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DNA sequence of the control region of phage D108: the N-terminal amino acid sequences of repressor and transposase are similar both in phage D108 and in its relative, phage Mu.

We have determined the DNA sequence of the control region of phage D108 up to position 1419 at the left end of the phage genome. Open reading frames for the repressor gene, ner gene, and the 5' part of the A gene (which codes for transposase) are found in the sequence. The genetic organization of this region of phage D108 is quite similar to that of phage Mu in spite of considerable divergence, both in the nucleotide sequence and in the amino acid sequences of the regulatory proteins of the two phages. The N-terminal amino acid sequences of the transposases of the two phages also share only limited homology. On the other hand, a significant amino acid sequence homology was found within each phage between the N-terminal parts of the repressor and transposase. We propose that the N-terminal domains of the repressor and transposase of each phage interact functionally in the process of making the decision between the lytic and the lysogenic mode of growth.

Amino Acid Sequence

Hemolytic Vibrio cholerae O1 that is sensitive to Mukerjee's cholera phage IV and the phage produced by the hemolytic vibrio lysogenized with the infection of Mukerjee's cholera phage IV.

Strains of hemolytic Vibrio cholerae O1 (El Tor vibrio) which are sensitive to Mukerjee's cholera phage group IV were isolated from cholera patients in North-East Thailand in 1986. Plaques of the phage on these hemolytic V. cholerae O1 were usually translucent but almost transparent on some strains, just like the plaques on non-hemolytic V. cholerae O1 (classical vibrio). These hemolytic V. cholerae O1 were lysogenized with the infection of cholera phage IV, and the lysogenized strains produced phage different from cholera phage IV. These hemolytic strains were classified into Cured type in prophage typing of V. cholerae O1, El Tor, because they were also lysogenized with Kappa phage and were hemolytic. When Cured-type V. cholerae O1, El Tor previously isolated in various countries were examined for the sensitivity to cholera phage IV, some of the isolates were sensitive.

Bacteriophages

[Compatibility of transposable phages of Escherichia coli and Pseudomonas aeruginosa. I. Co-development of phages Mu and D3112 and integration of phage D3112 into RP4::Mu plasmid in Pseudomonas aeruginosa cells].

The possibility of using a model system (which included RP4::Mu plasmid and D3112 phage in Pseudomonas aeruginosa cells) for analysis of compatibility of transposable Escherichia coli phage Mu and P. aeruginosa phage D3112, as phages and transposons, was studied. No interaction was observed during the vegetative growth of phages. The majority of the hybrid RP4::Mu plasmids lost the Mu DNA after insertion of D3112 into RP4::Mu. The phenomenon was not a result of transposition immunity. We consider the loss of the Mu DNA as a consequence either of plasmid RP4::Mu instability in P. aeruginosa cells, because of the lack of functional Mu repressor, or of some D3112-encoded activity involved in its transposition. For the inambiguous conclusion on compatibility of two phages as transposons, it is necessary to modify the model system, eliminating the possibility of Mu phage replication--transposition.

Bacteriophages

[Genetic study of bacteriophage phi81. II. Gene localization in the right arm of the phage chromosome and a comparison of phage phi81 with phages lambda and phi80 in regard to gene functions].

Data on genetic investigation on lambdoid bacteriophage phi81 made possible to localize the cos site on the prophage genetic map. Four essential genes and the gene c1 are located in the right arm of the phage genetic map. Regulatory genes of phage phi81 are found to be uncapable of functional substitution of lambda phages regulatory genes N and Q. It is discovered that some late genes of phage phi80 can be substituted with respective phage phi81 genes. No substitution possibility was observed for a number of early genes of phage phi80.

Chromosome Mapping

The strategy of infection as a criterion for phylogenetic relationships of non-coli phages morphologically similar to phage T7.

