Evolution of gene regulatory connections – Another lesson learned from phages

Gene regulation provides the basis for all organismal functions. Our study shows how very simple biophysical interactions between DNA and regulatory proteins provide the basis for gene regulatory evolution in microbes. We used genetic parts of bacteriophages - viruses that attack bacteria – to study how individual regulatory connections can change over time.
Evolution of gene regulatory connections – Another lesson learned from phages

The paper in Nature Ecology & Evolution is here: go.nature.com/2oWxv9d

Bacteriophages, and especially the model phage Lambda and its relatives, have been at the forefront of basic biological discoveries from gene regulation to bacterial virulence for almost 70 years now.  They are still providing new insights into molecular genetics as well as population ecology and evolution, and our study adds the evolution of gene regulatory networks to that list.

‘All the world is a phage’ © 2015, Forest Rohwer Laboratory, San Diego State University

Phage Lambda is a temperate phage, which means that besides just killing a bacterial cell, it can also choose a quiescent lifestyle by integrating itself into the genome of the infected host. The switch determining this lifestyle is a complex regulatory circuit that has fascinated many researchers and is understood in great molecular detail. Moreover, genetic switches of temperate phages are likely to be related through extensive horizontal gene transfer, but on the sequence level they are highly divergent from each other. We set out to investigate the molecular mechanisms driving the potential for this diversification. 

Evolutionary path between two repressor binding sites
By introducing mutations into the binding site of one repressor, we mutated to the other repressor binding sequence; changing the strength of binding for both repressors in opposite ways.

To this end, we measured binding of two bacteriophage transcriptional regulators along an artificial evolutionary path between their switches, which allowed us to estimate the potential of these regulators to alter their regulatory connectivity. We were surprised by how differently the two regulators responded to mutations in their DNA binding sites. It was therefore encouraging that using a simple model was enough to recapitulate those differences. More importantly, this model, which uses elementary thermodynamic principles to predict gene expression levels, allowed us to describe how the biophysical binding properties of regulators determine their evolutionary potential.

Biophysical parameters affect the evolutionary potential for rewiring
Model parameters that affect the evolutionary potential; most importantly the repressor binding parameters: offset (wild-type binding energy) and the energy matrix (mutation-dependent energy penalty).

Even though our study supplies a stepping-stone for understanding the evolution of gene regulatory connections, it also points to several intriguing questions:

-) Why are the two genetic switches of related bacteriophages so different? Did their evolutionary trajectories diverge by chance or were some selective factors in their genomic background responsible for this development?

-) How does the fitness landscape for regulatory evolution relate to the underlying molecular mechanisms that determine protein-DNA binding?

-) Our study indicates that a simple model cannot account for the phenotypic diversity produced by the presence of several DNA binding sites for regulatory proteins. Is this diversity an outcome of selection, or an inevitable property of biophysical laws?

By relating molecular and biophysical mechanisms to their evolutionary consequences, our study provides an example of how simple and well-studied model systems can still offer very valuable insights into basic biological questions.