Dual Active Site in the Endolytic Transglycosylase gp144 of Bacteriophage phiKZ.

Lytic transglycosylases are abundant peptidoglycan lysing enzymes that degrade the heteropolymers of bacterial cell walls in metabolic processes or in the course of a bacteriophage infection. The conventional catalytic mechanism of transglycosylases involves only the Glu or Asp residue. Endolysin gp144 of Pseudomonas aeruginosa bacteriophage phiKZ belongs to the family of Gram-negative transglycosylases with a modular composition and C-terminal location of the catalytic domain. Glu115 of gp144 performs the predicted role of a catalytic residue. However, replacement of this residue does not completely eliminate the activity of the mutant protein. Site-directed mutagenesis has revealed the participation of Tyr197 in the catalytic mechanism, as well as the presence of a second active site involving Glu178 and Tyr147. The existence of the dual active site was supported by computer modeling and monitoring of the molecular dynamics of the changes in the conformation and surface charge distribution as a consequence of point mutations.

[The properties of the Pseudomonas aeruginosa bacteriophage phiPMG1].

The properties of the isolated Pseudomonas aeruginosa bacteriophage phiPMG1 include the lytic infection cycle, and the formation of a broad halo (semi-transparent zone) around the plaques. We consider phiPMG1 as a potential member of therapeutic cocktails of live phages, and as a source of peptidoglycan and lipopolysaccharide degrading enzymes. Partial sequencing of phiPMG1 genome has revealed high similarity with known temperate P. aeruginosa phage D3. An open reading frame encoding lytic transglycosilase was identified in the genome. This enzyme PMG MUR was obtained in recombinant form, and its activity and substrate specificity has been studied.

[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.

Properties of the endolytic transglycosylase encoded by gene 144 of Pseudomonas aeruginosa bacteriophage phiKZ.

Bacteriophage endolysins degrading bacterial cell walls are prospective enzymes for therapy of bacterial infections. The genome of the giant bacteriophage phiKZ of Pseudomonas aeruginosa encodes two endolysins, gene products (g.p.) 144 and 181, which are homologous to lytic transglycosylases. Gene 144 encoding a 260 amino acid residue protein was cloned into the plasmid expression vector. Recombinant g.p. 144 purified from Escherichia coli effectively degrades chloroform-treated P. aeruginosa cell walls. The protein has predominantly alpha-helical conformation and exists in solution in stoichiometric monomer : dimer : trimer equilibrium. Antibodies against the protein bind the phage particle. This demonstrates that g.p. 144 is a structural component of the phiKZ particle, presumably, a phage tail.

Functional role of the N-terminal domain of bacteriophage T4 gene product 11.

Bacteriophage T4 late gene product 11 (gp11), the three-dimensional structure of which has been solved by us to 2.0 A resolution, is a part of the virus' baseplate. The gp11 polypeptide chain consists of 219 amino acid residues and the functionally active protein is a three-domain homotrimer. In this work, we have studied the role of gp11 N-terminal domain in the formation of a functionally active trimer. Deletion variants of gp11 and monoclonal antibodies recognizing the native conformation of gp11 trimer have been selected. Long deletions up to a complete removal of the N-terminal domain, containing 64 residues, do not affect the gp11 trimerization, but considerably change the protein structure and lead to the loss of its ability to incorporate into the baseplate. However, the deletion of the first 17 N-terminal residues results in functionally active protein that can complete the 11(-)-defective phage particles in in vitro complementation assay. This region of the polypeptide chain is probably essential for gp11-gp10 stable complex formation at the early stages of phage baseplate assembly in vivo. A study of the gp10 deletion variants suggests that the central domain of gp10 trimer is responsible for the interaction with gp11.

[Comparison of genomes of new gigantic Pseudomonas aeruginosa phages from native populations from different regions]. / Sravnenie genomov novykh gigantskikh fagov Pseudomonas aeruginosa iz priorodnykh populiatsii raznykh regionov.

To study the genome diversity of bacteriophages from geographically distant natural populations, new giant phi KZ-like Pseudomonas aeruginosa phages isolated in two different regions were compared with earlier known phages of three species (phi KZ, Lin68, EL). A broad spectrum of lytic activity was demonstrated for all phi KZ-like phages. Phages of the phi KZ species proved to be common in natural populations of various regions, while IL- and Lin68-related phages were extremely rare. Most phi KZ-related phages had unique DNA restriction patterns, but the differences between these were only minor, and the genomes did not contain nonhomologous fragments. The spectrum of capsid polypeptides proved to be conserved in each species, and was proposed as a character necessary and sufficient for express classification of phages with an accuracy of species. Phages isolated in different geographical regions showed no substantial difference. Some phages only slightly differing in DNA restriction pattern from phi KZ may be used to study the origin of phi KZ genes coding for orthologs of proteins of unrelated species (other phages, pathogenic bacteria, eukaryotes).

Molecular architecture of bacteriophage T4.

In studying bacteriophage T4--one of the basic models of molecular biology for several decades--there has come a Renaissance, and this virus is now actively used as object of structural biology. The structures of six proteins of the phage particle have recently been determined at atomic resolution by X-ray crystallography. Three-dimensional reconstruction of the infection device--one of the most complex multiprotein components--has been developed on the basis of cryo-electron microscopy images. The further study of bacteriophage T4 structure will allow a better understanding of the regulation of protein folding, assembly of biological structures, and also mechanisms of functioning of the complex biological molecular machines.

[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.

Structure and folding of bacteriophage T4 gene product 9 triggering infection. II. Study Of conformational changes of gene product 9 mutants using monoclonal antibodies.

Gene product 9 (gp9) of bacteriophage T4, whose spatial structure we have recently solved to 2.3 A resolution, is a convenient model for studying the folding and oligomerization mechanisms of complex proteins. The gp9 polypeptide chain consists of 288 amino acids forming three domains. Three monomers, packed in parallel, assemble to a functionally active protein. The main aim of this work was to study conformational changes and trimerization of gp9 deletion mutants using monoclonal antibodies (mAbs). We selected a set of mAbs interacting with the amino, middle, and carboxyl regions of the protein, respectively. Eighteen mAbs bind to native as well as to denatured protein, and two mAbs bind to denatured protein only. Using mAbs, we found that deletions of the gp9 N-terminal region result in conformational changes in the middle and C-terminal domains. The study of mAb binding to the CDelta. truncated mutant by competitive ELISA and immunoblotting shows that the C-terminus of the gp9 sequence is essential for protein trimerization and stability. A single point substitution of the Gln282 residue causes formation of a labile trimer that has significant conformational changes in the protein domains. The results of our study show that folding and trimerization of gp9 is a cooperative process that involves all domains of the protein.