Replication of Bacteriophage SH-133 in the Facultative Autotroph Hydrogenomonas facilis I. Bacteriophage Synthesis Under Heterotrophic, Autotrophic, and Mixotrophic Growth Conditions

The ability of bacteriophage SH-133 to replicate in heterotrophically (H-) and autotrophically (A-) grown Hydrogenomonas facilis was examined. Both the synthesis of infectious phage particles and the efficiency of plating (EOP) were reduced by 90% in A-grown cells. Adsorption of phage and lethal effects on H. facilis were identical in both systems. One-step growth experiments showed that cell lysis preceded the appearance of infectious particles in A-grown cells. Burst size studies with mixotrophically grown cells did not indicate the presence of an inhibitor of phage synthesis indigenous to autotrophic metabolism. DNA synthesis was identical in H- and A-grown infected cells; however, protein synthesis was significantly reduced in A-grown infected cells when compared with protein synthesis in H-grown infected cells. The data suggest that the reduction in EOP and phage synthesis in A-grown cells is caused by a defect in viral protein synthesis which results in the limited production of an essential viral protein at the time of cell lysis.     

Deoxyribonucleic Acid Homologies of Some So-Called “Hydrogenomonas” Species

Evidence based on deoxyribonucleic acid homology supports the abandonment of the genus Hydrogenomonas. Pseudomonas facilis (formerly Hydrogenomonas facilis) is closely related to the nonautotrophic species P. delafieldii. P. facilis and Alcaligenes eutrophus (often called H. eutropha) are not related to each other or to other hydrogen bacteria and pseudomonads studied.     

Effect of Growth Conditions on Morphology of Hydrogenomonas facilis and on Yield of a Phospholipoprotein

Hydrogenomonas facilis grown heterotrophically on fructose with very low aeration eventually ceased to divide and produced elongated forms. Short forms were obtained from fructose-grown long forms by increasing the availability of oxygen to the organisms. A phospholipoprotein, the protein moiety of which is known to be present in the cell envelope, precipitated upon lowering the ionic strength of extracts from cells in the earlier stages of elongation (i.e., in the middle and late log phase of growth). The maximal yield of the protein moiety of the phospholipoprotein precipitate (i.e., grams of protein/grams of soluble protein x 100) was 2%. Poly-beta-hydroxybutyric acid accumulated as growth on fructose progressed, the accumulation being more marked with lower aeration.     

Study of lysogeny in Bacillus thuringiensis and B. cereus]

Forty-eight strains of Bacillus thuringiensis and 12 strains of B. cereus were treated with ultraviolet light and mitomycin C. The former agent was the more effective inducer. Bacillus thuringiensis produces at least seven different phage particles with long, non-contractile tails. The frequencies of lysogeny and polylysogeny are 83 and 25% respectively. Morphologically defective phages occur in 25% of strains, whereas five of them produce low molecular-weight bacteriocins. One strain of B. cereus harbors "killer-particles." There is no apparent correlation between the presence of phage-like particles, phage senstivity, and serotypes, biotypes, or the origin of B. thuringiensis strains.     

Autotrophic and Heterotrophic Metabolism of Hydrogenomonas I. Growth Yields and Patterns Under Dual Substrate Conditions

Auxotrophic mutants of Hydrogenomonas eutropha and H. facilis requiring utilizable amino acids were employed to demonstrate the simultaneous utilization of H(2) and an organic substrate for growth. The ratio of the cell yields under dual substrate conditions compared to heterotrophic conditions indicated the relative contributions of the autotrophic and heterotrophic systems to the growth of the organism. Wildtype H. eutropha grown under simultaneous conditions exhibited a dicyclic growth pattern, the first cycle representing either heterotrophic or simultaneous growth and the second cycle representing autotrophic growth. The duration of the changeover period was either very short with no plateau or long with a plateau up to 8 hr, depending upon the organic substrate. The growth rate under simultaneous conditions with some organic substrates was faster than either the autotrophic or heterotrophic rate, but was not the sum of the two rates. The data suggest that, in the presence of both organic and inorganic substrates, heterotrophic metabolism functions normally but autotrophic metabolism is partially repressed.     

