Pseudomonas Phage MD8: Genetic Mosaicism and Challenges of Taxonomic Classification of Lambdoid Bacteriophages.

Pseudomonas phage MD8 is a temperate phage isolated from the freshwater lake Baikal. The organisation of the MD8 genome resembles the genomes of lambdoid bacteriophages. However, MD8 gene and protein sequences have little in common with classified representatives of lambda-like phages. Analysis of phage genomes revealed a group of other Pseudomonas phages related to phage MD8 and the genomic layout of MD8-like phages indicated extensive gene exchange involving even the most conservative proteins and leading to a high degree of genomic mosaicism. Multiple horizontal transfers and mosaicism of the genome of MD8, related phages and other λ-like phages raise questions about the principles of taxonomic classification of the representatives of this voluminous phage group. Comparison and analysis of various bioinformatic approaches applied to λ-like phage genomes demonstrated different efficiency and contradictory results in the estimation of genomic similarity and relatedness. However, we were able to make suggestions for the possible origin of the MD8 genome and the basic principles for the taxonomic classification of lambdoid phages. The group comprising 26 MD8-related phages was proposed to classify as two close genera belonging to a big family of λ-like phages.

Pseudomonas Phage PaBG-A Jumbo Member of an Old Parasite Family.

Bacteriophage PaBG is a jumbo Myoviridae phage isolated from water of Lake Baikal. This phage has limited diffusion ability and thermal stability and infects a narrow range of Pseudomonas aeruginosa strains. Therefore, it is hardly suitable for phage therapy applications. However, the analysis of the genome of PaBG presents a number of insights into the evolutionary history of this phage and jumbo phages in general. We suggest that PaBG represents an ancient group distantly related to all known classified families of phages.

Structure of an Acinetobacter Broad-Range Prophage Endolysin Reveals a C-Terminal α-Helix with the Proposed Role in Activity against Live Bacterial Cells.

Proteins that include enzymatic domain degrading the bacterial cell wall and a domain providing transport through the bacterial outer membrane are considered as prospective compounds to combat pathogenic Gram-negative bacteria. This paper presents an isolation and study of an enzyme of this class naturally encoded in the prophage region of Acinetobacter baumannii AB 5075 genome. Recombinant protein expressed in E. coli exhibits an antimicrobial activity with respect to live cultures of Gram-negative bacteria reducing the population of viable bacteria by 1.5⁻2 log colony forming units (CFU)/mL. However the protein becomes rapidly inactivated and enables the bacteria to restore the population. AcLys structure determined by X-ray crystallography reveals a predominantly α—helical fold similar to bacteriophage P22 lysozyme. The С-terminal part of AcLys polypeptide chains forms an α—helix enriched by Lys and Arg residues exposed outside of the protein globule. Presumably this type of structure of the C-terminal α—helix has evolved evolutionally enabling the endolysin to pass the inner membrane during the host lysis or, potentially, to penetrate the outer membrane of the Gram-negative bacteria.

The unique two-component tail sheath of giant Pseudomonas phage PaBG.

Myoviridae bacteriophages have a special contractile tail machine that facilitates high viral infection efficiency. The major component of this machine is a tail sheath that contracts during infection, allowing delivery of viral DNA into the host cell. Tail sheaths of Myoviridae phages are composed of multiple copies of individual proteins. The giant Pseudomonas aeruginosa phage PaBG is notable in its possession of two tail sheath proteins. These tail sheath proteins are encoded by orf 76 and 204, which were cloned and expressed individually and together in Escherichia coli. We demonstrate that only co-expression of both genes results in efficient assembly of thermostable and proteolytically resistant polysheaths composed of gp76 and gp204 with approximately 1:1 stoichiometry. Both gp76 and gp204 have been identified as structural components of the virion particle. We conclude that during PaBG morphogenesis in vivo two proteins, gp76 and gp204, assemble the tail sheath.

Complete Genome Sequence of the Novel Giant Pseudomonas Phage PaBG.

The novel giant Pseudomonas aeruginosa bacteriophage PaBG was isolated from a water sample of the ultrafreshwater Lake Baikal. We report the complete genome sequence of this Myoviridae bacteriophage, comprising 258,139 bp of double-stranded DNA containing 308 predicted open reading frames.

Crystal structure and location of gp131 in the bacteriophage phiKZ virion.

