Specialized transduction of the ilvD-thyB-ilvA region mediated by Bacillus subtilis bacteriophage SP beta

Specialized transducing SP beta particles were found that carried the Bacillus subtilis genes lying to the left of the prophage attachment site. Three classes of transducing particles were differentiated, depending upon whether they carried ilvA only, thyB and ilvA, or ilvD, thyB, and ilvA. Lysates prepared by the induction of strains that carried both a transducing phage and a plaque-forming phage contained the two particles in a ratio of about 1:3,000. When the transducing particles were used to transduce a phage-sensitive auxotrophic strain to prototrophy, some of the transductants carried only the transducing phage genomes which, by themselves, were defective. One putative nondefective transducing phage (for ilvA only) is also described. SP beta can mediate specialized transduction even in the absence of the major (recE) bacterial recombination system.     

The electrooptical parameters of suspensions of Escherichia coli XL-1 cells interacting with helper phage M13K07

The electrophysical properties of Escherichia coli XL-1 cells interacting with helper phage M13K07 were studied as a function of the phage-to-cell ratio and the contact time. The electro-optical signal of bacterial cells changed considerably as soon as 10 min after the onset of their incubation with phage particles, presumably due to phage adsorption on the cell surface. The maximum changes in the orientational spectra of cell suspensions were observed when the phage-to-cell ratio was 20. Selectivity studies showed that E. coli XL-1 cells interacting with the helper phage M13K07 in the presence of foreign microflora, such as E. coli K-12 or Azospirillum brasilense Sp7, can be identified by using their electrophysical properties. Changes in the orientational spectra of cell suspensions are interpreted with the stage of phage-bacterium interaction taken into account. The results obtained can probably be used to devise a new rapid method for identification of microorganisms and to study the particular stages of cell infection by bacteriophages.     

Bacteriophages of lactobacilli

Lactobacilli are members of the bacterial flora of lactic starter cultures used to generate lactic acid fermentation in a number of animal or plant products used as human or animals foods. They can be affected by phage outbreaks, which can result in faulty and depreciated products. Two groups of phages specific of Lactobacillus casei have been thoroughly studied. 1. The first group is represented by phage PL-1. This phage behaves as lytic in its usual host L. casei ATCC 27092, but can lysogenize another strain, L. casei ATCC 334. Bacterial receptors of this phage are located in a cell-wall polysaccharide and rhamnose is the main component of the receptors. Ca2+ and adenosine triphosphate (ATP) are indispensable to ensure the injection of the phage DNA into the bacterial cell. The phage DNA is double-stranded, mostly linear, but with cohesive ends which enables it to be circularized. The vegetative growth of PL-1 proceeds according to the classical mode. Cell lysis is produced by an N-acetyl-muramidase at the end of vegetative growth. 2. The second group is represented by the temperate phage phi FSW of L. casei ATCC27139. It has been shown how virulent phages originate from this temperate phage in Japanese dairy plants. The lysogenic state of phi FSW can be altered either by point mutations or by the insertion of a mobile genetic element called ISL 1, which comes from the bacterial chromosome. This is the first transposable element that has been described in lactobacilli. Lysogeny appears to be widespread among lactobacilli since one study showed that 27% of 148 strains studied, representing 15 species, produced phage particles after induction by mitomycin C. Similarly, 23 out of 30 strains of Lactobacillus salivarius are lysogenic and produce, after induction by mitomycin C, temperate phages, killer particles, or defective phages. Temperate phages have also been found in 10 out of 105 strains of Lactobacillus bulgaricus or Lactobacillus lactis after induction by mitomycin C. Phages so far studied of the latter 2 and closely related lactobacilli, either temperate or isolated as lytic, may be divided into 4 unrelated groups called a, b, c and d. Most of these phages are found in group a and an unquestionable relationship has already been shown between lytic phages and temperate phages that belong to this group. Lytic phage LL-H of L. lactis LL 23, isolated in Finland, is one of the most representative of those of group a and has been extensively studied on the molecular level.     

