The origin of phage virology.

The history of bacteriophage (phage) had its start in 1915, when Twort isolated an unusual filterable and infectious agent from excrete of patients struck by diarrhoea; this discovery was followed by an analogous, and probably independent, finding of d'Hérelle in 1917. For several years phage research made scant progress but great attention was paid to the question of phage nature, which saw the contrast between d'Hérelle and Bordet's views (living against chemical nature, respectively). This situation changed with the independent discovery of lysogeny, in 1925, thanks to Bordet and Bail: this phenomenon was considered of genetical origin, a view that Wollman interpreted by assimilating the properties of phage to those of gene (according to a previous idea of Muller). In the 1930s, Burnet's work opened a new era by demonstrating the occurrence of several species of phages and their antigenic property. In the same period, the physical and chemical characteristics of these viruses were disclosed thanks, in particular, to the work of Schlesinger, who first demonstrated that a virus (phage) was constituted of nucleoproteins. The peculiarity of phage was finally shown after the invention of electron microscope: H. Ruska, in 1940, and Anderson and Luria in the next years, obtained the first images of tailed phages, a finding that strongly helped the investigation on the first steps of the infection process. The decisive impulse to phage virology came from Delbrück, a physicist who entered biology giving it a new arrangement. The so-called "phage group" assembled brilliant minds (Luria, Hershey and Delbrück himself, and later a dozen of other scientists): this group faced three fundamental questions of phage virology, i.e., the mechanisms of attack, multiplication and lysis. In ten years' time, phage virology became an integrant part of molecular biology, also thanks to the discovery of the genetical properties of DNA: in such scientific context, Delbrück, Luria and Hershey's works emerged for the absolute excellence of their results, which led such scientists to Nobel prize. Lysogeny was however neglected by the phage group: this singular property shared by bacteria and phages was instead investigated by Lwoff's group, in Paris, and explained in its fundamental features during the 1950s. The "phage's saga" has gone on being an important division of molecular biology till today, and its history is far from being over.     

Geometry of phage head construction.

The process of phage capsid assembly is reviewed, with particular attention to the probable role of curvature in helping to determine head size and shape. Both measures of curvature (mean curvature and Gaussian curvature, explained in Appendix I), should act best when the assembling shell is spherical, which could account for procapsids having this shape. Procapsids are also relatively thick, which should help head size determination by the mean curvature. The accessory role of inner and outer scaffolds in size determination and head nucleation is also reviewed.Nucleation failure generates various malformations, including non-closure, but the most common is the tube or polyhead, where the subunits' inherent curvature is expressed as a constant mean curvature. This induces lattice distortions that only partly understood. An extra tubular section in normal heads leads to the prolate shape, with a more complex and variable geometry.Newly assembled procapsids are both enlarged and toughened by the head transformation. In the procapsid the Gaussian curvature is uniformly distributed. But toughening tends to equalize bond lengths, so all the Gaussian curvature gets concentrated in the vertices, being zero elsewhere. This explains head angularization. Because of this change in Gaussian curvature, the regular subunit packing in the polyhedral head cannot be mapped onto the procapsid. This explains part of the hexon distortions found in this region.The implications of translocase-induced DNA twist, end rotation and the coiling of packaged DNA, are discussed.The symmetry mismatches between the head, connector and tail are discussed in relation to the possible alpha-helical structures of their DNA channels.     

The genome of B. subtilis phage SPP1: physical arrangement in phage genes.

Summary 41 genes of SPP1 have been delineated by using complementation analyses of 75 conditionally lethal (ts and sus) mutations. The physical locations of these genes on the SPP1 chromosome have been determined by transfection/marker rescue experiments in which restriction endonuclease generated fragments of SPP1 DNA were used as donor DNA. The physical order of these fragments has been previously established (Ratcliff et al., 1979).Part of this work is from the doctoral thesis submitted by M. Behncke to the Freie Universität Berlin (1973).     

Growth and phage production of B. megatherium. I. Growth of cells after infection with C phage. II. Rate of growth,phage yield,and RNA content of cells. III. Effect of various substances on growth rate and phage production

