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Isolation and Properties of a Pseudomonas acidovorans Bacteriophage
Abstract
SUMMARY The lytic bacteriophage ~SW-I 4 was isolated from sewage using Pseudo- monas acidovorans no. I4 as host. The phage had an icosahedral head some 85 nm. in diameter and a contractile tail some I4o nm. long. ~W-I4 formed plaques on only a few strains of P. acidovorans. The phage gave biphasic ab- sorption kinetics, with an adsorption constant of I'9 x io -9 ml./min. The latent period was 6o rain. and the burst size was about 3oo. The burst size was dependent upon culture age. The kuv. for inactivation of ~W-I 4 was 4"35 min--1. P. acidovorans was shown to possess a potent photoreactivating system. The heat of inactivation of~W- 14 was calculated to be 75,7oo cal./mole. The phage gave biphasic thermal inactivation kinetics at 55 ° and 6o ° but not at 65 °. The phage mutated spontaneously to a different plaque type. This mutation affected the adsorptive properties, the thermal sensitivity and the burst size of the phage.
... The bacterial strains used are listed in Table 1. They were maintained as described previously (9), except for strain 3L, for which the medium was supplemented with 1 mg of thymidine and 15 jg of trimethoprim per ml. Bacteriophage 4W-14 stocks (9) were stored in dilute mannitol broth (DMB) at 4 C over chloroform. ...
... They were maintained as described previously (9), except for strain 3L, for which the medium was supplemented with 1 mg of thymidine and 15 jg of trimethoprim per ml. Bacteriophage 4W-14 stocks (9) were stored in dilute mannitol broth (DMB) at 4 C over chloroform. ...
... Media. The media used were mannitol broth (MB) (9); DMB, which was MB diluted 10-fold; and minimal medium M29 (6). Solid media contained 1.5% agar (Difco). ...
The alpha-putrescinylthymine (putThy) in bacteriophage phiW-14 DNA is synthesized at the mononucleotide level: it is labeled by uracil or deoxyuridine but not by thymidine, and it appears in the acid-soluble pool of infected cells before the onset of phage DNA synthesis. The methylene group at the C-5 position of the pyrimidine moiety of putThy is derived in vivo from a C(1) unit. Extracts of a phage infected thymidine auxotroph of the host, Pseudomonas acidovorans, apparently contain a phage-specific thymidylate synthetase and a phage-specific activity which forms 5-hydroxymethyl dUMP from N(5), N(10)-methylene-tetrahydrofolate and dUMP.
... A statistical t-test confirmed that the observed reduction of free phages between t = 0 and t = 2 is significant (p < 0.001). The finding of residual 4HA13 phage particles that had a slower adsorption rate supports previously reported biphasic adsorption kinetics and population heterogeneity in other phages [45][46][47][48]. The observed heterogeneity in adsorption kinetics of T4 phage population was suggested to be a result of two point mutations in long-tail fiber-encoding genes [49]. ...
... No detectable presence of antimicrobial resistance, virulence, or integrase-coding genes was observed. supports previously reported biphasic adsorption kinetics and population heterogeneity in other phages [45][46][47][48]. The observed heterogeneity in adsorption kinetics of T4 phage population was suggested to be a result of two point mutations in long-tail fiber-encoding genes [49]. ...
Shiga toxin-producing Escherichia coli (STEC) is one of the leading causes of foodborne illnesses in North America and can lead to severe symptoms, with increased fatality risk for young children. While E. coli O157:H7 remains the dominant STEC serotype associated with foodborne outbreaks, there has been an increasing number of non-O157 STEC outbreaks in recent years. For the food industry, lytic bacteriophages offer an organic, self-limiting alternative to pathogen reduction—one that could replace or reduce the use of chemical and physical food processing methods. From EHEC-enriched sewage, we isolated a novel bacteriophage, vB_EcoM-4HA13 (4HA13). Phenotypic characterizations revealed 4HA13 to possess a myoviral morphotype, with a high specificity to non-motile O111 serotype, and a long latent period (90 min). Through genomic analyses, this 52,401-bp dsDNA phage was found to contain 81 CDS, but no detectable presence of antibiotic resistance, integrase, or virulence genes. A BLASTn search for each of the identified 81 CDS yielded homologues with low levels of similarity. Comparison of RNA polymerase and terminase large subunit amino acid sequences led to the proposal and acceptance of a new bacteriophage family, Chaseviridae, with 4HA13 representing a new species and genus. The discovery of this phage has broadened our current knowledge of bacteriophage diversity.
... Phage strain f, from P. fluorescens, failed to lyse any of three known strains of the same species. Some Pseudomnonas and Xanthomonas phages have been shown to react across species and even generic lines (15,18), whereas other phages of Pseudomonas were species to strain specific (11,12). Eleven of our Pseudomonas cultures, representing four of the five species, are common food contaminants. ...
Thirty-eight bacteriophage-host systems were isolated from 22 of 45 refrigerated food products examined under psychrophilic conditions. Isolates were obtained from ground beef, pork sausage, chicken, raw skim milk, and oysters, whereas no isolations were made from liquid egg whites and processed meat products. Thirty of the 38 psychrophilic bacterial hosts were gram-negative rods, and 27 of these were classified within the genus Pseudomonas; three were members of the family Enterobacteriaceae. The remaining eight were gram-positive cocci, which were tentatively classified as Leuconostoc. Plate counts of psychrophilic bacteria were greater than 2.2 × 10⁵/ml (g) in all but one sample which contained phage, whereas phage titers ranged from less than 100 to 6.3 × 10⁶ plaque-forming units/ml (g). Phage isolates showed limited host ranges usually attacking only those hosts upon which they were isolated. Of eight phages tested against 13 cultures of known identity, one showed lytic action, and this was against strains of P. fragi.