Factors influencing the replication of somatic coliphages in the water environment

The potential replication of somatic coliphages in the environment has been considered a drawback for their use as viral indicators, although the extent to which this affects their numbers in environmental samples has not been assessed. In this study, the replication of somatic coliphages in various conditions was assayed using suspensions containing naturally occurring somatic coliphages and Escherichia coli WG5, which is a host strain recommended for detecting somatic coliphages. The effects on phage replication of exposing strain WG5 and phages to a range of physiological conditions and the effects of the presence of suspended particles or other bacteria were also assayed. Phage replication was further tested using a strain of Klebsiella terrigena and naturally occurring E. coli cells as hosts. Our results indicate that threshold densities of both host bacterium and phages should occur simultaneously to ensure appreciable phage replication. Host cells originating from a culture in the exponential growth phase and incubation at 37 degrees C were the best conditions for phage replication in E. coli WG5. In these conditions the threshold densities required to ensure phage replication were about 10(4) host cells/ml and 10(3) phages/ml, or 10(3) host cells/ml and 10(4) phages/ml, or intermediate values of both. The threshold densities needed for phage replication were higher when the cells proceeded from a culture in the stationary growth phase or when suspended particles or other bacteria were present. Furthermore E. coli WG5 was more efficient in supporting phage replication than either K. terrigenae or E. coli cells naturally occurring in sewage. Our results indicate that the phage and bacterium densities and the bacterial physiological conditions needed for phage replication are rarely expected to be found in the natural water environments.     

Variability of Bacillus thuringiensis under various growth conditions

When a lysogenic culture of Bacillus thuringiensis subsp. galleriae 69-6 was grown under the batch conditions, 93-99% of cells in the population produced R-form colonies and ca. 1% yielded S-form colonies. The amount of spore-forming cells was 99% in R-variants and 8% in S-variants. The quantity of S-variants rose abruptly to 99% when the culture was grown under the chemostat conditions. The number of S-variants increased with the rate and the duration of growth. The process was influenced by growth-limiting factors. Temperate phage variants capable of host culture lysis on solid media (i.e. h-mutants) were not found under the conditions of batch cultivation. However, such phage particles (h-mutants) appeared under the conditions of chemostat. The titre of these phage particles reached 10(8), 10(7) and 10(4) particles per 1 ml at limitation with yeast extract, glucose and phosphorus, respectively. Under the conditions of chemostat, the particles behaved as temperate ones and their growth was not found. Irrespective of the limitation, the phage titre did not correlate with the ratio of R and S-forms in the population. When the growth was limited with phosphorus, the quantity of S-forms increased abruptly while the spontaneous induction of the phage was inhibited. The quantity of cells capable of spore formation decreased in the cultures isolated from the chemostat and grown on MPA: 69-80% of the cells in R-forms and merely 8% in S-forms.     

Bacteriophage K1-5 encodes two different tail fiber proteins, allowing it to infect and replicate on both K1 and K5 strains of Escherichia coli

A virulent double-stranded DNA bacteriophage, Phi K1-5, has been isolated and found to be capable of infecting Escherichia coli strains that possess either the K1 or the K5 polysaccharide capsule. Electron micrographs show that the virion consists of a small icosohedral head with short tail spikes, similar to members of the Podoviridae family. DNA sequence analysis of the region encoding the tail fiber protein showed two open reading frames encoding previously characterized hydrolytic phage tail fiber proteins. The first is the K5 lyase protein gene of Phi K5, which allows this phage to specifically infect K5 E. coli strains. A second open reading frame encodes a protein almost identical in amino acid sequence to the N-acetylneuraminidase (endosialidase) protein of Phi K1E, which allows this phage to specifically infect K1 strains of E. coli. We provide experimental evidence that mature phage particles contain both tail fiber proteins, and mutational analysis indicates that each protein can be independently inactivated. A comparison of the tail gene regions of Phi K5, Phi K1E, and Phi K1-5 shows that the genes are arranged in a modular or cassette configuration and suggests that this family of phages can broaden host range by horizontal gene transfer.     

