Mutations upstream from and in uncover a complex interplay between the cAMP receptor protein and different sigma factors.

In , one of the best understood microorganisms, much can still be learned about the basic interactions between transcription factors and promoters. When a cAMP-deficient mutant is supplied with maltose as the main carbon source, mutations develop upstream from the two genes and . Here, we explore the regulation of the two promoters, using fluorescence-based genetic reporters in combination with both spontaneously evolved and systematically engineered -acting mutations.

Restoring Global Gene Regulation through Experimental Evolution Uncovers a NAP (Nucleoid-Associated Protein)-Like Behavior of Crp/Cap.

How do hierarchical gene regulation networks evolve in bacteria? Nucleoid-associated proteins (NAPs) influence the overall structure of bacterial genomes, sigma factors and global transcription factors (TFs) control thousands of genes, and many operons are regulated by highly specific TFs that in turn are controlled allosterically by cellular metabolites. These regulatory hierarchies have been shaped by millions of years of evolution to optimize fitness in response to changing environmental conditions, but it is unclear how NAPs and TFs relate and have evolved together. Cyclic AMP (cAMP) receptor protein (Crp) is the paradigmatic global TF in Escherichia coli, and here we report that mutations in the gene compensate for loss of cAMP, showing that the interplay between Crp and the supercoiling status of promoters is key to global stress response.

Temporal evolution of master regulator Crp identifies pyrimidines as catabolite modulator factors.

The evolution of microorganisms often involves changes of unclear relevance, such as transient phenotypes and sequential development of multiple adaptive mutations in hotspot genes. Previously, we showed that ageing colonies of an E. coli mutant unable to produce cAMP when grown on maltose, accumulated mutations in the crp gene (encoding a global transcription factor) and in genes involved in pyrimidine metabolism such as cmk; combined mutations in both crp and cmk enabled fermentation of maltose (which usually requires cAMP-mediated Crp activation for catabolic pathway expression).

Tailoring the evolution of BL21(DE3) uncovers a key role for RNA stability in gene expression toxicity.

Gene expression toxicity is an important biological phenomenon and a major bottleneck in biotechnology. Escherichia coli BL21(DE3) is the most popular choice for recombinant protein production, and various derivatives have been evolved or engineered to facilitate improved yield and tolerance to toxic genes. However, previous efforts to evolve BL21, such as the Walker strains C41 and C43, resulted only in decreased expression strength of the T7 system.

Surface display as a functional screening platform for detecting enzymes active on PET.

Poly(ethylene terephthalate) (PET) is the world's most abundant polyester plastic, and its ongoing accumulation in nature is causing a global environmental problem. Currently, the main recycling processes utilize thermomechanical or chemical means, resulting in the deterioration of the mechanical properties of PET. Consequently, polluting de novo synthesis remains preferred, creating the need for more efficient and bio-sustainable ways to hydrolyze the polymer.