Green approach to phytopathogen: Characterization of lytic bacteriophages of Pseudomonas sp., an etiology of the bacterial blight of pomegranate.

Two morphologically different bacteriophages were isolated from the river and soil samples from various locations of Maharashtra, India against the phytopathogen Pseudomonas sp. that was recently reported to cause a new bacterial blight of pomegranate. Both the phages belonged to the order Caudovirales representing the families Siphoviridae (vB_Psp.S_PRɸL2) and Myoviridae (vB_Psp.M_SSɸL8). The multiplicity of infection ranged from 0.01 to 0.1, phage adsorption rate from 39% to 66%, latent period from 10 to 20 min with a burst size of 24-85 phage particles per infected host cell. The genome size of phages PRɸL2 and SSɸL8 was approximately 25.403 kb and 29.877 kb respectively. Restriction digestion pattern of phage genomic DNA was carried out for phage PRɸL2, Eco RI resulted in two bands and Hind III resulted in three bands while for phage SSɸL8, both Eco RI and Hind III each resulted in three bands. SDS-PAGE protein profile showed six bands for PRɸL2 and nine bands for SSɸL8 of different proteins. Phages showed high pH stability over a range of 4-9, temperature stability over a range of 4-50 °C and UV radiation showed a reduction up to 89.36% for PRɸL2 and 96% for SSɸL8. In short, the present research work discusses for the first time in-detailed characterization of phages of a phytopathogen Pseudomonas sp. from Maharashtra, India, which can be further efficiently used for biological control of the causative agent of a new bacterial blight disease of pomegranate.

The phage L capsid decoration protein has a novel OB-fold and an unusual capsid binding strategy.

The major coat proteins of dsDNA tailed phages (order Caudovirales) and herpesviruses form capsids by a mechanism that includes active packaging of the dsDNA genome into a precursor procapsid, followed by expansion and stabilization of the capsid. These viruses have evolved diverse strategies to fortify their capsids, such as non-covalent binding of auxiliary 'decoration' (Dec) proteins. The Dec protein from the P22-like phage L has a highly unusual binding strategy that distinguishes between nearly identical three-fold and quasi-three-fold sites of the icosahedral capsid. Cryo-electron microscopy and three-dimensional image reconstruction were employed to determine the structure of native phage L particles. NMR was used to determine the structure/dynamics of Dec in solution. The NMR structure and the cryo-EM density envelope were combined to build a model of the capsid-bound Dec trimer. Key regions that modulate the binding interface were verified by site-directed mutagenesis.

Three-dimensional structures of bacteriophage neck subunits are shared in Podoviridae, Siphoviridae and Myoviridae.

Tailed bacteriophages (Caudovirales) are divided into three families: Myoviridae with long contractile tails, Siphoviridae with long noncontractile tails and Podoviridae with short noncontractile tails. All have an icosahedral head with a portal vertex connected to a neck structure followed by a tail. Bacteriophage Mu belongs to the Myoviridae family. Herein, the gp29 portal subunit and neck subunits gp35, gp36 and gp37 of the Mu phage were purified to elucidate their arrangement in the neck. Both gp29 and gp36 were monomeric in solution, like the corresponding subunits of Podoviridae P22 and Siphoviridae SPP1. X-ray crystal structure of gp36 showed structural similarity to neck subunits of Siphoviridae and Podoviridae. The gp36 structure has a characteristic aromatic hydrophobic core, and the structure of the ring form of the Mu phage connector deduced from the Siphoviridae and Podoviridae connector showed that this feature builds the contact surface between gp36 subunits. Structural comparison with the neck of Siphoviridae and Podoviridae also implies direct interaction between gp36 and gp29. Because gp35 and gp36 form a stable complex, we predict that the head-portal ring (gp29), the connector complex (gp36 and gp35), the tail terminator (gp37) and the tube (gp40) are arranged in the Mu phage neck in this order.

Identification of bacteriophages for biocontrol of the kiwifruit canker phytopathogen Pseudomonas syringae pv. actinidiae.

