Conformation and Segregation of Nucleoids Accompanying Cell Length Extension After Completion of a Single Round of DNA Replication in Germinated and Outgrowing Bacillus subtilis Spores

Abstract

When germinating spores of the temperature-sensitive DNA initiation mutant of Bacillus subtilis TsB134 are shifted to the restrictive temperature at a time such that just one or two rounds of replication are accomplished, the completed, nonreplicating nucleoids that form eventually adopt a doublet conformation. This conformation has now been observed after fixation by glutaraldehyde or osmium tetroxide, as well as by Formalin as found previously. The doublet was observed in media of different degrees of richness and under both light and electron microscopes. Electron micrographs of serial sections through the doublet were consistent with its formation by the gradual pulling apart of a single mass of DNA into two lobes. A systematic study was made of the effect of the time of shifting from the permissive to the restrictive temperature and of the restrictive temperature used on the number of nucleoids segregating within the outgrowing rod. It was established that the doublet nucleoid behaved as a single unit in replication control and segregation in both rich and poor media. Measurement of the relative position of the two segregating nucleoids within the outgrowing rod after completion of just one round of replication yielded quantitative information on the segregation and cell length extension processes. Segregation was accompanied by cell length extension at approximately equal rates on both sides of each nucleoid. Furthermore, the data were consistent with an exponential increase in such an extension with time over the early and major portion of the period studied, but it was not possible to rule out other models of length extension.