Engineering Nanowires in Bacteria to Elucidate Electron Transport Structural-Functional Relationships

Bacterial pilin nanowires are protein complexes, suggested to possess electroactive capabilities forming part of the cells’ bioenergetic programming. Their role is thought to be linked to facilitating electron transfer with the external environment to permit metabolism and cell-to-cell communication. There is a significant debate, with varying hypotheses as to the nature of the proteins currently lying between type-IV pilin-based nanowires and polymerised cytochrome-based filaments. Importantly, to date, there is a very limited structure-function analysis of these structures within whole bacteria. In this work, we engineered Cupriavidus necator H16, a model autotrophic organism to express differing aromatic modifications of type-IV pilus proteins to establish structure-function relationships on conductivity and the effects this has on pili structure. This was achieved via a combination of high-resolution PeakForce tunnelling atomic force microscopy (PeakForce TUNA™) technology, alongside conventional electrochemical approaches enabling the elucidation of conductive nanowires emanating from whole bacterial cells for the first time. This work is the first example of functional type-IV pili protein nanowires produced under aerobic conditions using a CN chassis. This work has far-reaching consequences in understanding the basis of bio-electrical communication between cells and with their external environment. Graphical abstract displaying theoretical PilA monomer models (left), PeakForce TUNA atomic force microscopy contact current images (right) of wild-type (top) and modified with increased tyrosine content (bottom) PilA filaments expressed by Cupriavidus necator H16 cells.