Effect of endogenous carotenoids on "adaptive" mutation in Escherichia coli FC40

Abstract

The appearance over many days of Lac(+) frameshift mutations in Escherichia coli strain FC40 incubated on lactose selection plates is a classic example of apparent "adaptive" mutation in an episomal gene. We show that endogenously overproduced carotenoids reduce adaptive mutation under selective conditions by a factor of around two. Carotenoids are known to scavenge singlet oxygen suggesting that the accumulation of oxidative base damage may be an integral part of the adaptive mutation phenomenon. If so, the lesion cannot be 7,8-dihydro-8-oxoguanine since adaptive mutation in FC40 is unaffected by mutM and mutY mutations. If active oxygen species such as singlet oxygen are involved in adaptive mutation then they should also induce frameshift mutations in FC40 under non-selective conditions. We show that such mutations can be induced under non-selective conditions by protoporphyrin photosensitisation and that this photodynamic induction is reduced by a factor of just over two when endogenous carotenoids are present. We argue that the involvement of oxidative damage would in no way be inconsistent with current understanding of the mechanism of adaptive mutation and the role of DNA polymerases.

Fig. 1

Effect of endogenous carotenoids on the number of Lac⁺ mutants arising as a function of time of incubation on selective agar. (a) FC40 (●) and FC40pPL376 (□); (b) FC40pPL376 (□) and FC40pART53 (■); (c) CM1423 (rpoS) (□) and CM1424 (rpoS pPL376) (▲). FC40pPL376 is the only strain expressing carotenoids. Means and standard errors of at least three experiments.

Fig. 2

Appearance of mutants on lactose selection plates in CM1461, a hemH1 derivative of FC40 overexpressing protoporphyrin IX, following exposure to fluorescent light for 0, 10, 20, 30, or 40 min. The first 2 days were at 37°C, the subsequent 3 days on the bench (approximately 25°C). (a) Panel shows raw data from a representative experiment; (b) panel shows the number of mutants on plates of exposed bacteria minus the number on plates of unexposed bacteria.

Fig. 3

Survival and induction of Lac⁺ mutants in CM1461 (circles), and a derivative containing pPL376 (squares) as a function of time of exposure to fluorescent light. Mutation data adjusted to 10⁸ viable cells plated. Each point is the mean and standard error of three experiments.

Fig. 4

Survival of CM1461 and its carotenoid-containing derivative CM1464pPL376 following exposure to 254 nm UV light. Cell density approximately 2 × 10⁹ per ml (representative experiment).

Fig. 5

Adaptive mutation in FC40 and derivatives carrying mutations in mutM (CM1443) or mutY (CM1429). Means and standard errors of three experiments.