Five phages which are morphologically similar to coliphage T7 but attack other host bacteria have been compared to T7 and to its relative, T3, by the following criteria: (a) cross-reactivity with antisera against T7 and T3, (b) DNA base sequence homologies, as determined by the C0t technique, (c) synthesis of two phage-coded enzymes: RNA polymerase and SAMase, (d) patterns of phage-directed protein synthesis, as determined by SDS-polyacrylamide gel electrophoresis of phage coat subunits. As judged by all these criteria, Pseudomonas phage PX3 is not related to T7; thus, morphological similarity was attributed to convergent evolution. The other phages, i.e. Serratia phage IV, Psuedomonas phage gh-1, Citrobacter phage ViIII and Klebsiella phage No. 11, were considered to be related to T7 on the basis of similarities in the patterns of phage-coded proteins and because, early after infection, these phages induced, as T7 does, an RNA polymerase which specifically transcribes the DNA of thehomologous phage. Phages IV and No. 11 also induced the early synthesis of SAMase (previously only known to occur upon T3 infection). With the exception of phage IV, however, DNA base sequence homologies with T7 or T3 seem to be poor or non-existent. The tested phages, again with the exception of phage IV, did not react with antiserum against T3 or T7. It is concluded that a particular pattern of phage-directed protein synthesis (as characterized by polyacrylamide gel electrophoresis and enzyme tests) may provide evidence for phylogenetic relationships between phages, even in cases where other criteria, such as genetic recombination, serological cross-reaction, and DNA base sequence homologies, fail to indicate relatedness.

Adenosylmethionine Decarboxylase

Biological and biophysical characteristics of phages isolated from Clostridium botulinum type C and D strains, and physicochemical properties of the phage DNAs.

Toxin-converting phages CE beta and d-16 phi and non-converting phages CE gamma and d-1', isolated from toxigenic strains C-468 and D-CB16 of Clostridium botulinum types C and D, respectively, were characterized biologically and biophysically. DNAs isolated from these four phages were studied physicochemically. Phages CE beta, d-16 phi, CE gamma and d-1' were adsorbed to their susceptible cells at the constant rates of 1.1 x 10(-8), 3.3 x 10(-8), 1.4 x 10(-8) and 1.4 x 10(-8) ml/min, and grew after latent periods of 35, 45, 35 and 35 min at the burst sizes of 38, 85, 35 and 35, respectively. Converting phages were more susceptible than non-converting phages to physical and chemical treatments such as temperature, pH, time, UV-irradiation and organic solvents. GC% of phage DNAs were determined by melting temperature, buoyant density and HPLC analysis to be 26, 26, 29 and 29% for CE beta, d-16 phi, CE gamma and d-1' phage DNAs, respectively. The restriction digestion profiles of phage DNAs with seven endonucleases were compared by agarose gel electrophoresis, revealing that the two converting phage DNAs were similar in regard to five enzyme digestion profiles, but different in the other two enzymatic profiles. Non-converting phage DNAs produced the same restriction digestion profiles with all seven endonucleases. The molecular sizes determined from the size of restriction enzyme digestion fragments were about 110 kb for converting phage CE beta and d-16 phi DNAs and 65 kb for non-converting phage CE gamma and d-1' DNAs. Dot blot hybridization experiments revealed that DNA homology between the converting phages CE beta and d-16 phi was 50-75%, while that between non-converting phages CE gamma and d-1' was about 100%. Converting phage and non-converting phage DNAs did not hybridize at all.

Adsorption

Exploring phage-host interactions in Burkholderia cepacia complex bacterium to reveal host factors and phage resistance genes using CRISPRi functional genomics and transcriptomics.

