Cable bacteria embed a network of conductive protein fibers in their cell envelope that efficiently guides electron transport over distances spanning up to several centimeters. This form of long-distance electron transport is unique in biology and is mediated by a metalloprotein with a sulfur-coordinated nickel (Ni) cofactor. However, the molecular structure of this cofactor remains presently unknown. Here, we applied multi-wavelength Raman microscopy to identify cell compounds linked to the unique cable bacterium physiology, combined with stable isotope labeling, and orientation-dependent and ultralow-frequency Raman microscopy to gain insight into the structure and organization of this novel Ni-cofactor. Raman spectra of native cable bacterium filaments reveal vibrational modes originating from cytochromes, polyphosphate granules, proteins, as well as the Ni-cofactor. After selective extraction of the conductive fiber network from the cell envelope, the Raman spectrum becomes simpler, and primarily retains vibrational modes associated with the Ni-cofactor. These Ni-cofactor modes exhibit intense Raman scattering as well as a strong orientation-dependent response. The signal intensity is particularly elevated when the polarization of incident laser light is parallel to the direction of the conductive fibers. This orientation dependence allows to selectively identify the modes that are associated with the Ni-cofactor. We identified 13 such modes, some of which display strong Raman signals across the entire range of applied wavelengths (405–1,064 nm). Assignment of vibrational modes, supported by stable isotope labeling, suggest that the structure of the Ni-cofactor shares a resemblance with that of nickel bis(1,2-dithiolene) complexes. Overall, our results indicate that cable bacteria have evolved a unique cofactor structure that does not resemble any of the known Ni-cofactors in biology.@en

Cable bacteria are filamentous, multicellular microorganisms that display an exceptional form of biological electron transport across centimeter-scale distances. Currents are guided through a network of nickel-containing protein fibers within the cell envelope. Still, the mechanism of long-range conduction remains unresolved. Here, we characterize the conductance of the fiber network under dry and wet, physiologically relevant, conditions. Our data reveal that the fiber conductivity is high (median value: 27 S cm⁻¹; range: 2 to 564 S cm⁻¹), does not show any redox signature, has a low thermal activation energy (E_a = 69 ± 23 meV), and is not affected by humidity or the presence of ions. These features set the nickel-based conduction mechanism in cable bacteria apart from other known forms of biological electron transport. As such, conduction resembles that of an organic semi-metal with a high charge carrier density. Our observation that biochemistry can synthesize an organo-metal-like structure opens the way for novel bio-based electronic technologies.

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Cable bacteria are multicellular, Gram-negative filamentous bacteria that display a unique division of metabolic labor between cells. Cells in deeper sediment layers are oxidizing sulfide, while cells in the surface layers of the sediment are reducing oxygen. The electrical coupling of these two redox half reactions is ensured via long-distance electron transport through a network of conductive fibers that run in the shared cell envelope of the centimeter-long filament. Here we investigate how this unique electrogenic metabolism is linked to filament growth and cell division. Combining dual-label stable isotope probing (¹³C and ¹⁵N), nanoscale secondary ion mass spectrometry, fluorescence microscopy and genome analysis, we find that the cell cycle of cable bacteria cells is highly comparable to that of other, single-celled Gram-negative bacteria. However, the timing of cell growth and division appears to be tightly and uniquely controlled by long-distance electron transport, as cell division within an individual filament shows a remarkable synchronicity that extends over a millimeter length scale. To explain this, we propose the “oxygen pacemaker” model in which a filament only grows when performing long-distance transport, and the latter is only possible when a filament has access to oxygen so it can discharge electrons from its internal electrical network.

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Filamentous cable bacteria display long-range electron transport, generating electrical currents over centimeter distances through a highly ordered network of fibers embedded in their cell envelope. The conductivity of these periplasmic wires is exceptionally high for a biological material, but their chemical structure and underlying electron transport mechanism remain unresolved. Here, we combine high-resolution microscopy, spectroscopy, and chemical imaging on individual cable bacterium filaments to demonstrate that the periplasmic wires consist of a conductive protein core surrounded by an insulating protein shell layer. The core proteins contain a sulfur-ligated nickel cofactor, and conductivity decreases when nickel is oxidized or selectively removed. The involvement of nickel as the active metal in biological conduction is remarkable, and suggests a hitherto unknown form of electron transport that enables efficient conduction in centimeter-long protein structures.