I. Lysogenic B. megatherium 899a (de Jong, 1931) produces two types of phage (Gratia, 1936 c) T and C. The T phage forms cloudy plaques and gives rise to fresh lysogenic strains (Gratia, 1936 b) when added to the sensitive strain of megatherium. It may or may not cause lysis, depending on the media (Northrop, 1951). The C phage occurs very rarely) forms clear plaques, does not give rise to lysogenic strains, and causes complete lysis of the sensitive strain under all conditions tested, provided infection occurs. If C phage is added to the sensitive strain, and the mixture allowed to stand, or made into a hanging drop preparation, the infected cells stop growing and lyse completely after 60 to 80 minutes with the liberation of from 50 to 200 phage particles per cell. If, however, C phage is added to a rapidly growing culture of B. megatherium and the suspension shaken at 34°, the cells continue to grow and divide for 50 to 60 minutes, after infection has occurred. They then lyse, with the liberation of from 1000 to 2500 phage particles per cell. II. The following determinations have been made on megatherium sensitive cells growing in 5 per cent peptone at different stages of growth. (1) Growth rate of infected and uninfected cells; (2) RNA, DNA, and protein content; (3) volume of the cell; (4) phage yield per cell by plaque count; (5) phage yield per cell by cell and plaque count; (6) lysis time. The growth rate decreases as the cell concentration increases. The lysis time and the protein N per cell are nearly independent of the growth rate; all the other values increase as the growth rate increases. The ratio See PDF for Equation is nearly constant. RNA and DNA per cell increase less rapidly than the volume, so that NA per unit volume is not constant, but decreases as the size of the cell increases. The phage yield measured under conditions in which the infected cells do not grow (by plaque count) is very nearly proportional to the size of the cell. The phage yield per cell, under conditions in which the infected cells do grow, increases more rapidly than the size of the cells. The phage yield per cell under these conditions may be calculated by the equation See PDF for Equation The determining factor for the variation in phage yield is the growth rate of the cells. This, in turn, is determined by the composition of the medium. III. The growth and phage production of megatherium 899a have been determined in the presence of the following substances: aureomycin, bacitracin, chloromycetin, gramicidin, Merck AB631, Merck AB191, Merck AB624, penicillin, streptomycin, terramycin, tyrothricin, usnic acid, acetone, chloroform, ethyl alcohol, formaldehyde, gentian violet, glycerin, maleic hydrazide, methyl alcohol, phenyl mercuric acetate, sodium fluoride, sulfanilamide, toluene, and urethane. In every case, the lowest concentration of the substance which completely inhibits growth, is also the lowest concentration which completely inhibits phage production. One antibiotic, Merck AB81, causes increased phage production in concentrations which partially inhibit growth, and low phage production in concentrations which completely inhibit growth (as determined by turbidity). Short exposure to ultraviolet light also decreases the growth rate, with increase in phage production. Longer exposure, which completely inhibits growth (as determined by turbidity) results in lysis and phage liberation.     

Characterization of Lactococcus lactis phage antigens.

Phage phi 197 is representative of a widespread lactococcal phage group characterized by a particular morphology (prolate head with a noncontractile tail). In order to develop an immunoenzymatic phage detection test, fusion proteins containing beta-galactosidase fused to epitopes of phage phi 197 structural proteins were constructed by cloning random DNA fragments from the phage genome upstream of a lacZ gene on a plasmid vector. Recombinant plasmids containing certain fragments encoded the synthesis of fusion proteins which react with polyclonal antibodies against the phage and confer a Lac+ phenotype on Escherichia coli. Three different epitopes were represented; phage-specific DNA fragments encoding these epitopes were mapped at three locations on the phage genome, and their nucleotide sequences were determined. Two fused phage antigens were conformational epitopes, whereas the phage epitope of protein encoded by the recombinant plasmid designated pOA17 was a denaturation-resistant epitope. This epitope was very immunogenic. Protein encoded by plasmid pOA17 was synthesized in large amounts from a strong promoter. Antibodies raised against this hybrid protein were used to identify the 46-kDa minor phage protein which provides the epitope. Antibody cross-reactivity of phages related to phi 197 showed that this epitope is well conserved in this genetic group.     

Characterization of Lactococcus lactis phage antigens.

Phage phi 197 is representative of a widespread lactococcal phage group characterized by a particular morphology (prolate head with a noncontractile tail). In order to develop an immunoenzymatic phage detection test, fusion proteins containing beta-galactosidase fused to epitopes of phage phi 197 structural proteins were constructed by cloning random DNA fragments from the phage genome upstream of a lacZ gene on a plasmid vector. Recombinant plasmids containing certain fragments encoded the synthesis of fusion proteins which react with polyclonal antibodies against the phage and confer a Lac+ phenotype on Escherichia coli. Three different epitopes were represented; phage-specific DNA fragments encoding these epitopes were mapped at three locations on the phage genome, and their nucleotide sequences were determined. Two fused phage antigens were conformational epitopes, whereas the phage epitope of protein encoded by the recombinant plasmid designated pOA17 was a denaturation-resistant epitope. This epitope was very immunogenic. Protein encoded by plasmid pOA17 was synthesized in large amounts from a strong promoter. Antibodies raised against this hybrid protein were used to identify the 46-kDa minor phage protein which provides the epitope. Antibody cross-reactivity of phages related to phi 197 showed that this epitope is well conserved in this genetic group.     