... A number of bacteriophages (phages) specific for organisms of the genus Pseudomonas have been isolated from a variety of sources (2,3,6,7,(13)(14)(15)17,18,24,25). Some of these phages have been shown to be psychrotrophic, capable of lysing their bacterial hosts at temperatures approaching 0°C (6,7,17,18,24,25). ...
A total of 40 beef spoilage pseudomonads was used as bacterial hosts for the isolation of psychrotrophic bacteriophages (phages) from spoiled rib steaks. Thirty-eight homologous phages, lytic for 25 of these hosts, were isolated and purified. An additional 12 bacterial isolates were susceptible to heterologous phage lysis and only three of the bacteria examined were resistant to lysis by any of the phage tested. On the basis of heterologous cross-sensitivity to phages, the meatborne Pseudomonas strains and 4 identified ATCC Pseudomonas hosts were differentiated into 37 distinct phage lysotypes. In addition, Pseudomonas phages were found to inhibit bacterial growth in tryptic soy broth by significantly extending the lag phase under psychrotrophic conditions (7°C). However, the incubation of bacteria with phages resulted in the selection of phage resistant bacterial mutants.
... Initial conditions: aggregators to serve as a reservoir for the phage during gentamicin treatment. The biphasic shape of the SA5 adsorption curve is unexpected, but these kinetics have been reported for other bacteriophage [52,53,54,55] . The two phases of adsorption have been hypothesized to be due to heterogeneity in the virus population, heterogeneity of receptors in the cell population, and/or fast, reversible attachment facilitating slower, irreversible attachment. ...
With the increasing frequency of antibiotic resistance and the decreasing frequency of new antibiotics entering the market, interest has returned to developing bacteriophage as a therapeutic agent. Acceptance of phage therapy, however, is limited by the unknown pharmacodynamics of a replicating agent, as well as the potential for the evolution of resistant bacteria. One way to overcome some of these limitations is to incorporate phage and antibiotics into a dual therapy regimen; however, this increases the complexity of the pharmacodynamics. The aim of this study is to develop an experimental system to evaluate the pharmacodynamics of dual phage-drug therapy. A continuous culture system for Staphylococcus aureus is used to simulate the pharmacokinetics of periodic antibiotic dosing alone and in combination with lytic phage. A computer model representation of the system allows further evaluation of the conditions governing the observed pharmacodynamics. The results of this experimental/modeling approach suggest that dual therapy can be more efficacious than single therapies, particularly if there is an overlap in the physiological pathways targeted by the individual agents. In this case, treatment with gentamicin induces a population of cells with a strong aggregation phenotype. These aggregators also have an increased ability to form biofilm, which is a well-known, non-genetic mechanism of drug resistance. However, the aggregators are also more susceptible than the parental strain to the action of the phage. Thus, dual treatment with gentamicin and phage resulted in lower final cell densities than either treatment alone. Unlike in the phage-only treatment, phage-resistant isolates were not detected in the dual treatment.
... All of these core genes except for gene 34, encoding the proximal arm of the tail fiber, are present in some form in CBA120, AG3 and ViI, whose structural-protein genes are often more closely related to those of the T4-like cyanobacterial phages than to similar phages from enteric sources. Nearly 70 of their genes are related to genes of D. acidovorans phage W-14 [47] (GenBank accession number GQ357915.1). ...
Phage vB_EcoM_CBA120 (CBA120), isolated against Escherichia coli O157:H7 from a cattle feedlot, is morphologically very similar to the classic phage ViI of Salmonella enterica serovar Typhi. Until recently, little was known genetically or physiologically about the ViI-like phages, and none targeting E. coli have been described in the literature. The genome of CBA120 has been fully sequenced and is highly similar to those of both ViI and the Shigella phage AG3. The core set of structural and replication-related proteins of CBA120 are homologous to those from T-even phages, but generally are more closely related to those from T4-like phages of Vibrio, Aeromonas and cyanobacteria than those of the Enterobacteriaceae. The baseplate and method of adhesion to the host are, however, very different from those of either T4 or the cyanophages. None of the outer baseplate proteins are conserved. Instead of T4's long and short tail fibers, CBA120, like ViI, encodes tail spikes related to those normally seen on podoviruses. The 158 kb genome, like that of T4, is circularly permuted and terminally redundant, but unlike T4 CBA120 does not substitute hmdCyt for cytosine in its DNA. However, in contrast to other coliphages, CBA120 and related coliphages we have isolated cannot incorporate 3H-thymidine (3H-dThd) into their DNA. Protein sequence comparisons cluster the putative "thymidylate synthase" of CBA120, ViI and AG3 much more closely with those of Delftia phage φW-14, Bacillus subtilis phage SPO1, and Pseudomonas phage YuA, all known to produce and incorporate hydroxymethyluracil (hmdUra).
