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

Abstract

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.