A broad-host-range vibriophage, KVP40, isolated from sea water.

A broad-host-range vibriophage, KVP40, was isolated from sea water by using Vibrio parahaemolyticus 1010 (EB101) as the indicator host. The host range of KVP40 extended over at least 8 Vibrio and 1 Photobacterium species. KVP40 was a large tailed phage containing double-stranded DNA and belonged to Ackermann's morphotype A2. KVP40 DNA was cleaved by 11 different type II restriction endonucleases including EcoRI and HindIII, but not by 17 other enzymes including BamHI, KpnI and SalI.     

Studies on synonymous codon and amino acid usage biases in the broad-host range bacteriophage KVP40

In this study, the relative synonymous codon and amino acid usage biases of the broad-host range phage, KVP40, were investigated in an attempt to understand the structure and function of its proteins/protein-coding genes, as well as the role of its tRNAs. Synonymous codons in KVP40 were determined to be ATrich at the third codon positions, and their variations are dictated principally by both mutational bias and translational selection. Further analysis revealed that the RSCU of KVP40 is distinct from that of its Vibrio hosts, V. cholerae and V. parahaemolyticus. Interestingly, the expression of the putative highly expressed genes of KVP40 appear to be preferentially influenced by the abundant host tRNA species, whereas the tRNAs expressed by KVP40 may be required for the efficient synthesis of all its proteins in a diverse array of hosts. The data generated in this study also revealed that KVP40 proteins are rich in low molecular weight amino acid residues, and that these variations are influenced primarily by hydropathy, mean molecular weight, aromaticity, and cysteine content.     

A 26-kDa outer membrane protein, OmpK, common to Vibrio species is the receptor for a broad-host-range vibriophage, KVP40

Abstract KVP40 is a broad-host-range vibriophage forming plaques on strains of at least eight Vibrio and one Photobacterium species. A spontaneous KVP40-resistant mutant, R4000, derived from Vibrio parahaemolyticus 1010 lacked a 26-kDa outer membrane protein designated OmpK. KVP40 was inactivated by outer membrane and OmpK prepared from 1010, but not by outer membrane from R4000. These results strongly suggest that OmpK is the receptor for KVP40. Immunoblotting analyses using an anti-OmpK rabbit serum revealed that OmpK or its homologs of molecular masses 25–29 kDa were distributed widely among Vibrio and Photobacterium strains including those naturally resistant to KVP40.     

Vibriophages Differentially Influence Biofilm Formation by Vibrio anguillarum Strains

Vibrio anguillarum is an important pathogen in marine aquaculture, responsible for vibriosis. Bacteriophages can potentially be used to control bacterial pathogens; however, successful application of phages requires a detailed understanding of phage-host interactions under both free-living and surface-associated growth conditions. In this study, we explored in vitro phage-host interactions in two different strains of V. anguillarum (BA35 and PF430-3) during growth in microcolonies, biofilms, and free-living cells. Two vibriophages, ΦH20 (Siphoviridae) and KVP40 (Myoviridae), had completely different effects on the biofilm development. Addition of phage ΦH20 to strain BA35 showed efficient control of biofilm formation and density of free-living cells. The interactions between BA35 and ΦH20 were thus characterized by a strong phage control of the phage-sensitive population and subsequent selection for phage-resistant mutants. Addition of phage KVP40 to strain PF430-3 resulted in increased biofilm development, especially during the early stage. Subsequent experiments in liquid cultures showed that addition of phage KVP40 stimulated the aggregation of host cells, which protected the cells against phage infection. By the formation of biofilms, strain PF430-3 created spatial refuges that protected the host from phage infection and allowed coexistence between phage-sensitive cells and lytic phage KVP40. Together, the results demonstrate highly variable phage protection mechanisms in two closely related V. anguillarum strains, thus emphasizing the challenges of using phages to control vibriosis in aquaculture and adding to the complex roles of phages as drivers of prokaryotic diversity and population dynamics.     