... Often, the origin of the names is difficult to trace, or is entirely lacking in the scientific literature. For example, Delftia phage jW-14 was named after Westbrook building room 14 where it was isolated at the University of British Columbia (Kropinski and Warren, 1970). Pseudomonas phage LKA1 is named after the research institute (K.U.Leuven), the area from which the phage was isolated (Kasteelpark Arenberg) and the isolate number (1) (Ceyssens et al., 2006). ...
... Thereafter the process continued at a much lower rate with an adsorption constant of 3 × 10-~°/ml/min. No satisfactory explanation is readily available for the biphasic nature of adsorption for any phage studied so far (Feary et al., 1964; Kropinski & Warren, 1970; Schade & Adler, 1967). The possibility that the phage preparations contained a significant population of slowly adsorbing contaminant phage particles was ruled out by the fact that the phage recovered from the supernatant after the first rapid phase of adsorption or phage purified from a single plaque showed identical biphasic adsorption kinetics. ...
Biophysical characteristics of Vibrio eltor phage e4, a key phage in the Vibrio cholerae typing scheme were studied. This icosahedral phage was found to contain 12 structural polypeptides with mol. wt. ranging from 25,000 to 120,000. One of these polypeptides of mol. wt. 50,000 accounted for most of the structural proteins present and was probably the major phage capsid protein. The phage genome comprised a single linear, double-stranded DNA molecule, 69.2 kbp in length (45.6 X 10(6) mol. wt.) as determined by electron microscopy and restriction fragment analyses. The G + C content was 34.6%. Electron microscopy data indicated that unlike the DNAs of other cholera phages, phage e4 DNA is not circularly permuted. Adsorption under normal conditions was biphasic with rate constants of 1.02 X 10(-9)/ml/min up to 60% adsorption and 3 X 10(-10)/ml/min thereafter. Intracellular phage multiplication was characterized by a latent period of 27 min. The burst size was approximately 100 phage particles per infected cell.
... The media and growth conditions were described previously (10,11). The minimal medium is referred to as M29. ...
Pseudomonas acidovorans lacks a number of enzymes of the salvage pathways of nucleic acid metabolism, including uridine phosphorylase, purine nucleoside phosphorylase, cytidine (deoxycytidine) deaminase and thymidine phosphorylase, and probably uridine kinase and deoxycytidine kinase. Its growth is inhibited by adenosine and deoxyadenosine. The level of aspartate transcarbamylase is the same in extracts of P. acidovorans grown in minimal medium ± 25 μg uracil/ml, and the enzyme appears to be insensitive to nucleotides which affect this enzyme in other bacteria. Growth of two pyrimidine-requiring mutants of P. acidovorans is supported by uracil or cytosine but not by their nucleosides nor by intermediates of the de novo pyrimidine biosynthetic pathway. Concentrations of uracil greater than 50 μg/ml have the unusual effect of lengthening the lag period of the mutants. The wild-type strain is not inhibited by uracil.
... c Final concentration of thymidine in mannitol broth (6). thymidine per ml for growth. ...
The aminopterin technique was adapted for the isolation of thymidine auxotrophs of Pseudomonas acidovorans. All the mutants isolated responded to thymidine but not to thymine.
The effects of stress and resuscitation on selective enumeration of coliforms, Escherichia coli and enterococci in mechanically deboned raw poultry meat and in dried foods were studied using a hydrophobic grid membrane filter (HGMF) technique. The effects of four different stresses, sublethal heating, freezing, acid pH and drying, were examined on 25 to 30 samples per indicator organism for each type of stress. Counts obtained with resuscitation were compared statistically to direct selective counts for each series of samples. Also, both the direct and resuscitative HGMF results were compared to a 5-tube most probable number method for coliforms and E. coli and to a spread plate method for the enterococci. The use of appropriate resuscitation procedures not only produced a significant increase in counts over the direct HGMF procedure, but also yielded HGMF results that were statistically equivalent to those obtained by conventional methods.
Expression of the Bla+ phenotype of the incompatibility group P-1 plasmid RP1 appears to be a variable phenomenon inPseudomonas acidovorans strains, although all plasmid-bearing strains examined synthesize the RP1-encoded TEM 2 ß-lactamase. There is also evidence suggesting that plasmid-encoded alterations to the outer membrane could be affecting the degree of resistance observed to ß-lactam drugs in at least some strains.
Lack of an active transport system prevents Pseudomonas acidovorans taking up putrescine under normal condition of growth. At pH 9.5, however, putrescine does enter the cell. That putrescine enters the intracellular pool is shown by its conversion to 2-hydroxyputrescine and spermidine after the cells are returned to pH 7.0. The accumulated putrescine can be used to label specifically the α-putrescinylthymine residues of bacteriophage DNA.
Bacteriophage φW-14 is unusual because its DNA contains 12 mol% of the hypermodified pyrimidine, α-putrescinylthymine. The φW-14 virion is similar in morphology to T4, except that the φW-14 head is isometric rather than prolate, there is no collar-whisker structure associated with the neck, the tail fibers are short (∼15 nm), and the base plate terminates in small plates or knobs rather than spikes. The contractile tail sheath of φW-14 appears to have a right-handed helical arrangement of subunits with a pitch in the extended form of ∼20 nm. The “stacked disk” appearance of the tail sheath visible on negatively stained particles has a periodicity of 3–4 nm. The protein shell of the head has a similar thickness (2–3 nm) to that of T4. The φW-14 virion contains at least 17 different polypeptide species. Based on measurements from electron micrographs of negatively stained phage particles on the same grid square, the volume of the φW-14 head was estimated to be approximately 72% that of the T4 head. Surprisingly, however, the lengths of the DNA molecules released from φW-14 and T4 heads by osmotic shock were 59.6 ± 1.9 and 62.1 ± 2.4 μm, respectively. am42 is an amber mutant of φW-14 in which there is only 5 mol% putThy in the DNA made in the nonpermissive host. am42 virions are morphologically normal, but the length of the DNA released from these virions is only 53.1 ± 3.1 μm. We conclude that φW-14 DNA is packed much more compactly than T4 DNA into a virion of similar morphology and comparable complexity and that the tight packing is a consequence of, and dependent upon, the presence of putThy in φW-14 DNA.