Bacteriophage P22-mediated specialized transduction in Salmonella typhimurium: identification of different types of specialized transducing particles.

The temperate bacteriophage P22 mediates both generalized and specialized transduction in Salmonella typhimurium. Specialized transduction by phage P22 is different from, and less restricted than, the well characterized specialized transduction by phage lambda, due to differences in the phage DNA packaging mechanism. Phage lysates produced by induction of lysogenic strains contain very high frequencies of supQ newD- and proA,B-specialized transducing particles (10(-2)/PFU and 10(-3)/PFU, respectively), most of which are produced by independent aberrant excision events of various types. In a model, 12 different modes of transduction mechanisms were characterized by: (i) the structure of the specialized transducing genomes after injection into a new host cell, i.e., linear or circular, and (ii) the requirements for the transduction process, i.e., host recombination functions, phage integration functions, or presence of a prophage. By using different recipient strains and phage helper strains, it was possible to show that most specialized transducing particles (ca. 99%) contain linear genomes that cannot circularize upon injection into a new host cell and that require the presence of an integrated prophage as a site for a recombinational event to give rise to a transductant. Only 0.1% of all specialized transducing particles were shown to transduce by integration, suggesting that transducing genomes containing terminally redundant ends represent only a minor fraction of all transducing particles that are produced. However, it should be pointed out that the frequency (approximately 10(-5)/PFU) of these specialized transducing genomes that can circularize upon injection into a new host cell is as high as or even higher than the frequency of specialized transducing particles of phage lambda. The remaining approximately 1% of all specialized transducing particles can transduce by any one of the other mechanisms described.     

Role of the pneumococcal autolysin (murein hydrolase) in the release of progeny bacteriophage and in the bacteriophage-induced lysis of the host cells.

The pneumococcal bacteriophage Dp-1 seems to require the activity of the N-acetylmuramic acid-L-alanine amidase of the host bacterium for the liberation of phage progeny into the medium. This conclusion is based on a series of observations indicating that the exit of progeny phage particles is prevented by conditions that specifically inhibit the activity of the pneumococcal autolysin. These inhibitory conditions are as follows: (i) growth of the bacteria on ethanolamine-containing medium; (ii) growth of the cells at pH values that inhibit penicillin-induced lysis of pneumococcal cultures and lysis in the stationary phase of growth; (iii) addition of trypsin or the autolysin-inhibitory pneumococcal Forssman antigen (lipoteichoric acid) to the growth medium before lysis; (iv) infection of an autolysin-defective pneumococcal mutant at a multiplicity of infection less than 10 (treatment of such infected mutant bacteria with wild-type autolysin from without can liberate the entrapped progeny phage particles); (v) release of phage particles and culture lysis can also be inhibited by the addition of chloramphenicol to infected cultures just before the time at which lysis would normally occur. Bacteria infected with Dp-1 under conditions nonpermissive for culture lysis and phage release secrete into the growth medium a substantial portion of their cellular Forssman antigen in the form of a macromolecular complex that has autolysin-inhibitory activity. We suggest that a phage product may trigger the bacterial autolysin by a mechanism similar to that operating during treatment of pneumococci with penicillin (Tomasz and Waks, 1975).     

Dissociation-independent selection of high-affinity anti-hapten phage antibodies using cleavable biotin-conjugated haptens

The engineering of hapten-specific antibodies with affinity constant higher (K(a) values >10(10)M(-1)) than those of conventional antibodies promises hapten immunoassays exhibiting sub-femtomole range sensitivity, based on the conventional competitive assay principle. Here we report a simple method to select phage particles displaying anti-hapten antibody fragments with exceptionally high affinity. 11-Deoxycortisol (11-DC), selected as a model target hapten, was covalently conjugated to biotin via a spacer that included a reductively cleavable disulfide bond. Phage particles displaying high-affinity, single-chain Fv fragments (scFvs) specific for 11-DC (K(a)1.3 x 10(10)M(-1)) were incubated with the "cleavable biotin"-conjugated 11-DC, and the resulting complexes was captured on immobilized NeutrAvidin. Mild reductive conditions that did not decrease phage infectivity easily cleaved the disulfide bond, allowing the recovery of target phage particles; this process is fully independent of the dissociation of the antigen-antibody interaction. Five serial rounds of selection enabled the isolation and enrichment of the anti-11-DC phage (specific phage ratio >90%) from among a 100,000-fold excess of nonspecific phage particles. This method will be applicable for selection of extra-high-affinity phage antibodies against a wide variety of haptens.     