ORF334 in Vibrio phage KVP40 plays the role of gp27 in T4 phage to form a heterohexameric complex

KVP40 is a T4-related phage, composed of 386 open reading frames (ORFs), that has a broad host range. Here, we overexpressed, purified, and biophysically characterized two of the proteins encoded in the KVP40 genome, namely, gp5 and ORF334. Homology-based comparison between KVP40 and its better-characterized sister phage, T4, was used to estimate the two KVP40 proteins' functions. KVP40 gp5 shared significant homology with T4 gp5 in the N- and C-terminal domains. Unlike T4 gp5, KVP40 gp5 lacked the internal lysozyme domain. Like T4 gp5, KVP40 gp5 was found to form a homotrimer in solution. In stark contrast, KVP40 ORF334 shared no significant homology with any known proteins from T4-related phages. KVP40 ORF334 was found to form a heterohexamer with KVP40 gp5 in solution in a fashion nearly identical to the interaction between the T4 gp5 and gp27 proteins. Electron microscope image analysis of the KVP40 gp5-ORF334 complex indicated that it had dimensions very similar to those of the T4 gp5-gp27 structure. On the basis of our biophysical characterization, along with positional genome information, we propose that ORF334 is the ortholog of T4 gp27 and that it plays the role of a linker between gp5 and the phage baseplate.     

Cloning and sequence analysis of Vibrio parahaemolyticus ompK gene encoding a 26-kDa outer membrane protein, OmpK, that serves as receptor for a broad-host-range vibriophage, KVP40

Abstract The ompK gene of Vibrio parahaemolyticus 1010 (RIMD 2210001) encoding an outer membrane protein (OMP), OmpK, which serves as the receptor for a broad-host-range vibriophage, KVP40, was cloned and sequenced. The gene consisted of 789 nucleotides encoding 263 amino acids. Since the first 20 amino acids most likely constitute the signal peptide, mature OmpK would consist of 243 amino acids with a calculated molecular mass of 27458 Da. Sequence comparisons indicate that OmpK is unique among Vibrio OMPs so far sequenced, but may be distantly related to Tsx of enteric bacteria and is homologous to an Aeromonas hydrophila OMP, protein IV.     

Environmental vibrio phage–bacteria interaction networks reflect the genetic structure of host populations

Phages depend on their bacterial hosts to replicate. The habitat, density and genetic diversity of host populations are therefore key factors in phage ecology, but our ability to explore their biology depends on the isolation of a diverse and representative collection of phages from different sources. Here, we compared two populations of marine bacterial hosts and their phages collected during a time series sampling program in an oyster farm. The population of Vibrio crassostreae, a species associated specifically to oysters, was genetically structured into clades of near clonal strains, leading to the isolation of closely related phages forming large modules in phage–bacterial infection networks. For Vibrio chagasii, which blooms in the water column, a lower number of closely related hosts and a higher diversity of isolated phages resulted in small modules in the phage–bacterial infection network. Over time, phage load was correlated with V. chagasii abundance, indicating a role of host blooms in driving phage abundance. Genetic experiments further demonstrated that these phage blooms can generate epigenetic and genetic variability that can counteract host defence systems. These results highlight the importance of considering both the environmental dynamics and the genetic structure of the host when interpreting phage–bacteria networks.     

Characterization of a novel Vibrio parahaemolyticus phage, KVP241, and its relatives frequently isolated from seawater

A vibriophage, KVP241, and six of its relatives were isolated independently from seawater using Vibrio parahaemolyticus as the host. All of the phages had the same morphology (a hexagonal head and a tail with a contractile sheath) and the same host range (specific for some V. parahaemolyticus strains). DNA-DNA hybridization experiments elucidated that their genomes are highly homologous to each other. Analyses of amino acid sequences of putative major capsid proteins indicated that KVP241 may be weakly related to T4-type phages having a more elongated head.     

Isolation and partial characterization of a bacteriophage T5 mutant unable to induce thymidylate synthetase and its use in studying the effect of uracil incorporation into DNA on early gene expression.

A mutant of phage T5 which is unable to induce thymidylate synthetase was isolated. T5 thy mutants synthesized less DNA than did wild-type T5, and the burst size of progeny phage was correspondingly reduced two- to threefold in thy+ Escherichia coli. No DNA or progeny phage were made in E. coli thy hosts grown in the absence of exogenous thymine. When the T5 thy mutation was recombined with a T5 dut mutation (unable to induce dUTPase), replication resulted in progeny which contained significant amounts of uracil in their DNA, and these phage failed to produce plaques unless the plating host was deficient in uracil-DNA glycosylase. T5 phage containing various amounts of uracil in their DNA were prepared and used to determine the effect of uracil on the induction of the early enzyme dTMP kinase. The presence of uracil in the parental DNA increased the rate of induction of this enzyme by about 2.5-fold. The T5 thy gene was mapped and is located near the T5 frd gene on the B region of the T5 genome.     

