Phage Host Range Research Articles

Genetic basis of infectivity evolution in a bacteriophage

Antagonistic coevolution between hosts and parasites is probably ubiquitous. However, very little is known of the genetic changes associated with parasite infectivity evolution during adaptation to a coevolving host. We followed the phenotypic and genetic changes in a lytic virus population (bacteriophage; phage Φ2) that coevolved with its bacterial host, Pseudomonas fluorescens SBW25. First, we show the rapid evolution of numerous unique phage infectivity phenotypes, and that both phage host range and bacterial resistance to individual phage increased over coevolutionary time. Second, each of the distinct phage phenotypes in our study had a unique genotype, and molecular evolution did not act uniformly across the phage genome during coevolution. In particular, we detected numerous substitutions on the tail fibre gene, which is involved in the first step of the host-parasite interaction: host adsorption. None of the observed mutations could be directly linked with infection against a particular host, suggesting that the phenotypic effects of infectivity mutations are probably epistatic. However, phage genotypes with the broadest host ranges had the largest number of nonsynonymous amino acid changes on genes implicated in infectivity evolution. An understanding of the molecular genetics of phage infectivity has helped to explain the complex phenotypic coevolutionary dynamics in this system.

Bacteriophage biocontrol has the potential to reduce enterococci on hospital fabrics, plastic and glass

Enterococci, particularly vancomycin resistant enterococci (VRE), are a cause of continuing concern in hospitals due to their intrinsic resistance to many antibiotics and the increasing prevalence of acquired resistance to vancomycin. Furthermore, enterococci are able to survive for prolonged periods on surfaces making disinfection more difficult. This study investigated bacteriophage biocontrol as a novel means of disinfecting hard and porous surfaces contaminated with enterococci. Four enterococcal bacteriophages, designated ΦSUT1, ΦSUT2, ΦSUT3 and ΦSUT4, were isolated from sewage and landfill leachate, characterised and used as biocontrol agents in studies investigating the efficacy of phage in the disinfection of vancomycin susceptible enterococci (VSE) and VRE on glass and fabrics commonly found in hospitals. Sterile glass slides and swatches of fabrics were inoculated with 105 cfu enterococci and 109 pfu/ml phage. Inoculated surfaces were incubated at ambient temperature for 2 h and bacterial populations were recovered by plating onto KF streptococcus agar. ΦSUT1 produced significant reductions in growth of Enterococcus faecalis on all surfaces tested, while ΦSUT4 produced significant reductions in growth of VRE (M168600) on all surfaces except for polyester. Phage cocktails containing ΦSUT1 and ΦSUT6 produced greater reductions in growth of VRE (M252807) on all surfaces compared to treatments using either phage alone. Although the results from this study varied considerably, the promising findings from ΦSUT1 studies indicated that phage biocontrol of enterococci should be further explored using broader host range phages.

In Vitro Management of Hospital Pseudomonas aeruginosa Biofilm Using Indigenous T7-Like Lytic Phage

Pseudomonas aeruginosa, a human pathogen capable of forming biofilm and contaminating medical settings, is responsible for 65% mortality in the hospitals all over the world. This study was undertaken to isolate lytic phages against biofilm forming Ps. aeruginosa hospital isolates and to use them for in vitro management of biofilms in the microtiter plate. Multidrug resistant strains of Ps. aeruginosa were isolated from the hospital environment in and around Pimpri-Chinchwad, Maharashtra by standard microbiological methods. Lytic phages against these strains were isolated from the Pavana river water by double agar layer plaque assay method. A wide host range phage bacterial virus Ps. aeruginosa phage (BVPaP-3) was selected. Electron microscopy revealed that BVPaP-3 phage is a T7-like phage and is a relative of phage species gh-1. A phage at MOI-0.001 could prevent biofilm formation by Ps. aeruginosa hospital strain-6(HS6) on the pegs within 24h. It could also disperse pre-formed biofilms of all hospital isolates (HS1-HS6) on the pegs within 24h. Dispersion of biofilm was studied by monitoring log percent reduction in cfu and log percent increase in pfu of respective bacterium and phage on the peg as well as in the well. Scanning electron microscopy confirmed that phage BVPaP-3 indeed causes biofilm reduction and bacterial cell killing. Laboratory studies prove that BVPaP-3 is a highly efficient phage in preventing and dispersing biofilms of Ps. aeruginosa. Phage BVPaP-3 can be used as biological disinfectant to control biofilm problem in medical devices.