Complex interactions of bacteriophages with their bacterial hosts determine phage host range and infectivity. While phage defense systems and host factors have been identified in model bacteria, they remain challenging to predict in non-model bacteria. In this paper, we integrate functional genomics and transcriptomics to investigate phage-host interactions, revealing active phage resistance and host factor genes in Burkholderia cenocepacia K56-2. Burkholderia cepacia complex species are commonly found in soil and are opportunistic pathogens in immunocompromised patients. We studied infection of B. cenocepacia K56-2 with Bcep176, a temperate phage isolated from Burkholderia multivorans. A genome-wide dCas9 knockdown library targeting B. cenocepacia K56-2 was constructed, and a pooled infection experiment identified 63 novel genes or operons coding for candidate host factors or phage resistance genes. The activities of a subset of candidate host factor and resistance genes were validated via single-gene knockdowns. Transcriptomics of B. cenocepacia K56-2 during Bcep176 infection revealed that expression of genes coding for host factor and resistance candidates identified in this screen was significantly altered during infection by 4 h post-infection. Identifying which bacterial genes are involved in phage infection is important to understand the ecological niches of B. cenocepacia and its phages, and for designing phage therapies.IMPORTANCEBurkholderia cepacia complex bacteria are opportunistic pathogens inherently resistant to antibiotics, and phage therapy is a promising alternative treatment for chronically infected patients. Burkholderia bacteria are also ubiquitous in soil microbiomes. To develop improved phage therapies for pathogenic Burkholderia bacteria, or engineer phages for applications, such as microbiome editing, it's essential to know the bacterial host factors required by the phage to kill bacteria, as well as how the bacteria prevent phage infection. This work identified 65 genes involved in phage-host interactions in Burkholderia cenocepacia K56-2 and tracked their expression during infection. These findings establish a knowledge base to select and engineer phages infecting or transducing Burkholderia bacteria.

Bacteriophages

[New lambdoid phages of Escherichia coli. II. Comparison of several genetic characteristics with lambda phages].

Functions of some newly isolated lambdoid phages and phage lambda genes were compared by their ability to interact with unrelated phages and the product of the bacterial gene gro P. 19 of 23 lambdoid phages studied interfere with prophage P2, that points out the presence of functionally active genes, essential for spi+ phenotype in their genomes. The development of 4 lambdoid phages with spi- phenotype is independent on the prophage P2 presence. Most of lambdoid phages show the reduced growth ability on the C600 groP- bacterial lawn. This indicates that they have a function similar to the gene P of phage lambda. The development of phage phi M417 in bacterial mutants groP- is not disturbed, which indicates that the gene P of phage phi M417 is different from that of the phage lambda. Newly isolated phages, that are homoimmune to phage lambda, restrict the development of T4 rII phage. The rest lambdoid phages have no rex function. The growth efficiency of lampodid phages on E. coli C cells, carrying Eco R1 plasmid, varies from 10(-6) to 10(-8) that presumbaly indicates on different amounts of restionci strites in DNA of these phages.

Coliphages

Filamentous cheater phages drive bacterial and phage populations to lower fitness.

Many bacteria carry phage genome(s) in their chromosome, which intertwines the fitness of the bacterium and the phage. Most Pseudomonas aeruginosa strains carry filamentous phages called Pf that establish chronic infections and do not require host lysis to spread. However, spontaneous mutations in the Pf repressor gene (pf5r) can allow extreme phage production that slows bacterial growth and increases cell death, violating an apparent détente between bacterium and phage. We observed this paradoxical outcome in an evolution experiment with P. aeruginosa in media simulating nutrients from the cystic fibrosis airway. Bacteria containing pf5r mutant phage grow to a lower density but directly outcompete their ancestor and convert them into pf5r mutants via phage superinfection. Reduced fitness therefore spreads throughout the bacterial population, driven by weaponized Pf. Yet high intracellular phage replication facilitates another evolutionary conflict: "cheater miniphages" lacking capsid genes and the superinfection exclusion gene (pfsE) invade populations of full-length phages within cells. Although bacteria containing both full-length phages and miniphages are most immune to superinfection by limiting the Pf receptor, this hybrid vigor is extremely unstable, as a classic Tragedy of the Commons scenario ensues that causes complete prophage loss. The entire cycle - from phage hyperactivation to miniphage invasion to prophage loss - can occur within 24h, showcasing rapid coevolution between bacteria and their filamentous phages. This study demonstrates that P. aeruginosa, and potentially many other bacterial species that carry filamentous prophages, risk being exploited by these phages in a runaway process that reduces fitness of both host and virus.

