Properties of Thymineless Strains of Bacillus megaterium

Both Bacillus megaterium KM:T(-)R(1), a strain partially resistant to thymineless death, and strain KM:T(-), the parent strain, can satisfy their thymine requirement with either thymidine, 5-methyldeoxycytidine, or 5-methyluridine. Neither strain can use 5-methylcytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, or 5-aminouracil for this purpose. Strain KM:T(-)R(1) requires as little as 0.01 mM thymine for maximum growth, whereas strain KM:T(-) requires 0.10 to 0.20 mM thymine. Lysogenic KM:T(-)R(1) dies more rapidly in the presence of mitomycin C than the corresponding phage-sensitive strain. Unexpectedly, the lysogenic strain was found to be less sensitive to thymineless death than the phage-sensitive strain. Lysogenic KM:T(-)R(1) is induced by exposure to mitomycin C and by thymineless incubation. It is concluded that thymineless death occurs by a mechanism which is unrelated to phage induction and that a major lethal effect of mitomycin C is probably a consequence of phage induction.     

Thymineless death in Bacillus megaterium

Wachsman, J. T. (University of Illinois, Urbana), S. Kemp, and L. Hogg. Thymineless death in Bacillus megaterium. J. Bacteriol. 87:1079-1086. 1964.-Strain KM:T(-), a thymine auxotroph of Bacillus megaterium strain KM, rapidly loses the ability to multiply when incubated in the absence of thymine, on an otherwise sufficient medium. At 37 C, there is a lag of approximately 60 min, prior to the onset of exponential death (decrease of 1 decade per 50 min). The extent of the decrease in viable count varies from 4 to 5 decades after 5 hr of starvation. The cells die more slowly at 30 C (decrease of 1 decade per 120 min) after a lag of approximately 90 min. Thymine starvation permits substantial net ribonucleic acid (RNA) and protein synthesis, but only slight deoxyribonucleic acid synthesis. In contrast with the changes occurring at 30 C, thymineless death at 37 C is eventually accompanied by a rapid hydrolysis of RNA and by cell lysis. Chloramphenicol inhibits thymineless death at 37 C. Strain T(-)R(1), a derivative of strain KM:T(-), undergoes a very low rate of thymineless death at 37 C (decrease of 1 decade per 240 min). Neither hydrolysis of RNA nor cell lysis occurs during 8 hr of thymine starvation. Strain KM:T(-)H(-) (doubly auxotrophic for thymidine and histidine) requires histidine for maximal thymineless death at 37 C. Preincubation of this strain on the basal medium supplemented with thymidine alone enables the population to become increasingly immune to subsequent thymineless death.     

Thymineless Death in Escherichia coli 15T− and Recombinants of 15T− and Escherichia coli K-12

Thymineless death was examined in Escherichia coli 15T(-) and recombinants of 15T(-) and E. coli K-12. Those strains that were very sensitive to thymine deprivation were also very sensitive to a variety of inducing agents (mitomycin C, ultraviolet light, hydroxyurea, and nalidixic acid). Those strains that were relatively resistant to thymineless death were also relatively resistant to the inducing agents. After exposure to thymineless death and the inducing agents, sensitive strains lysed, produced colicin, and had phage particles in their lysates. These strains also showed an increase in the 6-methyladenine content of their deoxyribonucleic acid (DNA) and an increase in the DNA methylase activity of their crude extracts under these conditions. None of these effects was noted in the strains relatively resistant to thymineless death and the inducing agents. These data indicate that there are two types of thymineless death. One is represented by the strains that are very sensitive to thymine deprivation and other inducing agents and is secondary to the induction of phage psi. The strains more resistant to thymine deprivation and the other inducing agents undergo a non-phage-mediated thymineless death. The mechanism of this latter process is currently under study.     