Genetically Engineered Virulent Phage Banks in the Detection and Control of Emergent Pathogenic Bacteria

Natural outbreaks of multidrug-resistant microorganisms can cause widespread devastation, and several can be used or engineered as agents of bioterrorism. From a biosecurity standpoint, the capacity to detect and then efficiently control, within hours, the spread and the potential pathological effects of an emergent outbreak, for which there may be no effective antibiotics or vaccines, become key challenges that must be met. We turned to phage engineering as a potentially highly flexible and effective means to both detect and eradicate threats originating from emergent (uncharacterized) bacterial strains. To this end, we developed technologies allowing us to (1) concurrently modify multiple regions within the coding sequence of a gene while conserving intact the remainder of the gene, (2) reversibly interrupt the lytic cycle of an obligate virulent phage (T4) within its host, (3) carry out efficient insertion, by homologous recombination, of any number of engineered genes into the deactivated genomes of a T4 wild-type phage population, and (4) reactivate the lytic cycle, leading to the production of engineered infective virulent recombinant progeny. This allows the production of very large, genetically engineered lytic phage banks containing, in an E. coli host, a very wide spectrum of variants for any chosen phage-associated function, including phage host-range. Screening of such a bank should allow the rapid isolation of recombinant T4 particles capable of detecting (ie, diagnosing), infecting, and destroying hosts belonging to gram-negative bacterial species far removed from the original E. coli host.

Identification of Tail Genes in the Temperate Phage 16 - 3 of Sinorhizobium meliloti 41

Genes encoding the tail proteins of the temperate phage 16-3 of the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti 41 have been identified. First, a new host range gene, designated hII, was localized by using missense mutations. The corresponding protein was shown to be identical to the 85-kDa tail protein by determining its N-terminal sequence. Electron microscopic analysis showed that phage 16-3 possesses an icosahedral head and a long, noncontractile tail characteristic of the Siphoviridae. By using a lysogenic S. meliloti 41 strain, mutants with insertions in the putative tail region of the genome were constructed and virion morphology was examined after induction of the lytic cycle. Insertions in ORF017, ORF018a, ORF020, ORF021, the previously described h gene, and hII resulted in uninfectious head particles lacking tail structures, suggesting that the majority of the genes in this region are essential for tail formation. By using different bacterial mutants, it was also shown that not only the RkpM and RkpY proteins but also the RkpZ protein of the host takes part in the formation of the phage receptor. Results for the host range phage mutants and the receptor mutant bacteria suggest that the HII tail protein interacts with the capsular polysaccharide of the host and that the tail protein encoded by the original h gene recognizes a proteinaceous receptor.

Genome Organization and Characterization of the Virulent Lactococcal Phage 1358 and Its Similarities to Listeria Phages

Virulent phage 1358 is the reference member of a rare group of phages infecting Lactococcus lactis. Electron microscopy revealed a typical icosahedral capsid connected to one of the smallest noncontractile tails found in a lactococcal phage of the Siphoviridae family. Microbiological characterization identified a burst size of 72 virions released per infected host cell and a latent period of 90 min. The host range of phage 1358 was limited to 3 out of the 60 lactococcal strains tested. Moreover, this phage was insensitive to four Abi systems (AbiK, AbiQ, AbiT, and AbiV). The genome of phage 1358 consisted of a linear, double-stranded, 36,892-bp DNA molecule containing 43 open reading frames (ORFs). At least 14 ORFs coded for structural proteins, as identified by SDS-PAGE coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The genomic organization was similar to those of other siphophages. All genes were on the same coding strand and in the same orientation. This lactococcal phage was unique, however, in its 51.4% GC content, much higher than those of other phages infecting this low-GC Gram-positive host. A bias for GC-rich codons was also observed. Comparative analyses showed that several phage 1358 structural proteins shared similarity with two Listeria monocytogenes phages, P35 and P40. The possible origin and evolution of lactococcal phage 1358 is discussed.

Phage Therapy for Plant Disease Control

Bacteria cause a number of economically important plant diseases. Bacterial outbreaks are generally problematic to control due to lack of effective bactericides and to resistance development. Bacteriophages have recently been evaluated for controlling a number of phytobacteria and are now commercially available for some diseases. Major challenges of agricultural use of phages arise from the inherent diversity of target bacteria, high probability of resistance development, and weak phage persistence in the plant environment. Approaches for resistance management--by applying phage mixtures and host-range mutant phages and, for increasing residual activity, by employing protective formulations, avoiding sunlight, and utilizing propagating bacterial strains--resulted in better efficacy and reliability. Deployment of phage therapy as part of an integrated disease management strategy, which includes the use of genetic control, cultural control, biological control, and chemical control, also has been investigated and will likely increase in the future.

