Team:Valencia Biocampus/Results3

Our experience with the cheating bacteria began when we realized that a mutation was produced in the Bac2 construction (that consisted of the groE promoter fused to the coding sequence AsRed2). We used to grow them all over the weekend in the orbital shaker that was supposedly at 37ºC. The real temperature of the shaker in the Valencia hot summer was about 39ºC. Since Bac2 has a promoter that is sensitive to heat shock (the transcription starts when the temperature is higher than 37ºC), this high temperature was mimicking the “Are you hot?” question. In other words, the cultures were “being asked” continuously because of this high temperature. This led to mutant selection and our mutants were found to lack Bac2 inserts (Figure 1), and exhibited larger molecular weights on a native electrophoresis (Figure 2), suggesting that the inserts were lost and that two plasmids without inserts were relegated in the mutant clones.

Figure 1. Fluorescence intensity (FI) normalized by the optical density (OD) of the culture. Measures were taken 60 min after the resuspension in a medium containing no nitrogen (pink) or 10 g/L ammonium sulphate (grey).		Figure 2. Fluorescence intensity (FI) normalized by the optical density (OD) of the culture. Measures were taken 150 min after the resuspension in a medium containing no nitrogen (pink) or 10 g/L ammonium sulphate (grey).

We decided to isolate and characterize those mutants “cheaters” and compare them with standard, “honest” clones. We compared their behavior in terms of growth in ampicillin-containing medium and we also performed experimental evolution tests. We started by comparing the growth of the original Bac2 strain and the cheaters, at different concentrations of antibiotic.

In all cases the curves were similar: Bac2 and the cheaters grew at a same rate up to 265 minutes, after which a variation in the growth pattern was observed: the cheaters started to grow faster.

Figure 3. Growth pattern of Bac2 and the cheaters in separated cultures.

After that we decided to characterize mixed cultures of both Bac2 and the cheaters (50% each, adjusted in terms of O.D), both let grown together. Under these conditions, the growth pattern was as follows.

Figure 2. Growth pattern of Bac2 and the cheaters mixed in the same proportion in the same culture.

We took aliquots at time 0’, 165’, 340’ and 24h and spread them on solid medium in order to check which was the predominating population. We performed the same experience at 37ºC and 40ºC and we carried out colony PCR with oligos amplifying the Bac2 insert in order to tell between honest and mutant colonies. We obtained the following results:

- At 37ºC, and after 165 and 340 minutes, Bac2 were clearly predominant over the cheaters (the initial rate honest:cheaters was 1:1). That suggests that at this temperature the growth of the cheaters is not favoured, probably because the larger size of the plasmid of the mutant strain, which is bigger than the original Bac2, or the presence of un-optimal sequences, such as two replication origins.

Figure 4. Percentages of Bac2 and the cheaters at time 165, 340 and 1440 min at 37ºC determined by colony PCR.

- In the 40ºC experience, at time 0 minutes, as expected both clones appeared in the same number, but after 165 minutes the percentage of Bac2 had started to slightly decrease (44%) and after 340 minutes they only appeared in a very low rate (4%). These tendency was confirmed after 24 h, when no Bac2 were detected.

Figure 5. Percentages of Bac2 and the cheaters at time 165, 340 and 1440 min at 37ºC determined by colony PCR.

Taken together, these results clearly indicate that both original Bac2 and cheater clones exhibit different growth rates and, when combined at high temperature, mutants tend to grow faster and/or inhibit the growth of Bac2. This leads to a progressive, yet very fast, substitution of Bac2 by mutant clones under selection pressure.