Immunologically Cross-Reacting Proteins in Cell Walls of Many Bacteria

Antibodies to a protein present in the cell envelope of Hydrogenomonas facilis agglutinate many gram-negative bacteria and a few gram-positive bacteria. Immunodiffusion studies of extracts from the various organisms tested indicate the presence of components sharing antigenic determinants with the cell envelope protein in all bacteria which can be agglutinated and a few that cannot. Cross-reacting components were not detected in extracts of Mycoplasma laidlawii, Anacystis nidulans, and several eukaryotic organisms.     

Isolation and preliminary characterization of bacteriophage phi-mu-4

Shafia, Fred (University of Nebraska, Lincoln), and T. L. Thompson. Isolation and preliminary characterization of bacteriophage phimu-4. J. Bacteriol. 87:999-1002. 1964.-Bacteriophage phimu-4 was isolated from lysogenic Bacillus stearothermophilus NU strain 4, and was propagated in strain 10 of the same species. The phage was extremely host-specific. One of the 23 strains of thermophiles screened for susceptibility supported phage replication. Plaque-forming efficiency of phimu-4 depended on the agar medium employed and the temperature of incubation. Generally, media which resulted in restricted growth of the host cells on agar plates were most satisfactory for plaque formation. A latent period of 35 min was terminated in cell lysis and release of about 175 plaque-forming units (PFU) per infected cell. The phage particles, propagated in cells cultured in Trypticase (BBL)-yeast extract-dextrose-calcium chloride broth, were routinely removed from the lysate by 0.6 ammonium sulfate saturation in the cold. Phage purification was by ultracentrifugation followed by sucrose density-gradient. The phage particles appeared spherical, in electron micrographs. Particle size determination by electron micrographs indicated a diameter of less than 100 A.     

Isolation and morphology of temperature Agrobacterium tumefaciens bacteriophage

Zimmerer, Robert P. (The Pennsylvania State University, University Park), Robert H. Hamilton, and Christine Pootjes. Isolation and morphology of temperate Agrobacterium tumefaciens bacteriophage. J. Bacteriol. 92:746-750. 1966.-Lysogeny was detected in 14 strains of Agrobacterium tumefaciens among 130 bacterial strains tested with strain B-6 used as the host. Partial lysis was observed with 13 additional bacterial strains. Morphological studies of five strains showed that the phage had similar features. A typical phage (Lv-1) had a polyhedralshaped head, approximately 71 by 63 mmu, and a tail, approximately 211 mmu by 9.5 mmu. The phage nucleic acid was found to be deoxyribonucleic acid. The bacteriophage have been designated L (for lysogenic) followed by the bacterial strain designation in lower case letters.     

Studies on the origin of bacterial viruses. I-IV

I. The Incidence of Phage-Producing Cells in Various B. megatherium Cultures Analyses of small samples containing a few cells each show that lysogenic B. megatherium produces phage particles in groups of from 10 to 1000 depending on the megatherium strain and the culture medium. These groups probably correspond to the number of particles produced by a single cell. The proportion of such phage-producing cells varies from <1 x 10(-10) to about 1 x 10(-2) depending on the megatherium strain and the culture medium. If a culture produces two types of phage, the different types usually appear in separate samples. If mixed samples occur, the number of such samples is about what would be expected for the probability that two separate groups would appear in one sample. This result indicates that the appearance of a distinct phage type is the result of a change in the bacterial cell rather than a change in a phage particle, since in the latter case a mixture of the two types would result. II. The Effect of Ultraviolet Light on the Incidence of Phage-Producing and of Terramycin-Resistant Cells in Various B. megatherium Cultures Low intensity of ultraviolet light increases the proportion of both phage-producing cells and of terramycin-resistant mutants. The increase in phage-producing cells is greater than the increase in terramycin-resistant cells. High intensities of ultraviolet light cause practically all the cells of some B. megatherium strains to produce phage. The number of terramycin-resistant mutants cannot be determined under these conditions. The effect of ultraviolet light varies, depending on the megatherium strain and the culture medium. III. The Effect of Hydrogen Peroxide on the Incidence of Phage-Producing and of Terramycin-Resistant Cells in Various B. megatherium Cultures Low concentrations of hydrogen peroxide increase the number of phage-producing cells and of terramycin-resistant cells, concomitantly, from two to five times. High concentrations of hydrogen peroxide cause almost all the cells of some strains of megatherium to produce phage. IV. Calculation of the Incidence of Phage-Producing Cells The time rate of the appearance of phage particles in normal cultures, or in cultures treated with ultraviolet light or hydrogen peroxide, may be calculated by the same equations which predict the occurrence of terramycin-resistant mutants in B. megatherium cultures. These equations predict that the number of mutants will increase more or less in proportion to the concentration of mutagenic agent, so long as the mutation rate remains very small compared to the growth rate. As the mutation rate approaches the growth rate, there will be a very rapid increase in the proportion of mutants. This explains the striking effect of higher concentrations of mutagenic agents. In order to calculate the results after exposure to strong ultraviolet light or hydrogen peroxide, it is necessary to assume that the change from normal to phage-producing cell occurs without cell division.     