Inoviridae

[New lambdoid Escherichia coli phages. I. Isolation, group immunity and recombination with lambda phage].

550 bacterial strains were isolated from sewage. 69 of them were lysogenic by phages active on Escherichia coli. The phages were divided into two groups on the basis of UV-inducibility, the ability to form plaques on Rep-E. coli mutants and particle morphology: lambdoid (23 phages) and related to P2 (46 phages). Hybrid phages isolated from the crosses of lambdoid phages with phage lambda harboured the region imm lambda and the gene of adsorption specificity from other parent. Ten groups of heteroimmune phages were found in the collection of new temperate phages are homoimmune with known phages: lambda, phi 80, phi 81, 434. Another 7 phages involved in 6 groups of immunity which were heteroimmune to known phages. Diversity of lambdoid phage genes determining the structure of repressor is discussed.

Coliphages

[Two loci in the genome of the transposable phage B39 of Pseudomonas aeruginosa affecting the process of integration. I. Study of the properties of phages with the Pde- phenotype and mapping of the rms site].

Mutants and recombinants of transposable Pseudomonas aeruginosa bacteriophage B39 with a specific phenotype Pde- (pleiotropic developmental effect) were studied. Pde- phages produce clear minute plaques on lawns of P. aeruginosa PAO1 and fail to grow in cells of PAO1 harbouring Rms 163 (Inc P5) plasmid. Pde+ character is under control of the two loci in phage genome which were designated pdeX and pdeY. In hybrid phages the pdeX and pdeY loci originating from different transposable phages (pdeX from B39 and pdeY from PH132) do not accomplish their function and, as a result, the hybrid phages have the Pde- phenotype. The frequency of integration (f.o.i.) of Pde- phages into bacterial chromosome is lower than f.o.i. for Pde+ phages, as well as the frequency of stable lysogenization of infected bacteria; lytic development of the Pde- phages is also limited. The great difference among the transposable phages in their reaction to the presence of Rms163 plasmid is caused by some differences in the specific rms site in the phage genome. The site is located inside the interval 1.1-3.9 kb of the physical genome map, being closely linked to cI gene of phage B39. The growth of Pde- phages in cells with Rms163 can be restored, due to additional mutations in phage genes affecting lysogenization.

Bacteriophages

Phage and defective phage of strains of Myxococcus.

1. Phage-like particles were found in the supernatants of cultures of strains of Myxococcus xanthus, M. virescens and M. fulvus. The largest number of such particles was associated with M. virescens V2. Most of the particles were similar in morphology to the virulent Myxococcus phage, MX-1. 2. Several new phages were isolated from soil and animal droppings. A new phage was isolated from cultures of M. virescens V2. All resembled phage MX-1 in morphology and were related to phage MX-1 serologically. One of these phage, om, was characterized by fractionation of its proteins by SDS-polyacrylamide gel electrophoresis and by analysis of restriction fragments of its DNA. The very close relatedness with MX-1 was confirmed by these techniques. Phage om, was found to exist in a state of pseudolysogeny with strains of M. virescens and M. fulvus. 3. Two types of bacteriocin-like activity were found associated with Myxococcus strains. In one case, the activity was extracted from chloroformkilled or from sonicated cells. In the second case it was associated with extracellular material. Strains of Salmonella and Cytophaga were found to be good indicators for this latter activity. These strains were found to be killed by phage MX-1. 4. The significance of these data for origin of the phages of myxococci are discussed and it is proposed that MX-1 and the newly isolated phages may be virulent mutants of a family of lysogenic phages.

Bacteriocins

Phage-typing of Salmonella weltevreden based on lysogeny. II. Epidemiological usefulness of the system and geographical distribution of its phage-types.

