Isolation and Characterization of Two Basic Internal Proteins from the T-Even Bacteriophages

Two species of basic internal proteins were found in osmotic shock supernatant solutions of bacteriophages T4B, T4D, T2H, T2L, and T6. The major species of protein isolated had a molecular weight of approximately 21,000 daltons, whereas the minor protein molecular weight was near 9,500 daltons. The two protein species exhibited unique isoelectric points and amino acid compositions. The 21,000-dalton protein of T2L showed major electrophoretic and compositional differences from the other 21,000-dalton proteins isolated. Similarities between the 21,000-dalton proteins and phage lysozyme are discussed.     

Head Proteins from T-Even Bacteriophage: I. Molecular Weight Characterization 1

T-even bacteriophage capsid proteins were separated on 6% agarose columns by use of 6 m guanidine hydrochloride containing 5 mm dithiothreitol both to dissociate and to elute the proteins. The head capsids of T2H, T4B, T4B01, T4D, and T6r(+) contained at least three structural proteins with molecular weights of 40,000, 18,000, and 11,000 daltons, amounting to 76, 2, and 8%, respectively, of the total capsid protein. On the other hand, T2L head capsids contained only two structural proteins with molecular weights of 40,000 and 18,000 daltons (81 and 2.5%, respectively, of the total protein). A discussion of the possible role of these structural head proteins and a T-even phage head model suggesting a structural arrangement of the 40,000 dalton subunit are presented.     

Characterization of T-Even Bacteriophage Substructures: II. Tail Plates

Tail plates obtained from T4D amber mutants were examined with respect to sedimentation behavior, subunit molecular weights, amino acid composition, isoelectric points, and morphology. Intact plates had an S_20,w of 77S from pH 5 to 9. The only conformational change noted was that below pH 5 tail plates readily dimerized yielding vis-à-vis dimers with an S_20,w of 124S. Dissociated plates consisted of three major proteins with molecular weights of 53 K ± 5, 31 K ± 3, and 17 K ± 2 daltons. The amino acid analyses indicated that plates had a composition distinct from fibers and tubes and were relatively rich in tryptophan. Degradation studies with dimethyl sulfoxide (DMSO) indicated that tail plates had a unique biological structure. After treatment with DMSO, and to some extent without DMSO, or from lysates of defective mutants, tetrad structures were observed in the electron microscope. These structures had an amino acid content and relative amounts of types of subunits similar but not identical to intact plates. It was proposed that plates were composed of nine such tetrads giving rise to a structure with six- and threefold symmetry.     

Bacteriophage Tail Components: I. Pteroyl Polyglutamates in T-Even Bacteriophages

A pteroylpolyglutamate has been found to be a constituent of all Escherichia coli T-even bacteriophages and has been characterized with regard to its oxidation state, molecular weight, origin, and location on the phage particle. The phage compound has been shown to be a dihydropteroyl penta- or hexaglutamate on the basis of its chemical and physical properties. Analyses of extracts of uninfected and T2L-infected E. coli have indicated that the phage dihydropteroyl polyglutamate was present only in infected cells. Its synthesis was sensitive to the addition of chloramphenicol before infection, and the compound appeared to be specifically induced by phage infection. Analyses of isolated phage ghosts and tail substructures have shown that each phage particle contains between two and six phage-specific pteroyl derivatives and that the juncture of the phage tail plate with the tail tube is the most likely site of binding of the phage-induced pteroyl compound.     

Isolation and Characterization of Bacteriophages from Fermenting Sauerkraut

This paper presents the first report of bacteriophage isolated from commercial vegetable fermentations. Nine phages were isolated from two 90-ton commercial sauerkraut fermentations. These phages were active against fermentation isolates and selected Leuconostoc mesenteroides and Lactobacillus plantarum strains, including a starter culture. Phages were characterized as members of the Siphoviridae and Myoviridae families. All Leuconostoc phages reported previously, primarily of dairy origin, belonged to the Siphoviridae family.     

Isolation and Characterization of Campylobacter Bacteriophages from Retail Poultry

The ability of phages to survive processing is an important aspect of their potential use in the biocontrol of Campylobacter in poultry production. To this end, we have developed a procedure to recover Campylobacter bacteriophages from chilled and frozen retail poultry and have validated the sensitivity of the method by using a characterized Campylobacter phage (i.e., NCTC 12674). By using this method, we have shown that Campylobacter phages can survive on retail chicken under commercial storage conditions. Retail chicken portions purchased in the United Kingdom were screened for the presence of endogenous Campylobacter phages. Thirty-four Campylobacter bacteriophages were isolated from 300 chilled retail chicken portions, but none could be recovered from 150 frozen chicken portions. The phage isolates were characterized according to their lytic profiles, morphology, and genome size. The free-range products were significantly more likely to harbor phages (P < 0.001 by single-factor analysis of variance) than were standard or economy products. This study demonstrates that Campylobacter bacteriophages, along with their hosts, can survive commercial poultry processing procedures and that the phages exhibited a wide range of recovery rates from chicken skin stored at 4°C.     

