Kinetic analysis of growth rate, ATP, and pigmentation suggests an energy-spilling function for the pigment prodigiosin of Serratia marcescens

Abstract

Serratia marcescens is a gram-negative environmental bacterium and opportunistic pathogen. S. marcescens expresses prodigiosin, a bright red and cell-associated pigment which has no known biological function for producing cells. We present here a kinetic model relating cell, ATP, and prodigiosin concentration changes for S. marcescens during cultivation in batch culture. Cells were grown in a variety of complex broth media at temperatures which either promoted or essentially prevented pigmentation. High growth rates were accompanied by large decreases in cellular prodigiosin concentration; low growth rates were associated with rapid pigmentation. Prodigiosin was induced most strongly during limited growth as the population transitioned to stationary phase, suggesting a negative effect of this pigment on biomass production. Mathematically, the combined rate of formation of biomass and bioenergy (as ATP) was shown to be equivalent to the rate of prodigiosin production. Studies with cyanide inhibition of both oxidative phosphorylation and pigment production indicated that rates of biomass and net ATP synthesis were actually higher in the presence of cyanide, further suggesting a negative regulatory role for prodigiosin in cell and energy production under aerobic growth conditions. Considered in the context of the literature, these results suggest that prodigiosin reduces ATP production by a process termed energy spilling. This process may protect the cell by limiting production of reactive oxygen compounds. Other possible functions for prodigiosin as a mediator of cell death at population stationary phase are discussed.

FIG. 1.

Growth and ATP per cell of E. coli in base complex broth at 37°C. E. coli K-12 was pregrown in base complex broth with aeration at 37°C and diluted into fresh broth to an initial OD₇₅₀ of 0.10 for continued growth. Culture aliquots were removed at 0.25-h intervals for assay of ATP concentration and cell concentration.

FIG. 2.

Prodigiosin-per-cell rate of change as a function of low-density growth rate. S. marcescens Nima was pregrown at 26°C in solid or liquid base complex medium with a single carbohydrate (citrate, inositol, glycerol, glucose, or maltose). Pregrowth was diluted into the corresponding broth to an initial OD₇₅₀ of 0.05 to 0.2 for continued growth at low density. Samples were removed at 0.25-h intervals for assay of cell and prodigiosin concentrations.

FIG. 3.

(A) ATP and prodigiosin during low-density growth in base complex broth. S. marcescens Nima was pregrown on a base complex medium slant at 26°C and washed into base complex broth to an initial OD₇₅₀ of 0.12. Growth was continued with aeration. Samples were removed at 0.25-h intervals for assay of cell, ATP, and prodigiosin concentrations. Vertical arrows identify low density as 1.50 through 5.75 h. The vertical double line separates low-density phase 1 (1.50 to 3.00 h) from phase 2 (3.25 to 5.75 h). (B) ATP per cell and prodigiosin per cell during low-density growth in base complex broth. Data for ATP per cell and prodigiosin per cell from the experiment in panel A are presented.

FIG. 4.

(A) ATP and prodigiosin during low-density growth in inositol complex broth. S. marcescens Nima pregrown for 1 day on an inositol complex medium slant was washed into inositol complex broth to an initial OD₇₅₀ of 0.15. Growth was continued with aeration at 26°C. Samples were removed at 0.25-h intervals for assay of cell, prodigiosin, and ATP concentrations. Asterisks denote ATP minima preceding ATP synthesis. (B) ATP per cell and prodigiosin per cell during low-density growth in inositol complex broth. Data for ATP per cell and prodigiosin per cell from the experiment in panel A are presented.

FIG. 5.

ATP per cell and prodigiosin per cell during low-prodigiosin growth in base complex broth. S. marcescens Nima pregrown for 1 day on a base complex medium slant at 37°C was washed into base complex broth to an initial OD₇₅₀ of 0.04. Growth was continued with aeration at 37°C. Samples were removed at 0.25-h intervals for assay of cell, prodigiosin, and ATP concentrations. Vertical arrows denote the logarithmic growth phase.

FIG. 6.

(A) Prodigiosin production throughout the population cycle in inositol complex broth at 26°C. S. marcescens Nima was pregrown in inositol complex broth and inoculated to an initial OD₇₅₀ of 0.05. Growth was continued with aeration at 26°C. Samples were removed at 0.25-h intervals for assay of cell concentration and prodigiosin concentration. Vertical double lines separate low-density (0.25 to 5.25 h), high-density (5.50 to 8.00 h), and stationary (8.25 to 11.75 h) phases. (B) Cell growth and prodigiosin per cell throughout the population cycle in inositol complex broth at 26°C. Data for cell concentration and prodigiosin per cell from the experiment described for panel A are presented.