Recovering from a bad start: rapid adaptation and tradeoffs to growth below a threshold density

Abstract

Background: Bacterial growth in well-mixed culture is often assumed to be an autonomous process only depending upon the external conditions under control of the investigator. However, increasingly there is awareness that interactions between cells in culture can lead to surprising phenomena such as density-dependence in the initiation of growth.

Results: Here I report the unexpected discovery of a density threshold for growth of a strain of Methylobacterium extorquens AM1 used to inoculate eight replicate populations that were evolved in methanol. Six of these populations failed to grow to the expected full density during the first couple transfers. Remarkably, the final cell number of six populations crashed to levels 60- to 400-fold smaller than their cohorts. Five of these populations recovered to full density soon after, but one population remained an order of magnitude smaller for over one hundred generations. These variable dynamics appeared to be due to a density threshold for growth that was specific to both this particular ancestral strain and to growth on methanol. When tested at full density, this population had become less fit than its ancestor. Simply increasing the initial dilution 16-fold reversed this result, revealing that this population had more than a 3-fold advantage when tested at this lower density. As this population evolved and ultimately recovered to the same final density range as the other populations this low-density advantage waned.

Conclusions: These results demonstrate surprisingly strong tradeoffs during adaptation to growth at low absolute densities that manifest over just a 16-fold change in density. Capturing laboratory examples of transitions to and from growth at low density may help us understand the physiological and evolutionary forces that have led to the unusual properties of natural bacteria that have specialized to low-density environments such as the open ocean.

Figure 1

Transient crash in final cell densities over the course of adaptation. Colony counts from dilutions of the final population sizes at various transfers indicated that six of eight E populations (shown in black) crashed before recovering. The E2 population (indicated with large circular symbols and a dotted line) stayed at moderately low densities until after 120 generations. In contrast, the F populations (grey, first 48 generations displayed), whose medium was supplemented with the rapidly-consumed substrate succinate for the first growth cycle to assure full density was reached from single colonies, did not exhibit this pattern.

Figure 2

Fitness of the E2 population tested at standard density had decreased by 84 generations. Unlike the other seven populations which either improved or were indistinguishable from the ancestor, the E2 population fitness decreased by half when assayed against the ancestor with both competitors starting at the standard initial density (grey bars). ANC represents the control of the two color variants of the EM ancestor competed against each other (white bar). Data represent the mean and standard error.

Figure 3

Growth dynamics in 96-well plates indicate that the E2 population is slower than the ancestor. Although much slower than wild-type (green), all populations other than E2 had increased their growth rate between generation 60 and 120 (light and dark blue). The E2 population, however, was slower than the EM ancestor (red) at both 60 and 120 generations (light and dark purple). Note that below a threshold OD₆₀₀ of ~0.002, cultures other than E2 fail to recover, including one of the EM ancestors. Data represent the mean of three replicates on the plate.

Figure 4

Population E2 adapted to low density growth with tradeoffs at the standard high density. Competitions against the EM ancestor were performed using the same set of acclimation cultures that were combined 1:1 and diluted to varying degrees (indicated by the shade of grey). Note E2 fitness at generation 60 is more than four-fold higher at the lowest versus the highest starting density, and that this low-density specificity waned as they evolved. Data represent the mean and standard error.