Objective 1. Determination of the phenotypic effects of resistance mutations:
Task 1.1. Biosynthetic capacity and expression profile.
Biosynthesis tested via two parallel methods: fluorescence intensity of a constitutively expressed reporter, and using an inducible reporter. We observed that biosynthetic capacity does not correlate well with the fitness cost of resistance mutations.
Task 1.2. Genome instability.
We measured genome instability using a fluorescent reporter that activates upon the generation of DNA breaks. We observed a significant overlap between the epistatic effects of double resistance with respect to their fitness cost and the SOS induction, suggesting a causal relationship. Importantly, we found a strong correlation between the genome instability and the cost of resistance.
Task 1.3. Proteostasis imbalance.
Not completed. The limitations imposed by the Covid-19 crisis during half of the time of the fellowship, together with the extraordinary results obtained in Task 1.2 caused us to focus on the most relevant phenotype (genome instability), and therefore withdraw the experiments associated to this task from our plans.
Task 1.4. The effects of antibiotics on the above phenotypes in wild-type bacteria.
We analyzed the effects of rifampicin and streptomycin on two phenotypes (genome instability and fitness), obtaining two interesting conclusions: first, that antibiotics can indeed cause genome instability, and second, that the fitness cost of mutants affected in transcription can be increased by using sub-MIC concentrations of an antibiotic targeting translation, and vice-versa.
Objective 2. Study of the phenotypes in tasks 1.1-1.3 in evolved strains carrying compensatory mutations.
Task 2.1. Experimental evolution, sequencing and determination of compensatory mutations.
We evolved several resistant genotypes, analyzed the pace at which compensation occurs and identified compensatory mutations for all of them. As previously observed, in double mutants we found compensatory mutations compensating specific for the epistasis.
Task 2.2. Study of the phenotypes in tasks 1.1-1.3 in the evolved lines.
As explained above, task 1.3 was withdrawn from our plans. We did perform experimental evolution, and measured fitness cost and genome instability in the ancestral and evolved clones (see section 1.2.2.1). We concluded that the mechanisms causing genome instability are being repeatedly targeted by compensatory evolution.
Objective 3. Integration of the results obtained into general hypotheses to explain the cause/s of the epistases. Test of the elaborated hypotheses.
We concluded that coordination between transcription and translation, and its ultimate consequences (DNA breaks) are key factor both for the fitness cost of resistance mutations and for the epistasis between these mutations.