Induktive Bildung partikelgebundener Uricase bei Hydrogenomonas H16 und anderen aeroben Bakterien

Zusammenfassung Induktive Bildung von Uricase (E.C. 1.7.3.3. Urat: O₂-Oxidoreductase) wurde während des Wachstums mit Harnsäure als alleiniger Kohlenstoff-und Stickstoffquelle bei Hydrogenomonas H 16, Micrococcus denitrificans und Pseudomonas aeruginosa beobachtet.Das Enzym wurde in der Partikelfraktion nachgewiesen, die aus Ultraschallextrakten bei 100 000 g sedimentiert.Gewaschene Partikeln aus Hydrogenomonas H 16 verbrauchten 0,45 Mole O₂ je Mol Harnsäure, gemessen in Boratpuffer beim Optimum von pH 9,0. Die Reaktion ist empfindlich gegenüber Cyanid; 4 · 10^-6 m KCN führt zu einer 50%igen Hemmung.

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Inductive biosynthesis of particle-bound uricase in Hydrogenomonas H 16 and other aerobic bacteria

Summary Induced biosynthesis of uricase (E.C. 1.7.3.3 Urate: O₂ oxidoreductase) was observed during growth with uric acid as the only carbon and nitrogen source in strains of Hydrogenomonas H 16, Micrococcus denitrificans and Pseudomonas aeruginosa.The enzyme was located in particles, sedimenting from ultrasonic preparations at 100000 g.An average of 0.45 moles of oxygen were utilized during the oxidation of one mole of uric acid by washed particles from Hydrogenomonas H 16 in borate buffer at the pH optimum of 9.0. The reaction is sensitive to cyanide; 4 · 10^-6 m KCN caused a 50% inhibition.

     

Bacteriophage P22-mediated specialized transduction in Salmonella typhimurium: high frequency of aberrant prophage excision.

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 mechanisms. Based on the properties of the DNA packaging mechanism of phage P22 a model for the generation of various types of specialized transducing particles is presented that suggests generation of substantial numbers of specialized transducing genomes which are heterogeneous but only some of which have terminally redundant ends. The primary attachment site, ataA, for phage P22 in S. typhimurium is located between the genes proA,B and supQ newD. (The newD gene is a substitute gene for the leuD gene, restoring leucine prototrophy of leuD mutant strains.) The proA,B and supQ newD genes are very closely linked and thus cotransducible by generalized transducing particles. Specialized transducing particles can carry either proA,B or supQ newD but not both simultaneously, and thus cannot give rise to cotransduction of the proA,B and supQ newD genes. This difference is used to calculate the frequency of generalized and specialized transducing particles from the observed cotransduction frequency in phage lysates. By this method, very high frequencies of supQ newD (10(-2)/PFU)- and proA,B (10(-3)/PFU)-specialized transducing particles were detected in lysates produced by induction of lysogenic strains. These transducing particles most of which would have been produced by independent aberrant excision events (which include in situ packaging), were of various types.     