Pseudomonas syringae pv. actinidiae is a reemerging pathogen which causes bacterial canker of kiwifruit (Actinidia sp.). Since 2008, a global outbreak of P. syringae pv. actinidiae has occurred, and in 2010 this pathogen was detected in New Zealand. The economic impact and the development of resistance in P. syringae pv. actinidiae and other pathovars against antibiotics and copper sprays have led to a search for alternative management strategies. We isolated 275 phages, 258 of which were active against P. syringae pv. actinidiae. Extensive host range testing on P. syringae pv. actinidiae, other pseudomonads, and bacteria isolated from kiwifruit orchards showed that most phages have a narrow host range. Twenty-four were analyzed by electron microscopy, pulse-field gel electrophoresis, and restriction digestion. Their suitability for biocontrol was tested by assessing stability and the absence of lysogeny and transduction. A detailed host range was performed, phage-resistant bacteria were isolated, and resistance to other phages was examined. The phages belonged to the Caudovirales and were analyzed based on morphology and genome size, which showed them to be representatives of Myoviridae, Podoviridae, and Siphoviridae. Twenty-one Myoviridae members have similar morphologies and genome sizes yet differ in restriction patterns, host range, and resistance, indicating a closely related group. Nine of these Myoviridae members were sequenced, and each was unique. The most closely related sequenced phages were a group infecting Pseudomonas aeruginosa and characterized by phages JG004 and PAK_P1. In summary, this study reports the isolation and characterization of P. syringae pv. actinidiae phages and provides a framework for the intelligent formulation of phage biocontrol agents against kiwifruit bacterial canker.

Isolation and characterization of numerous novel phages targeting diverse strains of the ubiquitous and opportunistic pathogen Achromobacter xylosoxidans.

The clinical relevance of nosocomially acquired infections caused by multi-resistant Achromobacter strains is rapidly increasing. Here, a diverse set of 61 Achromobacter xylosoxidans strains was characterized by MultiLocus Sequence Typing and Phenotype MicroArray technology. The strains were further analyzed in regard to their susceptibility to 35 antibiotics and to 34 different and newly isolated bacteriophages from the environment. A large proportion of strains were resistant against numerous antibiotics such as cephalosporines, aminoglycosides and quinolones, whereas piperacillin-tazobactam, ticarcillin, mezlocillin and imipenem were still inhibitory. We also present the first expanded study on bacteriophages of the genus Achromobacter that has been so far a blank slate with respect to phage research. The phages were isolated mainly from several waste water treatment plants in Germany. Morphological analysis of all of these phages by electron microscopy revealed a broad diversity with different members of the order Caudovirales, including the families Siphoviridae, Myoviridae, and Podoviridae. A broad spectrum of different host ranges could be determined for several phages that lysed up to 24 different and in part highly antibiotic resistant strains. Molecular characterisation by DNA restriction analysis revealed that all phages contain linear double-stranded DNA. Their restriction patterns display distinct differences underlining their broad diversity.

Isolation and characterization of ZZ1, a novel lytic phage that infects Acinetobacter baumannii clinical isolates.

BACKGROUND: Acinetobacter baumannii, a significant nosocomial pathogen, has evolved resistance to almost all conventional antimicrobial drugs. Bacteriophage therapy is a potential alternative treatment for multidrug-resistant bacterial infections. In this study, one lytic bacteriophage, ZZ1, which infects A. baumannii and has a broad host range, was selected for characterization. RESULTS: Phage ZZ1 and 3 of its natural hosts, A. baumanni clinical isolates AB09V, AB0902, and AB0901, are described in this study. The 3 strains have different sensitivities to ZZ1, but they have the same sensitivity to antibiotics. They are resistant to almost all of the antibiotics tested, except for polymyxin. Several aspects of the life cycle of ZZ1 were investigated using the sensitive strain AB09V under optimal growth conditions. ZZ1 is highly infectious with a short latent period (9 min) and a large burst size (200 PFU/cell). It exhibited the most powerful antibacterial activity at temperatures ranging from 35°C to 39°C. Moreover, when ZZ1 alone was incubated at different pHs and different temperatures, the phage was stable over a wide pH range (4 to 9) and at extreme temperatures (between 50°C and 60°C). ZZ1 possesses a 100-nm icosahedral head containing double-stranded DNA with a total length of 166,682 bp and a 120-nm long contractile tail. Morphologically, it could be classified as a member of the Myoviridae family and the Caudovirales order. Bioinformatic analysis of the phage whole genome sequence further suggested that ZZ1 was more likely to be a new member of the Myoviridae phages. Most of the predicted ORFs of the phage were similar to the predicted ORFs from other Acinetobacter phages. CONCLUSION: The phage ZZ1 has a relatively broad lytic spectrum, high pH stability, strong heat resistance, and efficient antibacterial potential at body temperature. These characteristics greatly increase the utility of this phage as an antibacterial agent; thus, it should be further investigated.

