Studies on the Mechanism of Transduction by Bacteriophage ?_entitystart_#947_entit I. Genetic Characterization of the Transducing Segment

The bacteriophage Φγ, though related to the lambdoid phage ϕ80, has unusual features in its specialized transduction and is being investigated to determine the mechanism of the transduction process. Genetic analysis of the transducing element gives evidence for a relatively long and uniform linear segment, up to about 1% of the E. coli chromosome, extending in either direction from the prophage attachment site, e.g., on the right side: att80-tonB-trpABCDE-cysB-pryF. The att end includes a variable amount of phage genome, probably very short in most particles. In a small fraction of the transducing particles the phage segment may be more extensive and, conversely, the bacterial segment is shorter, ending around cysB. The transducing segment from modificationless bacteria carries a site susceptible to the K-restriction system which affects the efficiency of transduction.     

Effects of Furazolidone on the Infection of Vibrio cholerae by Bacteriophage φ149

Furazolidone in concentrations which had little effect on the growth of host organisms greatly reduced the yield of phage 149 from the host Vibrio cholerae OGAWA 154. This phage was resistant to the in vitro action of the drug. The phage yield of infected bacteria depended significantly on the time of addition or withdrawal of the drug. The average burst size of the drug-treated and infected bacteria decreased exponentially with increase in drug concentration. The latent period of phage multiplication and also the eclipse period did not change significantly from the control values. A concentration of 0.05 μg of furazolidone per ml inhibited DNA synthesis by about 50% in phage-infected cells and only by about 18% in noninfected ones, relative to the respective controls. RNA and protein synthesis were affected by a much smaller degree both in infected and noninfected cells. Quantitative deduction of the length of furazolidone-treated cells from their phage adsorption characteristics and its agreement with previous electron microscopy data indicated that furazolidone did not affect the phage receptors.     

Studies on the Decomposition of Raffinose by α-Galactosidase of Actinomycetes

α-Galactosidase was isolated from the culture broth of Streptomyces olivaceus and was partially purified by chromatography on a DEAE-sephadex column. The optimum pH of the preparation was found to be 5.2 for raffinose and the preparation was inactivated completely by maintaining it at 60°C for 15 minutes. p-Chloromercuribenzoate, HgCl₂ and AgNO₃ caused complete inhibition of the enzyme activity at 2 × 10^?5 M concentration. The preparation showed transglycosylase activity. A sugar spot, chromatographically identical with that of stachyose, appeared in the digest of raffinose. However, the preparation hydrolyzed raffinose completely into galactose and sucrose after a prolonged incubation.

A simple raffinose estimation method was developed using the enzyme preparation, and it was found that the method allowed to estimate 125~500 μg of raffinose with an accuracy of ±5%. The method was applied to the estimation of raffinose in beet molasses.     

The Mechanism of Inactivation of Bacteriophage ϕX174 by Autoxidizable Synthetic Polysaccharides

Autoxidizable synthetic polysaccharides prepared by polycondensation of reducing aldose or ketose in dimethyl sulfoxide containing pohsphorus pentaoxide [Polymer, 13, 190 (1972)] inactivated phage ?X174. Another autoxidizable polysaccharides obtained by oxidation of natural glucans with the same oxidant also inactivated ?X174. The ?X174 inactivation was due to strand scission of viral DNA in the virion. The inactivation reaction was stimulated by Cu²⁺ and inhibited by EDTA, Superoxide dismutase, catalase and several radical scavengers. These results suggest that oxygen radicals produced during autoxidation of polysaccharides are responsible for ?X174 inactivation.     

Studies on type II glycogenosis. Effects of cortisone derivatives on acid α-glucosidase

Cortisone causes a marked increase in the activity of liver acid alpha-glucosidase 2h after injection into male Wistar rats. Studies on rat liver tissue slices, isolated lysosomes and cultured skin fibroblasts have demonstrated similar elevations of acid alpha-glucosidase activity after incubation with cortisone. Cortisone-treated human liver tissue, obtained by needle biopsy, also shows an increase in acid alpha-glucosidase activity. Neutral alpha-glucosidase activity was not stimulated by cortisone in vivo or in liver slices.     

Studies on the In Vitro Assembly of Bacteriophage φ80 and φ80-Lambda Hybrids

Suppressor-sensitive (sus) mutants of bacteriophage 80 defective in late functions were classified, by means of in vitro assembly tests, into two complementation groups: head donors and tail donors. Each group of mutants was subdivided, by means of two-factor crosses, into six cistrons. Deletion mapping revealed clustering of tail and also of head cistrons. The two clusters were located in the left arm of vegetative 80 (the tail specifying cluster being distal). In vitro cross complementation between 80 and lambda sus mutants revealed that whereas lambda heads could quite efficiently bind 80 tails to form viable phage, the union of 80 heads and lambda tails was very much less efficient. Deletion mapping of the 80 sus mutants, using both 80 and i⁸⁰h^λ deletion lysogens indicated congruent gross gene arrangement in the two related bacteriophages.     