Starting with stochastic rate equations for the fundamental interactions
between microbes and their viruses, we derive a mean field theory for the
population dynamics of microbe-virus systems, including the effects of
lysogeny. In the absence of lysogeny, our model is a generalization of that
proposed phenomenologically by Weitz and Dushoff. In the presence of lysogeny,
we analyze the possible states of the system, identifying a novel limit cycle,
which we interpret physically. To test the robustness of our mean field
calculations to demographic fluctuations, we have compared our results with
stochastic simulations using the Gillespie algorithm. Finally, we estimate the
range of parameters that delineate the various steady states of our model.
Three spontaneously arising rough mutants of Pseudomonas aeruginosa have been isolated by selection for resistance to virulent lipopolysaccharide (LPS) specific bacteriophages. In addition, the first phages specific for rough mutants of P. aeruginosa were isolated. Using these phage and autoagglutination patterns in 4% NaCl and acriflavine, these mutants could be clearly distinguished from the wild-type strain and each other. Chemical analysis of the LPS together with chromatographic resolution of the polysaccharide moieties showed alterations in both O-specific side chains and core regions.
Almost 50% of the clones of Pseudomonas acidovorans(pLM2) selected for resistance to tetracycline supported the growth of an amber mutant of bacteriophage PRD1.
Receptors for phages specific to Pseudomonas aeruginosa strain PAO were studied. Phages 16, 44, 109, F8, and PBI are lipopolysaccharide (LPS) specific as shown by neutralization tests. The PhI50's of the LPS, adsorption rate constants with strain PAO and the plaque morphologies of these five phages were quite similar. Phages 1214 and 7 also appear to be LPS-specific on the basis of host-range studies. Phage 73 is pilus-specific, while phages 21 and 68 fall into a group which does not attach to pili, flagella, or LPS. A theoretical approach to the interpretation of phage-cell interactions is presented.
The isolation and some properties of a lipopolysaccharide (LPS)-specific phage isolated against Pseudomonas aeruginosa is reported. The phage, designtaed omega PLS-I, is a Bradley A type phage with a head diameter of 70 nm and a contractile tail 120 nm long. The average adsorption rate constant for omegaPLS-I is 4-48 X 10(-9) ml/min. omegaPLS-I is inactivated by purified LPS from P. aeruginosa strain 1-1A, showing a PhI50 value of 1-25 mug/ml.
Bacteriophage phi W-14 is sensitive to osmotic shock. It contain sufficient free putrescine, 2-hydroxyputrescine and spermidine to neutralize about 15% of the DNA phosphates. The alpha-putrescinylthymine residues of the DNA could neutralize a further 25% of the phosphates. Label is transferred from ornithine to the alpha-putrescinyl residues of phi W-14 DNA. The rates of polyamine synthesis in Pseudomonas acidovorans are increased by phi W-14 infection.
Characterization studies were performed on two psychrophilic phages which were isolated from ground beef samples. Phage inactivation by exposure to heat, low pH, osmotic shock conditions, and freezing showed that these two isolates were different. One-step growth experiments indicated that one isolate had a burst size five times as large (500) and a latent period two times as long (4 hr) as the other when tested at 7 C. Nucleic acid type was 2-deoxyribonucleic acid for both. Electron micrographs showed one to belong to Bradley's phage group A and the other to phage group C.
Pseudomonas acidovorans contains putrescine, 2-hydroxyputrescine, and spermidine.
Pseudomonas acidovorans 29 synthesizes the polyamines putrescine, 2-hydroxyputrescine, and spermidine from glutamate and (or) ornithine but not from arginine. It is permeable to arginine and ornithine, and incorporates these compounds into acid-insoluble material; it is impermeable to putrescine. The conversion of labeled glutamate to the polyamines is not decreased in the presence of arginine or ornithine.
Thirty-eight bacteriophage-host systems were isolated from 22 of 45 refrigerated food products examined under psychrophilic conditions. Isolates were obtained from ground beef, pork sausage, chicken, raw skim milk, and oysters, whereas no isolations were made from liquid egg whites and processed meat products. Thirty of the 38 psychrophilic bacterial hosts were gram-negative rods, and 27 of these were classified within the genus Pseudomonas; three were members of the family Enterobacteriaceae. The remaining eight were gram-positive cocci, which were tentatively classified as Leuconostoc. Plate counts of psychrophilic bacteria were greater than 2.2 x 10(5)/ml (g) in all but one sample which contained phage, whereas phage titers ranged from less than 100 to 6.3 x 10(6) plaque-forming units/ml (g). Phage isolates showed limited host ranges usually attacking only those hosts upon which they were isolated. Of eight phages tested against 13 cultures of known identity, one showed lytic action, and this was against strains of P. fragi.