Diverse temperate bacteriophage carriage in Clostridium difficile 027 strains.

BACKGROUND: The hypervirulent Clostridium difficile ribotype 027 can be classified into subtypes, but it unknown if these differ in terms of severity of C. difficile infection (CDI). Genomic studies of C. difficile 027 strains have established that they are rich in mobile genetic elements including prophages. This study combined physiological studies, electron microscopy analysis and molecular biology to determine the potential role of temperate bacteriophages in disease and diversity of C. difficile 027. METHODOLOGY/PRINCIPAL FINDINGS: We induced prophages from 91 clinical C. difficile 027 isolates and used transmission electron microscopy and pulsed-field gel electrophoresis to characterise the bacteriophages present. We established a correlation between phage morphology and subtype. Morphologically distinct tailed bacteriophages belonging to Myoviridae and Siphoviridae were identified in 63 and three isolates, respectively. Dual phage carriage was observed in four isolates. In addition, there were inducible phage tail-like particles (PT-LPs) in all isolates. The capacity of two antibiotics mitomycin C and norfloxacin to induce prophages was compared and it was shown that they induced specific prophages from C. difficile isolates. A PCR assay targeting the capsid gene of the myoviruses was designed to examine molecular diversity of C. difficile myoviruses. Phylogenetic analysis of the capsid gene sequences from eight ribotypes showed that all sequences found in the ribotype 027 isolates were identical and distinct from other C. difficile ribotypes and other bacteria species. CONCLUSION/SIGNIFICANCE: A diverse set of temperate bacteriophages are associated with C. difficile 027. The observed correlation between phage carriage and the subtypes suggests that temperate bacteriophages contribute to the diversity of C. difficile 027 and may play a role in severity of disease associated with this ribotype. The capsid gene can be used as a tool to identify C. difficile myoviruses present within bacterial genomes.

Diversity of tailed phages in Baltic Sea sediment: large number of siphoviruses with extremely long tails.

We present the first attempt at quantitative analysis of morphological diversity of tailed viruses obtained from marine sediments without ultracentrifugation or enrichment on specific host strains. Sandy mud samples were collected in the Gulf of Gdansk in the spring, autumn and winter. VLPs were analyzed by transmission electron microscopy. The distribution of three groups of tailed phages was similar in all seasons (Siphoviridae: 52% on average; Myoviridae: 42%; Podoviridae: 6%). 19% of siphoviruses had prolate heads. Interestingly, 11% of siphoviral particles had tails longer than 300 nm, and 6% longer than 600 nm.

Bias in bacteriophage morphological classification by transmission electron microscopy due to breakage or loss of tail structures.

Virtually every study that has used transmission electron microscopy (TEM) to estimate viral diversity has acknowledged that loss of phage tails during sample preparation may have biased the results. However, the magnitude of this potential bias has yet to be constrained. To characterize biases in virus morphological diversity due to tail loss, six phage strains representing the order Caudovirales were inoculated into sterile sediments and soils. Phage particles were then extracted using standard methods. Morphologies of extracted phage particles were compared to those of unmanipulated control samples to determine the extent of tail breakage incurred by extraction procedures. Podoviruses exhibited the smallest frequency of tail loss during extraction (1.2-14%), myoviruses were moderately susceptible to tail breakage (15-40%), and siphoviruses were highly susceptible (32-76%). Thus, TEM assessments of viral diversity in soils or sediments by distribution of tail morphologies may be biased toward podoviruses and virions lacking tails, while simultaneously underestimating the abundance of siphoviruses. However, since the majority of viral capsids observed under TEM were intact, estimates of viral diversity based on the distribution of capsid diameters may provide a more reliable basis for morphological comparisons within and across ecosystems.

The Staphylococci phages family: an overview.