Concepts in antibody phage display.

This paper introduces the reader to antibody phage display and its use in combinatorial biochemistry. The focus is on overviewing phage display formats, library design and selection technology, which are the prerequisites for the successful isolation of specific antibody fragments against a diverse set of target antigens.     

To Shigella sonnei phage typing.

The epidemiological significance of phage types changes in the course of years. In Czechoslovakia, in the years 1967-1971, the most frequent phage types were 2,6, 3, 30 and 65, and, in the Middle-Bohemian Region, 2,30, 6, 3 and 65. In the epidemiological years 1972-1973 the sequence of the most frequent S. sonnet phage types changed in the Middle-Bohemian region to: 65, 23, 6, 2 and 12. Some problems concerning the instability of phage types and the part of further auxiliary tests (biochemical differentiation according to Bojlen and drug sensitivity pattern) are discussed.     

A phage in Bartonella bacilliformis.

Bacteriophage-like particles were found in Bartonella bacilliformis culture. The particles consisted of head (icosahedral), 40 nm in diameter, and tail, 16 nm in length.     

N-acetylmuramyl-L-alanine amidase of Vi phage III.

1. A lytic enzyme was isolated from Vi phage III-induced lysate of Salmonella typhi, and purified about 200-fold by chromatography on IRC-50, CM-cellulose, and Sephadex G-75 columns. 2. Both E. coli B murein and muropeptide C6 were digested on incubation with the lytic enzyme. The main product of murein and muropeptide C6 digestion is identical with tetrapeptide Ala-Glu-DAP-Ala. The release of amino groups during digestion was not accompanied by the appearance of either reducing groups or hexosamines. 3. It is concluded that Vi phage III-induced lytic enzyme is N-acetylmuramyl-L-alanine amidase, which cleaves the amide bond between N-acetylmuramic acid and L-alanine.     

European regulatory conundrum of phage therapy.

The treatment of infectious diseases with antibiotics is becoming increasingly challenging. Very few new antimicrobials are in the pharmaceutical industry pipeline. One of the potential alternatives for antibiotics is phage therapy. Major obstacles for the clinical application of bacteriophages are a false perception of viruses as 'enemies of life' and the lack of a specific frame for phage therapy in the current Medicinal Product Regulation. Short-term borderline solutions under the responsibility of a Medical Ethical Committee and/or under the umbrella of the Declaration of Helsinki are emerging. As a long-term solution, however, we suggest the creation of a specific section for phage therapy under the Advanced Therapy Medicinal Product Regulation.     

Bacteriolytic enzymes of Vi phage III lysate.

Vi phage III infected Salmonella typhi cells were shown to contain two activities which lyse the chloroform-killed E. coli B cells. These enzymes have been separated by chromatography on CM-cellulose column and identified as the D-alanyl-meso-DAP endopeptidase and the N-acetylmuramyl-L-ala-nine amidase. The substrate specificity of these enzymes was investigated using low molecular weight muropeptides C3 and C6. It has been shown that muropeptide C3, the cross-linking unit in E coli B murein is completely resistant to the amidase action. This property of Vi phage III amidase suggested that this enzyme does not possess the ability to cause lysis, at the end of the production cycle, of host-bacteria infected with this phage.     

W-reactivation and W-mutagenesis of gamma-irradiated phage lambda.

UV irradiation of Escherichia coli wild-type cells manifested the phenomena of W-reactivation (WR) and W-mutagenesis (WM) of phage lambda irradiated by 60Co gamma-rays in broth. WR of gamma-irradiated phage was half as efficient as that of UV-irradiated phage, although the frequency of c mutations in conditions of WR was about the same in both phages. The xthA and recBrecC sbcB mutants were practically identical with wild-type cells in respect of WR and WM of UV- and gamma-irradiated phage. As in UV-irradiated phage, WR and WM of gamma-irradiated phage were absolutely dependent on the recA+ and lexA+ genes of the host cell. WR and WM required much smaller doses of UV radiation for induction in polA1 and uvrB mutants. The lig-ts mutant, temperature sensitive in polynucleotide ligase, was deficient in WR and WM of UV- and gamma-irradiated phage at the semi-permissive temperature of 37 degrees. The uvrE502 mutant and the allelic recL152 strain were absolutely deficient in WR and WM of gamma-irradiated phage. In UV-irradiated phage WR was reduced, but not eliminated, in the uvrE mutant, and WM was entirely suppressed. This is another example of uncoupling of WR and WM which shows that several repair systems are active in WR but only some of them are mutagenic.