The Vertical Distribution and Diversity of Marine Bacteriophage at a Station off Southern California

Sixty-two bacteriophages were isolated on eight indigenous bacteria from a Pacific Ocean station spanning 887-m vertical depth, on two occasions between 1999 and 2000. On the basis of 16S rRNA sequences, six hosts were tentatively identified to be in the genus Vibrio and the other two were closely related to Altermonas macleodii (W9a) and Pseudoalteromonas spp. (W13a). Restriction fragment length polymorphism (RFLP) analysis of phage genomes using AccI and HapI showed that 16 phages infecting host C4a (Vibrio) displayed 14 unique RFLP patterns. However, identical phages infecting host C4b, C6a, and C6b (all Vibrio) were obtained from both the surface layer and the hypoxic zone at 850 m. Most phage isolates from the second year had a different RFLP pattern but shared genetic similarity to the phages infecting the same host from the previous year based on a hybridization study using phage genome probes. Cluster analysis of RFLP patterns and hybridization results also indicated that phages infecting the same or genetically related hosts, in general, shared higher degrees of homology in spite of the diverse RFLP patterns. Pulsed field gel electrophoresis (PFGE) analysis of native viral genomes indicated a range in genome size from less than 40 to 200 kb, and the dominant band shifted up by about 5-10 kb in the deep samples compared to the shallow ones. Hybridization of phage genome probes with total viral community DNA from various depths suggests these isolates, or at least some of their genes, represent a detectable portion of the natural viral community and were distributed throughout the water column. Thus, the results of this study demonstrated that the genetic diversity of bacteriophage in the ocean is far greater than that of their bacterial hosts. However, host range may have contributed to the evolution of the diverse phage population in the marine environment.     

Conditional Temperature-sensitive Restriction of Pseudomonas Bacteriophage CB3

Restriction of Pseudomonas bacteriophage CB3 growth on some Pseudomonas aeruginosa hosts was studied. On restricting hosts, growth of this phage was severely inhibited below 32 C and hence was temperature-sensitive. Investigation of this phenomenon revealed that restricting hosts were not killed as a consequence of their infection under nonpermissive conditions. The ability of some hosts to restrict showed segregation in sexual crosses between restricting and nonrestricting hosts. However, the pattern of restriction among various hosts differed with the phage in question when other phages were compared with CB3. Temperature-shift experiments indicated that blockage of an early event in the phage lytic cycle occurred when restricting conditions were imposed on cells infected with CB3. This blockage could be eliminated by holding at permissive conditions until the cold-sensitive step was bypassed or by pulsing restricting cells for 5 min at 37 C.     

On the mechanism of production of tandem genetic duplications in phage lambda

We have asked whether the mechanism by which tandem genetic duplications arise in the chromosome of phage lambda is inter- or intramolecular. Two parental phages carrying genetic markers at opposite ends of the phage chromosome have been grown in mixed infection, and progeny phages carrying newly-arising tandem duplications have been analysed to determine whether they carry the markers in parental or recombinant configuration. Ordinary genetic recombination of the markers has been prevented by mutations in the phage and host. Phages carrying tandem duplications are isolated by use of CsCl density gradients and an Escherichia coli strain that does not plate deletion phages. Of the duplication mutants isolated under these conditions, 13% carry the input markers in recombinant configuration. This suggests that tandem duplications can be produced via an intermolecular route which joins sequences originally present on different DNA molecules.     

Isolation of a bacterial host selective for bacteriophage T4 containing cytosine in its DNA.

An Escherichia coli B strain, B834 galU56, has been isolated which supports growth of bacteriophage T4 with cytosine in its DNA while restricting growth of T4 with hydroxymethylcytosine. This host is partially deficient in uridine diphosphoglucose as determined by the ability of DNA isolated from T4 grown on it to accept glucose in an in vitro assay. In this mutant an intact rgl restriction system recognizes unglucosylated hydroxymethylcytosine residues in phage DNA, while the absence of a functional rB restriction function prevents degradation of unmodified DNA containing cytosine.     