Due to their crucial role in pathogenesis and virulence, phages of Staphylococcus aureus have been extensively studied. Most of them encode and disseminate potent staphylococcal virulence factors. In addition, their movements contribute to the extraordinary versatility and adaptability of this prominent pathogen by improving genome plasticity. In addition to S. aureus, phages from coagulase-negative Staphylococci (CoNS) are gaining increasing interest. Some of these species, such as S. epidermidis, cause nosocomial infections and are therefore problematic for public health. This review provides an overview of the staphylococcal phages family extended to CoNS phages. At the morphological level, all these phages characterized so far belong to the Caudovirales order and are mainly temperate Siphoviridae. At the molecular level, comparative genomics revealed an extensive mosaicism, with genes organized into functional modules that are frequently exchanged between phages. Evolutionary relationships within this family, as well as with other families, have been highlighted. All these aspects are of crucial importance for our understanding of evolution and emergence of pathogens among bacterial species such as Staphylococci.

Intestinal colonization by enteroaggregative Escherichia coli supports long-term bacteriophage replication in mice.

Bacteriophages have been known to be present in the gut for many years, but studies of relationships between these viruses and their hosts in the intestine are still in their infancy. We isolated three bacteriophages specific for an enteroaggregative O104:H4 Escherichia coli (EAEC) strain responsible for diarrhoeal diseases in humans. We studied the replication of these bacteriophages in vitro and in vivo in a mouse model of gut colonization. Each bacteriophage was able to replicate in vitro in both aerobic and anaerobic conditions. Each bacteriophage individually reduced biofilms formed on plastic pegs and a cocktail of the three bacteriophages was found to be more efficient. The cocktail was also able to infect bacterial aggregates formed on the surface of epithelial cells. In the mouse intestine, bacteriophages replicated for at least 3 weeks, provided the host was present, with no change in host levels in the faeces. This model of stable and continuous viral replication provides opportunities for studying the long-term coevolution of virulent bacteriophages with their hosts within a mammalian polymicrobial ecosystem.

[Detection of bacteriophages of siphoviridae family in Erwinia carotovora subsp. carotovora].

It was established that the polylysogenic phage system of culture Erwinia carotovora subsp. carotovora 91P includes: a) defective bacteriophages of Myoviridae family, which are displayed as macromolecular carotovoricins b) valuable highly unstable temperate phage, which can be attributed to the family Myoviridae, and which, perhaps, is an analogue of phage ZF40 [6], and c) resistant to osmotic shock temperate phage of family Siphoviridae. This phage, called TIRI, consists of isometric head 50 nm in diameter and a rigid tail structure 203 nm long. A characteristic feature of the phage tail is an evident transverse striation, which is also indicative for the tail-like particle of the defective temperate phage of the strain 48A-7/4b. In general, the phage system of E carotovora subsp. carotovora is similar to Pseudomonas aeruginosa with its R- and F-bacteriocins, and phages of the families Myoviridae and Siphoviridae.

Novel Bacteriophages in Enterococcus spp.

Most of the bacteriophages (phages) currently reported in Enterococcus spp. belong to tailed families of bacteriophages Podoviridae, Siphoviridae, and Myoviridae. There is a little information on non-tailed bacteriophages isolated from enterococci. Samples of sewage and piggery effluents were tested on pig and chicken isolates of Enterococcus faecalis, E. faecium and E. gallinarum for lytic phages. In addition, isolates were exposed to mitomycin C to induce lysogenic phages. Bacteriophages that were detected were visualized by electron microscopy. Ten bacteriophages were of isometric shape with long flexible or non-flexible tails, while one had a long head with a long flexible tail; all contained double-stranded DNA molecules. Seven Polyhedral, filamentous, and pleomorphic-shaped phages containing DNA or RNA were also observed. The pleomorphic phages were droplet- or lemon-shaped in morphology. This study is the first report on polyhedral phages in Enterococcus spp. of animal origin and also the first report of filamentous and pleomorphic phages in enterococci.

Diversity of temperate bacteriophages induced in bovine and ovine Mannheimia haemolytica isolates and identification of a new P2-like phage.