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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.

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Dark carbon fixation (DCF) by chemoautotrophic microorganisms can sustain food webs in the seafloor by local production of organic matter independent of photosynthesis. The process has received considerable attention in deep sea systems, such as hydrothermal vents, but the regulation, depth distribution, and global importance of coastal sedimentary DCF have not been systematically investigated. Here we surveyed eight coastal sediments by means of stable isotope probing (¹³C-DIC) combined with bacterial biomarkers (phospholipid-derived fatty acids) and compiled additional rates from literature into a global database. DCF rates in coastal sediments range from 0.07 to 36.30 mmol C m⁻² day⁻¹, and there is a linear relation between DCF and water depth. The CO₂ fixation ratio (DCF/CO₂ respired) also shows a trend with water depth, decreasing from 0.09 in nearshore environments to 0.04 in continental shelf sediments. Five types of depth distributions of chemoautotrophic activity are identified based on the mode of pore water transport (advective, bioturbated, and diffusive) and the dominant pathway of microbial sulfur oxidation. Extrapolated to the global coastal ocean, we estimate a DCF rate of 0.04 to 0.06 Pg C year⁻¹, which is less than previous estimates based on indirect measurements (0.15 Pg C year⁻¹), but remains substantially higher than the global DCF rate at deep sea hydrothermal vents (0.001–0.002 Pg C year⁻¹).

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Microphytobenthos forms an important part of the diet of macrofauna (macrozoobenthos) in many intertidal ecosystems. It is unclear, however, whether the dependence of macrofauna on microphytobenthos varies spatially within and among tidal systems. We aim (1) to assess the spatial variability in the importance of microphytobenthos in the diet of macrofauna (i.e., between and within two tidal basins and as function of elevation), (2) to quantify grazing pressure of the macrofaunal community on different potential food sources (microphytobenthos, phytoplankton and terrestrial organic material) for several sites in two tidal basins and (3) to compare microphytobenthic production and summer/autumn grazing of the total macrofaunal community and grazing pressure per feeding type, with potential microphytobenthic production estimated from rates in early spring, when grazing was low. Using a natural stable isotope approach, we identified microphytobenthos as a more important food source for macrofauna than phytoplankton and terrestrial organic material. Microphytobenthos dependency differed between tidal basins for the genera Bathyporeia (sand digger shrimp), Macoma (Baltic tellin), and Peringia (mudsnail) and for sampled individuals of all genera combined, and did not vary as function of elevation. We showed that macrofaunal grazing on microphytobenthos is quantitatively important and, in some cases, approached microphytobenthic production rates in early spring. No positive relation between microphytobenthic production in early spring and macrofaunal grazing in summer/autumn was observed. This suggests that the studied consumer-resource interactions are coupled on a larger spatial scale (i.e., mesoscale, ≈10 to 100 km), rather than the fine (mm to m) scale.@en

Multicellularity is a key evolutionary innovation, leading to coordinated activity and resource sharing among cells, which generally occurs via the physical exchange of chemical compounds. However, filamentous cable bacteria display a unique metabolism in which redox transformations in distant cells are coupled via long-distance electron transport rather than an exchange of chemicals. This challenges our understanding of organismal functioning, as the link among electron transfer, metabolism, energy conservation, and filament growth in cable bacteria remains enigmatic. Here, we show that cells within individual filaments of cable bacteria display a remarkable dichotomy in biosynthesis that coincides with redox zonation. Nanoscale secondary ion mass spectrometry combined with ¹³C (bicarbonate and propionate) and ¹⁵N-ammonia isotope labeling reveals that cells performing sulfide oxidation in deeper anoxic horizons have a high assimilation rate, whereas cells performing oxygen reduction in the oxic zone show very little or no label uptake. Accordingly, oxygen reduction appears to merely function as a mechanism to quickly dispense of electrons with little to no energy conservation, while biosynthesis and growth are restricted to sulfide-respiring cells. Still, cells can immediately switch roles when redox conditions change, and show no differentiation, which suggests that the “community service” performed by the cells in the oxic zone is only temporary. Overall, our data reveal a division of labor and electrical cooperation among cells that has not been seen previously in multicellular organisms.