Pseudomonas phage ϕKZ and its two close relatives ϕPA3 and 201ϕ2-1 are very large bacteriophages that form a separate branch in phage classification because their genomes are very different from the rest of GenBank sequence data. The contractile tail of ϕKZ is built from at least 32 different proteins, but a definitive structural function is assigned to only one of them-the tail sheath protein. Here, we report the crystal structure of the C-terminal domain of another phiKZ tail protein, gene product 131 (gp131C). We show that gp131 is located at the periphery of the baseplate and possibly associates with fibers that emanate from the baseplate. Gp131C is a seven-bladed ß-propeller that has a shape of a skewed toroid. A small but highly conserved and negatively charged patch on the surface of gp131C might be important for substrate binding or for interaction with a different tail protein.

Expression and functional characterization of the first bacteriophage-encoded chaperonin.

Chaperonins promote protein folding in vivo and are ubiquitously found in bacteria, archaea, and eukaryotes. The first viral chaperonin GroEL ortholog, gene product 146 (gp146), whose gene was earlier identified in the genome of bacteriophage EL, has been shown to be synthesized during phage propagation in Pseudomonas aeruginosa cells. The recombinant gp146 has been expressed in Escherichia coli and characterized by different physicochemical methods for the first time. Using serum against the recombinant protein, gp146's native substrate, the phage endolysin gp188, has been immunoprecipitated from the lysate of EL-infected bacteria and identified by mass spectrometry. In vitro experiments have shown that gp146 has a protective effect against endolysin thermal inactivation and aggregation, providing evidence of its chaperonin function. The phage chaperonin has been found to have the architecture and some properties similar to those of GroEL but not to require cochaperonin for its functional activity.

Characterization of tail sheath protein of giant bacteriophage phiKZ Pseudomonas aeruginosa.

The tail sheath protein of giant bacteriophage phiKZ Pseudomonas aeruginosa encoded by gene 29 was identified and its expression system was developed. Localization of the protein on the virion was confirmed by immunoelectron microscopy. Properties of gene product (gp) 29 were studied by electron microscopy, immunoblotting and limited trypsinolysis. Recombinant gp29 assembles into the regular tubular structures (polysheaths) of variable length. Trypsin digestion of gp29 within polysheaths or extended sheath of virion results in specific cleavage of the peptide bond between Arg135 and Asp136. However, this cleavage does not affect polymeric structure of polysheaths, sheaths and viral infectivity. Digestion by trypsin of the C-truncated gp29 mutant, lacking the ability to self-assemble, results in formation of a stable protease-resistant fragment. Although there is no sequence homology of phiKZ proteins to proteins of other bacteriophages, some characteristic biochemical properties of gp29 revealed similarities to the tail sheath protein of bacteriophage T4.

Comparative analysis of the widespread and conserved PB1-like viruses infecting Pseudomonas aeruginosa.

We examined the genetic diversity of lytic Pseudomonas aeruginosa bacteriophage PB1 and four closely related phages (LBL3, LMA2, 14-1 and SN) isolated throughout Europe. They all encapsulate linear, non-permuted genomes between 64 427 and 66 530 bp within a solid, acid-resistant isometric capsid (diameter: 74 nm) and carry non-flexible, contractile tails of approximately 140 nm. The genomes are organized into at least seven transcriptional blocks, alternating on both strands, and encode between 88 (LBL3) and 95 (LMA2) proteins. Their virion particles are composed of at least 22 different proteins, which were identified using mass spectrometry. Post-translational modifications were suggested for two proteins, and a frameshift hotspot was identified within ORF42, encoding a structural protein. Despite large temporal and spatial separations between phage isolations, very high sequence similarity and limited horizontal gene transfer were found between the individual viruses. These PB1-like viruses constitute a new genus of environmentally very widespread phages within the Myoviridae.

The genome and structural proteome of YuA, a new Pseudomonas aeruginosa phage resembling M6.

