Figure 1.
The Wrinkly Spreader (WS) produces biofilms at the air-liquid interface and wrinkled colonies on agar plates.
The ecological success of the WS in static 6ml liquid King's B (KB) microcosms is the ability to produce a substantial (3.8 cm2, 1–1.5 mm depth) biofilm at the air-liquid interface (left). The WS genotype produces a wrinkled colony morphology on KB agar plates in contrast to the smooth colonies of the wild-type P. fluorescens SBW25 strain (middle). After a long period of incubation, SM-like revertants appear at the edge of the WS colony (right). On KB plates containing Congo Red, the central WS-like portion of the colony binds the dye and has a strongly-coloured red rim whilst the SM-like outer ring is pale and does not bind significant amounts of dye.
Figure 2.
The degree of WS reversion on KB agar plates was higher when incubated at 28°C than at 10°C.
The percentage of cells still showing the WS phenotype (WS Stability) recovered from colonies incubated for varying periods (left) and the total cell numbers (right) are indicated. Note that WS stability (the retention of the WS genotype) is the inverse of WS reversion to SM-LR (the loss of the WS genotype). Colonies were incubated on KB plates at 28°C for 3-days before sampling. Mean±SE indicated.
Figure 3.
The relative fitness (W) of the WS compared to SM/SM-LR in mixed colonies is sensitive to population density and less sensitive to nutrient levels.
Shown are W values for WS in mixed WS/SM/SM-LR colonies grown on normal KB plates inoculated at high (A) and low (B) cell densities, and for mixed colonies grown on plates containing 0.1× proteose peptone inoculated at high (C) and low (D) cell densities. (A) and (B) are significantly different to (C) and (D) (Tukey-Kramer HSD, q = 3.46172, α = 0.05). The inoculum consisted of a 40∶60 WS∶SM mixture at 106 cfu (high density, 1×) and 103 cfu (low density, i.e. 0.001×) applied as a 5 µl drop to the centre of the plate. Note that SM-LR lineages arise from WS cells, producing a mixed colony of WS, SM and SM-LR cells; W of WS is therefore the relative fitness of WS compared to (SM plus SM-LR). W values were determined after 3-days incubation at 28°C. Mean±SE indicated.
Table 1.
Reversion rates (instability) in WS mutants can identify the nature of the maladaptive characteristic.
Figure 4.
The agar plate can be used as mega-colony or multiple-colony agar microcosms (MEGAM/MUCAM) in which bacterial adaptation and colony interactions can be observed.
In the MEGAM microcosm, a single mixed colony is incubated in the centre of the plate. The colony is harvested and an appropriate dilution is used to inoculate a second plate, again as a single colony. In the MUCAM microcosm, the inoculation is spread across the surface of the plate to allow the development of multiple colonies with differing degrees of separation and overlap. The colonies are pooled together on harvest to produce the inoculation for the subsequent plate, which again is spread across the surface of the entire plate.
Figure 5.
The WS genotype was maintained on agar plates at low population densities in the mega-colony agar microcosm (MEGAM) experiment, but was rapidly lost at high population densities.
The mega-colony was recovered from plates every 2 or 4-days and used to inoculate fresh plates with a single 5 µl drop (a third set of populations was incubated for 8-days without transfer). The percentage of cells still showing the WS phenotype (WS Stability) after each period is indicated. Populations were transferred at three densities, high (H) (∼107 cfu per drop), medium (M) (∼104 cfu per drop), and low (L) (∼10 cfu per drop). Colonies were incubated on KB plates at 28°C before transfer to new plates. Mean±SE indicated.
Figure 6.
The WS genotype was maintained on agar plates at low population densities in the multiple-colony agar microcosm (MUCAM), but was rapidly lost at high population densities.
Colonies were recovered from plates every 5-days as a single population and used to inoculate fresh plates. The percentage of cells still showing the WS phenotype (WS Stability) after each 5-day period is indicated. Populations were transferred at three densities, high (H) (1,000–2,000 cfu per plate), medium (M) (100–200 cfu per plate), and low (L) (10–20 cfu per plate). Colonies were incubated on KB plates at 28°C before transfer to new plates. Mean±SE indicated.
Figure 7.
Plate-adapted Hardy WS isolates from the MUCAM experiment show a lowered degree of WS reversion, enhanced WS stability and different colony sizes compared to the ancestral WS strain.
The percentage of cells still showing the WS phenotype (WS Stability) recovered from colonies is graphed against the colony diameter for each isolate obtained from high (white circles) and medium-density (grey circles) evolved populations, plus the WS (black circle). Colonies were incubated on KB plates at 28°C for 3-days before sampling. Mean±SE indicated.