Cryo-EM study of the Pseudomonas bacteriophage phiKZ

The phiKZ virus is one of the largest known bacteriophages. It infects Pseudomonas aeruginosa, which is frequently pathogenic in humans, and, therefore, has potential for phage therapy. The phiKZ virion consists of an approximately 1450 A diameter icosahedral head and an approximately 2000 A long contractile tail. The structure of the phiKZ tail has been determined using cryo-electron microscopy. The phiKZ tail is much longer than that of bacteriophage T4. However, the helical parameters of their contractile sheaths, surrounding their tail tubes, are comparable. Although there is no recognizable sequence similarity between the phiKZ and T4 tail sheath proteins, they are similar in size and shape, suggesting that they evolved from a common ancestor. The phiKZ baseplate is significantly larger than that of T4 and has a flatter shape. Nevertheless, phiKZ, similar to T4, has a cell-puncturing device in the middle of its baseplate.     

Structural conservation of the myoviridae phage tail sheath protein fold

Bacteriophage phiKZ is a giant phage that infects Pseudomonas aeruginosa, a human pathogen. The phiKZ virion consists of a 1450?? diameter icosahedral head and a 2000??-long contractile tail. The structure of the whole virus was previously reported, showing that its tail organization in the extended state is similar to the well-studied Myovirus bacteriophage T4 tail. The crystal structure of a tail sheath protein fragment of phiKZ was determined to 2.4?? resolution. Furthermore, crystal structures of two prophage tail sheath proteins were determined to 1.9 and 3.3?? resolution. Despite low sequence identity between these proteins, all of these structures have a similar fold. The crystal structure of the phiKZ tail sheath protein has been fitted into cryo-electron-microscopy reconstructions of the extended tail sheath and of a polysheath. The structural rearrangement of the phiKZ tail sheath contraction was found to be similar to that of phage T4.     

Genome comparison of Pseudomonas aeruginosa large phages

Pseudomonas aeruginosa phage EL is a dsDNA phage related to the giant phiKZ-like Myoviridae. The EL genome sequence comprises 211,215 bp and has 201 predicted open reading frames (ORFs). The EL genome does not share DNA sequence homology with other viruses and micro-organisms sequenced to date. However, one-third of the predicted EL gene products (gps) shares similarity (Blast alignments of 17-55% amino acid identity) with phiKZ proteins. Comparative EL and phiKZ genomics reveals that these giant phages are an example of substantially diverged genetic mosaics. Based on the position of similar EL and phiKZ predicted gene products, five genome regions can be delineated in EL, four of which are relatively conserved between EL and phiKZ. Region IV, a 17.7 kb genome region with 28 predicted ORFs, is unique to EL. Fourteen EL ORFs have been assigned a putative function based on protein similarity. Assigned proteins are involved in DNA replication and nucleotide metabolism (NAD+-dependent DNA ligase, ribonuclease HI, helicase, thymidylate kinase), host lysis and particle structure. EL-gp146 is the first chaperonin GroEL sequence identified in a viral genome. Besides a putative transposase, EL harbours predicted mobile endonucleases related to H-N-H and LAGLIDADG homing endonucleases associated with group I intron and intein intervening sequences.     

Comparison of DNA sizes in a group of giant Pseudomonas aeruginosa phages by the PFGE method

Genome sizes of Pseudomonas aeruginosa phages phiKZ and EL earlier determined by sequence analysis were shown to correspond to sizes of their DNAs assessed by pulse-electrophoresis (PFGE). Putative "redundant" genes in phiKZ phage genome are supposed to control functions promoting vigorous growth of the phage belonging to this species, compared to phages of EL species.     