The infection of bacilli by Mu cts phage integrated into the plasmid

The infection of Bacillus thuringiensis, B. cereus, B. mesentericus and B. polymyxa strains with temperate E. coli bacteriophage Mu cts62 integrated into plasmid RP4 under conditions of conjugative transfer is shown possible. The investigated strains of bacilli are not able to produce intact phage particles but they acquire the thermosensitive property determined by the phage genome. Gel electrophoresis and blot hybridization of DNA have confirmed the transfer of Mu cts62 genome or a part of it in the investigated strains of bacilli.     

Temperature-sensitive mutants of bacteriophage SH-133 specific for the hydrogen bacterium Pseudomonas facilis: isolation, complementation, and partial characterization.

Seventeen temperature-sensitive mutants of bacteriophage SH-133 have been isolated following mutagenesis with UV-light, nitrosoguanidine, and ethyl methanesulfonate. The mutants were classified into 15 complementation groups according to their ability to complement each other at 32 degrees C, the nonpermissive temperature. Each mutant was studied with regard to the relationship between its ability to multiply in heterotrophically (H-) and autotrophically (A-) grown Pseudomonas facilis cells. At 27 degrees C, the permissive temperature, the plaque-forming ability of the 17 mutants and wild-type phage was reduced 10-fold in A-grown cells. At 32 degrees C, mutants belonging to 10 groups exhibited identical levels of multiplicity-dependent leak under both modes of growth. However, the infection of A-grown cells by mutants belonging to the remaining five groups resulted in as much as 500-fold inhibition of multiplicity-dependent leak when contrasted with the infection of cells grown heterotrophically. These observations indicate that the expression of five SH-133 phage cistrons is defective when multiplication proceeds under autotrophic metabolism. Seven mutants were found to differ from the wild-type phage with regard to thermal stability at 56 degrees C which suggests that they possess altered structural proteins. Four of the seven thermosensitive mutants exhibited reduced levels of multiplicity-dependent leak in A-grown cells. The data suggest that the reduction in plaque-forming ability of SH-133 in A-grown cells is caused by a defect in the expression of specific phage structural components.     

Phage Typing Reactions on Brucella Species

The nature of the phage typing reactions on Brucella species was determined by rates of adsorption and infection, one-step growth experiments, and susceptibility to lysis from without. The highest rates of adsorption and infection were obtained on smooth B. abortus cultures, and large clear plaques were produced. One or a few phage particles per B. neotomae cell killed about one-half of the cells, but some went through an infective cycle and released mature phage that resulted in production of small clear plaques. With B. suis, more phage particles per cell were required to kill, replication did not occur, and plaques were not observed. Still greater numbers of phage particles were required to cause some inhibition of growth of B. melitensis lawns. Rough Brucella cultures and species, such as B. ovis and B. canis, were not affected by the highest concentrations of phage. B. abortus cultures of intermediate colonial morphology adsorbed phage, but only a few infected cells (after a delayed latent period) released mature phage. An infected culture or colony appeared normal until spontaneous phage mutants appeared which could penetrate the cell wall more effectively than the parent phage. The mutant phage multiplied more rapidly, and the colony changed to a sticky white form.     

A new scheme for phage typing Salmonella bareilly and characterization of typing phages

A new phage typing scheme using wild bacteriophages isolated from sewage for phage typing Salmonella bareilly is described. Six hundred and thirty-seven strains of Salm. bareilly could be separated into 11 different phage types using five wild phages. Overall typability was 94.5%. These phages belonged to two different morphotypes. A1 and B1, and showed varying host range.     

A new scheme for phage typing Salmonella bareilly and characterization of typing phages

A new phage typing scheme using wild bacteriophages isolated from sewage for phage typing Salmonella bareilly is described. Six hundred and thirty-seven strains of Salm. bareilly could be separated into 11 different phage types using five wild phages. Overall typability was 94˙5%. These phages belonged to two different morphotypes, A1 and B1, and showed varying host range.     

