Therefore new methods need to be developed where individual filaments can be labeled and the position of cells along a filament can be traced back to either the suboxic or the oxic zone

Therefore new methods need to be developed where individual filaments can be labeled and the position of cells along a filament can be traced back to either the suboxic or the oxic zone. this article are made available in the Supplementary Datasheet 1 provided in the Supplementary Material. Abstract 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 SETDB2 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. (Trojan et al., 2016), which also contains single-celled sulfate-reducing and sulfur disproportionating bacteria. Genomic analysis suggests that cable bacteria oxidize sulfide by reversing the canonical sulfate reduction pathway and use the WoodCLjungdahl pathway for inorganic carbon uptake (CO2 fixation), but also have the potential to additionally assimilate organic carbon (Kjeldsen et al., 2019). Stable isotope probing (SIP) experiments using 13C-labeled CO2 and propionate followed by either community lipid analysis (Vasquez-Cardenas et al., 2015) or analysis of individual cells and filaments by nanoscale secondary ion mass spectrometry GSK 525762A (I-BET-762) (nanoSIMS) (Geerlings et al., 2020) have confirmed that cable bacteria incorporate both inorganic and organic carbon. Cable bacteria can thus be categorized as facultative chemoautotrophs (Vasquez-Cardenas et al., 2015; Kjeldsen et al., 2019; Geerlings et al., 2020). Interestingly, carbon fixation in cable bacteria appears to be strongly dependent on the redox environment, where only the sulfide-oxidizing cells assimilate carbon whereas the oxygen-reducing cells do not assimilate carbon (Geerlings et al., 2020). Thus, the dichotomy that characterizes the energy metabolism in cable bacteria is also directly reflected in their carbon metabolism. Consequently, it appears that the cathodic cells dispense electrons as quickly as possible via oxygen reduction without any energy conservation, while biosynthesis and growth remain restricted to the anodic cells, which are able to generate metabolic energy from sulfide oxidation (Kjeldsen et al., 2019; Geerlings et al., 2020). A cable bacterium filament is linear (not branched) and typically consists of thousands of cells. Although the cells are separated from each other by a rigid septum, they share a periplasmic space that contains the network of conductive fibers, which run along the longitudinal axis of the filament (Pfeffer et al., 2012; Jiang et al., 2018; Meysman et al., 2019) and are inter-connected between adjacent cells by a cartwheel-shaped structure located within the septum (Cornelissen et al., 2018; Thiruvallur Eachambadi et al., 2020). Cable GSK 525762A (I-BET-762) bacterium filaments hence display a complex metabolism and architecture, but little is presently known about how these filaments grow and elongate. Previous observations by fluorescence microscopy have indicated GSK 525762A (I-BET-762) that filament growth is too fast to be exclusively apical, and hence cell division must occur continuously along the filament (Schauer et al., 2014). Here, we combine SIP-nanoSIMS, fluorescence microscopy and genomic data to gain insights into the cell cycle of cable bacteria and the process of filament elongation. Previously, the SIP-nanoSIMS technique has shown that the rates of inorganic carbon and nitrogen assimilation are remarkably homogeneous among the cells of individual filaments that perform the sulfide-oxidizing half-reaction (Geerlings et al., 2020). Here, we use these previously published data and expand it with three-dimensional reconstructions of stable isotope incorporation to gain more detailed insights into the biomass synthesis and growth of cable bacteria. We show that, on the level of individual cells, the process of cell division in cable bacteria appears to be highly comparable to that of the Gram-negative model species Yet, on the filament level, cable bacteria display unique characteristics, where the cells performing sulfide oxidation show synchronized cell division along the filament over millimeter-scale lengths. We propose a.