Bacteriophage and bacteriophage-like structures carried by Bacillus medusa and their effect on sporulation.

Bacillus medusa was found to carry three phages or phagelike structures named phi med-1, phi med-2, and phi med-3. phi med-1 is a minute, 25-nm-diameter particle without a tail. It was extracted from the sporulation lysate of a phi med-2-minus strain of B. medusa and purified by differential centrifugation. The nucleic acid from this structure was shown to be orcinol positive, alkali sensitive, RNase sensitive, and DNase resistant. An RNase-resistant core of nucleic acid was not found, indicating that it was single-stranded RNA. A host strain has not yet been found for phi med-1. Phage phi med-3 was induced with mitomycin C or UV light and consisted of empty heads of 57 nm in diameter, whereas phi med-2 induced with mitomycin was a phage of 60-nm head diameter and 220-nm tail length. The sporulation sequence proceeded faster in those mutants lacking phi med-2, and when the phage was reintroduced to B. medusa the extended wild-type sporulation sequence was observed. B. thuringiensis var. schwetzova was sensitive to phi med-2 and yielded small turbid plaques. B. medusa produced small numbers of phi med-2 during growth. The other phage may be produced at the same time but were not detected. Phi med-1 was found in sporulating cells by electron microscopy techniques. Its release from these was demonstrated by both electron microscopy techniques and a radioactive assay. It appears to participate in the formation of a surface layer on the parasporal inclusion of B. medusa.

Enumeration of sublethally heated staphylococci in some dried foods.

The effect of 45 substances to restore the salt tolerance of sublethally heat-injured Staphylococcus aureus was tested. Sodium pyruvate, yeast extract, L-histine, casitone (Difco), adenosine triphosphate, and acetylphosphate were effective. For enumeration a repair medium was first used, containing sodium pyruvate and penicillin in 1% skim milk. This step was followed by counting on Baird-Parker agar with penicillinase. This method was selective; fewer than 100 staphylococci/g food could be enumerated and it gave counts about 8 times higher than the method of Giolitti and Cantoni used as a five-tube most probable number technique. Heat injury sensitized S. aureus to polymyxin.

Exclusion of induced bacteriophage from cells of a lysogenic Bacillus megaterium committed to sporulation.

Spontaneous release of the temperate bacteriophage T (phiT), carried by Bacillus megaterium 899a, occurred during early growth of the host cells. Rejuvenated cells (accomplished by a 5x dilution in fresh medium) and unrejuvenated cells were induced by mitomycin C during the course of sporulation and subsequent phage phiT production measured by burst size. Induction of sequential samples of unrejuvenated cells resulted in burst sizes that fell to zero as T(0) sporulation time in the main culture was approached. This drop in burst size was not considered a sporulation event, as it also occurred during analogous stages of growth in an asporogenous mutant. Rejuvenated, induced portions of the culture of sporulating cells of B. megaterium 899a gave large burst sizes until T(+2), when the burst sizes fell to zero. The stage I asporogenous mutant, treated in a similar manner, gave lower, but still substantial, burst sizes; thus, the sharp decline in burst size of induced rejuvenated sporulating cells appeared to be a sporulation event. Sporulating cells induced at times shortly after T(1.5) formed spores in which the induced phage were trapped until germination of the spores, which formed infectious centers. This induced phage-trapping was maximal when the sporulating cultures were induced at T(2.25). Commitment to sporulation could be defined by our system as that point beyond which rejuvenated sporulating cells were unable to support the replication of the phage. This point also correlated with the increase in induced phage-trapping by spores. Two other methods gave a similar commitment time. Commitment to sporulation, in spite of added glucose or fresh complex medium, occurred at the same time. Electron micrography showed that the committed cell was still undergoing engulfment. The fate of induced phage phiT was determined at different points during growth and sporulation. Induction at times prior to T(0), which were longer than the eclipse period of the phage, resulted in a burst size of approximately 50. At times prior to T(0), shorter than the phage eclipse period, induction led to lysis with low burst sizes, approaching zero. The pattern of spontaneous phage release during growth was similar. From T(0) up to the point of commitment to sporulation, induction resulted in the blocking of spore formation without lysis. At the commitment point, induced phage were trapped and carried into spores which germinated to give infectious centers. The spontaneous derepression of phage at a time which blocked spore formation led to 7 x 10(4) infectious centers per ml and would not normally be noticed. Derepression at the time of phage entrapment was not observed to occur without induction with mitomycin C.

Characteristics of phi T, the temperate bacteriophage carried by Bacillus megaterium 899a.

Phage T was the only phage observed in lysates of Bacillus megaterium 899a induced with mitomycin C, 0.35 mug/ml. The phage adsorbed slowly to its host in nutrient agar, giving rise to plaques of varying sizes and turbidity. Only clear plaques were observed when the phage and host cells were preincubated in an adsorption buffer and plated under optimum conditions. Plaque turbidity was caused by either the addition of 0.5 x 10(-2) to 1.0 x 10(-2) M CaCl(2) to the phage assay medium, or by raising the incubation temperature to 34 C. Phage T purified on a CsCl gradient had a density of 1.48 g/ml in CsCl and the extracted phage DNA had a buoyant density in CsCl of 1.6975 g/ml, equivalent to 38.2% guanine plus cytosine. The phage was rapidly inactivated at 75 C and was unstable in the presence of chloroform at 4 C, but it was stable in buffer stored in ice. When stage I sporulating cells were induced with mitomycin C, phage were carried into spores which when germinated lyse with the release of phi T. The burst size on induction of early-log vegetative cells was 52, whereas the burst size of induced T(0) sporulating cells, diluted in fresh medium, was 47 for a sporulating strain and 140 for an asporogenous mutant. A typical phage T had a long, noncontracting tail 240 nm long, 9 to 11 nm wide, with a repeating disk unit along the tail, 4 nm in size center to center. The tail ended in a small disk (15 nm wide) which is presumably for attachment to the host. The hexagonal head measures 68 by 57 nm and is composed of donut-shaped units 9 nm in diameter.

Disruptive action of potassium phosphotungstate on phiT, the temperate bacteriophage of Bacillus megaterium 899a.

Although untreated phiT may be dried on grids without damage, they were disrupted when negatively stained with potassium phosphotungstate. The potassium phosphotungstate reacted with phage suspended in phosphate buffer, and the altered but still-viable phage was disrupted only on drying. Stability of altered phage was not recovered by dialysis against 10(-3) M Mg(2+).