Thymineless Death in Bacillus subtilis: Correlation Between Cell Lysis and Deoxyribonucleic Acid Breakdown

Bacillus subtilis carrying an inducible defective phage is several times more sensitive to thymineless death than a mutagenized derivative that behaves as a nonlysogen. When the integrity of the deoxyribonucleic acid (DNA) of both strains was examined during thymine starvation by transformation experiments, sedimentation studies, and measurements of acid-soluble DNA degradation products, it was shown that extensive DNA breakdown occurred only in the lysogenic strain. During thymine starvation of this strain, there is a progressive proclivity to lysis, followed by leakage of DNA and DNA degradation products. Such leakage was not observed in the nonlysogen. A correlation between proclivity to lysis and extensive DNA degradation is indicated.     

Use of thymineless death to enrich for doubly auxotrophic mutants of Bacillus megaterium

Wachsman, J. T. (University of Illinois, Urbana), and L. Hogg. Use of thymineless death to enrich for doubly auxotrophic mutants of Bacillus megaterium. J. Bacteriol. 87:1118-1122. 1964.-When strain KM:T(-), a thymine auxotroph of Bacillus megaterium strain KM, is allowed to undergo thymineless death on a minimal medium, the survivors are greatly enriched in polyauxotrophic mutants. Cells were irradiated with ultraviolet light, grown in the presence of thymidine and a complete amino acid mixture, and then starved for thymidine in the absence of amino acids. Doubly auxotrophic mutants (thymine(-) amino acid(-)) may account for more than 90% of the survivors. The most reproducible results were obtained when sucrose (0.4 m) was added to both growth and starvation media. Although the percentage of mutants among the survivors increases with the time of thymine starvation, the absolute number of double auxotrophs per milliliter decreases. It is probable that the extent of cross-feeding determines both the mutant yield and the mutants types. Substrains of KM:T(-) having additional requirements for each of the following amino acids have been isolated: histidine, threonine, tyrosine, tryptophan, arginine, isoleucine, methionine, serine, and cysteine.     

Potential Significance of Lysogeny to Bacteriophage Production and Bacterial Mortality in Coastal Waters of the Gulf of Mexico

The potential effect that induction of lysogenic bacteria has on bacteriophage production and bacterial mortality in coastal waters was investigated, and we present estimates for the percentage of lysogenic cells in a natural aquatic bacterial community. Various concentrations of mitomycin C and exposure times to UV C radiation (UV-C) (wavelength of 254 nm) were used to induce the lytic cycle in lysogenic cells of natural communities of marine bacteria. UV-C treatment occasionally resulted in phage production, but phage production induced by UV-C was always less than that caused by the addition of mitomycin C. There was no evidence that high growth rates of bacteria resulted in lysogenic phage production. The burst size of cells induced by mitomycin C was determined by transmission electron microscopy and ranged from 11 to 45. Dividing the induced phage production by the burst size provided an estimate of the number of lysogenic bacterial cells, which ranged from 0.07 to 4.4% (average, 1.5%) of the total bacterial population. The percentages of lysogenic bacteria that were induced by mitomycin C were similar for samples collected nearshore from the pier of the Marine Science Institute (chlorophyll a, 1.6 to 2.9 (mu)g liter(sup-1)) and in relatively oligotrophic water (chlorophyll a, 0.2 to 0.9 (mu)g liter(sup-1)) collected 25 to 100 km offshore. By using a steady-state model, if all lysogenic bacteria were induced simultaneously, 0.14 to 8.8% (average, 3.0%) of the total bacterial mortality would result from induction of lysogenic cells. If mitomycin C induces all or the majority of lysogenized cells, our results imply that lysogenic phage production is generally not an important source of phage production or bacterial mortality in the coastal waters of the western Gulf of Mexico.     

