Mutants of bacteriophage T4 deficient in the ability to induce nuclear disruption: II. Physiological state of the host nucleoid in infected cells

Abstract

Studies with ndd mutants of phage T4, deficient in the ability to induce nuclear disruption, the movement of the host DNA from a largely central location in the cell into close association with the cell membrane, show that nuclear disruption is not essential for host DNA breakdown. Degradation of prelabeled host DNA to acid-soluble products occurs at the same rate in the absence of nuclear disruption as it does in its presence. Moreover, the absence of nuclear disruption results in an alternative pathway of slow degradation of host DNA independent of phage endonuclease II.M-band analyses of association between DNA andmembrane (Earhart et al., 1968) indicate that endonuclease II is required for the release of host DNA from the membrane when nuclear disruption occurs normally, and that the product of at least one of the genes rIIA, rIIB, D1 or D2a (probably D2a, which is necessary for the synthesis of endonuclease IV) is required for DNA release when nuclear disruption does not occur.Analyses of the sizes of host DNA single strands at various times after infection by means of alkaline sucrose density-gradients show that the presence or absence of nuclear disruption has little, if any, effect on the rate of accumulation of single-strand nicks. Neutral sucrose density-gradient analyses suggest that a limited number of double-strand breaks can accumulate in host DNA when endonuclease IV is active, but few, if any, occur when neither endonuclease II or IV is active.Gentle lysis of ndd-infected cells and subsequent sedimentation analysis of the host DNA in neutral sucrose density-gradients reveal that the host chromosomes become “unfolded” within five minutes after infection. Thin-section electron microscopy shows that the host DNA becomes widely dispersed throughout the cytoplasm of cells at late times after infection with ndd mutants. These observations make it very unlikely that nuclear disruption is a passive process which occurs whenever the forces or structures which maintain the normal state of the Escherichia coli nucleoid are altered.All of our data are consistent with a mechanism of nuclear disruption which involves multiple attachment of the host DNA to the cell membrane under the control of the D2b gene of phage T4. We propose that in ndd-infected cells this multiple attachment does not occur, with the result that a limited number of double-strand breaks release much of the host DNA from the cell membrane.