Picture a telephone pole, its tall frame holding up wires with an electric current, directing the electricity to where it needs to go. If you were asked which part is carrying the current, you would quickly say it’s carried by the wires. However, when looking at an organic electrical system that is completely new to us, what structure is carrying the current and what structure is supporting it isn’t nearly as obvious. This time around, if you assume the things that look like wires are carrying the current, you might just be mistaken. Microbiological research can help us test our assumptions and figure out how these novel systems really work.
Geobacter is a genus of Gram-negative bacteria with a particularly interesting quality: it can transfer electrons. This process plays many important biological roles, including oxidizing metals such as iron, an ability that has attracted much interest from researchers, particularly those studying bioremediation and biofuel development. Electron transfer occurs in Geobacter species as a part of the anaerobic respiration process, where ATP is formed in the absence of oxygen by using other electron acceptors such as ferric iron, nitrate, or sulfur. This transfer helps in breaking down toxic materials such as heavy metals and electrons can even potentially be used as a current for electrical systems. The exact mechanism Geobacter species use to transfer electrons is still being determined, with many studies focusing on which parts of the cell structure are involved. This varies from species to species, and researchers have found diverse methods of electron transfer in different Geobacter species.
A new study, Gu et al., 2021, challenged assumptions about the role of pili in Geobacter sulfurreducens electron transfer. Pili are filamentous structures found in bacteria and archaea that can serve different purposes in different cells, including being involved in cell adhesion and conjugation. Pili are also used for a kind of movement known as twitching, where they are extended like a grappling hook to attach to a surface and then retract, pulling the cell forwards. Gram-negative bacterial pili are classified into five types, which fulfill different roles in the cell, and pili were originally suspected to play a direct role in G. sulfurreducens electron transfer. Earlier studies theorized that G. sulfurreducens create Type IV pili, which functioned as “nanowires,” and were responsible for electron transfer from the cell surface to the surface of electron acceptors. However, there was only circumstantial evidence for this theory, so Gu et al. set out to examine the true role of pili structures in this species.
Examining the pili in this bacterium, the researchers saw inconsistencies with typical structure of Type IV pili, which extend outside of the cell, and the pili structures in G. sulfurreducens and suggest that they are closer to Type II pseudopili, which secrete substrates, rather than building an extracellular pilus themselves.
Figure 1 from Melville and Craig illustrating Type IV vs Type II Pili
Type II secretion systems promote the secretion of proteins between the cell membranes within the periplasm, as compared to Type IV secretion systems, which secrete molecules outside of the bacterial cell into other cells or the extracellular space. G. sulfurreducens pili are made up of the proteins PiLA-N and PiLA-C, which combine to form a PilA-N-C filament. In their experiments, Wild Type cells showed no PilA-N-C filaments in extracellular space. Immunoblotting isolated nanowire filaments did not show either protein was present: instead, analysis showed that both proteins are associated with the inner membrane of the cell. The same observations were also found in Wild Type cells. As displayed in the representative model in Figure 1 from Melville and Craig 2014, PilA-N-C units were only found to be periplasmic, or in the region between the inner and outer cell membranes
Figure 1c from Gu et al. showing an immunoblot of subcellular fractionation for PilA-N and PilA-C in Wild Type cells. There is no pilA in the cytoplasm or outer membrane, but there is in the inner membrane and parts of the periplasm.
EC: extracellular | PP: periplasm | CY: cytoplasm | IM: inner membrane | OM: outer membrane.
The researchers also found that G. sulfurreducens pili are fragile and sensitive to lower temperatures, which is typical of Type II pseudopili. PilA-N-C filaments do not show any of the other characteristic functions of Type IV pili, such as twitching motility or adhesion, likely because they are so fragile. Furthermore, the pili were also more loosely associated with the cell than would be expected from Type IV pili, and were easily removed when induced to grow extracellularly. This further supports the idea that these are not Type IV pili, as a filament excreted outside of the cell would need to be more stable and durable than one excreted within it. So, with all this evidence, can we close the case and say G. sulfurreducens has Type II pili? Not yet. The observed similarity to Type II pseudopili does not necessarily mean the researchers found Type II pseudopili in G. sulfurreducens. Using phylogenetic analysis, the researchers determined that the G. sulfurreducens pili, along with the pili of many other Geobacter species, are evolutionarily distinct from both Type II and Type IV secretion systems in other gram-negative bacteria. Deleting genes that are usually crucial for the Type II secretion system also did not affect secretion of nanowires, potentially placing G. sulfurreducens in a new category of Type II-esque pseudopili.
Instead of the pili carrying these charges, this paper suggests that the nanowire capacity of G. sulfurreducens actually comes from excreted cytochromes, which are proteins with heme groups that contain an iron atom. In G. sulfurreducens, those cytochromes, which are found in the extracellular space, are OmcS and OmcZ.
Figure 1f and 1g from Gu et al. TEM image of OmcS nanowires vs Pili filaments in G. sulfurreducens
When analyzing the current-carrying capacity of these cytochrome nanowires compared to the pili, the nanowires at their greatest conductivity were able to exceed 20,000x the current-conducting limits of the pili. In a related experiment, another strong piece of evidence against PilA-N-C filaments acting as nanowires in G. sulfurreducens was their low conductivity in in-vitro experiments. PilA-N-C filaments had incredibly low levels of both voltage they could and their level of conductivity, meaning OmcS massively overshadows PilA-N-C filaments in both categories. These pili may look like wires, but they just don’t work like them.
Figure 7h and 7i from Gu et al. showing the large current and conductivity potential of OmcS cytochrome compared to PilA-N-C
Despite being a poor candidate for the wires of this structure, the PilA-N-C subunits still have a part to play in the electron-transferring nanowire system. Rather than being secreted, these pseudopili are anchored to the inner membrane. When the pilA gene itself is knocked out, no OmcS or OmcZ cytochromes are secreted, showing it is necessary for successful secretion of extracellular nanowires. Rather than being the wire itself, perhaps a better comparison is that they are akin to telephone poles: anchored and required for nanowires to be hung in the air (or in this case, secreted extracellularly). Gu. et al came up with a new model for how PilA-N-C could be structured in relation to the cell walls and the excretion of OmcS and OmcZ nanowires:
Figure 3e from Gu et al. of a model of structure of nanowire secretion with PilA-N-C in yellow/blue and OmcS in red
PilA-N-C playing a less-direct role in electron transfer greatly changes the general understanding of Geobacter nanowires and how they work in the cell. This model is similar to Type II pili secretion, but is evolutionarily distinct from known models, despite resembling them on the surface. Gu et al. did not explore whether this analogous system is entirely novel to known pili types or if there are other examples of Type II-like secretion in other species. This presents a clear avenue for future research, as there may be something more complicated going on with this secretion system, even if the researchers describe it as “analogous” to Type II secretion systems in the article. Alternatively, it could be a case of convergent evolution, when evolutionarily distant organisms develop the same traits separately from one another, similar to how birds, bats, and insects have all developed wings. Solving part of this puzzle helps scientists looking into practical applications of nanowires be better prepared to work with the cellular machinery in newly developed biotechnology.
About the Authors:
Lauren Comeau '23 and Fiona Quigley '23 are both seniors in the Biology Department at Mount Holyoke College taking BIOL-327 Microbiology with Dr. Amy Camp. Lauren is majoring in Biology and English, and plans to pursue research in entomology following her graduation. Fiona is majoring in Biology and English and looking to continue to do work in the field of microbiological research post-graduation.
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