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Biological electron transport is classically thought to occur over nanometre distances, yet recent studies suggest that electrical currents can run along centimetre-long cable bacteria. The phenomenon remains elusive, however, as currents have not been directly measured, nor have the conductive structures been identified. Here we demonstrate that cable bacteria conduct electrons over centimetre distances via highly conductive fibres embedded in the cell envelope. Direct electrode measurements reveal nanoampere currents in intact filaments up to 10.1 mm long (>2000 adjacent cells). A network of parallel periplasmic fibres displays a high conductivity (up to 79 S cm-1), explaining currents measured through intact filaments. Conductance rapidly declines upon exposure to air, but remains stable under vacuum, demonstrating that charge transfer is electronic rather than ionic. Our finding of a biological structure that efficiently guides electrical currents over long distances greatly expands the paradigm of biological charge transport and could enable new bio-electronic applications.

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Coastal seas like the North Sea have been subject to major changes in nutrient inputs over the last decades, resulting in shifts of limiting nutrients for phytoplankton communities. Here, we investigated the seasonal and spatial distribution and synthesis patterns of individual amino acids and distinct fatty acid groups and show how these were affected by different nutrient limitations in natural coastal phytoplankton communities. Nitrogen limited communities exhibited substantially slower synthesis of essential amino acids compared to synthesis of nonessential amino acids. In short-term nutrient addition experiments, this trend was reversed immediately after N addition to levels found under not limiting conditions. On the contrary, phosphorus limited communities showed no such shift in amino acids. Both N and P limitation induced a shift from structural to storage fatty acids with a concurrent decrease in the synthesis of poly-unsaturated fatty acids. Reversed effects in fatty acid synthesis after N or P addition were only apparent after 72 h, when they could be found in both fatty acid biosynthesis and concentrations. The different strategies of qualitative and quantitative regulation of different biomolecule synthesis under nutrient scarcity may have far-reaching consequences for the phytoplankton's nutritional value. Higher trophic levels may have to cope with the loss of essential amino acids and poly-unsaturated fatty acids in nutrient limited phytoplankton, which could induce changes in the structure of food webs.

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Petroleum hydrocarbons reach the deep-sea following natural and anthropogenic factors. The process by which they enter deep-sea microbial food webs and impact the biogeochemical cycling of carbon and other elements is unclear. Hydrostatic pressure (HP) is a distinctive parameter of the deep sea, although rarely investigated. Whether HP alone affects the assembly and activity of oil-degrading communities remains to be resolved. Here we have demonstrated that hydrocarbon degradation in deep-sea microbial communities is lower at native HP (10 MPa, about 1000 m below sea surface level) than at ambient pressure. In long-term enrichments, increased HP selectively inhibited obligate hydrocarbon-degraders and downregulated the expression of beta-oxidation-related proteins (i.e., the main hydrocarbon-degradation pathway) resulting in low cell growth and CO₂ production. Short-term experiments with HP-adapted synthetic communities confirmed this data, revealing a HP-dependent accumulation of citrate and dihydroxyacetone. Citrate accumulation suggests rates of aerobic oxidation of fatty acids in the TCA cycle were reduced. Dihydroxyacetone is connected to citrate through glycerol metabolism and glycolysis, both upregulated with increased HP. High degradation rates by obligate hydrocarbon-degraders may thus be unfavourable at increased HP, explaining their selective suppression. Through lab-scale cultivation, the present study is the first to highlight a link between impaired cell metabolism and microbial community assembly in hydrocarbon degradation at high HP. Overall, this data indicate that hydrocarbons fate differs substantially in surface waters as compared to deep-sea environments, with in situ low temperature and limited nutrients availability expected to further prolong hydrocarbons persistence at deep sea.