Nine hundred and forty-six strains of Salmonella weltevreden isolated in different states of India during 1958-1974 and 124 strains from Australia, Burma, Holland, Hong Kong, New Zealand, Papua New Guinea, the Philippines, Thailand, the United States and Vietnam during 1953-1971 were phage-typed according to the phage-typing scheme described in the first part of this paper (Sood and Basu, 1977). The epidemiological incidence and geographical distribution of phage-types of Salmonella weltevreden were studied. All the phage-types were present in India, the predominant phage-types being b, d and i. Phage-type g was isolated exclusively from India. All the 14 strains from Hawaii belonged to phage-type i. Phage-type h was the most predominant phage-type in Vietnam. The 15 strains isolated from Papua New Guinea in 1965, which were supposed to have originated from a single source, belonged to 3 phage-types. Except these cultures all the available epidemiologically related strains were of uniform phage-types - a finding which establishes the epidemiological validity of the scheme.

Animals

Morphology of Pseudomonas aeruginosa phages from the sputum of cystic fibrosis patients and from the phage typing set. An electron microscopy study.

Sixteen phage suspensions isolated from the sputum of sixteen cystic fibrosis patients and five phages from the present phage typing set were studied by electron microscopy. All sputum samples contained at least one type of bacteriophage (range: 1-4) which could be classified by the morphology and dimensions of the virion. All phages isolated from sputum as well as the four typing phages were tailed phages. The clinical phages belonged either to the Myoviridae, Siphoviridae or Podoviridae family. The four typing phages belonged to the Myoviridae family. The detection of the presence of tailed phages in sputum further supports the prevailing theory that phages play a role in the phenotypical change of Pseudomonas aeruginosa during the chronic lung infection of cystic fibrosis patients, since only tailed phages are known to be temporate and thus mediate transduction and conversion.

Bacteriophage Typing

Phage typing of Vibrio cholerae using a new collection of phages.

The authors formed a collection of typing phages in order to differentiate cholera vibrios of both biotypes. The collection consists of 7 phages proposed by Mukerjee for typing vibrios of the classical biotype and 3 El Tor phages isolated at the period of the 7th pandemy of cholera. In forming the typing collection the authors observed the following principles: 1) to crate a single set of typing phages and a single scheme of phage type differentiation of classical cholera and El Tor vibrios; 2) to use virulent phages; 3) to form a collection of serologically different phage types. The proposed collection allows to reveal a larger number of phage types as compared with phages proposed by Mukerjee phage typing a larger number of strains enhancing thus the epidemiological significance of the method of phage typing cholera vibrios.

Bacteriophage Typing

A new Vi-phage-type of Salmonella typhi and preparation of the typing phage.

A new Vi-phage-type of Salmonella typhi, type F(7), isolated in Poland in 1963, is described. The homologous typing phage was prepared from phage F(1). The new Vi-phage-type belongs to the F group and its "precursor" is type F(1). No temperate type-determining phage has so far been isolated from this phage-type. The homologous typing phage F(7) is a phenotypically modified host-range mutant of Vi-phage II; its phenotype is F(1) and the genotype is that found in phage 35, and is related to phage C(11).

Bacteriophage Typing

Effects of the phage P1 restriction system on coliphage phi W: degradation and complex formation of phage phi W DNA.

Growth of phages phi W and T7 was restricted in Escherichia coli lysogenic for phage P1. Only a fraction of the infected cells gave burst of phages. Cells permitting phage growth gave normal burst size. Host strains carrying P1 mutants with defective endonuclease gave no restriction of phages T7 and phi 3, the latter a host-range mutant of phi W. Degradation but not modification of parental phage DNA could be demonstrated. Although no DNA, RNA or protein was synthesized in phi W infected P1 lysogenic cells, the parental phage DNA was found in increasingly larger complexes during the course of infection. At early times after infection, parental phage DNA was found to sediment about twice as fast as mature phage DNA. At later times during the infection the parental phage DNA was recovered as a very rapidly sedimenting material. Such material was also found in alkaline sucrose gradient centrifugation after treatment of the cell extract with sodium dodecyl sulphate, pronase digestion and phenol extractions.

Coliphages