Pseudomonas aeruginosa phage YuA (Siphoviridae) was isolated from a pond near Moscow, Russia. It has an elongated head, encapsulating a circularly permuted genome of 58,663 bp, and a flexible, noncontractile tail, which is terminally and subterminally decorated with short fibers. The YuA genome is neither Mu- nor lambda-like and encodes 78 gene products that cluster in three major regions involved in (i) DNA metabolism and replication, (ii) host interaction, and (iii) phage particle formation and host lysis. At the protein level, YuA displays significant homology with phages M6, phiJL001, 73, B3, DMS3, and D3112. Eighteen YuA proteins were identified as part of the phage particle by mass spectrometry analysis. Five different bacterial promoters were experimentally identified using a promoter trap assay, three of which have a sigma54-specific binding site and regulate transcription in the genome region involved in phage particle formation and host lysis. The dependency of these promoters on the host sigma54 factor was confirmed by analysis of an rpoN mutant strain of P. aeruginosa PAO1. At the DNA level, YuA is 91% identical to the recently (July 2007) annotated phage M6 of the Lindberg typing set. Despite this level of DNA homology throughout the genome, both phages combined have 15 unique genes that do not occur in the other phage. The genome organization of both phages differs substantially from those of the other known Pseudomonas-infecting Siphoviridae, delineating them as a distinct genus within this family.

Structure, stability, and biological activity of bacteriophage T4 gene product 9 probed with mutagenesis and monoclonal antibodies.

Gene product (gp) 9 connects the long tail fibers and triggers the structural transition of T4 phage baseplate at the beginning of infection process. Gp9 is a parallel homotrimer with 288 amino acid residues per chain that forms three domains. To investigate the role of the gp9 amino terminus, we have engineered a set of mutants with deletions and random substitutions in this part. The structure of the mutants was probed using monoclonal antibodies that bind to either N-terminal, middle, or C-terminal domains. Deletions of up to 12 N-terminal residues as well as random substitutions of the second, third and fourth residues yielded trimers that failed to incorporate in vitro into the T4 9(-)-particles and were not able to convert them into infectious virions. As detected using monoclonal antibodies, these mutants undergo structural changes in both N-terminal and middle domains. Furthermore, deletion of the first twenty residues caused profound structural changes in all three gp9 domains. In addition, N-terminally truncated proteins and randomized mutants formed SDS-resistant "conformers" due to unwinding of the N-terminal region. Co-expression of the full-length gp9 and the mutant lacking first 20 residues clearly shows the assembly of heterotrimers, suggesting that the gp9 trimerization in vivo occurs post-translationally. Collectively, our data indicate that the aminoterminal sequence of gp9 is important to maintain a competent structure capable of incorporating into the baseplate, and may be also required at intermediate stages of gp9 folding and assembly.

A three-dimensional cryo-electron microscopy structure of the bacteriophage phiKZ head.

The three-dimensional structure of the Pseudomonas aeruginosa bacteriophage phiKZ head has been determined by cryo-electron microscopy and image reconstruction to 18A resolution. The head has icosahedral symmetry measuring 1455 A in diameter along 5-fold axes and a unique portal vertex to which is attached an approximately 1800 A-long contractile tail. The 65 kDa major capsid protein, gp120, is organized into a surface lattice of hexamers, with T = 27 triangulation. The shape and size of the hexamers is similar to the hexameric building blocks of the bacteriophages T4, phi29, P22, and HK97. Pentameric vertices of the capsid are occupied by complexes composed of several special vertex proteins. The double-stranded genomic DNA is packaged into a highly condensed series of layers, separated by 24 A, that follow the contour of the inner wall of the capsid.

The genome of bacteriophage phiKMV, a T7-like virus infecting Pseudomonas aeruginosa.

The complete DNA sequence of a new lytic T7-like bacteriophage phiKMV is presented. It is the first genome sequence of a member of the Podoviridae that infects Pseudomonas aeruginosa. The linear G + C-rich (62.3%) double-stranded DNA genome of 42,519 bp has direct terminal repeats of 414 bp and contains 48 open reading frames that are all transcribed from the same strand. Despite absence of homology at the DNA level, 11 of the 48 phiKMV-encoded putative proteins show sequence similarity to known T7-type phage proteins. Eighteen open reading frame products have been assigned, including an RNA polymerase, proteins involved in DNA replication, as well as structural, phage maturation, and lysis proteins. Surprisingly, the major capsid protein completely lacks sequence homology to any known protein. Also, the strong virulence toward many clinical P. aeruginosa isolates and a short replication time make phiKMV attractive for phage therapy or a potential source for antimicrobial proteins.