Isolation of lytic bacteriophage againstVibrio harveyi

The isolation of lytic bacteriophage of Vibrio harveyi with potential for phage therapy of bacterial pathogens of phyllosoma larvae from the tropical rock lobster Panulirus ornatus. Water samples from discharge channels and grow-out ponds of a prawn farm in northeastern Australia were enriched for 24 h in a broth containing four V. harveyi strains. The bacteriophage-enriched filtrates were spotted onto bacterial lawns demonstrating that the bacteriophage host range for the samples included strains of V. harveyi, Vibrio campbellii, Vibrio rotiferianus, Vibrio parahaemolyticus and Vibrio proteolyticus. Bacteriophage were isolated from eight enriched samples through triple plaque purification. The host range of purified phage included V. harveyi, V. campbellii, V. rotiferianus and V. parahaemolyticus. Transmission electron microscope examination revealed that six purified phage belonged to the family Siphoviridae, whilst two belonged to the family Myoviridae. The Myoviridae appeared to induce bacteriocin production in a limited number of host bacterial strains, suggesting that they were lysogenic rather than lytic. A purified Siphoviridae phage could delay the entry of a broth culture of V. harveyi strain 12 into exponential growth, but could not prevent the overall growth of the bacterial strain. Bacteriophage with lytic activity against V. harveyi were isolated from prawn farm samples. Purified phage of the family Siphoviridae had a clear lytic ability and no apparent transducing properties, indicating they are appropriate for phage therapy. Phage resistance is potentially a major constraint to the use of phage therapy in aquaculture as bacteria are not completely eliminated. Phage therapy is emerging as a potential antibacterial agent that can be used to control pathogenic bacteria in aquaculture systems. The development of phage therapy for aquaculture requires initial isolation and determination of the bacteriophage host range, with subsequent creation of suitable phage cocktails.

Genetically engineered phage harbouring the lethal catabolite gene activator protein gene with an inducer-independent promoter for biocontrol ofEscherichia coli

Complications of chemotherapy, such as appearance of multidrug resistance, have persuaded researchers to consider phage therapy as a new method to combat bacterial infections. In vitro experiments were performed to assess the therapeutic value of genetically modified phages for controlling gastrointestinal Escherichia coli O157:H7 cells in Luria-Bertani (LB) media and contaminated cow milk. We constructed a modified nonreplicating M13-derived phage expressing a lethal catabolite gene activator protein (CAP) that is a Glu181Gln mutant of CAP. The modified phagemid was propagated in the lethal CAP-resistant strain XA3DII. Time-kill assay experiments showed a considerable reduction in the number of surviving bacteria in both LB media and contaminated cow milk. Our further study using other test strains demonstrated that the host range of lethal phage is limited to E. coli strains that produce pili. This study provides a possible strategy for the exploitation of genetically engineered nonlytic phages as bactericidal agents by minimizing the risk of release of progeny phages and endotoxins into the environment. The phage was engineered to remain lethal to its bacterial target, but incapable of replicating therein. Furthermore, the addition of an inducer to express the lethal protein is not required.

Site-specific recombination of T2 phage using IP008 long tail fiber genes provides a targeted method for expanding host range while retaining lytic activity

The application of bacteriophages (phages) in therapy urgently requires the production of wide-host-range recombinant phages that possess strong lytic activity. The wide-host-range IP008 phage was classified by transmission electron microscopy analysis as an A2 morphotype member of the Myoviridae family of the order Caudovirales. IP008 showed a high homology (99.4% similarity in the amino acid alignment of the major capsid protein Gp 23) with KEP10, another wide-host-range phage. The long tail fiber genes (genes 37 and 38) from the genome of T2 were replaced with those of the IP008 phage by homologous recombination. The host range of the recombinant phages was identical to that of IP008. Furthermore, the recombinant phage bacterial lytic activity was restored. Future analyses of host-range mutants of the closely related phages T2 and IP008 could lead to a more precise localization of the genetic factors responsible for receptor specificity.

