The structural imaging in this manuscript is fantastic. My concern is that the wording of some portions of the text may give a reader that is not a specialist in bacterial nanowires the incorrect impression that: 1) the filaments that were investigated are conductive along their length under hydrated, physiologically relevant conditions; and that 2) Shewanella oneidensis has been shown to be capable of cell-to-cell electron transfer. Examples are listed blow.
1. The non-specialist is likely to be confused by the first sentence of the Abstract: “Bacterial nanowires have garnered recent interest as a proposed Extracellular Electron Transfer (EET) pathway that links the bacterial electron transport chain to solid-phase electron acceptors away from the cell.” It is true that electron transport along the length of nanowires produced by some bacteria has been shown to be involved in extracellular electron transfer. However, this is not the case for the Shewanella filaments studied in this investigation. Electron transport along the length of the Shewanella filaments has never been demonstrated under hydrated, physiologically relevant conditions. The Shewanella filaments have not been shown to be involved in electron transport to electrodes or other cells. Although the filaments do contain cytochromes that are involved in electron transport to minerals, there is no evidence that electron transport along the length of the filaments is important for mineral reduction.
2. The last sentence of the Abstract is also likely to confuse the non-specialist. The statement begins: “We therefore propose that electron transfer along nanowires involves…”. This phrase suggests that there is some evidence that the filaments that were studied with cryo-EM are known to be conductive along their length. There is no evidence for this. Measurements of conductivity along some form of Shewanella filaments were obtained with dried preparations (El-Naggar et al. 2012). The drying substantially shrinks the filaments which artificially compresses the distances between the cytochromes. Therefore, although dried filaments may be conductive, there is no evidence that the hydrated filaments that were investigated in this study were conductive. Many readers only read abstracts. It is important not to confuse readers by implying in the Abstract that the filaments that were studied by cryo-EM are known to be conductive along their length.
3. Lines 73-76 will probably confuse the non-specialist: “The Shewanella multiheme cytochromes MtrC and OmcA have also been found to localize along conductive outer membrane extensions known as bacterial nanowires, which are associated with Outer Membrane Vesicles (OMVs)20-22.” It should be clearly stated that the cytochromes are found throughout the outer-membrane of the cell. As now written, the logical conclusion of a non-specialist would be that the cytochromes are specifically localized in the membrane extensions because it is not mentioned that the cytochromes are found anywhere else.
4. Lines 76-78 could give the non-specialist the idea that Shewanella has properties that have not been documented: “Nanowires may offer a pathway for extending the respiratory electron transport chain of cells up to micrometers away from the inner membrane, possibly even to other cells22.” There is no evidence that Shewanella can transfer electrons to other cells.
5. Lines 162-163 could easily give the non-specialist the wrong impression of the properties of the filaments being studied: “It has been shown previously that the conductance of nanowires depends on the presence of the outer membrane multiheme cytochromes MtrC and OmcA21, and these cytochromes have been detected using immunofluorescence along membrane-stained nanowires from perfusion flow cultures22, so the nanowires we observe are likely conductive.” There is no evidence that the filaments that were observed were conductive. Again, conductivity has never been demonstrated with hydrated filaments.
6. Lines 341-344 could easily confuse the non-specialist: “This irregular packing of cytochromes means that EET along whole nanowires likely involves a combination of direct electron transfer within segments of closely packed cytochromes and cytochrome diffusion to bridge larger length scales.” This statement discusses electron transfer along the hydrated filaments as if this were a fact. It is not. As noted in the previous comments, such electron transfer has not been demonstrated. To suggest otherwise leaves the non-specialist with the wrong impression.
The general reader would be better served if the study was presented in a manner consistent with the findings in previous studies. Simply state that cytochromes have been proposed to be charge carriers for electron transport along the filaments, but previous conductivity measurements were made with dried filaments. In order to further evaluate the cytochrome hypothesis the filaments were examined with cryo-EM. The cryo-EM demonstrates that under physiologically relevant conditions the cytochromes are spaced too far apart for long-range conduction along the length of the filaments.
The study would greatly benefit from a control study with a mutant in which key cytochrome genes were deleted in order to definitively identify the electron-dense objects in the membrane.