The diversity of temperate bacteriophages was examined in 32 Mannheimia haemolytica, six Mannheimia glucosida and four Pasteurella trehalosi isolates. Phage particles were induced and identified by electron microscopy in 24 (75%) M. haemolytica isolates, but in only one (17%) M. glucosida and one (25%) P. trehalosi isolate. The M. haemolytica phages were relatively diverse as seven Siphoviridae, 15 Myoviridae and two Podoviridae-like phages were identified; the Myoviridae-type phages also exhibited structural variation of their tails. The bacteriophages induced in M. glucosida and P. trehalosi were of the Myoviridae type. Restriction endonuclease (RE) analysis identified nine distinct RE types among the M. haemolytica bacteriophages, providing further evidence of their relative diversity. A limited number of phages caused plaques on indicator strains and the phages exhibited a narrow host range. A subgroup of 11 bovine serotype A1 and A6 isolates contained Myoviridae-type phages of the same RE type (type A), but these differed in their abilities to infect and form plaques on the same panel of indicator strains. A P2-like phage (phiPHL213.1), representative of the RE type A phages, was identified from the incomplete M. haemolytica genome sequence. The phiPHL213.1 genome contains previously unidentified genes and represents a new member of the P2 phage family.

Common ancestry of herpesviruses and tailed DNA bacteriophages.

Comparative analysis of capsid protein structures in the eukaryote-infecting herpesviruses (Herpesviridae) and the prokaryote-infecting tailed DNA bacteriophages (Caudovirales) revealed a characteristic fold that is restricted to these two virus lineages and is indicative of common ancestry. This fold not only serves as a major architectural element in capsid stability but also enables the conformational flexibility observed during viral assembly and maturation. On the basis of this and other emerging relationships, it seems increasingly likely that the very diverse collection of extant viruses may have arisen from a relatively small number of primordial progenitors.

Main features on tailed phage, host recognition and DNA uptake.

Phage nucleic acid transport is atypical among membrane transport and thus poses a fascinating problem: transport is unidirectional; it concerns a unique molecule the size of which may represent 50 times that of the bacterium. The rate of DNA transport can reach values as high as 3 to 4 thousands base pairs/sec. This raises many questions, which will be addressed in this review. Is there a single mechanism of transport for all types of phages? How does the phage genome overcome the hydrophobic barrier of the host envelope? Is DNA transported as a free molecule or in association with proteins? Is such transport dependent on phage and/or host cell components? What is the driving force for transport? Data will be presented for a few selected tailed phages, which are the most common type of phages and for which DNA transport has been most extensively studied. Part of the review is devoted to recent in vitro data which have allowed to partly decipher the mechanism of phage T5 DNA transport.

Bacteriophage observations and evolution.

Bacteriophages are classified into one order and 13 families. Over 5100 phages have been examined in the electron microscope since 1959. At least 4950 phages (96%) are tailed. They constitute the order Caudovirales and three families. Siphoviridae or phages with long, noncontractile tails predominate (61% of tailed phages). Polyhedral, filamentous, and pleomorphic phages comprise less than 4% of bacterial viruses. Bacteriophages occur in over 140 bacterial or archaeal genera. Their distribution reflects their origin and bacterial phylogeny. Bacteriophages are polyphyletic, arose repeatedly in different hosts, and constitute 11 lines of descent. Tailed phages appear as monophyletic and as the oldest known virus group.

Study of the potential relationship between the morphology of infectious somatic coliphages and their persistence in the environment.

The proportions of different morphological types of infectious somatic coliphages were determined in faecally polluted freshwaters. Myoviridae, followed by Siphoviridae, were the most frequently isolated morphological types in raw sewage, treated sewage and river water collected a few metres downstream from a sewage outfall. However, in river water collected further downstream from the pollution point, in river water after 'in situ' inactivation experiments and in chlorinated raw and treated sewage significant changes in the proportions of the different somatic coliphage morphological types occurred. In all cases, Siphoviridae, especially those with flexible and curled tails, became more abundant to the detriment of Myoviridae.

Bacteriophage diversity in the North Sea.