Comparison of new giant bacteriophages OBP and Lu11 of soil pseudomonads with bacteriophages of phiKZ-supergroup of Pseudomonas aeruginosa

Study of two recently isolated giant bacteriophages Lu11 and OBP that are active on Pseudomonas putida var. Manila and Pseudomonas fluorescens, respectively, demonstrated their similarity in morphology, genome size, and size of phage particles, with giant bacteriophages of Pseudomonas aeruginosa assigned to the supergroup of phiKZ-like phages of the family Myoviridae designated in this manner according to the best studied phage phiKZ that belongs to the species of this group widely distributed in nature. Comparison of major polypeptide sizes of mature particles suggests the similarity of certain proteins in the phages examined. In OBP particles visualized with an electron microscope, an "inner body" was detected, which points to the specific DNA package intrinsic to phages of phiKZ group. In the meantime, phages Lul11 and OBP do not exhibit resemblance among themselves or with any of earlier described phiKZ-like phages in respect to other traits; particularly, they have no detectable DNA homology. Note that phage Lu11 of P. putida var. Manila exhibits very slight homology with phage Lin68 of the family of P. aeruginosa phiKZ-like phages detected only in blot hybridization. This suggests the possible involvement of these phages in interspecies recombination ("gene shuffling") between phages of various bacterial species. Results of partial sequencing of phage genomes confirmed the phylogenetic relatedness of phage OBP to phages of the phiKZ-supergroup, whereas phage Lu11 most probably belongs to a novel species that is not a member of supergroup phiKZ composition. The results of the study are discussed in terms of the evolution of these phages.     

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.     

Peptidoglycan lytic activity of the Pseudomonas aeruginosa phage phiKZ gp144 lytic transglycosylase

The gp144 endolysin gene from the Pseudomonas aeruginosa phage phiKZ was cloned and studies of gp144 expression into Escherichia coli showed host cell lysis. The gp144 protein was purified directly from the culture supernatant and from the bacterial cell pellet and showed in vitro antibacterial lytic activity against P. aeruginosa bacteria and degraded purified peptidoglycan of Gram-negative bacteria. MS analysis identified the gp144 peptidoglycan cleavage site and confirmed a lytic transglycosylase enzyme. Studies of gp144 expression in the presence of sodium azide (NaN(3)), an inhibitor of the protein export machinery, and into an E. coli MM52 secA(ts) mutant at permissive and restrictive temperatures showed that gp144 was secreted independently of the Sec system. The solution conformation of purified gp144 analyzed by circular dichroism spectroscopy was 61% in alpha-helical content, and showed a 72% decrease when interacting with dimyristoylphosphatidylglycerol (DMPG), one of the major components of bacterial membranes and less than 10% with dimyristoylphosphatidylcholine (DMPC) found in eukaryotic membranes. Membrane vesicles of DMPG anionic lipids containing calcein indicated that gp144 caused a rapid release of fluorescent calcein when interacting with synthetic membranes. These results indicated that gp144 from phiKZ is a lytic transglycosylase capable of interacting with and disorganizing bacterial membranes and has potential as an antipseudomonal in phage therapy.     

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 α-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. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 3, pp. 379–385.     

Isolation and characterization of novel giant Stenotrophomonas maltophilia phage phiSMA5

Stenotrophomonas maltophilia is one of the most prevalent opportunistic bacteria causing nosocomial infections. It has become problematic because most of the isolates are resistant to multiple antibiotics, and therefore, development of phage therapy has attracted strong attention. In this study, eight S. maltophilia phages were isolated from clinical samples including patient specimens, catheter-related devices, and wastewater. These phages can be divided into four distinct groups based on host range and digestibility of the phage DNAs with different restriction endonucleases. One of them, designated phiSMA5, was further characterized. Electron microscopy showed it resembled Myoviridae, with an isometric head (90 nm in diameter), a tail (90 nm long), a baseplate (25 nm wide), and short tail fibers. The phiSMA5 double-stranded DNA, refractory to digestion by most restriction enzymes, was tested and estimated to be 250 kb by pulsed-field gel electrophoresis. This genome size is second to that of the largest phage, phiKZ of Pseudomonas aeruginosa. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 25 virion proteins were visualized. N-terminal sequencing of four of them suggested that each of them might have had its N terminus cleaved off. Among the 87 S. maltophilia strains collected in this study, only 61 were susceptible to phiSMA5, indicating that more phages are needed toward a phage therapy strategy. Since literature search yielded no information about S. maltophilia phages, phiSMA5 appears to be the first reported.     

