Myoviridae Phages Research Articles

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 1450Å diameter icosahedral head and a 2000Å-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 2.4Å resolution. Furthermore, crystal structures of two prophage tail sheath proteins were determined to 1.9 and 3.3Å 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.

Sequencing and Characterization of Pseudomonas aeruginosa phage JG004

BackgroundPhages could be an important alternative to antibiotics, especially for treatment of multiresistant bacteria as e.g. Pseudomonas aeruginosa. For an effective use of bacteriophages as antimicrobial agents, it is important to understand phage biology but also genes of the bacterial host essential for phage infection.ResultsWe isolated and characterized a lytic Pseudomonas aeruginosa phage, named JG004, and sequenced its genome. Phage JG004 is a lipopolysaccharide specific broad-host-range phage of the Myoviridae phage family. The genome of phage JG004 encodes twelve tRNAs and is highly related to the PAK-P1 phage genome. To investigate phage biology and phage-host interactions, we used transposon mutagenesis of the P. aeruginosa host and identified P. aeruginosa genes, which are essential for phage infection. Analysis of the respective P. aeruginosa mutants revealed several characteristics, such as host receptor and possible spermidine-dependance of phage JG004.ConclusionsWhole genome sequencing of phage JG004 in combination with identification of P. aeruginosa host genes essential for infection, allowed insights into JG004 biology, revealed possible resistance mechanisms of the host bacterium such as mutations in LPS and spermidine biosynthesis and can also be used to characterize unknown gene products in P. aeruginosa.

Isolation and characterization of a new bacteriophage MMP17 from Meiothermus

Thermophiles and their viruses are extraordinarily important because of their roles in processes of evolution, biogeochemistry and ecology. Species of the genus Meiothermus share close relationship with genus Thermus, but no Meiothermus bacteriophage has been reported yet. In this work, a new thermophilic bacteriophage named MMP17 (Meiothermus Myoviridae phage 17) was isolated from a Meiothermus strain and was further characterized. MMP17 was a typical myovirus with an icosahedral head (42 nm in diameter) and a tail (120 nm in length and 17 nm in width). Its DNA was about 33.5-39.5 kb in size. MMP17 was very stable at 55-60°C and pH 6-7. According to the one-step growth curve, the latent period and the burst period were 60 and 30 min, respectively. An average of 15 phage particles was released from each infected cell. Four major bands were detected in purified virion preparation by SDS-PAGE. As MMP17 was a thermophilic bacteriophage with lower production temperature, its characterization and the relationship between MMP17 and Thermus bacteriophages deserved for further study.

How viruses infect bacteria?

Viruses are minuscule infectious particles composed of a protein coat and a nucleic acid core. They exist in a huge variety of forms and infect practically all living creatures: animals, plants, insects and bacteria. Insight into the infection process could facilitate new therapeutic strategies for viral and bacterial diseases as well as food preservation. An article by Aksyuk et al (2009) published in this issue sheds light on the still mysterious infection process. It reports the first crystal structure of a significant portion of the bacteriophages T4 tail sheath protein. Together with fittings into existing cryo‐EM reconstructions, it suggests a mechanism of genome delivery into the host cell for the Myoviridae phages.

Characterization of Temperate Lactobacillus gasseri Phage LgaI and Its Impact as Prophage on Autolysis of Its Lysogenic Host Strains

We show by electron microscopy that Lactobacillus gasseri phage LgaI, a temperate phage residing in the chromosome of Lactobacillus gasseri ATCC33323, belongs to the family of Myoviridae phages. The LgaI DNA is packed by the "head-full" mechanism, as demonstrated by analysis of restriction patterns of heated (74 degrees C) or non-heated DNA. By isolating prophage-cured cells, we were able to demonstrate phage LgaI to be responsible for the strong autolytic phenotype observed for Lactobacillus gasseri ATCC33323. In addition, we show that a copy of the LgaI prophage resides in the chromosome of Lactobacillus gasseri NCK102. The LgaI prophage was not inducible in L. gasseri NCK102-adh by mitomycin C, however, it apparently contributed to the autolytic phenotype of this strain.

Bacteriophages reduce experimental contamination of hard surfaces, tomato, spinach, broccoli, and ground beef by Escherichia coli O157:H7.