RP 1-Phagen sind wenig stabil. Durch Chloroform werden sie rasch inaktiviert, whrend entsprechende Konzentrationen an ther ohne Titerverlust toleriert werden; pH-Werte, kleiner als 5 und grer als 8,5 fhren zur Inaktivierung, ebenso Temperaturen ber 30C. Die temperaturabhngige Inaktivierungskinetik ist bei 46C bi-phasisch. Der Phage hat eine Latenzzeit von 90 bis 100 min, eine rise period von 2 Std und eine Wurfgre von 10–12.Anhand von Ultradnnschnitten konnte gezeigt werden, da die intracellulre Phagenentwicklung im peripheren Cytoplasma abluft. Durch Fluorescenzfrbung und Einbau von 3H-Thymidin konnte 2-DNS als Phagennucleinsure bestimmt werden.Isolation and enrichment of the Rp1-bacteriophage of Rhodopseudomonas palustris, strain 1 e5, resulted in a yield of 10 to 20% of the total amount of phage particles in the lysate and a titer of about 109 pfu/ml. The high lost of phage particles may be due to an unspecific adsorption to the membranes of the bacterial cell. The bacteriophage Rp 1 is quickly inactivated at a pH lower than 5 and higher than 8.5 and a temperature higher than 30C. The inactivation curve at 46C shows two phases. The phage is rapidly inactivated by chloroform but not by ether. The one-step-growth curve shows a latency period of 90 to 100 min, a rise period of 2 h, and a burst size of 10 to 12 pfu/cell. The development of Rp1 was demonstrated to be in the peripheral region of the cytoplasm. By fluorescence staining and incorporation of 3H-thymidine the nucleic acid of Rp 1 was found to be a double-stranded DNA. The phage is tentatively put into the group C of Bradley.
Cholera bacteriophages have been classified into four distinct groups by Mukerjee (1963a). Of these, the group IV phages can differentiate Vibrio cholerae and V. eltor organisms (Mukerjee, 1963b) and are of practical importance. The hitherto unreported physiological and physico-chemical properties of the group IV cholera phage, φ149, have been investigated by us and the results are presented below.
Cholera bacteriophage φ149 was propagated on the host V. cholerae strain ogawa 154 following in general the method of Hershey, Kalmanson & Bronfenbrenner (1943). The phage stock thus obtained usually contained about 1011 p.f.u./ml. as assayed by double agar layer technique (Adams, 1959). This phage yielded clear plaques of diameter between 0.5 and 1.5 mm. after 16 hr incubation at 37°. Longer incubation produced a surrounding halo. In all cases, bacteria were grown in nutrient broth (NB) containing, in 1 l. of distilled water: bactopeptone (Difco), 10g.; NaCl, 5 g.; beef extract (ACAS, Italy), 10 g.; pH 7.4.
The amount of initiation protein (IP) produced by Clostridium perfringens was increased by approximately one-third by modification of Duncan-Strong sporulation medium to include 0.5% glucose, 2% proteose peptone, and 2% sodium phosphate. IP but no spores were produced by the wild type when grown at 46 degrees C and by a mutant blocked at stage 0 of sporulation. IP appears to be a vegetative cell product, perhaps an overproduced autolysin or pathogenicity factor.
This chapter discusses the physical, chemical, and biological properties of the vibriophages and vibriocins. On the basis of the lytic patterns, vibriophages can be differentiated into four distinct groups: I, II, III, and IV. The vibriophages possess a polyhedral head and a tail. The first step in the interaction between a vibriophage particle and its host cell is specific adsorption. The bacteriocins, in general, were earlier considered as antibiotics with a narrow bacterial host range and of activity depending on their accessibility to suitable surface receptors on the sensitive bacteria. The conditions for vibriocin production are closely related to those for bacteriophage induction. Vibriocin are proteinaceous in nature and are heat resistant. The reaction of vibriocin with a sensitive cell is expected to be biphasic in nature. The first phase involves the adsorption of the particles to specific receptors at the cell surface (adsorption phase). This is followed by the second phase when pathological changes leading to death of the cell (lethal phase) occurs. At the molecular level, vibriocin inhibits DNA synthesis in sensitive cells and promotes DNA degradation leading to the leakage of nucleotides. The vibriophages and vibriocins find its application in typing of bacteria and several therapeutic and prophylactic uses.
The DNA synthesized in the nonpermissive host by the noncomplementing mutants am36 and am42 of bacteriophage phi W-14 contains about half the wild-type level of alpha-putrescinylthymine (putThy) and a correspondingly greater level of thymine. The mechanisms whereby thymine nucleotides are excluded from replicating DNA are functional in both mutants because neither of them incorporates exogenous thymidine into DNA. It is proposed that (i) in wild-type phi W-14, the conversion of hydroxymethyluracil to putThy at the polynucleotide level is sequence specific, but that to thymine is nonspecific; and (ii) in the mutants, the sequence-specific recognition is impaired so that more thymine and less putThy are formed. The thymine-rich DNA can be packaged into phage particles. In the case of am42, the phage particles are morphologically indistinguishable from and have essentially the same polypeptide composition as wild-type particles. However, the DNA molecules they contain are about 11% shorter than those in wild-type phage, am42rev4, a revertant of am42, contains DNA with about 70% of the normal level of putThy; these molecules are about 3% shorter than wild-type DNA. The properties of am42 and am42rev4 are consistent with the suggestion that putThy facilitates the very tight packing of phi W-14 DNA (Scraba et al., Virology 124:152-160, 1983). It also appears that the putThy content of phi W-14 DNA can be reduced by no more than 30% without adversely affecting the production of viable progeny; for example, the burst size of am42rev4 is about 25% of that of the wild type.
