Bet-hedging during bacterial diauxic shift

Abstract

When bacteria grow in a medium with two sugars, they first use the preferred sugar and only then start metabolizing the second one. After the first exponential growth phase, a short lag phase of nongrowth is observed, a period called the diauxie lag phase. It is commonly seen as a phase in which the bacteria prepare themselves to use the second sugar. Here we reveal that, in contrast to the established concept of metabolic adaptation in the lag phase, two stable cell types with alternative metabolic strategies emerge and coexist in a culture of the bacterium Lactococcus lactis. Only one of them continues to grow. The fraction of each metabolic phenotype depends on the level of catabolite repression and the metabolic state-dependent induction of stringent response, as well as on epigenetic cues. Furthermore, we show that the production of alternative metabolic phenotypes potentially entails a bet-hedging strategy. This study sheds new light on phenotypic heterogeneity during various lag phases occurring in microbiology and biotechnology and adjusts the generally accepted explanation of enzymatic adaptation proposed by Monod and shared by scientists for more than half a century.

Keywords: Gram-positive bacteria; metabolic fitness; phenotypic heterogeneity.

Fig. 1.

L. lactis diauxic shift. Growth (OD₆₀₀) of L. lactis M1 in chemically defined medium with 0.1% glucose (red line), 1% cellobiose (blue line), or a mixture of 0.1% glucose and 1% cellobiose (green line) is shown; maximal growth rates (r) measured along the growth curves of the cultures are shown in the right lower corner. During biphasic growth in a medium with a mixture of glucose and cellobiose, cells first consume glucose. The diauxie lag phase, which follows the switch point after glucose depletion, is generally thought to result from adaptation of the metabolism of cells to using the second sugar (in this case, cellobiose). During the second exponential growth phase, cellobiose is used.

Fig. 2.

Effects of initial glucose concentration on the L. lactis shift to growth on cellobiose. (A) Growth (OD₆₀₀) of L. lactis M1 in CDM, with various concentrations of glucose (0.05–0.25%; orange to red) and 1% cellobiose. (B) L. lactis M1gfp population growth rate expressed by the change in OD of a culture—i.e., the difference between two subsequent OD₆₀₀ measurements (shown in SI Appendix, Fig. S2) (black; df = 13; R² = 0.88; P = 2.267 × 10⁻⁷) after the switch point is negatively related to the initial glucose concentration in the medium. The same holds for the intensity of green fluorescence of the whole population (green; df = 13; R² = 0.93; P = 1.03 × 10⁻⁸). Curiously, just after glucose depletion, the culture density slightly drops for all glucose–cellobiose combinations, resulting in negative values for population growth (see also SI Appendix, Fig. S2). Based on our microscopy data, it cannot be explained by cell lysis. This drop in culture OD is always observed at the transition point before L. lactis enters the stationary phase and is not a specific characteristic of diauxie. Rather, it is an intrinsic L. lactis property, probably attributable to cell-structure changes. However, the level of the OD drop (lowest population growth value) and the population growth rate during the second exponential growth phase correlates well with initial glucose concentration. (C) The fraction of cellobiose-using cells, determined on the basis of fluorescent microscopy from liquid culture samples (of identical experiments to those in A and B), also decreases with the increase of the initial glucose concentration. See also SI Appendix, Fig. S2. (D) Snapshots of the time-lapse experiment, performed in glucose and cellobiose containing CDM, illustrating appearance (at 6 h 30 min) and coexistence of two stable phenotypes: cellobiose-consuming (green cells) and nongrowing (black cells) (Movie S1). Error bars are means ± SD of three independent measurements.

Fig. 3.

Deletion of ccpA, relA, or ldh from the chromosome of L. lactis M1gfp increases the fraction of Cel⁺ cells (green). Snapshots of time-lapse and -course experiments performed in G-C (0.1–1%) medium with different M1gfp deletion mutants. Overlays of phase-contrast and green fluorescence images are shown. The clumps of cells in the microscopy pictures resulted from their growth on agarose pads during time-lapse experiments and were chosen intentionally to show more cells in one picture. Neither L. lactis M1 nor its parent strain MG1363 forms aggregates under the conditions used in our experiments.

Fig. 4.

Putative mechanism underlying the phenotypic heterogeneity in L. lactis sugar utilization. At the moment of glucose exhaustion from the medium (the switch point), the CCR level in a cell decreases. Once the repression is relieved, a cell can start expressing to the cel cluster and switch to cellobiose consumption, but it must have sufficient energy (“metabolic state”) to do so. This switch, however, is only possible if the cell switches early enough. If the cell runs out of energy before it makes the switch, the stringent response locks the cell in a nongrowing state (Cel⁻). If a cell is able to make the switch, it continues to grow using cellobiose (Cel⁺).

Fig. 5.

Epigenetic effects influence the decision making of L. lactis cells. Comparison of population growth rate after the switch point for populations precultured in CDM with cellobiose (blue dots and line) and those precultured in CDM containing glucose (red dots and line) is shown. A higher fraction of cellobiose precultured cells switch to cellobiose consumption after the switch point for all initial glucose concentrations tested compared with glucose precultured cells (df = 17; R² = 0.7877; P_{initial glucose conc.} = 9.407 × 10⁻⁷; P_{preculture effect} = 0.014). See also SI Appendix, Fig. S2.

Fig. 6.

Heterogeneity as a bet-hedging strategy. (A) Cell-division rates calculated from the time-lapse movies (Movies S1, S2, and S6) for L. lactis M1 Cel⁺ and Cel⁻ cells (n = 3). (B) Geometric mean fitness (blue, low; red, high) as a function of the fraction of Cel⁺ cells produced by a genotype. The adaptive landscape shows the performance of genotypes (vertical axis) for various environmental conditions (horizontal axis) characterized by the probability P that conditions favor Cel⁺ cells. Dotted line indicates the best-performing genotypes. All genotypes that do not exclusively produce Cel⁻ or Cel⁺ cells represent bet-hedging strategies. The evolved genotypes, from agent-based simulation, are superimposed on the adaptive landscape (circles, average evolved genotype; error bars, SD; n = 100; see SI Appendix for details).