A bacteriophage cocktail (designated ECP-100) containing three Myoviridae phages lytic for Escherichia coli O157:H7 was examined for its ability to reduce experimental contamination of hard surfaces (glass coverslips and gypsum boards), tomato, spinach, broccoli, and ground beef by three virulent strains of the bacterium. The hard surfaces and foods contaminated by a mixture of three E. coli O157:H7 strains were treated with ECP-100 (test samples) or sterile phosphate-buffered saline buffer (control samples), and the efficacy of phage treatment was evaluated by comparing the number of viable E. coli organisms recovered from the test and control samples. Treatments (5 min) with the ECP-100 preparation containing three different concentrations of phages (10(10), 10(9), and 10(8) PFU/ml) resulted in statistically significant reductions (P = <0.05) of 99.99%, 98%, and 94%, respectively, in the number of E. coli O157:H7 organisms recovered from the glass coverslips. Similar treatments resulted in reductions of 100%, 95%, and 85%, respectively, in the number of E. coli O157:H7 organisms recovered from the gypsum board surfaces; the reductions caused by the two most concentrated phage preparations were statistically significant. Treatment with the least concentrated preparation that elicited significantly less contamination of the hard surfaces (i.e., 10(9) PFU/ml) also significantly reduced the number of viable E. coli O157:H7 organisms on the four food samples. The observed reductions ranged from 94% (at 120 +/- 4 h posttreatment of tomato samples) to 100% (at 24 +/- 4 h posttreatment of spinach samples). The data suggest that naturally occurring bacteriophages may be useful for reducing contamination of various hard surfaces, fruits, vegetables, and ground beef by E. coli O157:H7.

Temperate bacteriophage ΦO18P from an Aeromonas media isolate: Characterization and complete genome sequence

A group of 74 Aeromonas isolates from surface water of three ponds in Bielefeld, Germany was screened for prophage induction after UV irradiation. The phage ΦO18P was induced from the Aeromonas media isolate O18. ΦO18P belongs to the Myoviridae phage family. The complete nucleotide sequence of the double stranded DNA genome of bacteriophage ΦO18P consists of 33,985 bp. The genome has 5′ protruding cohesive ends of 16 bases. On the ΦO18P genome 46 open reading frames ( orfs) were identified which are organized in the modules integration and regulation, replication, head, packaging, tail and lysis. Additionally the phage DNA includes a methylase gene. Comparison of the genome architecture with those of other bacteriophages revealed significant similarities to the P2 phage family and especially to the prophages of Aeromonas salmonicida and the Vibrio cholerae phage K139.

Three Bacillus anthracis bacteriophages from topsoil.

Three Bacillus anthracis bacteriophages from Iowa topsoil are characterized as to latent period, morphology, structural proteins, DNA size, and restriction endonuclease digestion. Electron micrographs indicate that the three isolates include two members of the Myoviridae and one smaller phage belonging to the Podoviridae. Phages Nk and DB resemble Myoviridae phage SP50 in morphology, but host range studies, protein, and DNA analysis indicate that both differ from SP50. Phage MH is very similar to phage phi 29, but differs in terms of host range, structural protein, and DNA characteristics.

Observations on cyanobacterial population collapse in eutrophic lake water.

In two laboratory-scale enclosures of water from the shallow, eutrophic Lake Loosdrecht (the Netherlands), the predominating filamentous cyanobacteria grew vigorously for 2 weeks, but then their populations simultaneously collapsed, whereas coccoid cyanobacteria and algae persisted. The collapse coincided with a short peak in the counts of virus-like particles. Transmission electron microscopy showed the morphotype Myoviridae phages, with isometric heads of about 90 nm outer diameter and > 100-nm long tails, that occurred free, attached to and emerging from cyanobacterial cells. Also observed were other virus-like particles of various morphology. Similar mass mortality of the filamentous cyanobacteria occurred in later experiments, but not in Lake Loosdrecht. As applies to lakes in general, this lake exhibits high abundance of virus-like particles. The share and dynamics of infectious cyanophages remain to be established, and it is as yet unknown which factors primarily stabilize the host-cyanophage relationship. Observations on shallow, eutrophic lakes elsewhere indicate that the cyanophage control may also fail in natural water bodies exhibiting predominance of filamentous cyanobacteria. Rapid supply of nutrients appeared to be a common history of mass mortality of cyanobacteria and algae in laboratory and outdoor enclosures as well as in highly eutrophic lakes.