T4 phages against Escherichia coli diarrhea: Potential and problems

A combination of in vitro and in vivo experiments with comparative phage genomics was used for the rational design of a phage cocktail against E. coli diarrhea. Orally applied T4 coliphages representing three different subgroups (T4-, RB49- and JS98-like phages) had no negative impact on the murine gut microbiota. T4 phages were found with high titers in the cecum and colon and lower titers in the small intestine, but were not detected in the blood, liver or spleen. No adverse effects were observed after one-month exposure to phage nor were serum anti-T4 antibodies detected. T4 phages belonging to the same subgroup showed closely related genomes that differed by 12 (phage JS10 vs. JS98 reference) to 17 (phage JSE vs. RB49 reference) insertion/deletions mostly representing single small ORFs. Bioinformatic analysis did not reveal undesired genes in the T4 genomes. Sequence variability was seen over the tail fibre genes, but the variability did not correlate with phage host range. The investigated T4 phages were not only species- but also strain-specific, necessitating the use of phage cocktails consisting of 10 and 16 T4 phage isolates to cover half to two thirds of E. coli strains representing the five main pathotypes isolated from diarrhea patients.

Morphology, host range and phylogenetic diversity of Sphingomonas phages in the floodwater of a Japanese paddy field

Members of the Sphingomonas-related genera (Sphingomonas, Sphingobium, Novosphingobium and Sphingopyxis) are dominant in bacterial isolates from the floodwater of Japanese paddy fields. Fifty-eight Sphingomonas/Novosphingobium bacteriophages (phages) were isolated to elucidate their morphology, host range and phylogenetic diversity based on the capsid gene (g23) sequence. All of the phages were siphoviruses with isometric or elongated, icosahedral capsids and a long, non-contractile tail. The genomes were double-stranded DNA measuring either 40, 60, 100 or 160 kb. The host range of the phages was examined by infecting 16 bacterial isolates from the floodwater, belonging to Sphingomonas, Novosphingobium, Sphingopyxis and Porphyrobacter. The host range was widely different and varied between infection of only the host used for isolation and infection of hosts belonging to the three genera of Sphingomonas, Novosphingobium and Porphyrobacter. All phages had g23, indicating the ubiquity of the g23 gene among Myoviridae and Siphoviridae members. Every g23 sequence of the phages belonged to one of the six uncharacterized Paddy Groups proposed by Fujii et al. (2008). The g23 sequences were identical at the nucleotide level for several phages with isometric and elongated capsids with 60 and 160 kb genomes, and between some phages and the clones that were retrieved from distant paddy fields. This indicates the common occurrence of horizontal transfer of g23 in the paddy fields. The g23 sequence does not correlate with the host range of those phages. In addition, a larger degree of divergence of g23 from coliphage T4 in paddy fields compared to marine environments was estimated from the present study.

Optimal Foraging by Bacteriophages through Host Avoidance

Optimal foraging theory explains diet restriction as an adaptation to best utilize an array of foods differing in quality, the poorest items not worth the lost opportunity of finding better ones. Although optimal foraging has traditionally been applied to animal behavior, the model is easily applied to viral host range, which is genetically determined. The usual perspective for bacteriophages (bacterial viruses) is that expanding host range is always advantageous if fitness on former hosts is not compromised. However, foraging theory identifies conditions favoring avoidance of poor hosts even if larger host ranges have no intrinsic costs. Bacteriophage T7 rapidly evolved to discriminate among different Escherichia coli strains when one host strain was engineered to kill infecting phages but the other remained productive. After modifying bacteria to yield more subtle fitness effects on T7, we tested qualitative predictions of optimal foraging theory by competing broad and narrow host range phages against each other. Consistent with the foraging model, diet restriction was favored when good hosts were common or there was a large difference in host quality. Contrary to the model, the direction of selection was affected by the density of poor hosts because being able to discriminate was costly.

Detection of Escherichia coli with Fluorescent Labeled Phages That Have a Broad Host Range to E. coli in Sewage Water