Electron-microscopic study of the serological affinity between the antigenic components of phages T4 and DDVI.

In an investigation of the antigenic fine structure of phages T4 and DDVI with the use of the neutralization reaction and electron-microscopic observation of the phage-antibody complexes, it has been possible to establish that the head of phage T4 consists of proteins which have antigenic determinants of two types: The first type is identical to the antigens of the head of phage DDVI, and the second type is apparently absent in phage DDVI. The phage DDVI head contains mostly determinants which are common to the phage T4 head, since it was not possible to detect antigenically specific components in the phage DDVI head. The tail sheaths of phage T4 and DDVI appear to be identical in the antigenic respect. A difference has been observed in the fibers and the base plates of the phages investigated. The presence of the following three types of antigens has been established: 1) common to phages T2, T4, and DDVI, 2) common to phages T4 and DDVI, and 3) specific for each phage investigated.     

Muralytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages phiKZ and EL

Pseudomonas aeruginosa bacteriophage endolysins KZ144 (phage phiKZ) and EL188 (phage EL) are highly lytic peptidoglycan hydrolases (210 000 and 390 000 units mg(-1)), active on a broad range of outer membrane-permeabilized Gram-negative species. Site-directed mutagenesis indicates E115 (KZ144) and E155 (EL188) as their respective essential catalytic residues. Remarkably, both endolysins have a modular structure consisting of an N-terminal substrate-binding domain and a predicted C-terminal catalytic module, a property previously only demonstrated in endolysins originating from phages infecting Gram-positives and only in an inverse arrangement. Both binding domains contain conserved repeat sequences, consistent with those of some peptidoglycan hydrolases of Gram-positive bacteria. Fusions of these domains with green fluorescent protein immediately label all outer membrane-permeabilized Gram-negative bacteria tested, isolated P. aeruginosa peptidoglycan and N-acetylated Bacillus subtilis peptidoglycan, demonstrating the broad range of peptidoglycan-binding capacity by these domains. Specifically, A1 chemotype peptidoglycan and fully N-acetylated glucosamine units are essential for binding. Both KZ144 and EL188 appear to be a natural chimeric enzyme, originating from a recombination of a cell wall-binding domain encoded by a Bacillus or Clostridium species and a catalytic domain of an unknown ancestor.     

The Crystal Structure of Bacteriophage HK97 gp6: Defining a Large Family of Head-Tail Connector Proteins

The final step in the morphogenesis of long-tailed double-stranded DNA bacteriophages is the joining of the DNA-filled head to the tail. The connector is a specialized structure of the head that serves as the interface for tail attachment and the point of egress for DNA from the head during infection. Here, we report the determination of a 2.1 Å crystal structure of gp6 of bacteriophage HK97. Through structural comparisons, functional studies, and bioinformatic analysis, gp6 has been determined to be a component of the connector of phage HK97 that is evolutionarily related to gp15, a well-characterized connector component of bacteriophage SPP1. Whereas the structure of gp15 was solved in a monomeric form, gp6 crystallized as an oligomeric ring with the dimensions expected for a connector protein. Although this ring is composed of 13 subunits, which does not match the symmetry of the connector within the phage, sequence conservation and modeling of this structure into the cryo-electron microscopy density of the SPP1 connector indicate that this oligomeric structure represents the arrangement of gp6 subunits within the mature phage particle. Through sequence searches and genomic position analysis, we determined that gp6 is a member of a large family of connector proteins that are present in long-tailed phages. We have also identified gp7 of HK97 as a homologue of gp16 of phage SPP1, which is the second component of the connector of this phage. These proteins are members of another large protein family involved in connector assembly.     