Construction of intergeneric hybrids using bacteriophage P1CM: transfer of the Klebsiella aerogenes ribitol dehydrogenase gene to Escherichia coli.

Study of many of the interesting properties of Klebsiella aerogenes is limited by the lack of a well-characterized genetic system for this organism. Our investigations of the evolution of the enzyme ribitol dehydrogenase (EC 1.1.1.56) in K. aerogenes would be greatly facilitated by the availability of such a system, and we here report two approaches to developing one. We have isolated mutants sensitive to the coliphage P1, which will efficiently tranduce genetic markers between such sensitive strains and which will thus make detailed mapping studies possible. Derivatives of K. aerogenes lysogenic for P1 can be readily isolated by using the specialized transducing particle P1CMclr100. Bacteria lysogenic for this phage are chloramphenicol resistant and temperature sensitive. Phage particles produced by temperature induction of such lysogens can be used to transfer K. aerogenes genes to the natural host of P1 phage. Escherichia coli. We have used this method to prepare derivatives of E. coli K-12 carrying the K. aerogenes genes conferring the ability to metabolize the pentitols ribitol and D-arabitol. We have shown that these E. coli-K. aerogenes hybrids synthesize a ribitol dehydrogenase with the properties of the K. aerogenes enzyme and have mapped the position of the transferred gene on the E. coli chromosome. The ramifications of this methodology are discussed.     

Polylysogeny and prophage induction by secondary infection in Vibrio cholerae

Strains of Vibrio cholerae O1, biotypes El Tor and classical, were infected with a known temperate phage (PhiP15) and monitored over a 15-day period for prophage induction. Over the course of the experiment two morphologically and three genomically distinct virus-like particles were observed from the phage-infected El Tor strain by transmission electron microscopy and field inversion gel electrophoresis, respectively, whereas only one phage, PhiP15, was observed from the infected classical strain. In the uninfected El Tor culture one prophage was spontaneously induced after 6 days. No induction in either strain was observed after treatment with mitomycin C. Data indicate that El Tor biotypes of V. cholerae may be polylysogenic and that secondary infection can promote multiple prophage induction. These traits may be important in the transfer of genetic material among V. cholerae by providing an environmentally relevant route for multiple prophage propagation and transmission.     

Bacteriophage P22 virion protein which performs an essential early function. II. Characterization of the gene 16 function.

P16 is a virion protein and, as such, is incorporated into the phage head as a step in morphogenesis. The role of P16 in assembly is not essential since particles are formed without this protein which appear normal by electron microscopy. P16 is essential when the particle infects a cell in the following cycle of infection. In the absence of functional P16, the infection does not appear to proceed beyond release of phage DNA from the capsid. No known genes are expressed, no DNA is transcribed, and the host cell survives the infection, continuing to grow and divide normally. The P16 function is required only during infection for the expression of phage functions. Induction in the absence of P16 proceeds with the expression of early and late genes and results in particle formation. P16 must be incorporated during morphogenesis into progeny particles after both infection and induction for the progeny to be infectious. The P16 function is necessary for transduction as well as for infection. Its activity is independent of new protein synthesis and it is not under immunity control. P16 can act in trans, but appears to act preferentially on the phage or phage DNA with which it is packaged. The data from complementation studies are compatible with P16 release from the capsid with the phage DNA. In the absence of P16 the infection is blocked, but the phage genome is not degraded. The various roles which have been ruled out for P16 are: (i) an early regulatory function, (ii) an enzymatic activity necessary for phage production, (iii) protection of phage DNA from host degradation enzymes, (iv) any generalized alteration of the host cell, (v) binding parental DNA to the replication complex, and (vi) any direct involvement in the replication of P22 DNA. P16 can be responsible for: (i) complete release of the DNA and disengagement from the capsid, (ii) bringing the released DNA to some necessary cell site or compartment such as the cytoplasm, (iii) removal of other virion proteins from the injected DNA, and (iv) alterations of the structure of the injected DNA.     