Studies on the Biosynthesis of α-Putrescinylthymine in Bacteriophage φW-14-Infected Pseudomonas acidovorans

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.     

Studies with Bacteriophage φII. Events Following Infection of Male and Female Derivatives of Escherichia coli K-12

We studied the course of infection of the female-specific bacteriophage phiII in male and female cells isogenic except for the presence of the substituted sex factor, F'lac. Both male and female cells are killed by phiII; however, only limited phage replication occurs in male cells. Host macromolecular synthesis stops abruptly at 4 to 6 min after infection of male cells, and synthesis of phage components cannot be detected. Experiments with chloramphenicol indicate that phage deoxyribonucleic acid (DNA) penetrates into male cells, since protein synthesis after infection is required to stop synthesis of DNA in males. Phage DNA becomes membrane-associated in both female and male cells. In male cells, parental phage DNA does not dissociate from the membrane during the latent period as is the case with females, indicating a block in phage DNA replication. Isolation of nonrestricting F'lac mutations indicates involvement of a specific episome product in phiII restriction.     

Biochemical Studies on the χ Mutation of Bacteriophage T4: Differential Inhibition of χ+ and χ DNA Synthesis by Mitomycin C

Biochemical studies were carried out to determine the effect of chi mutation on T4 DNA synthesis. The rate and final extent of DNA synthesis are almost the same with T4D- and T4chi-infected cells, although the burst size of T4chi is about one-sixth that of the wild type. The DNA synthesis of T4chi-infected cells is more readily inhibited by mitomycin C than is that of T4 wild type. When mitomycin C was added during active phage growth, DNA synthesis of T4chi halted almost immediately. T4 DNA polymerases isolated from chi(+)- and chi-infected cells, however, exhibit no difference with regard to their sensitivities to mitomycin C, priming activities with alkylated or ultraviolet light-irradiated templates and other enzymatic properties.     

Cold-Sensitive Mutants of Bacteriophage φX174. II. Comparison of Two Cold-Sensitive Mutants

Cold-sensitive bacteriophage phiX174 mutants, another class of conditional lethals, were examined with regard to growth parameters, DNA synthesis, and particle properties. Two mutants, cs70 and cs82, were examined. Mutant cs70 was eclipse defective, showing altered eclipse kinetics at permissive temperature (40 C) and failing entirely to eclipse at restrictive temperature (25 C). Mutant cs70 replicated well at 25 C if allowed prior eclipse at 40 C. Mutant cs82 had wild-type eclipse at both temperatures but was defective in single-strand synthesis at 25 C, which led to delayed progeny phage appearance, decreased progeny phage synthesis rate, and greatly reduced burst size. The cs82 block could not be bypassed by temperature shift. Since complementation analysis of cs70 and cs82 was not feasible due to the unique properties of these mutants, those phiX174 properties affected by the virus coat were examined as an index of a mutation in a coat protein gene. Mutant cs70 had aberrant attachment kinetics at both 25 C and 40 C, evidence of a coat protein alteration. Mutant cs70 also exhibited significantly decreased thermal stability, further evidence of an altered virus structure. Mutant cs82 had increased thermal stability, but the difference was not sufficient to allow unequivocal assignment of this mutant to a coat protein gene. Both mutants had wild-type antiserum inactivation and host range, although cs70 was subject to less of (low-level) plating restriction by endogenous F(+) factors.     

Mechanism of Adsorption and Eclipse of Bacteriophage φX174 II. Attachment and Eclipse with Isolated Escherichia coli Cell Wall Lipopolysaccharide

A mixture of aqueous phenol, choloroform, and ether extracts the lipopolysaccharides (LPS) from the phiX174-sensitive strain, Escherichia coli C/1, and resistant strains, C/phiX and K12. Interaction of the C/1 LPS with phiX in a starvation buffer containing 10(-3) M CaCl(2) at 37 C, but not at 15 C, results in a first-order inactivation that is specific for C/1 LPS. After interaction for 60 min at 15 C, followed by centrifugation, 37 and 20% of a (14)C-phiX preparation are bound to the C/1 and C/phiX LPS pellets, respectively. The results for intact cells are 75 and 10%. Supporting the conclusion that this represents specific attachment of phiX to its receptor site in the LPS is the fact that EDTA-borate buffer is required to elute 85% of the (14)C-phiX from the C/1 LPS, whereas starvation buffer elutes the same amount from C/phiX LPS. Moreover, 95% of the PFU are found in the C/1 LPS pellets as compared with 50% in the resistant strain LPS pellets. When the products of interaction between phiX and LPS at 37 C are examined by sucrose density gradients in EDTA-borate, a single 60 to 90S peak is observed in the C/1 sample, and the single peak cosediments with the 120S marker phiX in the C/phiX sample. This change in S(20, w) is very similar to that reported for the eclipse of phiX in vivo. If the inactivation at 37 C is carried out on phiX-LPS complexes first formed at 15 C, the first-order kinetics are biphasic and nearly identical to that observed for the eclipse kinetics of phiX attached to intact cells. Thus, the phiX-LPS system is suitable for in vitro studies on the early events in phiX infection.