Bacteriophage SPO1 structure and morphogenesis. III. SPO1 proteins and synthesis.

The virion proteins of SPO1 have been determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis methods on purified phage components and on phage lysates. The phage head contains 16 proteins, and the connector or neck structure has an additional 3 proteins not found in the head. The proximal part of the tail, composed of sheath, tube and connecting components, contains six proteins. The distal baseplate is the most complex structure, with 28 proteins identifiable on sodium dodecyl sulfate gels. The maximum number of proteins found in phage subassemblies is 53, which would account for nearly half the coding capacity of the SPO1 genome.     

一株苏云金杆菌噬菌体的形态结构及其蛋白质性质

从武汉微生物药厂苏云金杆菌发酵裂解液中分离到1株具有独特形态结构的苏云金杆菌噬菌体GP-1。电镜观察发现,这株噬菌体的头部呈长六棱柱状,具一短直尾和一“衣领”状结构,并首次发现了“衣领”状结构是由8～10个颗粒亚单位组成。该株噬菌体所具有的这8～10个颗粒亚单位对于噬菌体牢固地吸附于宿主表面应具有很强的促进作用,对于进一步研究噬菌体与宿主之间的关系提供一个结构上的证据。该株噬菌体的蛋白经SDS-聚丙烯酰胺凝胶电泳法测定,呈现一条主带,分子量为58892 D,一条次主带和七条次带,表明该株噬菌体的蛋白是由9种蛋白质构成。     

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.     

Identification of messenger RNA for human type II collagen

The fluorescence polarization of acridine orange-stained, oriented lambda phages was measured. The parameters of DNA packing within the phage head cos2 theta and cos4 theta were calculated (theta, angle between the direction of a small segment of DNA and the phage axis). It is shown that simple models of lambda phage DNA tertiary structure are not consistent with calculated values. A new model is proposed.     

A new defective phage containing a randomly selected 8 kilobase-pairs fragment of host chromosomal DNA inducible in a strain of Bacillus natto

A new defective phage, designated PBND8, was induced in Bacillus natto strain IAM1207 with bleomycin and mitomycin C. PBND8 particles contained a randomly selected 8 kilobase-pairs (kbp) fragment of the host chromosomal DNA. Electron microscopy showed that PBND8 has a small head with a complex tail structure like PBSX, a defective phage of Bacillus subtilis 168. The PBND8 head, however, is clearly smaller than that of PBSX which contains 13-kbp fragments of the host chromosomal DNA. SDS-polyacrylamide gel electrophoretic analysis revealed that the structural proteins of PBND8 are distinct from those of PBSX and PBSY (PBSZ) of B. subtilis W23. PBND8 exhibited a bacteriocin-like killing activity to the other Bacillus cells.     

Bacteriophage lambda derivatives carrying two copies of the cohesive end site

A spontaneously arising tandem duplication derivative of bacteriophage lambda has been isolated, which carries two copies of the site where the cohesive ends are formed (designated cos). Its structure has been determined by electron microscopy of DNA heteroduplexes. These heteroduplexes reveal that the duplication is usually, but not always, carried on the left end of the chromosome. A second duplication phage having two copies of cos, constructed by Feiss &; Campbell (1974), has also been studied by electron microscopy and is found to have a similar property.Unlike most tandem duplication derivatives of phage λ, the mutant studied here is not stable during growth in the absence of generalized recombination, but segregates both the triplication and the parental phage. This verifies that both cos sites are functional. The triplication does not arise as a result of end-to-end aggregation of phage chromosomes or site-specific recombination catalyzed by the chromosome maturation system at cos. It must therefore result from the cutting of mature ι chromosomes from concatemeric replication intermediates. The pattern of cutting observed shows that the λ cohesive ends are not created by a free nuclease acting on unpackaged DNA. The cutting appears to be influenced by the amount of DNA previously packaged into a phage head. A model for λ packaging is presented which explains the results.The duplication phage of Feiss &; Campbell (1974) carries a novel addition containing self-complementary sequences.