To reach their site of action, it is essential for antibiotic molecules to cross the bacterial outer membrane. The progress of enhanced sampling techniques in molecular dynamics simulations enables us to understand these translocations at an atomic level. To this end, calculations of free energy surfaces for these permeation processes are of key importance. Herein, we investigate the translocation of a variety of anionic solutes through the outer membrane pore OprO of the Gram-negative bacterium Pseudomonas aeruginosa using the metadynamics and umbrella sampling techniques at the all-atom level. Free energy calculations have been performed employing these two distinct methods in order to illustrate the difference in computed free energies, if any. The investigated solutes range from a single atomic chloride ion over a multiatomic monophosphate ion to a more bulky fosmidomycin antibiotic. The role of complexity of the permeating solutes in estimating accurate free energy profiles is demonstrated by performing extensive convergence analysis. For simple monatomic ions, good agreement between the well-tempered metadynamics and the umbrella sampling approaches is achieved, while for the permeation of the monophosphate ion differences start to appear. In the case of larger molecules such as fosmidomycin it is a tough challenge to achieve converged free energy profiles. This issue is mainly due to neglecting orthogonal degrees of freedom during the free energy calculations. Nevertheless, the freely driven metadynamics approach leads to clearly advantageous results. Additionally, atomistic insights of the translocation mechanisms of all three solutes are discussed.

The unprecedented rise of high-throughput sequencing and assay technologies has provided a detailed insight into the non-coding sequences and their potential role as gene expression regulators. These regulatory non-coding sequences are also referred to as cis-regulatory elements (CREs). Genetic variants occurring within CREs have been shown to be associated with altered gene expression and phenotypic changes. Such variants are known to occur spontaneously and ultimately get fixed, due to selection and genetic drift, in natural populations and, in some cases, pave the way for speciation. Hence, the study of genetic variation at CREs has improved our overall understanding of the processes of local adaptation and evolution. Recent advances in high-throughput sequencing and better annotations of CREs have enabled the evaluation of the impact of such variation on gene expression, phenotypic alteration and fitness. Here, we review recent research on the evolution of CREs and concentrate on studies that have investigated genetic variation occurring in these regulatory sequences within the context of population genetics.