Importance of c-Type cytochromes for U(VI) reduction by Geobacter sulfurreducens

Somewhere beneath a former nuclear weapons facility, uranium-laced groundwater is moving. Slowly, but it is moving — spreading through an aquifer, invisible, carried by geology. The cleanup problem isn't just finding it; it’s locking it in place. The most promising tool for achieving that isn’t a chemical treatment or an engineered barrier. It’s a bacterium living in the sediment. Geobacter sulfurreducens breathes metals the way we breathe oxygen. Instead of passing electrons to oxygen, it passes them to metal ions — including uranium. That electron transfer converts soluble hexavalent uranium, U(VI), into insoluble tetravalent uranium, U(IV), which precipitates as the mineral uraninite and stays put. Field trials and laboratory incubations, described by Shelobolina and colleagues, showed that when you stimulate microbial metal reduction in contaminated sediments, soluble uranium disappears from the groundwater — and the microbial community shifts to be dominated by Geobacteraceae. The bacterium works. But Shelobolina's team wanted to know exactly how. Which molecular machinery does it use? And where, inside the cell, does the actual chemistry happen? The proteins at the center of this story are c-type cytochromes — heme-containing proteins that carry electrons from one place to another, like relay runners passing a baton through the cell. To understand why their location matters, you need a quick picture of the cell's architecture. Moving from inside to outside: there's the inner membrane, then a gap called the periplasm, then the outer membrane. Cytochromes can sit in the periplasm or be anchored to the outer membrane, and that location determines whether uranium reduction happens inside the cell envelope or right at the cell surface. That distinction sounds like a microscopy footnote. It isn’t. If reduction happens at the outer membrane, uranium never needs to enter the cell. If it happens in the periplasm, uranium has to cross the outer membrane first. These are different processes, with different implications for engineering bioremediation at scale. To figure out which cytochromes matter, Shelobolina and colleagues took a direct approach: they deleted the genes for specific cytochromes, one by one, and measured what happened to U(VI) reduction. Twelve cytochrome-deficient strains in total — eleven single mutants and one double mutant — each tested against the wild type. The pattern that emerged was clear but not simple. Start with the outer membrane. Deleting certain outer membrane cytochromes hit uranium reduction hard. Deletion of OmcE knocked reduction down by 45 percent. Two putative outer membrane cytochromes, coded by genes GSU1334 and GSU3332, produced similar results — the GSU1334 mutant reduced uranium at roughly 50 percent of the wild type rate, and the GSU3332 mutant was similarly impaired. Summing across these outer membrane deletions, Shelobolina and colleagues report that elimination of two confirmed and two putative outer membrane cytochromes decreased U(VI) reduction by approximately 50 to 60 percent. But — and this is the critical point — not all outer membrane cytochromes behaved this way. Deleting OmcB and OmcC, two other confirmed outer membrane cytochromes, had very little impact on uranium reduction. That result rules out a simple story in which any outer membrane cytochrome can do the job. The effect is specific. Particular proteins matter; others are apparently irrelevant to uranium. Among the periplasmic cytochromes, most deletions had modest effects — members of the Ppc family retained about 80 to 90 percent of wild type activity when removed. But one deletion stood apart. Knocking out macA, which encodes a protein proposed to transfer electrons from the inner membrane into the periplasm, reduced U(VI) reduction by 98 percent. This was near-total abolition. MacA appears to be essential for getting electrons into the periplasm in the first place, which would explain why losing it is so catastrophic for uranium reduction regardless of what the outer membrane cytochromes are doing. There was also one surprise in the other direction. A strain lacking GSU0616 showed roughly 30 percent greater U(VI)-reducing activity than the wild type — suggesting that this cytochrome may normally compete with or suppress the uranium reduction pathway. Now here is where the story gets genuinely interesting. Transmission electron microscopy of uranium-exposed cells showed something striking: dark deposits of uranium accumulating in the periplasm. In bright-field images, the cell wall outlines light up with uranium contrast. In dark-field, those same regions shine. High-resolution imaging and electron diffraction confirmed the deposits were amorphous — diffuse diffraction rings, not the sharp spots of