Characterization of temperate bacteriophages of Leuconostoc oenos and evidence for two prophage attachment sites in the genome of starter strain PSU-1

The close relatedness between 17 Leuconostoc oenos bacteriophages, induced with mitomycin C from strains isolated in different geographic regions, was inferred from their morphology, DNA homology and protein composition. The genome of all the phages had cohesive end termini and ranged in size from 36.4 to 40.9 kb. According to the restriction patterns obtained by digestion with five enzymes, the phages were divided in six groups. Lysogenization of a spontaneous phage-cured derivative of Leuc. oenos strain PSU-1 was achieved with 16 phages and the analysis of the lysogens showed that the phage DNA integrates in the host chromosome in one or two sites. The att B loci were located on the macrorestriction Asc I and Not I fragments of the recipient strain. A survey of Leuc. oenos strains with a phage DNA probe confirmed the lysogenic nature of several, but not all of the original phage hosts. These results are discussed in the light of evidence for the instability of some lysogenic PSU-1 derivatives.     

Isolation and characterization of a mutant of Escherichia coli K12 synthesizing DNA polymerase I and endonuclease I constitutively

A mutant of Escherichia coli K12, highly resistant to ultraviolet radiation, has been isolated. Preliminary tests show that this mutant is also resistant to mitomycin C, nalidixic acid, fluorouracil and thymineless death. The mutant strain apparently repairs its damaged DNA more efficiently than wild-type E. coli K12 strains and, to do so, constitutively produces 35 times more DNA polymerase I and 12 times more endonuclease I than the wild-type strain.     

苏云金芽孢杆菌两株溶原性噬菌体的生物学特性

研究苏云金芽孢杆菌(Bacillus thuringiensis)的溶原性及其噬菌体的生物学特性,从生产菌株MZ1中分离了两株溶原性噬菌体。MZ1经诱导后产生直径约为3mm和1mm的噬斑,分离获得属长尾噬菌体科的噬菌体MZTP01和MZTP02两株;分别对6株和7株不同亚种的Bt菌株具有侵染力;免疫血清与相应噬菌体的中和反应K值分别为45和326,且两者无相关抗原性。MZTP01抵抗酸、碱、紫外线和热的能力比MZTP02强,但抵抗有机溶剂的程度比MZTP02弱。MZTP01的潜伏期为80min,裂解量为55;MZTP02的潜伏期为40min,裂解量为175。核酸结构分析均表明为线性dsDNA分子。两基因组DNA的凝胶电泳表明分子量均在9.4~23kb之间,并被HindⅢ酶切分别产生8条和9条清晰条带。该菌株被证明为二元溶原菌,可能是造成生产损失的主要原因;为防治溶原性噬菌体提供了生物学信息。     

Bacteriophage Release in a Lysogenic Strain of Agrobacterium tumefaciens

Bacteriophage release in a lysogenic strain of Agrobacterium tumefaciens V-1 is temperature-sensitive. At 25 C and 30 C, phage was released in a ratio of 1 plaque-forming unit per 100 bacteria; at 35 C, although bacterial growth was not inhibited, phage release was suppressed. Phage synthesis was induced by heat shock, 42 C for 30 min, ultraviolet irradiation, and mitomycin C. Induction by ultraviolet light was unusual-an immediate rise in phage titer followed irradiation. A large increase occurred after a 90-min latent period. The lysogenic strain was cured of the phage by incubation at 37 C, and the cured strain produced plant tumors.     

Aflatoxin B1 Induction of Lysogenic Bacteria

A technique for biological verification of aflatoxin B(1) was developed based on toxin-mediated induction of lysis in a lysogenic strain of Bacillus megaterium NNRL B-3695. Reduction of culture turbidity was determined at various concentrations of toxin. Incubation of 1.1 x 10(-4) g (dry weight) of cells/ml of growth medium containing 25 mug of B(1) per ml at 37 C reduced initial turbidity 0.20 absorbance units in 4 hr. If the bacterial lysate of the lysogenic strain, after a 2-hr incubation with 25 mug of B(1) per ml, was plated with a sensitive B. megaterium strain (NRRL B-3694), plaque-forming units increased approximately 150 times relative to the control. Comparable testing of the effects of aflatoxin on the nonlysogenic, sensitive strain demonstrated that 75 mug of B(1) per ml neither induced lysis nor plaque-forming units. Although induction is not an exclusive property of aflatoxin B(1), the differential response of the lysogenic and sensitive Bacillus strains to B(1) offers a unique and rapid technique for biological verification of the toxin.     

