Date of Award

Spring 1982

Project Type

Dissertation

Program or Major

Microbiology

Degree Name

Doctor of Philosophy

Abstract

The ultrastructure of the lytic cycle of phage 41c in Bacillus subtilis 168 was examined by transmission electron microscopy. Thin sections of phage 41c-infected cells revealed that, prior to the appearance of phage structures, a marked rearrangement of the tightly packed, vesicular mesosomes was usually seen within the host cells. These mesosomes opened up into loosely arranged, lamellar forms. As phage accumulated, they appeared to be preferentially associated with the mesosomes. Towards the later portion of the lytic cycle, the mesosomes took on distinctive, loosely organized, spiral configurations. These ultrastructural changes occurred even in the presence of chloramphenicol, indicating that protein synthesis was not responsible for the alterations. Since it was possible that phage assembly was mesosome associated, infected cells were treated with lysozyme to form protoplasts. However, phage were never found within the extruded mesosomes. Furthermore, phage replication occurred in protoplasts, although the burst size was decreased. Sucrose-stabilized protoplasts only produced a burst of 1-2% of that in whole cells, while sorbitol-stabilized protoplasts produced a burst of 25-30% of that in whole cells. Results of energy-dispersive X-ray analysis indicated less potassium and phosphorus in infected cells than in noninfected cells. These results suggested that the mesosome alterations may have occurred due to differences in the ionic composition of the cells.

A characterization study of phage 41c was also conducted. Phage 41c produced a burst size of 10('3) phage per host cell, following a latent period of 50 minutes. In addition, phage 41c (a double-stranded DNA phage) was found to be remarkably similar to another B. subtilis phage, SSP1. They were almost identical in size, morphology, sensitivity of infection to deoxyribonuclease or streptomycin, plaquing efficiency, host range, and incorporation of ('3)H-thymidine into their DNA. Differences were detected in the molecular weights of their structural proteins and in their plaque morphologies. Phage SPP1 produced a series of changes in the ultrastructure of the host cell which was identical to that of 41c-infected cells. In contrast, a dissimilar phage, SP82G, did not produce such changes in the host cells.

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