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Anoxic mineralization of organic matter releases dissolved inorganic carbon and produces reduced mineralization products. The reoxidation of these reduced compounds is essential for biogeochemical cycling in sediments and is mainly performed by chemoautotrophic microbes, which synthesize new organic carbon by dark CO₂ fixation. At present however, the biogeochemical importance of chemoautotrophy in high-latitude sediments is largely unknown. Here, we determine the seasonal variation in sedimentary chemoautotrophic production in Kobbefjord (SW Greenland). Intact sediment cores from the fjord were incubated, and dark CO₂ fixation was quantified by combining bacterial phospholipid-derived fatty acid analysis with ¹³C stable isotope probing (PLFA-SIP). Our results reveal a distinct seasonal cycle in chemoautotrophic activity, which increases after the spring bloom and shows lowest activity in the late winter when the fjord is covered by sea ice. The depth distribution of chemoautotrophic activity also varied seasonally, likely due to seasonal variation in the bioturbation activity of sediment infauna. Although chemoautotrophy rates (0.4 ± 0.2 mmol C m⁻² d⁻¹) were in the low range for coastal sediments, they are comparable to those from intertidal sandflats and brackish tropical lagoons, and scale with the sulfide production through sulfate reduction in the fjord. Chemoautotrophic production in these fjord sediments thus appears to be mainly driven by sulfide oxidation and can re-fix 4% of the CO₂ produced by mineralization.

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Electron transport within living cells is essential for energy conservation in all respiring and photosynthetic organisms. While a few bacteria transport electrons over micrometer distances to their surroundings, filaments of cable bacteria are hypothesized to conduct electric currents over centimeter distances. We used resonance Raman microscopy to analyze cytochrome redox states in living cable bacteria. Cable-bacteria filaments were placed in microscope chambers with sulfide as electron source and oxygen as electron sink at opposite ends. Along individual filaments a gradient in cytochrome redox potential was detected, which immediately broke down upon removal of oxygen or laser cutting of the filaments. Without access to oxygen, a rapid shift toward more reduced cytochromes was observed, as electrons were no longer drained from the filament but accumulated in the cellular cytochromes. These results provide direct evidence for long-distance electron transport in living multicellular bacteria.

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The flexible regulation of feeding behaviour and nutrient metabolism is a prerequisite for consumers to grow and survive under variable food conditions. Thus, it is essential to understand the ecological trade-offs that restrict regulatory mechanisms in consumers to evaluate the consequences of nutrient limitations for trophic interactions. Here, we assessed behavioural and physiological adjustments to nutrient deficiencies in copepods and examined whether energy limitation, food digestibility or co-limitation with a second nutrient restricted compensatory mechanisms. A combination of ¹³C-labelling and compound-specific stable isotope measurements revealed that copepods compensated nitrogen deficiencies by raising retention efficiencies of amino acids (AA). The costs of higher retention efficiencies were reflected in the doubling of structural fatty acids (FA), probably required for morphological adaptations of the gut. A depletion of highly unsaturated FA in storage lipids and their selective retention suggested that these FA became co-limiting and restricted a further increase in AA retention efficiencies. Copepods feeding on phosphorus-limited algae showed a marked increase in ingestion rates but were not fully able to compensate dietary deficiencies. The increase in ingestion rates was thereby not restricted by higher foraging costs because energy storage in copepods increased. Instead, thicker cell walls of nutrient-limited algae indicated that algal digestion resistance restricted the extent of surplus feeding. The strongly nutrient-specific response of copepods had large implications for recycling rates, growth efficiencies and the potential top-down control at the plant–animal interface. Compensatory mechanisms to mitigate nutrient deficiencies are therefore an essential aspect of trophic interactions and have the potential to alter the structure of food web. A plain language summary is available for this article.

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We investigated seasonal changes in the production of extracellular polymeric substances (EPS) and short-chain organic acids (SCOA) exuded by benthic diatoms, and the use of these exudates as a carbon source by heterotrophic bacteria. An in situ ¹³C pulse-chase method was used to follow the fate of EPS for 5 consecutive days. These experiments were done at 2 mo intervals for 1 yr. The EPS were recovered from the sediment as 2 operationally defined fractions (i.e. water-extractable and EDTA-extractable EPS). Seasonal differences in EPS production correlated to light intensity and temperature. From February until June the biomass and production of diatoms and bacteria were closely coupled. It was concluded that SCOA were the most important substrates for the bacteria. Sulfate-reducing bacteria (SRB) in particular benefited from SCOA released by diatoms. From August on, the coupling of biomass and production of diatoms and bacteria weakened and was almost lost in December. During the period from August to December, EPS produced by diatoms promoted the growth of other bacterial taxa rather than SRB, and the production of SCOA was low. Thus, it appears that the seasonal variation in exudates produced by diatoms plays an important role in shaping community composition and maintaining the diversity of the associated bacteria.

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