Abstract
A review of the recent publication by Gu et al. (
Naturehttps://doi.org/10.1038/s41586-021-03857-w) reveals that important
and well-known previously published data that directly refutes the
primary conclusions of Gu. et al. were not discussed.
Main Text
The discovery of electrically conductive filaments emanating from the
electroactive microbe Geobacter sulfurreducens 1introduced new possibilities for microbial long-range electron transport
of importance to global biogeochemical cycles, interspecies syntrophy,
microbial conversion of wastes to useful energy sources, and the
fabrication of sustainably produced electronic devices with novel
functions2-6. The initial report of these
filaments1, as well as many subsequent studies
(see7,8 for recent reviews), have indicated that the
most abundant conductive filaments are comprised of the pilin monomer
protein PilA, also known as PilA-N. As detailed below, recent direct
observation of filaments emanating from cells provides additional
evidence that 90 % of G. sulfurreducens’ extracellular filaments
are comprised of PilA-N9.
Gu et al.10 state that these earlier conclusions were
wrong. They claim that PilA-N combines with the protein PilA-C to form
poorly conductive filaments. However, the 6.5 nm diameter PilA-N/PilA-C
filaments that Gu et al. studied are artifacts produced by genetically
modified cells. Filaments of this diameter have never been observed in
wild-type G. sulfurreducens 7-9. Not only do Gu
et al. provide no evidence for these filaments in wild-type cells they
also fail to discuss the previous contradictory finding that in
wild-type cells PilA-C forms a PilA-C trimer that is associated with the
inner membrane11.
The foundation of the Gu et al. study is the initial result reported:
“Purified filament preparations from wild-type cells grown under these
nanowire-producing conditions did not show either PilA-N or PilA-C using
immunoblotting”. However, similar previous studies reported PilA-N in
filament preparations from wild-type cells, as well as PilA-N antibody
reacting with filaments12-15. In most
studies12-14 additional strong detergent or acidic
conditions were required to recover PilA-N, indicating that PilA-N was a
component of a highly stable filament and not a component of more easily
disassociated filament types, such as cytochrome-based filaments or the
proposed PilA-N/PilA-C filaments. Gu et al. fail to mention these
previously published results that directly contradict their primary
finding even though the corresponding author of Gu et al. was an author
on papers reporting the recovery of PilA-N from filament
preparations13,15.
Gu et al. also neglected to mention additional lines of evidence for the
presence of PilA-N in extracellular filaments. For example, G.
sulfurreducens strains expressing PilA-N with peptide tags display
filaments with those tags16. Decreasing the aromatic
amino acid content of PilA-N variants expressed in G.
sulfurreducens decreases filament conductivity and increasing the
abundance of aromatic amino acids increases
conductivity9,17-21. One of the most striking examples
of such results is the finding that expression of the homologous PilA-N
gene from G. metallireducens in G. sulfurreducens yields
individual filaments with the same 3 nm diameter as the wild-typeG. sulfurreducens filaments, but with a conductivity that is
5000-fold higher21. Changing the amino acid content of
PilA-N would not influence the conductivity of filaments comprised of
cytochromes or DNA that Gu et al. suggest predominate, but would be
expected to tune the conductivity of filaments comprised of PilA-N.
Gu et al. state that G. sulfurreducens cannot produce filaments
from PilA-N because the cells lack the twitching motility attributed to
type IV pili in Pseudomonas aeruginosa . In making this claim Gu
et al. fail to acknowledge the fact that G. sulfurreducens ’ lack
of twitching motility was previously described in the initial report on
its electrically conductive pili1. Not all bacteria
that have type IV pili exhibit twitching motility. The expectation that
filaments comprised of PilA-N could only exist if they also conferred
twitching motility is not justified.
The fact that deleting the gene for PilA-N impacts on the localization
of potential filament-producing c -type cytochromes has been known
for some time22 and was the impetus for the
construction of G. sulfurreducens strain Aro-5, a strain
expressing a pilin with reduced aromatic amino acid content while
properly localizing outer-surface cytochromes17.
Strain Aro-5 expresses filaments with a morphology like that of
wild-type cells, but the conductivity of individual filaments is orders
of magnitude lower19. Again, the only logical
explanation is that the filaments being investigated are comprised of
PilA-N. The conductivity of cytochrome- or DNA-based filaments should
not be influenced by a change in the PilA-N sequence.
Direct observation of cells demonstrated that 90% of the filaments
emanating from G. sulfurreducens are not cytochrome-based
filaments, rather they have a morphology and conductance like the
filaments that E. coli displays when it heterologously expresses
the G. sulfurreducens PilA-N9. The most likely
explanation for these observations is that the filaments emanating fromG. sulfurreducens are also comprised of PilA-N. In strain Aro-5,
90% of the filaments have this PilA-N filament morphology, but their
conductance is more than 100-fold lower than the wild-type
filaments9. This result is also consistent with the
filaments being comprised of PilA-N and inconsistent with cytochrome- or
DNA-based filaments.
Gu et al. and related studies15,23 have argued that
the 3 nm diameter filaments with the morphology and conductance expected
for filaments comprised of PilA-N, which are readily observed emanating
from cells9, do not exist because they were not
observed in cryo-EM analysis of G. sulfurreducens filament
preparations. Capturing these filaments in cryo-EM preparations will be
the key to unlocking their likely unique structure and mechanisms for
long-range electron transport.