Fluorinated amino acids profoundly perturb cellular physiology because they enter the proteome while differing from their natural counterparts in subtle but functionally important ways. Here we investigated how Escherichia coli adapts to the biosynthesis and proteome-wide incorporation of fluorinated tryptophans derived from 4-, 5-, 6-, and 7-fluoroindoles using adaptive laboratory evolution (ALE). Whole-genome sequencing of independently evolved populations revealed convergent adaptive solutions. All 6- and 7-fluoroindole lineages acquired disruptive mutations in the stringent starvation regulator (sspA), effectively attenuating stress signaling and allowing continued expression of housekeeping functions despite proteotoxic pressure. In parallel, recurrent mutations in tryptophanyl-tRNA synthetase (trpS), which charges tRNATrp with tryptophan, pointed to translational tuning consistent with improved handling of fluorinated substrates, with Q27P emerging most prominently. In several 6-fluoroindole populations, additional defects in mutS, involved in DNA mismatch repair, allowed replication errors to accumulate, generating transient hypermutator states that accelerated evolutionary exploration but were not required for successful adaptation. Reconstruction experiments confirmed that loss of stringent response control and altered TrpRS function together increased fitness during fluorotryptophan incorporation. Together, these results suggest that adaptation does not appear to primarily rely on establishing a fundamentally new fluorine-based biochemistry, but rather on adjustment of stress-response regulation and translational control. More broadly, this work establishes a general framework for understanding, and ultimately engineering, microbial adaptation to non-natural metabolites through targeted modification of regulatory and translational control nodes rather than metabolic redesign.

Nature has scarcely evolved a biochemistry around fluorine. However, modern science has shown that fluorinated organic molecules are suitable building blocks for biopolymers, from peptides and proteins up to entire organisms. Here, we conducted adaptive laboratory evolution (ALE) experiments to introduce organofluorine into living microorganisms. By cultivating Escherichia coli with fluorinated indole analogs, we successfully evolved microbial cells capable of utilizing either 6-fluoroindole or 7-fluoroindole for growth. Our improved ALE protocols enabled us to overcome previous challenges and adaptation was achieved, enabling a former growth inhibiting unnatural molecule to become a substrate for the cell’s protein synthesis machinery to the extent that the entire proteome underwent Trp to F-Trp substitution. In the ALE experiments, we supplied fluoroindoles to Trp-auxotrophic E. coli bacteria, exerting strong selective pressure that led to microbial adaptation. Within the cells, these indoles were converted into the corresponding amino acids (6- and 7-fluorotryptophan) and globally incorporated into the proteome at tryptophan sites. This study is a first step and establishes a strong foundation for further exploration of the mechanisms underlying fluorine-based life and how a former antimetabolite can become a vital nutrient.

The chemistry of fluorinated compounds was essentially established by humans. Yet, fluorinated molecules have been present in our daily lives for a long time, be it as a nonstick coating for pans or as liquid crystals that are used in every smartphone, or as an important component of herbicides, pesticides, and pharmaceuticals. However these compounds are also released into nature and interact with plants and bacteria. Little is known about how nature adapts to increasing exposure to fluorinated molecules. In this chapter we want to shed light on the paths that fluorine takes in the ecosphere. We will discuss what nature does with these molecules and how nature itself produces fluorinated molecules. This is important in the context of the development of sustainable concepts in chemistry in order to conserve resources and tackle climate change.

The self-assembly of peptides onto the surface of gold nanoparticles has emerged as a promising strategy towards the creation of artificial enzymes. The resulting high local peptide density surrounding the nanoparticle leads to cooperative and synergistic effects, which result in rate accelerations and distinct catalytic properties compared to the unconjugated peptide. This Minireview summarizes contributions to and progress made in the field of catalytically active peptide-gold nanoparticle conjugates. The origin of distinct properties, as well as potential applications, are also discussed.

Multicomponent self-assembly of peptides is a powerful strategy to fabricate novel functional materials with synergetic properties that can be used for several nanobiotechnological applications. In the present study, we used a coassembly strategy to generate an injectable ultrashort bioactive peptide hydrogel formed by mixing a dipeptide hydrogelator with a macrophage attracting short chemotactic peptide ligand. Coassembly does not impede hydrogelation as shown by cryo-transmission electron microscopy (cryo-TEM), scanning electron microscopy, and rheology. Biocompatibility was shown by cytotoxicity assays and confocal microscopy. The hydrogels release the entrapped skin antibiotic ciprofloxacin, among others, in a slow and continuous manner. Such bioinspired advanced functional materials can find applications as wound dressing materials to treat chronic wound conditions like diabetic foot ulcer.

Infections caused by Staphylococcal and Micrococcal species represent a major public health burden. Although treatments do exist, these tend to be associated with cytotoxic effects; furthermore, the emergence of antimicrobial resistance presents an immediate challenge. New classes of active compounds are required to address these threats to human health. Here we present a de novo peptidomimetic strategy that produces self-assembling cationic antimicrobials. To identify a candidate compound with bactericidal activity, a small library of 8 peptidomimetics comprising ultrashort peptide sequences attached to a 3,5-diaminobenzoic acid scaffold was generated and tested against Micrococcus luteus and Staphylococcus aureus. Self-assembly appears to be the driving force for increased potency, likely by contributing to increased local surface charge density and peptide mass and producing a multivalent effect that enhances electrostatic interactions with negatively charged bacterial membranes, causing membrane disruption. The most active library member C7 forms patched micellar nanoparticles and has an activity higher than that of known natural antimicrobial peptides against M luteus. C7 also shows activity comparable to that of gramicidin S and the standard antibiotic vancomycin used in antibacterial therapy, but with a greater selectivity index. Importantly, C7 is also nontoxic and nonhemolytic, unlike the currently administered vancomycin, which can cause acute renal failure, and gramicidin S, which is highly hemolytic in nature. The short sequence length, ease of design, convenient synthesis strategy, and presence of a substitutable hydrophobic residue that enables self-assembly into different nanostructures make this model compound highly attractive for generating cost-effective, rapid-acting peptide-based antimicrobials.