crystalline uraninite, with an average scattering distance of 2.2 angstroms. X-ray energy-dispersive spectroscopy showed elevated uranium and phosphorus in the periplasm, with relatively little in the cytoplasm. So uranium is getting into the periplasm. That seems to support periplasmic reduction. But then Shelobolina and colleagues counted cells. Only 24 percent of wild type cells had detectable periplasmic uranium by electron microscopy. And here’s the tension: the U(VI) reduction-impaired mutants — strains that had lost 50 to 60 percent of their uranium-reducing capacity — showed approximately 30 percent of cells with periplasmic uranium accumulation. Nearly the same fraction. The DL1-MacA strain, which had essentially no uranium-reducing activity at all, still accumulated uranium in the periplasm in that same subpopulation of cells. The periplasmic uranium deposits look identical in the impaired mutants and the wild type. Same amorphous character. Same elemental signature. But the mutants can barely reduce uranium. That disconnect is the key result. Periplasmic uranium accumulation and uranium reduction are not the same thing, and they don't track together. What the data suggest is that uranium can cross the outer membrane and sit in the periplasm as an amorphous precipitate without ever being enzymatically reduced. The accumulation is not evidence of reduction. It may simply reflect passive penetration. This reframes what the electron microscopy images mean. Seeing uranium in the periplasm does not tell you that’s where reduction happened. The fact that outer membrane cytochrome deletions substantially impair uranium reduction — while periplasmic accumulation stays constant — points toward the cell surface, not the periplasm, as the primary site of enzymatic activity. The iron comparison drives this point home from a different angle. Geobacter is famous for reducing iron — it's the founding function of the genus. So Shelobolina and colleagues tested the same cytochrome deletion mutants against both iron three hydroxide and chelated iron three. What they found was no correlation between the impact on uranium and the impact on iron. OmcB deletion barely touches uranium reduction but cripples iron three reduction. OmcE deletion cuts uranium reduction by 45 percent while leaving iron reduction at wild type levels. The GSU1334 and GSU3332 mutants were deficient in both uranium and iron three hydroxide reduction but retained activity with soluble, chelated iron — a distinction that separates insoluble from soluble iron pathways. MacA deletion abolished uranium reduction and soluble iron three reduction, but the MacA mutant still reduced iron three hydroxide at 42 percent of the wild type rate. Across the panel, deletion of cytochromes rarely produced similar impacts on both metals. That lack of correlation has a straightforward implication: U(VI) and iron three do not share a single electron transfer pathway through the cell. They use overlapping but distinct molecular routes. This matters for how we think about using Geobacter in the field. A community thriving on iron reduction is not automatically optimized for uranium reduction. The two activities can be decoupled by the specific cytochrome composition of the cell. Shelobolina and colleagues are careful about the limits of their conclusions. Complementation data — the definitive test that restoring a deleted gene rescues activity — was incomplete for some mutants. Gene deletions can have indirect effects, altering the abundance of neighboring proteins; the omcF deletion, for instance, changed the levels of at least six other outer membrane cytochromes. So the precise mechanistic role of each individual cytochrome remains to be established. What the data establish firmly is this: c-type cytochromes are central to uranium reduction in Geobacter sulfurreducens. The reduction is specific — not every outer membrane cytochrome contributes, and the periplasmic cytochrome MacA plays an outsized role in feeding electrons into the system. The electron transfer routes to uranium and iron are distinct. Periplasmic uranium accumulation, visible and measurable by electron microscopy, is not the same as reduction — it may simply reflect uranium's capacity to cross the outer membrane and sit there. For bioremediation, that last point is the one to hold onto. If uranium reduction happens primarily at the cell surface, then the relevant engineering target is the outer membrane — the cytochromes anchored there, the electron flow reaching them, and the conditions that keep those proteins active in a contaminated aquifer. The bacterium is already in the sediment. The question is how to keep it working. This lecture was created by ennepō. Go to https://ennepo.ai to Discover, Create and Follow the latest research in your field. Read when you can. Listen when you want to.