Autoradiographic Studies of Bacterial Chromosome Replication in Amino-Acid Deficient Escherichia coli 15T-

Abstract

Autoradiographic experiments on amino-acid requiring strains of Escherichia coli T(-) have been performed with fully-labeled cells harvested in log-phase and after periods of amino-acid starvation. The simplest segregation of incorporated label among progeny grown on non-radioactive medium is into two packets. The result corroborates the two-unit model of E. coli DNA inferred from previous studies with partially labeled cells. Following amino-acid starvation, the distribution of label among clones derived from labeled cells indicates cells are grouped into classes having DNA contents in the ratios 1:2:4. The segregation of label among progeny isolated by micromanipulation from such starved cells supports the view that the chromosomes are brought to a state of completed synthesis with different cell classes containing different integral numbers of chromosomes. Infrequent clones interpretable as arising from cells with three chromosomes suggest that the control of replication of chromosomes lying in the same cytoplasm is on an individual basis. The block in DNA synthesis resulting from amino-acid starvation is not perfect. Nevertheless, such starvation permits characterization of the dispersive replication events for more homogeneously labeled and definable DNA units than otherwise possible. The size-frequency distribution of label among progeny following six rounds of chromosome replication is close to that expected from a model of random breakage of linear polymers. The frequency of dispersive events is estimated as 0.5 to 0.7 per chromosome per generation and is only slightly influenced by tritium decays occurring during bacterial growth.

Segregation of Deoxyribonucleic Acid in Bacteria: Association of the Segregating Unit with the Cell Envelope

“…This indicates that some chromosome fragmentation (which may be analogous to sister-chromatid exchange in higher cells) is occurring during replication. A similar situation has been observed with E. coli (6,7,13). The data do not justify an exact estimation of the extent of chromosome fragmentation in L. acidophilus.…”

“…However, the data in Fig. 3 can be best fitted by assuming that each labled nucleotide strand produces a fragment about once every 7 to 10 generations [a value similar to that reported in detailed studies of E. coli (6)]. Such fragmentation is reflected in the slow decrease in the amount of radioactivity per labeled cell in the values presented in Table 1 for pulse-labeled cells after three or more generations of growth in nonradioactive medium or for prelabeled cells after four or more generations of growth.…”

Spontaneous Lethal Sectoring, a Further Feature of Escherichia coli Strains Deficient in the Function of rec and uvr Genes

“…Tlhere is no way of ruling out the idea that SLS in Rec-strains may result from attempts of recombination between sister DNA molecules which might be lethal in such strains. This possibility of lethal recombination was considered by Clark et al (2) for Rec-strains, since there is evidence for recombination of sister chromosomes in Rec+ strains (5). The high level of SLS in the double mutant (uvrA6, recA13) relative to the uvr+ recA13 strain, however, is difficult to understand in these terms, since the uvrA6 mutation is known not to affect recombination (10).…”

Regulation of chromosome replication and segregation in bacteria.

“…This has been attributed to chromosome fragmentation (such as sister strand exchanges) and the subsequent distribution of these fragments into cells which would otherwise not receive label. Forro has presented experimental evidence for such fragmentation (7,8).…”