Enhanced Laterally Resolved ToF-SIMS and AFM Imaging of the Electrically Conductive Structures in Cable Bacteria

Abstract

Cable bacteria are electroactive bacteria that form a long, linear chain of ridged cylindrical cells. These filamentous bacteria conduct centimeter-scale long-range electron transport through parallel, interconnected conductive pathways of which the detailed chemical and electrical properties are still unclear. Here, we combine time-of-flight secondary-ion mass spectrometry (ToF-SIMS) and atomic force microscopy (AFM) to investigate the structure and composition of this naturally occurring electrical network. The enhanced lateral resolution achieved allows differentiation between the cell body and the cell−cell junctions that contain a conspicuous cartwheel structure. Three ToF-SIMS modes were compared in the study of so-called fiber sheaths (i.e., the cell material that remains after the removal of cytoplasm and membranes, and which embeds the electrical network). Among these, fast imaging delayed extraction (FI-DE) was found to balance lateral and mass resolution, thus yielding the following multiple benefits in the study of structure−composition relations in cable bacteria: (i) it enables the separate study of the cell body and cell-cell junctions; (ii) by combining FI-DE with in situ AFM, the depth of Ni-containing protein - key in the electrical transport - is determined with greater precision; and (iii) this combination prevents contamination, which is possible when using an ex situ AFM. Our results imply that the interconnects in extracted fiber sheaths are either damaged during extraction, or that their composition is different from fibers, or both. From a more general analytical perspective, the proposed methodology of ToF-SIMS in the FI-DE mode combined with in situ AFM holds great promise for studying the chemical structure of other biological systems. C able bacteria are multicellular microorganisms that form long unbranched filaments and belong to the Desulfobulbaceae family. 1 They are the focus of interdisciplinary research due to their unique capability of conducting electrical currents over centimeter distances, 2,3 a process also known as long-distance electron transport (LDET). Cable bacteria have been found to thrive in different environments such as fresh water 4,5 and marine sediments, 6,7 and have also been found in different parts of the world. 7 Cable bacteria display a distinct morphology with parallel ridges running along the length of the filament. 1,8,9 Scanning electron microscopy of the cross section of a cable bacteria revealed the presence of fibers of about 50 nm in diameter under the ridges and a cartwheel structure at the junctions. 8 These fibers are embedded in the periplasm (i.e., in space between the cytoplasmic membrane and the bacterial outer membrane) and were suspected to be the conductive structures 8 (Figure 1A−C). Recently, Meysman et al. experimentally investigated the conductivity of these fibers. 9 A sequential extraction procedure was developed 8 (see the Experimental Section) from which the fiber structures can be isolated from the cable bacterium filaments. 8 After chemical removal of cytoplasm and membranes, a so-called fiber sheath remains, which embeds the periplasmic fibers. 9−11 The fiber sheath flattens when air-dried (Figure 1D), and the top part of this fiber sheath mirrors the bottom part due to its cylindrical symmetry. 8 Meysman et al. demonstrated that fiber sheaths were indeed highly conductive. 9 Fiber sheaths were placed on top of two gold pads with a nonconductive oxide spacing, and when applying a potential difference between the two pads, a flow of current indicated that the periplasmic fibers are the conductive conduits. These results were subsequently confirmed by