Effect of chitosan oligomer on phage particles and reproduction of phage 1-97A in Bacillus thuringiensis culture]

The causes of bacteriophage 1-97A inactivation by the chitosan oligomer with a polymerization degree of 15 and the influence of the oligomer on the phage reproduction in the culture of Bacillus thuringiensis subsp. galleriae, strain 1-97, were studied. The study of the inactivation kinetics showed that, in 1 h, virtually all chitosan was bound to the phage particles, causing, as evidenced by electron microscopy, DNA release from the phage head, destruction of the phage particles, and agglutination of the phage particles or of their tails in the region of the endplate. High-polymeric chitosan caused more pronounced destruction of the phage particles than the oligomer. It was established that chitosan prevented the production of complete phage particles. One of the mechanisms of such an influence may be the production in the presence of chitosan of phage particles devoid of DNA.     

Influence of the Chitosan Oligomer on the Phage Particles and Reproduction of Phage 1-97A in the Culture of Bacillus thuringiensis

The causes of bacteriophage 1-97A inactivation by the chitosan oligomer with a polymerization degree of 15 and the influence of the oligomer on the phage reproduction in the culture of Bacillus thuringiensissubsp. galleriae, strain 1-97, were studied. The study of the inactivation kinetics showed that, in 1 h, virtually all chitosan was bound to the phage particles, causing, as evidenced by electron microscopy, DNA release from the phage head, destruction of the phage particles, and agglutination of the phage particles or of their tails in the region of the basal plate. High-polymeric chitosan caused more pronounced destruction of the phage particles than the oligomer. It was established that chitosan prevented the production of complete phage particles. One of the mechanisms of such an influence may be the production in the presence of chitosan of phage particles devoid of DNA.     

Agrobacterium tumefaciens Conn IV. Bacteriophage PB21 and Its Inhibitory Effect on Tumor Induction

Strain B2 of Agrobacterium tumefaciens Conn produces plaques when seeded against strain B6-806 of the same organism. From such a plaque, a highly virulent bacteriophage was obtained by use of D'Herelle's technique of selecting for virulent phage. On nutrient agar, this phage, PB2₁, produced large clear plaques which did not overgrow. Plaques produced on a glutamate medium and on White's plant tissue culture medium were even larger and in White's medium had a three-dimensional appearance. PB2₁ does not appear to be an oncogenic virus. To the contrary, the addition of phage under circumstances which insure mass lysis completely inhibited tumor initiation. Fewer than 10 phage particles present at the beginning of a 21-hr induction period were able, at times, to inhibit completely tumor induction by highly virulent bacteria (strain B6). The data lend further support to the concept that anything which interferes with the metabolic activity associated with the growth of the bacteria interferes with the tumor-inducing process. Attempts to use the phage to rid crown gall tissue of bacteria were unsuccessful.     

Growth and phage production of lysogenic B. megatherium

Cell multiplication and phage formation of lysogenic B. megatherium cultures have been determined under various conditions and in various culture media. 1. In general, the more rapid the growth of the culture, the more phage is produced. No conditions or culture media could be found which resulted in phage production without cell growth. 2. Cultures which produce phage grow normally, provided they are shaken. If they are allowed to stand, those which are producing phage undergo lysis. Less phage is produced by these cultures than by the ones which continue to grow. 3. Cells plated from such phage-producing cultures in liquid yeast extract medium grow normally on veal infusion broth agar or tryptose phosphate broth agar, which does not support phage formation, but will not grow on yeast extract agar. 4. Any amino acid except glycine, tyrosine, valine, leucine, and lysine can serve as a nitrogen source. Aspartic acid gives the most rapid cell growth. 5. The ribose nucleic acid content is higher in those cells which produce phage. 6. The organism requires higher concentrations of Mg, Ca, Sr, or Mn to produce phage than for growth. 7. The lysogenic culture can be grown indefinitely in media containing high phosphate concentrations. No phage is produced under these conditions, but the cells produce phage again in a short time after the addition of Mg. The potential ability to produce phage, therefore, is transmitted through cell division. 8. Colonies developed from spores which have been heated to 100°C. for 5 minutes produce phage and hence, infected cells must divide. 9. No phage can be detected after lysis of the cells by lysozyme.     