In recent years interest in bacteriophages in aquatic environments has increased. Electron microscopy studies have revealed high numbers of phage particles (10(4) to 10(7) particles per ml) in the marine environment. However, the ecological role of these bacteriophages is still unknown, and the role of the phages in the control of bacterioplankton by lysis and the potential for gene transfer are disputed. Even the basic questions of the genetic relationships of the phages and the diversity of phage-host systems in aquatic environments have not been answered. We investigated the diversity of 22 phage-host systems after 85 phages were collected at one station near a German island, Helgoland, located in the North Sea. The relationships among the phages were determined by electron microscopy, DNA-DNA hybridization, and host range studies. On the basis of morphology, 11 phages were assigned to the virus family Myoviridae, 7 phages were assigned to the family Siphoviridae, and 4 phages were assigned to the family Podoviridae. DNA-DNA hybridization confirmed that there was no DNA homology between phages belonging to different families. We found that the 22 marine bacteriophages belonged to 13 different species. The host bacteria were differentiated by morphological and physiological tests and by 16S ribosomal DNA sequencing. All of the bacteria were gram negative, facultatively anaerobic, motile, and coccoid. The 16S rRNA sequences of the bacteria exhibited high levels of similarity (98 to 99%) with the sequences of organisms belonging to the genus Pseudoalteromonas, which belongs to the gamma subdivision of the class Proteobacteria.

Tailed bacteriophages: the order caudovirales.

Tailed bacteriophages have a common origin and constitute an order with three families, named Caudovirales. Their structured tail is unique. Tailed phages share a series of high-level taxonomic properties and show many facultative features that are unique or rare in viruses, for example, tail appendages and unusual bases. They share with other viruses, especially herpesviruses, elements of morphogenesis and life-style that are attributed to convergent evolution. Tailed phages present three types of lysogeny, exemplified by phages lambda, Mu, and P1. Lysogeny appears as a secondary property acquired by horizontal gene transfer. Amino acid sequence alignments (notably of DNA polymerases, integrases, and peptidoglycan hydrolases) indicate frequent events of horizontal gene transfer in tailed phages. Common capsid and tail proteins have not been detected. Tailed phages possibly evolved from small protein shells with a few genes sufficient for some basal level of productive infection. This early stage can no longer be traced. At one point, this precursor phage became perfected. Some of its features were perfect enough to be transmitted until today. It is tempting to list major present-day properties of tailed phages in the past tense to construct a tentative history of these viruses: 1. Tailed phages originated in the early Precambrian, long before eukaryotes and their viruses. 2. The ur-tailed phage, already a quite evolved virus, had an icosahedral head of about 60 nm in diameter and a long non-contractile tail with sixfold symmetry. The capsid contained a single molecule of dsDNA of about 50 kb, and the tail was probably provided with a fixation apparatus. Head and tail were held together by a connector. a. The particle contained no lipids, was heavier than most viruses to come, and had a high DNA content proportional to its capsid size (about 50%). b. Most of its DNA coded for structural proteins. Morphopoietic genes clustered at one end of the genome, with head genes preceding tail genes. Lytic enzymes were probably coded for. A part of the phage genome was nonessential and possibly bacterial. Were tailed phages general transductants since the beginning? 3. The virus infected its host from the outside, injecting its DNA. Replication involved transcription in several waves and formation of DNA concatemers. Novel phages were released by burst of the infected cell after lysis of host membranes by a peptidoglycan hydrolase (and a holin?). a. Capsids were assembled from a starting point, the connector, and around a scaffold. They underwent an elaborate maturation process involving protein cleavage and capsid expansion. Heads and tails were assembled separately and joined later. b. The DNA was cut to size and entered preformed capsids by a headful mechanism. 4. Subsequently, tailed phages diversified by: a. Evolving contractile or short tails and elongated heads. b. Exchanging genes or gene fragments with other phages. c. Becoming temperate by acquiring an integrase-excisionase complex, plasmid parts, or transposons. d. Acquiring DNA and RNA polymerases and other replication enzymes. e. Exchanging lysin genes with their hosts. f. Losing the ability to form concatemers as a consequence of acquiring transposons (Mu) or proteinprimed DNA polymerases (phi 29). Present-day tailed phages appear as chimeras, but their monophyletic origin is still inscribed in their morphology, genome structure, and replication strategy. It may also be evident in the three-dimensional structure of capsid and tail proteins. It is unlikely to be found in amino acid sequences because constitutive proteins must be so old that relationships were obliterated and most or all replication-, lysogeny-, and lysis-related proteins appear to have been borrowed. However, the sum of tailed phage properties and behavior is so characteristic that tailed phages cannot be confused with other viruses.