Escherichia coli is used as an indicator microorganism in public health. The conventional way to detect E. coli requires several days to produce a result, because it requires incubation of cells. Therefore a rapid and sensitive detection method is needed. T4e-/GFP phage, characterized by suppression of lysozyme and fusion of GFP (green fluorescent protein) to its SOC (small outer capsid) protein, was constructed, and it was shown to be able to detect E. coli K12 sensitively within several hours. However, because the host range of T4 phage to E. coli present in sewage water and sea water is narrow, this phage cannot be used to detect E. coli in environmental water. Two phages named IP008 and IP052, which have a broad host range to E. coli present in sewage influent, were screened from sewage influent. Mixture of these two phages produced clear plaques on 50% of E. coli screened from sewage influent. To use these phages as a tool for detection of E. coli, gfp was inserted into gene e, which encodes a lytic enzyme, and thus lytic-activity-suppressed phages were constructed (IP008e-/GFP and IP052e-/GFP). However, the fluorescent intensity of E. coli cells infected with IP008e-/GFP and IP052e-/GFP was not enough for visualization of the cell. Therefore, in addition to the insertion of gfp into gene e, fusion of GFP to SOC of IP008e-/GFP and IP052e-/GFP was conducted to produce IP008e-/2xGFP and IP052e-/2xGFP. E. coli cells infected with IP008e-/2xGFP and IP052e-/2xGFP showed much stronger fluorescence intensity than E. coli cells infected by IP008e-/GFP and IP052e-/GFP. It is anticipated that, using these GFP-labeled phages, a broad range of E. coli present in sewage influent water can be detected rapidly.

Salmonella Host Range of Bacteriophages That Infect Multiple Genera

Conventionally, bacteriophages are considered viruses capable of amplification only in a narrow range of closely related bacteria. Presently, we selected bacteriophages with the ability to infect more than 1 bacterial genus. Initially, wild-type bacteriophages were selected for ability to form plaques in Salmonella enteritidis agar overlays. For determination of host specificity, a pool of 44 bacteriophages was combined with each bacterial isolate in tryptic soy broth. This mixture was incubated with fresh bacterial culture and media for 4 sequential passes, and the resulting bacteriophage titer was determined using S. enteritidis. One Klebsiella and 3 different Escherichia isolates successfully amplified some bacteriophage(s) from the S. enteritidis-selected bacteriophage pool (experiment 1). Amplification of bacteriophages in each species was confirmed by the formation of increased plaque forming units in a tryptic soy agar overlay with the enteric (alternative host) bacteria, Klebsiella or Escherichia (experiment 2). Two selected bacteriophages, confirmed to amplify in Escherichia or Klebsiella, were further evaluated for ability to amplify in 10 different Salmonella serovars by amplification in broth culture (experiment 3). One had the ability to amplify in 6 different Salmonella serovars, and the other had the ability to amplify in 2 different Salmonella serovars. These experiments suggest that bacteriophage host range is not always genera-restricted and that selection of subpopulations of bacteriophages capable of amplification in alternative genera may provide a tool for selection of broad host-range bacteriophages for the pathogen of interest. Selection of non-pathogenic host isolates to support replication of Salmonella bacteriophages may allow improved safety for bacteriophage application to poultry because this would reduce the necessity for 100% purification of the bacteriophages(s) from resistant host bacteria.

Large Variabilities in Host Strain Susceptibility and Phage Host Range Govern Interactions between Lytic Marine Phages and Their Flavobacterium Hosts

Phages are a main mortality factor for marine bacterioplankton and are thought to regulate bacterial community composition through host-specific infection and lysis. In the present study we demonstrate for a marine phage-host assemblage that interactions are complex and that specificity and efficiency of infection and lysis are highly variable among phages infectious to strains of the same bacterial species. Twenty-three Bacteroidetes strains and 46 phages from Swedish and Danish coastal waters were analyzed. Based on genotypic and phenotypic analyses, 21 of the isolates could be considered strains of Cellulophaga baltica (Flavobacteriaceae). Nevertheless, all bacterial strains showed unique phage susceptibility patterns and differed by up to 6 orders of magnitude in sensitivity to the same titer of phage. The isolated phages showed pronounced variations in genome size (8 to >242 kb) and host range (infecting 1 to 20 bacterial strains). Our data indicate that marine bacterioplankton are susceptible to multiple co-occurring phages and that sensitivity towards phage infection is strain specific and exists as a continuum between highly sensitive and resistant, implying an extremely complex web of phage-host interactions. Hence, effects of phages on bacterioplankton community composition and dynamics may go undetected in studies where strain identity is not resolvable, i.e., in studies based on the phylogenetic resolution provided by 16S rRNA gene or internal transcribed spacer sequences.

Biodiversity of Lactococcus lactis bacteriophages in Polish dairy environment.