Myoviridae bacteriophages of Pseudomonas aeruginosa: a long and complex evolutionary pathway.

Recently we have accomplished the entire DNA sequence of bacteriophage phiKZ, a giant virus infecting Pseudomonas aeruginosa. The 280334-bp of phiKZ genome is a linear, circularly permutated and terminally redundant, AT-rich dsDNA molecule that contains no sites for NotI, PstI, SacI, SmaI, XhoI and XmaIII endonucleases. Limited homology to other bacteriophages on the DNA and protein levels indicated that phiKZ represents a distinct branch of the Myoviridae family. In this work, we analyzed a group of six P. aeruginosa phages (Lin68, Lin21, PTB80, NN, EL, and RU), which are morphologically similar to phiKZ, have similar genome size and low G+C content. All phages have a broad host range among P. aeruginosa strains, and they are resistant to the inhibitory action of many P. aeruginosa plasmids. The analysis of the genomic DNA by restriction enzymes and DNA-DNA hybridization shows that phages are representative of three phiKZ-like species: phiKZ-type (phiKZ, Lin21, NN and PTB80), EL-type (EL and RU) and Lin68 which has a shorter tail than other phages. Except for related phages EL and RU, all phiKZ-like phages have identical N-terminal amino acid sequences of the major capsid protein. Random genome sequencing shows that the EL and RU phages have no homology to the phiKZ-like phages on DNA level. We propose that the phiKZ, Lin21, NN, PTB80 and Lin68 phages can be included in a new phiKZ genus, and that the EL and RU phages belong to a separate genus within the Myoviridae family. Based on the resistance to many restriction enzymes and the transduction ability, there are indications that over the long pathway of evolution, the phiKZ-like phages probably inherited the capacity to infect different bacterial species.

The genome of bacteriophage phiKZ of Pseudomonas aeruginosa.

Bacteriophage phiKZ is a giant virus that efficiently infects Pseudomonas aeruginosa strains pathogenic to human and, therefore, it is attractive for phage therapy. We present here the complete phiKZ genome sequence and a preliminary analysis of its genome structure. The 280,334 bp genome is a linear, circularly permutated and terminally redundant, A+T-rich double-stranded DNA molecule. The phiKZ DNA has no detectable sequence homology to other viruses and microorganisms, and it does not contain NotI, PstI, SacI, SmaI, XhoI, and XmaIII endonuclease restriction sites. The genome has 306 open reading frames (ORFs) varying in size from 50 to 2237 amino acid residues. According to the orientation of transcription, ORFs are apparently organized into clusters and most have a clockwise direction. The phiKZ genome also encodes six tRNAs specific for Met (AUG), Asn (AAC), Asp (GAC), Leu (TTA), Thr (ACA), and Pro (CCA). A putative promoter sequence containing a TATATTAC block was identified. Most potential stem-loop transcription terminators contain the tetranucleotide UUCG loops. Some genes may be assigned as phage-encoded RNA polymerase subunits. Only 59 phiKZ gene products exhibit similarity to proteins of known function from a diversity of organisms. Most of these conserved gene products, such as dihydrofolate reductase, ribonucleoside diphosphate reductase, thymidylate synthase, thymidylate kinase, and deoxycytidine triphosphate deaminase are involved in nucleotide metabolism. However, no virus-encoded DNA polymerase, DNA replication-associated proteins, or single-stranded DNA-binding protein were found based on amino acid homology, and they may therefore be strongly divergent from known homologous proteins. Fifteen phiKZ gene products show homology to proteins of pathogenic organisms, including Mycobacterium tuberculosis, Haemophilus influenzae, Listeria sp., Rickettsia prowazakeri, and Vibrio cholerae that must be considered before using this phage as a therapeutic agent. The phiKZ coat contains at least 40 polypeptides, and several proteins are cleaved during virus assembly in a way similar to phage T4. Eleven phiKZ-encoded polypeptides are related to proteins of other bacteriphages that infect a variety of hosts. Among these are four gene products that contain a putative intron-encoded endonuclease harboring the H-N-H motif common to many double-stranded DNA phages. These observations provide evidence that phages infecting diverse hosts have had access to a common genetic pool. However, limited homology on the DNA and protein levels indicates that bacteriophage phiKZ represents an evolutionary distinctive branch of the Myoviridae family.