Evolution of microbial diversity during prolonged starvation

Abstract

Models of evolutionary processes postulate that new alleles appear in populations through random spontaneous mutation. Alleles that confer a competitive advantage in particular environments are selected and populations can be taken over by individuals expressing these advantageous mutations. We have studied the evolutionary process by using Escherichia coli cultures incubated for prolonged periods of time in stationary phase. The populations of surviving cells were shown to be highly dynamic, even after many months of incubation. Evolution proceeded along different paths even when the initial conditions were identical. As cultures aged, the takeovers by fitter mutants were incomplete, resulting in the coexistence of multiple mutant forms and increased microbial diversity. Thus, the study of bacterial populations in stationary phase provides a model system for understanding the evolution of diversity in natural populations.

Figure 1

Consecutive generations of GASP mutants arise in the same culture. Progressively aged cultures were mixed. (A) One-day-old in the majority (solid line) vs. 10-day-old in the minority (broken line). (B) Ten-day-old in the majority (solid line) vs. 20-day-old in the minority (broken line). (C) Twenty-day-old in the majority (solid line) vs. 30-day-old in the minority (broken line). Asterisks indicate that cfu ml⁻¹ were below the limit of detection (<10² cfu ml⁻¹).

Figure 2

Dynamism in stationary phase cultures. (A) Five milliliter cultures of either nalidixic acid (Nal^R)- or streptomycin (Str^R)-resistant bacteria were incubated for 30 days and then mixed 1:1 to generate two new cultures. (B–D) Three representative pairs of mixed Nal^R and Str^R cells. Each culture of the pair is represented by either the solid or open symbols. Nal^R cells are represented by solid lines with squares, Str^R cells are represented by broken lines with circles. Asterisks indicate that cfu ml⁻¹ were below the limit of detection (<10² cfu ml⁻¹).

Figure 3

Initially isogenic strains evolve along different paths. (A) One Nal^R and two Str^R cultures were incubated for 30 days. The Nal^R culture was mixed 1:1 with cells from either the Str^R-1 or Str^R-2 cultures, creating two new mixed cultures. The two Str^R cultures (Str^R-1 and Str^R-2) were derived from the same colony and were initially isogenic. (B and C) Representative pair of mixed cultures. Nal^R cells (■), Str^R cells (○).

Figure 4

Coexistence of different colony morphotypes during long-term incubation in a single culture. (A) Colonies plated from a 150-day-old culture: normal or cream-colored colony morphotype (C), white morphotype (W), and minicolony morphotype (M). (B) Pulsed-field gel electrophoresis of SfiI digested chromosome of wild-type (WT) cells and the three new morphotypes (C, W, and M); a phage lambda (λ) size standard is at left. (C) Pie charts reflecting population composition (%) during different times of incubation (days) of each morphotype, each color represents a different colony morphotype (see text for descriptions).