Characterization of T-Even Bacteriophage Substructures: I. Tail Fibers and Tail Tubes

T-even bacteriophages were grown and purified in bulk quantities. The protein coats were disrupted into their component substructures by treatment with 67% dimethyl sulfoxide (DMSO). Tail fibers and tubes were purified on glycerol-CsCl-D(2)O gradients and examined with respect to sedimentation properties, subunit molecular weights, amino acid composition, isoelectric points, and morphology. It was found that intact tail fibers had a sedimentation coefficient of 12 to 13S and that dissociated fibers consisted of three classes of proteins having molecular weights of 150 K +/- 10, 42 K +/- 4, and 28 K +/- 3 daltons. A model was constructed in which the 150-K subunit folded back on itself twice to give a three-stranded rope. Each 150-K subunit then represented a half-fiber and it was proposed that the role of the 42- and 28-K subunits was to hold each half-fiber together as well as serve as a possible link with other substructures. Isoelectric point studies also indicated that there were three different proteins with pI values of 3.5, 5.7, and 8.0. Amino acid analyses indicated that fibers had a composition distinct from other phage substructures. In addition, a striking difference was noted in the content of tryptophan among the phages examined. T4B had three to five times more tryptophan than did T2L, T2H, T4D, and T6. Intact tail tubes had an S(20,w) of 31 to 38S and dissociated tubes consisted of three proteins of molecular weights 57 K +/- 5, 38 K +/- 4, and 25 K +/- 3 daltons. Based on degradation studies with DMSO, it was proposed that these three proteins were arranged in a helical array yielding the tube structure. Isoelectric point studies indicated that there were three major proteins in the tube whose pI values were 5.1, 5.7, and 8.5. No significant differences were observed in the amino acid content of tubes obtained from all the T-even bacteriophages.     

Escherichia coli Capsule Bacteriophages II. Morphology

The Escherichia coli capsule bacteriophages (K phages) described herein are specific for certain capsular strains of E. coli, all of them test strains for different E. coli K antigens. The phages are not adsorbed to the acapsular mutants of their host organisms nor to similar strains with serologically and chemically different capsular polysaccharides. Thirteen E. coli (and one Klebsiella) K phages were visualized in the electron microscope. Most viruses are similar to P22 and thus belong to Bradley group C; however, one each of group A (long, contractile tail) and group B (long, noncontractile tail) was also found. All K phages were seen to carry spikes but no tail fibers were detected. These results suggest that the structures responsible for the recognition of the thick (about 400 nm or more) capsular polysaccharide gels are located in these spikes.     

T-Even Bacteriophage-Tolerant Mutants of Escherichia coli B II. Nucleic Acid Metabolism

T-even bacteriophage-tolerant mutants are strains of Escherichia coli which can adsorb T-even phages but cannot support the growth of infective virus. Under some conditions, the infected cells are not killed. Mutant cells infected by phage T6 are able to carry out several metabolic functions associated with normal virus development, including arrest of bacterial nucleic acid and protein synthesis, incorporation of isotopic precursors into viral nucleic acids and proteins, synthesis of early enzymes of deoxyribonucleic acid (DNA) metabolism, formation of rapidly sedimenting DNA intermediates, and formation of normal levels of early and late messenger ribonucleic acid species. Phage are unable to mutate to forms capable of growth on these mutants. The nature of the biochemical alteration leading to tolerance is still unknown.     

Escherichia coli K Bacteriophages I. Isolation and Introductory Characterization of Five Escherichia coli K Bacteriophages

A set of five Escherichia coli K phages has been isolated. These phages are adsorbed to and lyse the capsular forms of the host bacteria, whereas their spontaneous, acapsular mutants are not affected. All host strains are heavily encapsulated test strains for E. coli K antigens of the thermostable A type and they readily segregate acapsular mutants. In four of the phage-host systems, all secondary growth obtained was found to be acapsular. When tested for host-range mutants on 38 strains of E. coli and Klebsiella, less than one mutant per 10(5) plaque-forming units was found. No cross-reacting neutralizing antibodies were obtained when rabbits were immunized with the K phages. The latent periods (between 16 and 30 min) and average burst sizes (between 145 and 580) were determined by one-step growth experiments.     

Characterization of Salmonella Bacteriophages Isolated from Swine Lagoon Effluent

Four Salmonella bacteriophages that had been originally isolated from swine manure lagoons were characterized and compared to each other and to well-known Salmonella phages P22 and Felix 01. Host ranges of the lagoon phages were similar to each other in spot tests on reference strains of Salmonella, but differed slightly from each other on a panel of Salmonella lagoon strains. In single-step growth at 35°C the lagoon phages had latent periods of 15 to 20 min and burst sizes from 100 to 230. The lagoon phages and P22 were purified by cesium chloride (CsCl) gradient centrifugation and used to produce specific antisera and DNA. The lagoon phages were indistinguishable from each other but distinct from P22 and Felix 01 in immunodiffusion and infectivity neutralization tests and in restriction digest analysis. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee, warranty, or endorsement by the USDA and does not imply its approval to the exclusion of other products that may be suitable.     