Flagellin inhibits Myoviridae phage phiCTX infection of Pseudomonas aeruginosa strain GuA18: purification and mapping of binding site.

PhiCTX is a double-stranded DNA phage of the Myoviridae family that converts Pseudomonas aeruginosa into a cytotoxin producer. A 42-kDa phiCTX-inhibiting protein was purified from the outer membrane fraction of P. aeruginosa strain GuA18 by octyl-beta-glucoside extraction, DEAE-chromatography, and mono-Q HPLC. This protein had an isoelectric point of 5.4 and bound specifically [125I]-labeled phiCTX. The N-terminal amino acid sequence of six out of seven Lys-C fragments was highly similar (87%) to that of the entire of type-a flagellin of P. aeruginosa strain PAK. At a concentration of 14 nM, purified flagellin protein caused a 50% decrease in the phage titer after a 20-min incubation at 37 degrees C (PhI50). The presence of ethanol was necessary to reconstitute the inhibitory activity. In contrast, no ethanol treatment was necessary for the inhibitory activity of the sheared flagellin filaments from P. aeruginosa strain GuA18, which consists of the 42-kDa flagellin subunits and the synthesized 17-mer phage-binding-peptide NGSNSDSERTALNGEAK, representing flagellin residues 100-116 of P. aeruginosa strain PAK. The PhI50 was 10 nM and 200 nM, respectively. Antisera against the flagellin filament protein as well as against the 17-mer peptide neutralized phage infection. These results indicated that the amino acid region 100-116 of the flagellin subunit of strain GuA18 is involved in phiCTX binding. This region might play a role in phage attachment.

An unexpected temporal pattern of coliphage isolation in groundwaters sampled from wells at varied distances from reclaimed water recharge sites

Potable and monitoring wells located in close proximity to a large groundwater rechargeproject which utilizes a blend of surface water and reclaimed wastewater for recharge were tested for coliphage over a period of 6 months to assess the potential for virus migration. During the first 3 months FRNA phage were detected once at a shallow monitoring well. In late summer, an unexpected pulse of phage was detected in all wells, including control sites, suggesting an ecological phenomenon independent of recharge operations. Cubic and filamentous F-specific coliphage, consistent with the Leviviridae and Inoviridae groups, and a noncontractile tailed phage consistent with the Siphoviridae family were detected. There was no discernible relationship between recharge operations and the pattern of phage populations detected. Phage were detected using a host designated HS12, a variant of HS(pFamp)R (Debartolomeis, J. and Cabelli, V. J. (1991) Evaluation of an Escherichia coli host strain for enumeration of f male-specific bacteriophages. Appl. Environ. Microbiol. 57, 1301.). During the study it was found that HS12 contained a temperate Myoviridae phage; Myoviridae phage were subsequently excluded from the results. A total of 26 production wells, including 3 control sites, were sampled monthly and 6 monitoring wells were sampled every two weeks. Water reclamation plant effluents and river water upstream of effluent discharges were randomly sampled. The concentration and distribution of phage isolated was quite different in chlorinated effluent compared to river water. The majority of isolates from reclaimed water were filamentous DNA F-specific phage suggesting this group was more resistant to chlorine. Groundwater samples were analyzed using a novel large volume enrichment technique that proved very sensitive for detecting low concentrations of phage.

Evaluation of seven experimental phages for inclusion in the international phage set for the epidemiological typing of Listeria monocytogenes.

The purpose of our study was to evaluate the inclusion of seven experimental phages into the international phage set for subtyping Listeria monocytogenes. The seven additional phages included the broad-host-range virulent Myoviridae phage A511 (M. J. Loessner, Appl. Environ. Microbiol. 57:1912-1918, 1991), three temperate phages from the Danish subsystem for typing serotype 1/2 strains (12682, 6223, and 5775) (P. Gerner-Smidt, V.T. Rosdahl, and W. Frederiksen, APMIS 101:160-167, 1993), and three temperate phages isolated by this laboratory in France (9425, 1313, and 197). A panel of 395 Listeria monocytogenes isolates (including 180 that were non-phage typeable by the international set) were used in the study for a comparison of the lytic spectra of the various bacteriophages. These results showed that the inclusion of five of the experimental phages contributed greatly to the overall typeability and discriminatory power of the system, especially for strains within serogroup 1/2.