Isolation and Characterization of a Lysogenic Strain of Nocardia erythropolis

A stable phage-carrying strain of Nocardia erythropolis was isolated from an infection with the nocardiophage phiEC. Growth of the strain in phage-specific antiserum for 48 hr produced cured organisms at a frequency of about 0.5%. Spontaneous curing, determined by serial single-colony isolations, was less than 0.4%. The strain could not be infected by phage phiEC nor by a closely related phage, phiC, although the cells were able to adsorb these phages. In cell populations, a frequency of 2.5 x 10(-4) cells spontaneously induced. The growth rate of the strain was comparable to that of the uninfected wild-type N. erythropolis. Ultraviolet irradiation or treatment with mitomycin C induced the strain to produce larger numbers of phage. It was concluded that the isolated strain was lysogenic.     

Interactions of Lactobacillus bulgaricus Temperate Bacteriophage 0448 with Host Strains

Lactobacillus bulgaricus LT4(0448) is a lysogenic strain from which a temperate bacteriophage can be induced by mitomycin C or UV irradiation. Lactobacillus lactis CNRZ 326 is an indicator strain for the temperate phage 0448, but this strain lyses only in the presence of Ca²⁺ ions. A resistant culture developed secondarily after phage lysis and grew normally in MRS broth but again lysed abruptly if Ca²⁺ ions were added after two or three transfers. This behavior of the secondary culture and its subcultures is explained by a heterogeneous and fluctuating bacterial population, including clones identical to L. lactis 326, which were sensitive to 0448 and which formed rough colonies, as does the indicator. The proportion of these clones increased in the course of transfers in MRS, explaining lysis when Ca²⁺ was added. The population also included clones which formed smooth colonies (S clones). SI clones, which could not be induced by mitomycin C, were the major type in the initial culture, although they were sensitive to temperate phage 0448. The SI population then decreased and was gradually replaced by SII clones, inducible by mitomycin C and resistant to 0448. These SII clones were lysogenized clones, 326(0448), whose stability was confirmed by growth in the presence of an antiphage serum. When L. bulgaricus LT4(0448) was treated with mitomycin C, several cured LT4 clones were obtained that were related to the clones of the indicator L. lactis 326; they formed rough colonies. They also became sensitive to lytic phages or temperate phages active against L. lactis 326 and insensitive to lytic phages which lysed L. bulgaricus LT4(0448). This suggests that phage 0448 can lead to a lysogenic conversion of host strain LT4.     

Isolation and physiological characterization of mitomycin C-sensitive/UV-sensitive mutants in Bacteroides fragilis

Mutants of Bacteroides fragilis sensitive to mitomycin C were isolated after mutagenesis with ethyl methane sulphonate. One mutant (MTC25) was markedly sensitive to mitomycin C but was unaffected as regards UV sensitivity; another mutant (UVS9) was sensitive to UV radiation but was only moderately sensitive to mitomycin C. Caffeine decreased the survival after UV-irradiation of the wild-type, MTC25 and UVS9 strains by the same relative amount. Aerobic liquid holding recovery occurred in each of the three strains. The MTC25 and UVS9 mutants showed reduced host cell phage reactivation. The wild-type, MTC25 and UVS9 strains all showed UV- and H2O2-induced phage reactivation. The physiological characterization of the MTC25 and UVS9 mutants indicates that it is possible to differentiate between mechanisms for the repair of mitomycin C- and UV-induced DNA damage in B. fragilis.     