Studies on Temperate Phages of Lactobacillus salivarius

Twenty-three temperate phages of Lactobacillus salivarius isolated from human feces were studied as to their morphological, biological, and serological properties. (1) Among 30 strains of L. salivarius tested, 23 strains were lysed by induction with mitomycin C (MC). In all these lysates, phage particles were detected by electron microscopic examination. (2) These phages were morphologically divided into three groups: particles with a regular hexagonal head and a long flexible tail; particles having a regular hexagonal head with or without a short tail-like structure; particles with an elongated head and a long noncontractile tail. (3) Only two, phage 223 having an elongated head and phage 227 with a regular hexagonal head and a long noncontractile tail, produced tiny and very turbid plaques on several host bacteria. Six phages could produce only inhibition zones, ranging from complete inhibition through partial inhibition to normal growth by a serial dilution spot test. (4) All these killer particles could also inhibit the growth of their producer cells. (5) A serological relationship was observed between temperate phages and killer particles, and this was somewhat consistent with the morphological groupings.     

CHARACTERISTICS OF A STAPHYLOCOCCAL PHAGE CAPABLE OF TRANSDUCTION.

Morse, M. L. (University of Colorado Medical Center, Denver) and J. W. LaBelle. Characteristics of a staphylococcal phage capable of transduction. J. Bacteriol. 83:775-780. 1962-The growth of staphylococcal phage 53, a temperate phage, has been studied. The latent periods in nutrient broth are: infected sensitive cells, 45 min; ultraviolet-induced lysogenic cells, 83 min. The burst size, as indicated by one-step growth studies and single-burst experiments, is 25 to 30 particles per lysing cell. Phage particles transducing streptomycin resistance and novobiocin resistance are produced at the same time as nontransducing particles. Some observations have been made on the host, Staphylococcus aureus NCTC 8511.     

3-phosphoglycerate kinase from Hydrogenomonas facilis

Phosphoglycerate kinase levels in Hydrogenomonas facilis were reasonably constant whether cells were utilizing or synthesizing hexose during growth. Specific enzyme activities (micromoles of 3-phosphoglycerate disappearing per minute per milligram of protein) at 30 C were 0.234, 0.391, 0.300, and 0.229 in the "soluble" fraction derived from cells grown on fructose, lactate, succinate, and glutamate, respectively. The enzyme was purified 300-fold from succinate-grown cells. The final preparation, which was not homogenous but was free from glyceraldehyde-3-phosphate dehydrogenase and adenylate kinase, had a specific activity at 30 C of 90 mumoles of 3-phosphoglycerate per min per mg of protein. K(m) values for adenosine triphosphate (ATP), 3-phosphoglycerate, and Mg(++) were 0.16, 0.83, and 0.4 mm, respectively, at pH 7.4 and 30 C. Adenosine monophosphate (AMP) inhibited 23% at a ratio of AMP to ATP of 2.4, and the possible physiological implications of this inhibition are discussed. No evidence was found for an enzyme which catalyzes ATP-dependent conversion of 3-phosphoglycerate to 1,3-diphosphoglycerate, AMP, and phosphate.     

Anaerobic mineralization of quaternary carbon atoms: isolation of denitrifying bacteria on dimethylmalonate.

The microbial capacity to degrade simple organic compounds with quaternary carbon atoms was demonstrated by enrichment and isolation of five denitrifying strains on dimethylmalonate as the sole electron donor and carbon source. Quantitative growth experiments showed a complete mineralization of dimethylmalonate. According to phylogenetic analysis of the complete 16S rRNA genes, two strains isolated from activated sewage sludge were related to the genus Paracoccus within the alpha-Proteobacteria (98.0 and 98.2% 16S rRNA gene similarity to Paracoccus denitrificans(T)), and three strains isolated from freshwater ditches were affiliated with the beta-Proteobacteria (97.4 and 98.3% 16S rRNA gene similarity to Herbaspirillum seropedicae(T) and Acidovorax facilis(T), respectively). Most-probable-number determinations for denitrifying populations in sewage sludge yielded 4.6 x 10(4) dimethylmalonate-utilizing cells ml(-1), representing up to 0.4% of the total culturable nitrate-reducing population.