amber-mutants (defective in the genes coding for heads and tails), the major part of distal and proximal fibril halves found within the cell appears to be unassociated. The interaction of distal and proximal fibril halves with the bacteriophage particle is necessary for the whole tail fibril formation to proceed effectively.