用凝胶电泳方法鉴定苏云金芽孢杆菌溶源性菌株

根据原噬菌体的可诱导性,将苏云金芽孢杆菌(Bacillus thuringiensis,简称B t)培养液用丝裂霉素C(MMC)诱导,诱导液经高速离心除菌和2.5×SDS-EDTA染料混合液处理后,琼脂糖凝胶电泳检测有无DNA带,以确定菌株的溶源性。实验证明,该DNA为溶源菌诱导出的噬菌体DNA,而非溶源菌以同样方法不能获得DNA。用此方法,可作为鉴定B t溶源性菌株的一个手段,有助于B t工业发酵中噬菌体污染的预防。     

Participation of Escherichia coli K-12 groE gene products in the synthesis of cellular DNA and RNA.

Cells having the temperature-sensitive mutation groES131(Ts) were isolated from Escherichia coli K-12 strain C600T by thymineless death selection at 44 degrees C. This conditionally expressed mutation affected both cellular DNA and RNA syntheses at nonpermissive temperature, in addition to rendering cells unable to propagate phage lambda at permissive temperature.     

Comparative Study of 35 Bacteriophages of Lactobacillus helveticus: Morphology and Host Range

This survey included 23 phages isolated from cheese whey and 12 temperate phages induced with mitomycin from their lysogenic host strains. All of the phages had an isometric head and a tail with a contractile sheath. In addition, short-tailed (160-nm-long) and long-tailed (260-nm-long) phages were distinguished. Short-tailed phages were by far the most widespread in French cheese factories (32 of the 35 phages studied). The study of phage relationships enabled two large groups of strains to be distinguished: those not or slightly sensitive to phages and those very sensitive to phages. There was an obvious relationship in the first group between phage sensitivity (or resistance) and the geographic origin of the strains. The second group contained primarily strains from large international collections and those isolated from commercial starters. The relationships among short-tailed phages, either temperate or isolated as lytic, suggest that lysogenic strains could be the major source of phages in French cheese factories.     

Bordetella pertussis bacteriophage

For the first time Bordetella pertussis bacteriophage was isolated, and its presence was confirmed by electron microscopy and by agar layer titration. The lysogenic strains were activated by their treatment with mitomycin C in a dose of 4.5 mg/ml. The phage system of the Bordetella genus, heretofore unknown, has been revealed: Bordetella pertussis phage lyzed all the tested strains of Bordetella parapertussis (25 strains) and could be passaged in these strains. The phage formed turbid and transparent negative colonies 0.1 mm and 0.15 mm in size. The phage titer (e. g., in strain No. 3865) was 1 X 10(10). The lysogenic variants of Bordetella pertussis, capable of spontaneous release of the phage, were obtained. These variants were characterized by changes in some of their phenotypical properties, e.g., the increased content of certain toxic substances and increased virulence.     

Incidence and properties of temperate bacteriophages induced from lactic streptococci.

Sixty-three strains of lactic streptococci isolated from commercial lactic streptococcal starter cultures were examined for lysogeny by treatment with ultraviolet light or mitomycin C. After treatment with the inducing agent, all strains, whether or not they lysed, were examined for evidence of phage release by electron microscopy. Thirty-eight strains yielded intact phages or phage particles of varying morphology. All the temperate phages had isometric heads and noncontractile tails; some had collars and structurally distinctive baseplates. Indicator host strains were found for phages induced from seven different strains. Three strains that released phages spontaneously yielded titers of 10(3) to 10(4) plaque-forming units per ml. When strains that spontaneously released phages were grown in mixed culture with indicator strains, increased phage titers of 10(6) to 10(7) plaque-forming units per ml were observed. These findings indicate that lysogenic lactic streptococcal strains may serve as a reservoir for phages that attack sensitive strains in mixed- or multiple-strain lactic starter cultures.