T-Even Bacteriophage-Tolerant Mutants of Escherichia coli B: I. Isolation and Preliminary Characterization

A general procedure is described for isolation of T-even phage-tolerant mutants of Escherichia coli. Two such mutants of E. coli B have been examined in some detail. These mutants adsorb T-even phages but are unable to release viable progeny. Under certain conditions, viability of the cells is completely unaffected by phage infection in one mutant, and there is but a slight decrease in colony-forming ability in the other. Phage deoxyribonucleic acid (DNA) is injected into these cells, as shown by the formation of phage-specific enzymes, but it is not degraded to acid-soluble material. Some phage DNA replication occurs in both strains. The mutants are both more resistant to ultraviolet light than is the parent strain.     

Characterization of Two Psychrophilic Pseudomonas Bacteriophages Isolated from Ground Beef

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.     

Chemical and Physical Characterization of Interfacial-Active Lipids from Rhodococcus erythropolis Grown on n-Alkanes

Lipophilic compounds of the culture suspension containing Rhodococcus erythropolis DSM43215 had surfactant properties when the bacteria were cultivated with n-alkanes as the sole carbon source. Thirteen main components from a dichloromethane-methanol extract of the R. erythropolis cultures were isolated and characterized to specify quantitatively their surfactant properties, e.g., minimum surface and interfacial tensions and critical micelle concentrations. The interfacial activity of the organic extract was dominated by α,α-trehalose-6,6′-dicorynomycolates which reduced interfacial tension from 44 to 18 mN/m. Phosphatidylethanolamines which were also present in the organic extract reduced the interfacial tension below 1 mN/m. The trehalose corynomycolates had extremely low critical micelle concentrations in high-salinity solutions, and the interfacial properties were stabile in solutions with a wide range of pH and ionic strength.     

Characterization of the Tumorlike (T) Antigen Induced by Type 12 Adenovirus : II. Physical and Chemical Properties

T antigen, purified from KB cells infected with type 12 adenovirus, was characterized by chemical and physical methods. The immunologically active molecule had an ultraviolet-absorption spectrum characteristic of a protein, and was partially inactivated (i.e., approximately 85%) by trypsin (final concentration of 150 mug/ml). Centrifugation analyses indicated that the purified antigen was a single molecular species with an average sedimentation coefficient of 2.40S (measured by zonal centrifugation in sucrose gradients) and an S(20,w) of 2.58 determined by analytical centrifugation. The antigen was inactivated at temperatures above 40 C, it was stable between pH 5.7 and 8.8, and it had an isoelectric point of approximately 5.0, as determined by electrophoresis on cellulose polyacetate membranes.     

The Chloroplast and Cytoplasmic Ribosomes of Euglena: II. Characterization of Ribosomal Proteins

Cytoplasmic and chloroplast ribosomal proteins were isolated from Euglena gracilis and analyzed on polyacrylamide gels. Cytoplasmic ribosomes appear to contain 75 to 100 proteins ranging in molecular weight from 10,200 to 104,000, while chloroplast ribosomes appear to contain 35 to 42 proteins with molecular weights ranging from 9,700 to 57,900. This indicates that the cytoplasmic ribosomes are similar in composition to other eucaryotic ribosomes, while chloroplast ribosomes have a protein composition similar to the 70S procaryotic ribosome. The kinetics of light-induced labeling of cytoplasmic ribosomal proteins during chloroplast development has been determined, and the results are compared with the kinetics of ribosomal RNA synthesis.     

Isolation and Characterization of Bacteriophages Infecting Staphylococcus epidermidis

Bacteriophages infecting Staphylococcus epidermidis were isolated by mitomycin C induction. Three distinct phages (vB_SepiS-phiIPLA5, vB_SepiS-phiIPLA6, and vB_SepiS-phiIPLA7)—defined by plaque morphology, structure, virion proteins pattern, DNA restriction bands, and host range—were obtained. One-step growth curves of bacteriophages under optimal growth conditions for S. epidermidis F12 revealed eclipse and latent periods of 5–10 and 10–15 min, respectively, with burst sizes of about 5 to 30 PFU per infected cell. Transmission electron microscopy revealed that the phages were of similar size and belonged to the Siphoviridae family. Phage phi-IPLA7 had the broadest host range infecting 21 out of 65 S. epidermidis isolates. Phage phi-IPLA5 seemed to be a virulent phage probably derived from phi-IPLA6. Phages phi-IPLA5 and phi-IPLA7 exhibited increasing plaques surrounded by a halo that could be indicative of a polysaccharide depolymerase activity. Viable counts, determined during the infection of S. epidermidis F12, confirmed that phi-IPLA5 had a potent lytic capability and reduced S. epidermidis population by 5.67 log units in 8 h of incubation; in the presence of the mixture of phi-IPLA6 and phi-IPLA7, however, a reduction of 2.27 log units was detected