Insight into the ruggedness of adaptive landscapes is central to understanding the mechanisms and constraints that shape the course of evolution. While empirical data on adaptive landscapes remain scarce, a handful of recent investigations have revealed genotype-phenotype and genotype-fitness landscapes that appeared smooth and single peaked. Here, we used existing in vivo measurements on lac repressor and operator mutants in Escherichia coli to reconstruct the genotype-phenotype map that details the repression value of this regulatory system as a function of two key repressor residues and four key operator base pairs. We found that this landscape is multipeaked, harboring in total 19 distinct optima. Analysis showed that all direct evolutionary pathways between peaks involve significant dips in the repression value. Consistent with earlier predictions, we found reciprocal sign epistatic interactions at the repression minimum of the most favorable paths between two peaks. These results suggest that the occurrence of multiple peaks and reciprocal epistatic interactions may be a general feature in coevolving systems like the repressor-operator pair studied here.

Repetitive proteins are thought to have arisen through the amplification of subdomain-sized peptides. Many of these originated in a non-repetitive context as cofactors of RNA-based replication and catalysis, and required the RNA to assume their active conformation. In search of the origins of one of the most widespread repeat protein families, the tetratricopeptide repeat (TPR), we identified several potential homologs of its repeated helical hairpin in non-repetitive proteins, including the putatively ancient ribosomal protein S20 (RPS20), which only becomes structured in the context of the ribosome. We evaluated the ability of the RPS20 hairpin to form a TPR fold by amplification and obtained structures identical to natural TPRs for variants with 2–5 point mutations per repeat. The mutations were neutral in the parent organism, suggesting that they could have been sampled in the course of evolution. TPRs could thus have plausibly arisen by amplification from an ancestral helical hairpin.DOI: http://dx.doi.org/10.7554/eLife.16761.001

Many of the gene regulatory networks used within the field of synthetic biology have extensively employed the AraC and LacI inducible transcription factors. However, there is no Escherichia coli strain that provides a proper background to use both transcription factors simultaneously. We have engineered an improved E. coli strain by knocking out the endogenous lacI from a strain optimal for AraC containing networks, and thoroughly characterized the strain both at molecular and functional levels. We further show that it enables the gradual and independent induction of both AraC and LacI in a simultaneous manner. This construct will be of direct use for various synthetic biology applications.