Bacterial Mutation in a Stationary Phase and the Question of Cell Turnover

Abstract

SUMMARY: During a stationary phase induced and maintained by the exhaustion of histidine, the total number of histidineless Escherichia coli (h -) remains constant as does the cytological appearance of the cells, If glucose is available to the starved bacteria they die at a rate of c. 10-2 per hr., while mutations t o a histidine-independent ( h + ) condition occur at a rate of c. 10-8 per bacterium per hr. Bacteria adapted to use lactose behave essentially the same way when it, instead of glucose, is available during starvation; but if the starved cells are not fully adapted, death does not occur or is very slow (c. 1 0 -5 per hr.) and the rate of mutation is c. 1O-lo. When no carbon source is available to the starved cells, mutations cannot be detected.The following predictions served as tests of the hypothesis of cell-turnover, wherein some bacteria lyse only to be replaced at the same rate by the growth of othersthe mutations are presumed to have occurred during this cryptic growth:(1) Mixtures of h-lac+ and h-lac-bacteria in lactose medium where the h-lac+ has a selective advantage should show population shifts if cryptic growth were occurring.(2) During the hypothesized lysis and growth the enzyme /?-galactosidase should, at predictable rates, be lost to the medium from'adapted cultures in the absence of lactose and developed in unadapted cultures in its presence.(3) Penicillin should kill those cells that grow to replace others, causing an accelerated death and preventing the mutations from taking place.(4) The lysis and death might be microscopically observable on an agar surface. The hypothesis of turnover did not withstand any of these tests. It was concluded, therefore, that. the bacteria under investigation were not dividing and that mutations -genotypic changes (Ryan, 1955 a)-were taking place among them.Reasons are given to suppose that the mutations result from errors in the replication of genetic material which is in the process of turnover within the non-dividing cells.

5-Methylcytosine is not a mutation hot spot in nondividing Escherichia coli

“…Mutation in nondividing E. coli was studied intensively by F. J. Ryan more than 40 years ago (32 direct evidence for DNA synthesis in starved bacteria, Ryan deduced that some DNA turnover must occur (33). Recent studies of mutation in starved bacteria suggest strongly that DNA synthesis in nondividing cells can account for these mutations (34,35).…”

Clusters of mutations from transient hypermutability

“…In addition to mutations arising in replicating cells, which dominate the entries in Table 1, mutation also occurs in nondividing stationary-phase cells (28,29). Some of these mutations also arise as multiples in a transiently hypermutating subpopulation of the mutating cells (30,31).…”

Back Mutation of Leucine-Requiring Auxotrophs of Salmonella typhimurium Induced by Diethylsulphate

“…Such a mechanism would be facilitated by the technically uncontrolled residual replication of the bacteria upon resuspension in minimal medium, Whatever the mechanism of delayed mutation, further complications may be expected to stem from indirect effects, such as the possible influence of neighbouring bases in the DNA molecule, on base substitution. It is not unlikely that similar mechanisms underlie spontaneous mutation in the stationary phase (Ryan, 1959) and of diethylsulphate-induced mutation. More direct evidence and better understanding of the role of base turnover in stationary-phase mutation (Ryan et al 1961) seem necessary for the interpretation of alkylationinduced mutagenesis.…”

“…It is not unlikely that similar mechanisms underlie spontaneous mutation in the stationary phase (Ryan, 1959) and of diethylsulphate-induced mutation. More direct evidence and better understanding of the role of base turnover in stationary-phase mutation (Ryan et al 1961) seem necessary for the interpretation of alkylationinduced mutagenesis. Experiments bearing on the uptake by bacteria of bases alkylated in vitro, and the pattern of consequent mutation, are underway now.…”

“…Nearly equal amounts of 7-ethylguanine and 3-ethyladenine are released initially during post-treatment storage (Lawley & Brookes, 1963). The apurinic gaps formed in this manner may account for the delayed mutations, by any of three mechanisms: (1) by taking in a 'wrong' base prior to replication (Ryan, Nakada & Schneider, 1961); (2) by the incorporation of the latter opposite the gap, during replication; (3) by being capable of coding for the 'right' messenger RNA, thus initiating growth and replication. Such processes are quite speculative, but the idea of an apurinic gap leading to mutation is supported by the mutagenic effects of treatment at low pH values on phage (Freese, 1959), and of heat to bacteria (Greer & Zamenhof, 1962).…”