The latent period of bacteriophage phi W-14 is approximately 65 min when the doubling time of its host, Pseudomonas acidovorans, is 85 min. Host protein synthesis is shut off relatively slowly, stopping approximately 25 min after infection. There are several phases of phage-specific polypeptide synthesis during the latent period: early polypeptides appear within 10 min after infection; middle polypeptides start to appear between 10 nd 30 min; late polypeptides appear after 30 min. The lengths of time for which individual polypeptides are synthesized vary widely. Several late polypeptides do not appear in the virion. DNA replication is not required for late gene expression. The hypermodified pyrimidine, alpha-putrescinylthymine, appears not to be required in both strands of the DNA duplex for transcription.
Of 42 amber mutants of bacteriophage phi W-14, 6 were defective in DNA synthesis. Three of the mutants synthesized DNA in the nonpermissive host, but were defective in post-replicational modification of the DNA. The DNA synthesized by two of these mutants, am36 and am42, contained more thymine and less alpha-putrescinylthymine than did wild-type DNA; that synthesized by the third mutant, am37, contained the normal amount of thymine, no alpha-putrescinylthymine, and hydroxymethyluracil. The properties of these mutants suggested that the presence of the normal amount of alpha-putrescinylthymine in phi W-14 DNA was essential for the production of viable progeny. Three of the mutants, am6, am35, and am45, failed to synthesize DNA in the nonpermissive host. These mutants were analogous to the DNA off mutants of T4. Nonpermissive cells infected with DNA off mutants accumulated dATP, dGTP, dCTP, and hydroxymethyl dUTP, but not dTTP or alpha-putrescinyldeoxythymidine triphosphate, confirming that both thymine and alpha-putrescinylthymidine in phi W-14 DNA are formed from hydroxymethyluracil at the polynucleotide level. The synthesis of phi W-14 DNA is unusual because (i) thymine is formed from hydroxymethyluracil at the polynucleotide level, (ii) the hypermodification forming alpha-putrescinylthymine is essential, and (iii) thymine and alpha-putrescinylthymine must be made in the correct proportions. Complementation tests showed that the mutants defined three genes involved in DNA polymerization and two genes involved in post-replicational modification.
Viruses have been found with many prokaryotes and eukaryotes. This paper is one of a series of taxonomical reports which attempts to classify prokaryotic viruses into workable groupings. Part 7 of Bergey's Manual, devoted to gram-negative aerobic rods and cocci, includes the following genera for which bacteriophage have been reported: Pseudomonas, Xanthomonas, Azotobacter, Rhizobium, Agrobacterium, Methylomonas, Halobacterium, Alcaligenes, Acetobacter, Brucella, Bordetella and the no longer taxonomically valid Hydrogenomonas. The phages surveyed in this review included only the tailed phages of Pseudomonas and the related genera Xanthomonas and Alcaligenes, as well as phages of the former genus Hydrogenomonas. Strains of this genus now belong to Pseudomonas and Alcaligenes. In addition, we included 19 phages from the former genus Achromobacter which included incompletely described strains of Pseudomonas, Alcaligenes and Comamonas. Our aim was to arrange these phages into the equivalent of 'species' and to propose 'type viruses' for each group. This was done to facilitate comparisons between known phages and new isolates. In addition, the proposal of species and type viruses should further the preservation of culture collections. In some cases, it was impossible to establish groups with reasonable certainty, but we then selected well-known isolates which were suitable for comparison.
Ten Pseudomonas phage were isolated by sewage enrichment. Five psychrophilic and five mesophilic phage were selected for a description of some of their biological properties. In addition to growth on psychrophilic hosts, the psychrophilic phage studied were also able to grow on a mesophilic host within its growth temperature range. Latent periods for psychrophilic phage at 3.5 C were 6 to 12 hr and at 25 C were 30 to 60 min. Mesophilic phage had a latent period of 85 to 190 min at 25 C and 35 to 85 min at 37 C. Psychrophilic phage were significantly more heat-sensitive than the mesophilic phage. Of all the parameters studied, only thermal sensitivity correlated with growth at 3.5 C. Phage used in this study had a deoxyribonucleic acid base composition ranging from 39.6 to 68.2% guanine plus cytosine, deduced from melting temperature measurements.
A study has been made of the effect of bactericidal agents on the phage adsorption properties of Phase II. Sh. sonnei. The ability of this microorganism to adsorb T(3), T(4), and T(7) phages can be altered by treating the bacteria with chemical and physical agents.
Feary, Thomas W. (Tulane University School of Medicine, New Orleans, La.), Earl Fisher, Jr., and Thelma N. Fisher. Isolation and preliminary characteristics of three bacteriophages associated with a lysogenic strain of Pseudomonas aeruginosa. J. Bacteriol.