应用噬菌体肽库筛选能封阻霍乱噬菌体VP1转染菌细胞的寡肽

噬菌体感染细菌首先要吸附于细菌表面受体 ,从目前报道的细菌与噬菌体相互作用的研究中发现 ,这些受体包括细菌细胞外膜上的蛋白、糖脂结构和鞭毛等。霍乱弧菌是霍乱的病原体 ,高守一等 (副霍乱资料汇编 ,1 984,2 37～ 2 4 5 .)从国内分离并选择出 5株噬菌体 (VP1～VP5 ) ,根据霍乱弧菌菌株对噬菌体的敏感性不同 ,将埃尔托型霍乱弧菌分为 32个噬菌体型。结合生物学分型方法 ,可区分埃尔托型霍乱弧菌的两类不同菌株 (流行株和非流行株 )和不同菌型。对各种来源的菌株进行分型 ,可作为一种追溯传染来源、传播途径和分析流行形式的流行病学研…     

MOLECULAR GENETICS OF ADAPTATION IN AN EXPERIMENTAL MODEL OF COOPERATION

The evolution of cooperation was studied in an empirical system utilizing a parasitic bacteriophage (f1) and a bacterial host. Infected cells were propagated by serial passage so that a phage could increase its representation among infected hosts only by enhancing the rate of growth of its host. Loss of infectivity was therefore without selective penalty, and phage benevolence could potentially evolve through a variety of genetic changes. The infected hosts evolved to grow faster over the course of the study, but the genetic bases of this phenotypic change were more difficult to anticipate. Two fundamentally different types of genetic changes in the phage were revealed. One involved the loss of some phage genes, resulting in a noninfectious plasmid that continued to replicate via the parental phage replicon. The second change involved integration of the phage genome into host DNA by a process that, at low frequency, could be reversed to produce infectious phage particles. Integration is a previously unknown property of wild-type f1, and in the system studied, may have resulted from the use of a phage bearing an insert containing nonfunctional DNA. The evolution of this novel function apparently depended only on the presence of a small region in the phage genome that provided some homology to the host DNA, with the host providing all necessary functions. Although f1 is one of the simplest phages known, these observations suggest that host-parasite interactions of the filamentous phages are more complicated than previously thought. More generally, the f1 system offers a useful model for many problems concerning the genetic basis of adaptation.     

The isolation of a staphylococcal phage variant susceptible to an unusual host control

A series of phage P₁ variants was isolated from plaques developing on S. aureus WF 145. One in particular, phage 14, was studied in detail because its host range appeared to be dependent on the previous host of production; i.e., it was subject to a host control. When this phage was produced on host K₁ its lysate assayed equally well on both 145 and K₁ cells. When produced on host 145, however, it assayed manyfold higher on 145 than on host K₁. All its particles adsorbed on K₁ cells, but only a small percentage were able to produce plaques. No differences could be found in adsorption rates, latent periods, or burst sizes of the phage on the two hosts. No extracellular inactivating substances could be detected which could account for such changes, nor could the results be explained readily on a mutational basis, since distinct phage strains could not be isolated. The change in virus properties was found to occur in the first burst of singly infected host 145 cells, regardless of the previous host or its prior lytic abilities. Heat inactivation destroyed activity for K₁ cells more rapidly than for 145 cells. This was found to be a property of both the stock phage P₁ and phage 14. Phage 14 could be heated until there remained particles which could multiply only on strain 145. When the plaques of such survivors were examined they were found to contain phage which could multiply on both hosts in a ratio characteristic of the original unheated preparation. The data suggest that the observed changes were caused by a host control over the formation of a phage material(s) necessary for successful infection of host K₁. Such a substance theoretically could be related to the labile material destroyed by heat and required for plaque formation on host K₁.     

High frequency of mutations that expand the host range of an RNA virus

The ability of a virus population to colonize a novel host is predicted to depend on the equilibrium frequency of potential colonists (i.e., genotypes capable of infecting the novel host) in the source population. In this study, we investigated the determinants of the equilibrium frequency of potential colonists in the RNA bacteriophage 6. We isolated 40 spontaneous mutants capable of infecting a novel Pseudomonas syringae host and sequenced their host attachment genes to identify the responsible mutations. We observed 16 different mutations in the host attachment gene and used a new statistical approach to estimate that 39 additional mutations were missed by our screen. Phenotypic and fitness assays confirmed that the proximate mechanism underlying host range expansion was an increase in the ability to attach to the novel host and that acquisition of this ability most often imposed a cost for growth rate on two standard hosts. Considered in a population genetic framework, our data suggest that host range mutations should exist in phage populations at an equilibrium frequency (3 x 10(-4)) that exceeds the phage mutation rate by more than two orders of magnitude. Thus, colonization of novel hosts is unlikely to be limited by an inability to produce appropriate mutations.