We present here the results of an exploration of the bacteriophage content of dairy wheys collected from milk plants localized in various regions of Poland. Thirty-three whey samples from 17 regions were analyzed and found to contain phages active against L. lactis strains. High phage titer in all whey samples suggested phage-induced lysis to be the main cause of fermentation failures. In total, over 220 isolated phages were examined for their restriction patterns, genome sizes, genetic groups of DNA homology, and host ranges. Based on DNA digestions the identified phages were classified into 34 distinct DNA restriction groups. Phage genome sizes were estimated at 14-35 kb. Multiplex PCR analysis established that the studied phages belong to two out of the three main lactococcal phage types--c2 and 936, while P335-type phages were not detected. Yet, analyses of bacterial starter strains revealed that the majority of them are lysogenic and carry prophages of P335-type in their chromosome. Phage geographical distribution and host range are additionally discussed.

Exclusion of Glucosyl-Hydroxymethylcytosine DNA Containing Bacteriophages Is Overcome by the Injected Protein Inhibitor IPI*

The Escherichia coli isolate CT596 excludes infection by the Myoviridae T4 ip1− phage that lacks the encapsidated IPI* protein normally injected into the host with the phage DNA. Screening of a CT596 genomic library identified adjacent genes responsible for this exclusion, gmrS (942 bp) and gmrD (708 bp) that are encoded by a cryptic prophage DNA. The two genes are necessary and sufficient to confer upon a host the ability to exclude infection by T4 ip1− phage and other glucosyl-hydroxymethylcytosine (glc-HMC) Tevens lacking the ip1 gene, yet allow infection by phages with non-glucoslyated cytosine (C) DNA that lack the ip1 gene. A plasmid expressing the ip1 gene product, IPI*, allows growth of Tevens lacking ip1 on E. coli strains carrying the cloned gmrS/gmrD genes. Members of the Teven family carry a diverse and, in some cases, expanded set of ip1 locus genes. In vivo analysis suggests a family of gmr genes that specifically target sugar-HMC modified DNA have evolved to exclude Teven phages, and these exclusion genes have in turn been countered by a family of injected exclusion inhibitors that likely help determine the host range of different glc-HMC phages.

Optimality models of phage life history and parallels in disease evolution

Optimality models constitute one of the simplest approaches to understanding phenotypic evolution. Yet they have shortcomings that are not easily evaluated in most organisms. Most importantly, the genetic basis of phenotype evolution is almost never understood, and phenotypic selection experiments are rarely possible. Both limitations can be overcome with bacteriophages. However, phages have such elementary life histories that few phenotypes seem appropriate for optimality approaches. Here we develop optimality models of two phage life history traits, lysis time and host range. The lysis time models show that the optimum is less sensitive to differences in host density than suggested by earlier analytical work. Host range evolution is approached from the perspective of whether the virus should avoid particular hosts, and the results match optimal foraging theory: there is an optimal “diet” in which host types are either strictly included or excluded, depending on their infection qualities. Experimental tests of both models are feasible, and phages provide concrete illustrations of many ways that optimality models can guide understanding and explanation. Phage genetic systems already support the perspective that lysis time and host range can evolve readily and evolve without greatly affecting other traits, one of the main tenets of optimality theory. The models can be extended to more general properties of infection, such as the evolution of virulence and tissue tropism.

Bacteriophage OP1, lytic for Xanthomonas oryzae pv. oryzae, changes its host range by duplication and deletion of the small domain in the deduced tail fiber gene

The entire genome of bacteriophage OP1, lytic for Xanthomonas oryzae pv. oryzae causing bacterial leaf blight of rice, and the partial genomes of related phages were sequenced and analyzed. The OP1 genome comprises double-stranded, 4785-bp long DNA with 51.1% G + C content. Fifty-nine open reading frames (ORFs) were detected. ORF25 had similarity with the tail fiber gene of phages, whose product is related to host specificity. The ORF25 regions were amplified from four host-range mutants (OP1h, OP1hC, OP1h2, and OP1h2C) by polymerase chain reaction, and their deduced amino acid sequences were compared. Three mutants (OP1hC, OP1h2, OP1h2C) had duplications of a small domain in the N-terminal portion, although there were slight differences in the position of the duplicated sequences. One mutant OP1h had substituted amino acids in the duplication region. New mutants isolated in the laboratory (OP1hC and OP1h2C from OP1 and OP1h2) acquired the ability to lyse strain N5874 belonging to phagovar (lysotype) C. However, they rapidly lost this lytic ability when incubated with other phagovars. This loss was always accompanied by a loss of the characteristic repeats, suggesting that the host range of OP1-related phages changed mainly through duplication and deletion of a small domain in ORF25.