87
196–208. 1964.—Three bacteriophages designated 7v, 7m, and 7s were isolated from a lysogenic strain of Pseudomonas aeruginosa designated Ps-7. The three viruses were found to be completely unrelated on the basis of plaque morphology, host range, serology, ultraviolet induction, sensitivity to heat, and particle morphology as revealed by electron microscopy. In addition, it was shown that the three phages were incapable of plaque formation on bacteria other than various strains of P. aeruginosa. Of the three phages, only phage 7v was capable of plaque formation on strain Ps-7. The growth of phage 7v on strain Ps-7 exhibited properties which suggest that this virus arises as the result of mutation in a temperate phage for which strain Ps-7 is lysogenic. Phages 7m and 7s are incapable of plaque formation on strain Ps-7, but are adsorbed at characteristic rates to cell suspensions of strain Ps-7. The relationship between phage 7m and strain Ps-7 was shown to meet the classical criteria for lysogeny. Because phage 7s contains ribonucleic acid as its nucleic acid component, it was concluded that its production by strain Ps-7 and the demonstration of immunity of strain Ps-7 to infection by phage 7s were not sufficient evidence to define the nature of the relationship between phage 7s and P. aeruginosa strain Ps-7. It was observed that under certain conditions the infectious titer of phage 7s preparations are markedly reduced in the presence of ribonuclease.
From a stock of varkappa phage grown on Salmonella, a host-range mutant which attacks Escherichia coli was isolated. As in the case of Salmonella, only motile strains of E. coli are sensitive to varkappa. The phage shows an eclipse period of 35 min and a minimal latent period of 52 min. The adsorption rate constant is 3 x 10(-9) ml/min. Adsorption shows a marked dependence on temperature. Bacteriophage varkappa was purified by differential centrifugation and CsCl density gradient centrifugation. It contains deoxyribonucleic acid (DNA) which is double-stranded. The DNA has a molecular weight of 42 million and a guanine plus cytosine content of 57%. Of 68 molecules of DNA inspected, 7 were circular. The phage particle weight is about 90 million.
Twelve lytic Pseudomonas aeruginosa bacteriophages have been characterized in detail. All phages were resistant to osmotic shock and stable from pH 5.0 to pH 11.0. Ultraviolet light of germicidal wavelength inactivated 80% of each strain within 75 seconds. Heating at 70° inactivated all but five “heat-resistant” strains. Studies on a representative heat-sensitive and heat-resistant strain indicated that this inactivation was not affected by the ionic environment of the heating medium. Adsorption and growth rates were studied in tryptic soy broth at 37°. Adsorption rates were low, with K values ranging from 1.2 to 33.0 × 10−11 ml−1. One-step growth curves showed phages to have latent periods of either 35–40 min or 65–70 min and burst sizes ranging from 10 to 200 PFU per infected bacterium.Phages contained DNA at concentrations of 1.1 to 4.6 × 10−16 g/PFU and RNA at concentrations less than 10−17 g/PFU. Each DNA was composed of the four major DNA bases, and purine to pyrimidine ratios indicate that each is double-stranded. The guanine-cytosine molar ratio determined by ultracentrifugation and by spectrophotometric analysis ranged from 46% to 63%, several phages having similar ratios.Serological relations were determined by two independent methods and results indicate that there are two serological subgroups plus three serologically unrelated phages.
Procedures are described for the isolation in pure form of the bacterial virus PM2. This virus isolated from seawater is shown to contain lipids by its sensitivity to ethyl ether and by chemical analysis of the purified virus.The virus has a buoyant density in CsCl of 1.28 g cm−3 and a sedimentation constant in 1 M NaCl of 230 S; it contains 14% by weight of DNA and a minimum of 10% by weight of lipids. Phosphatidylethanolamine is the main lipid present in weight percent. The virus is disrupted in 0.05% Sarkosyl or sodium dodecyl sulfate. Analysis in acrylamide gel electrophoresis indicates the presence in the virus of at least two proteins, probably lipoproteins since they can be stained with Black Sudan.The virus appears to be a polyhedral object 60 mμ in diameter; the coat seems to have an unusual structure, in that an apparent double-layered wall can be observed in the electron microscope.
Roberts, F. F., Jr. (University of Maryland, College Park), and R. N. Doetsch. Some singular properties of bacterial flagella, with special reference to monotrichous forms. J. Bacteriol.
91
414–421. 1966.—Heat (60 C for 30 min), 10 m acetamide, and 8 m urea all brought about rapid and complete dissolution of flagella from monotrichous bacteria; hence, these flagella respond similarly to those of peritrichous forms. Chloramphenicol (10³ μg/ml) inhibited regeneration of flagella in all peritrichously flagellated cultures; however, monotrichous forms were able to regenerate their flagella in a concentration 10² times that required to inhibit multiplication. Peritrichous bacteria did not synthesize flagella when infected by lytic bacteriophages. In these experiments, the time from infection to lysis was sufficient for uninfected controls to resynthesize their flagella. Monotrichous bacteria, however, in all but one instance, were able to resynthesize their flagella before lysis occurred. A study of flagella resynthesis in a non-nutritive milieu indicated that only a small amount of flagellum precursor is present in any given cell. The effect of temperature on synthesis of flagella indicated that, although some bacteria multiply and are motile at a given temperature, they are unable to resynthesize their flagella at that same temperature. This strongly suggests that initial flagellum synthesis and flagellum regeneration are not necessarily identical processes.