Assay for the Killing Properties of T2 Bacteriophage and Their "Ghosts".

Duckworth, Donna H. (Johns Hopkins University, Baltimore, Md.), and Maurice J. Bessman. Assay for the killing properties of T2 bacteriophage and their "ghosts." J. Bacteriol. 90:724-728. 1965.-A procedure for the assay of bacteriophage and their "ghosts" which is based on their ability to kill cells is described. The method is derived from the well-known ability of phage and ghosts to prevent the induction of beta-galactosidase. Conditions are described whereby a direct relationship is found between the decrease in beta-galactosidase and the number of phage or ghosts present during the induction period. The number of phage measured by this method was found to be identical with the number of plaque-forming units found in a fresh lysate. The method has been used to follow the fate of ghosts under several conditions and to measure killer (but nonviable) particles in various preparations of phage.     

Head length determination in bacteriophage T4: The role of the core protein P22

We have found that two different temperature-sensitive mutations in gene 22, tsA74 and ts22-2, produce high frequencies (up to 85%) of petite phage particles when grown at a permissive or intermediate temperature. Moreover, the ratio of petite to normal particles in a lysate depends upon the temperature at which the phage are grown. These petite phage particles appear to have approximately isometric heads when viewed in the electron microscope, and can be distinguished from normal particles by their sedimentation coefficient and by their buoyant density in CsCl. They are biologically active as detected by their ability to complement a co-infecting amber helper phage. Lysates of both mutants grown at a permissive temperature reveal not only a significant number of petite phage particles in the electron microscope, but also sizeable classes of wider-than-normal particles, particles having abnormally attached tails, and others having more than one tail.Striking protein differences exist between the purified phage particles of tsA74 or ts22-2 and wild-type T4. B1¹, a 61,000 molecular weight head protein, is completely absent from the phage particles of both mutants, and the internal protein IPIII¹ is present in reduced amounts as compared to wild type. The precursor to B1¹ is present in the lysates, but these mutations appear to prevent its incorporation into heads, so it does not become cleaved.The product of gene 22 (P22) is known to be the major protein of the morphogenetic core of the T4 head. Besides the mutations reported here, several mutations which affect head length have been found in gene 23, which codes for the major capsid protein (Doermann et al., 1973b). We suggest a model in which head length is determined by an interaction between the core (P22 and IPIII) and the outer shell (P23).     

The electrooptical parameters of suspensions of <Emphasis Type="Italic">Escherichia coli</Emphasis> XL-1 cells interacting with helper phage M13K07

The electrophysical properties of Escherichia coli XL-1 cells interacting with helper phage M13K07 were studied as a function of the phage-to-cell ratio and the contact time. The electro-optical signal of bacterial cells changed considerably as soon as 10 min after the onset of their incubation with phage particles, presumably due to phage adsorption on the cell surface. The maximum changes in the orientational spectra of cell suspensions were observed when the phage-to-cell ratio was 20. Selectivity studies showed that E. coli XL-1 cells interacting with the helper phage M13K07 in the presence of foreign microflora, such as E. coli K-12 or Azospirillum brasilense Sp7, can be identified by using their electrophysical properties. Changes in the orientational spectra of cell suspensions are interpreted with the stage of phage-bacterium interaction taken into account. The results obtained can probably be used to devise a new rapid method for identification of microorganisms and to study the particular stages of cell infection by bacteriophages.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 198–203.Original Russian Text Copyright © 2005 by Bunin, Ignatov, Guliy, Zaitseva, ONeil, Ivnitskii