Bacteriophages treated with NaClO4 release their DNA. The rate of release depends upon NaClO4 concentration and varies with phage type. There are apparently two mechanisms of release: (1) at low perchlorate concentrations the DNA is injected through the phage tail; (2) at high concentration the phage head is disrupted. For several phages examined only 50% of the particles will release by the injection mechanism. It has also been found that in some situations the DNA is injected but remains attached to the phage ghost by means of an unknown bond which is thermally labile and broken by high perchlorate concentrations. In all cases the perchloratereleased DNA is indistinguishable from DNA released by more standard methods.
Lee, Lucy F. (Michigan State University, East Lansing), and J. A. Boezi. Characterization of bacteriophage gh-1 for Pseudomonas putida. J. Bacteriol.
92
1821–1827. 1966.—Bacteriophage gh-1 of Pseudomonas putida A.3.12 was isolated and purified by differential centrifugation and diethylaminoethyl (DEAE) cellulose chromatography. An electron micrograph of the phage stained with uranyl acetate revealed a regular hexagonal outline about 50 mμ across with a short wedge-shaped tail attached at one corner of the head. The phage formed 10% as many plaques on P. putida C1S as on P. putida A.3.12, the organism used in the isolation procedure. No plaques were formed on P. fluorescens (ATCC 9712) or P. aeruginosa. The latent period of the infectious cycle was 21 min, and the average burst size was 103. The nucleic acid component of gh-1 is double-stranded deoxyribonucleic acid (DNA), with a base composition of 57.0% guanine plus cytosine (G + C) as determined by chemical analysis. The per cent G + C of P. putida A.3.12 DNA measured in a similar manner was 63.7%. The buoyant density of phage gh-1 measured by cesium chloride equilibrium centrifugation was 1.45 g/cm³, whereas that of gh-1 DNA, heat-denatured gh-1 DNA, and P. putida A.3.12 DNA was 1.716, 1.730, and 1.722 g/cm³, respectively. The per cent G + C of gh-1 DNA and P. putida A.3.12 DNA calculated from the buoyant densities was 57.1 and 63.3%, respectively. The sedimentation coefficients, S⁵⁰20,w, of gh-1 and the phenol-extracted gh-1 DNA, measured by the boundary sedimentation velocity method, were 460 and 18.9, respectively. The molecular weight of phenol-extracted gh-1 DNA, calculated by use of the equation of Burgi and Hershey, is 6 × 10⁶.
A collection of 267 strains, representing many of the principal biotypes among aerobic pseudomonads, has been subjected to detailed study, with particular emphasis on biochemical, physiological and nutritional characters. A total of 146 different organic compounds were tested for their ability to serve as sources of carbon and energy. Other characters that were studied included : production of extracellular hydrolases; nitrogen sources and growth factor requirements H-chemolithotrophy; denitrifying ability; pigment production; ability to accumulate poly-p-hydroxybutyrate as a cellular reserve material; biochemical mechanisms of aromatic ring cleavage; and nature of the aerobic electron transport system. The resultant data have revealed many hitherto unrecognized characters of taxonomic significance. As a consequence, it has become possible to recognize among the biotypes examined a limited number of species which can be readily and clearly distinguished from one another by multiple, unrelated phenotypic differences.
Kelner (1949), working with conidia of Streptomyces griseus, discovered that light belonging to the visible range is capable of reactivating biological material that has been rendered inactive by ultraviolet radiation (UV). Shortly after Kelner's discovery was known, a similar phenomenon in bacteriophages (bacterial viruses) was observed by accident. Plates of nutrient agar containing UV-inactivated phage and sensitive bacteria had been left for several hours on a table illuminated by a fluorescent lamp. After incubation it was noticed that the number of plaques was higher on these plates than on similar plates incubated in darkness. A short report of this phenomenon of "photoreactivation" (PHTR) has already been published (Dulbecco, 1949). The present paper contains the results of a first group of experiments concerning PHTR of seven bacteriophages of the T group active on Escherichia coli, strain B.
Effect of NaC104 on bacteriophage: release of DNA and evidence for popula-tion heterogeneity Characterization of bacteriophage gh-I for Pseudomonas putida
- D Freifelder
- L F Lee
- Boezt
FREIFELDER, D. (1966). Effect of NaC104 on bacteriophage: release of DNA and evidence for popula-tion heterogeneity. Virology 28, 742. LEE, L.F. ~ BOEZt, J.a. 0966). Characterization of bacteriophage gh-I for Pseudomonas putida
Purification and chemistry of bacteriophage X The aerobic Pseudomonads: a taxonomic study
- S Z Schade
- S Adler
- R V Stanier
- N S Palleroni
- M Doudoroff
SCHADE, S. Z. & ADLER, S. 0967). Purification and chemistry of bacteriophage X. Journal of Virology i, 591 • STANIER, R. V., PALLERONI, N. S. & DOUDOROFF, M. 0966). The aerobic Pseudomonads: a taxonomic study. Journal of General Microbiology 4:3, 159. (Received I7 July I969) 7-2
Studies on psychrophilic bacteriophages infectious for Pseudomonas putrefaciens
- R E Levln
- L Delisle
LEVlN, R. E. & DELISLE, a. L. (1969). Studies on psychrophilic bacteriophages infectious for Pseudomonas
putrefaciens. Bacteriological Proceedings p. 158.
Ultrastructure of bacteriophages and bacteriocins
BRADLEY, I). E. (1967). Ultrastructure of bacteriophages and bacteriocins. Bacteriological Reviews 3 r,
23 °.