Nielsen, Lars Peter

ABSTRACT Bacterial cells can vary greatly in size, from a few hundred nanometers to hundreds of micrometers in diameter. Filamentous cable bacteria also display substantial size differences, with filament diameters ranging from 0.4 to 8 µm. We analyzed the genomes of cable bacterium filaments from 11 coastal environments of which the resulting 23 new genomes represent 10 novel species-level clades of Candidatus Electrothrix and two clades that putatively represent novel genus-level diversity. Fluorescence in situ hybridization with a species-level probe showed that large-sized cable bacteria belong to a novel species with the proposed name Ca . Electrothrix gigas. Comparative genome analysis suggests genes that play a role in the construction or functioning of large cable bacteria cells: the genomes of Ca . Electrothrix gigas encode a novel actin-like protein as well as a species-specific gene cluster encoding four putative pilin proteins and a putative type II secretion platform protein, which are not present in other cable bacteria. The novel actin-like protein was also found in a number of other giant bacteria, suggesting there could be a genetic basis for large cell size. This actin-like protein (denoted big bacteria protein, Bbp) may have a function analogous to other actin proteins in cell structure or intracellular transport. We contend that Bbp may help overcome the challenges of diffusion limitation and/or morphological complexity presented by the large cells of Ca . Electrothrix gigas and other giant bacteria. IMPORTANCE In this study, we substantially expand the known diversity of marine cable bacteria and describe cable bacteria with a large diameter as a novel species with the proposed name Candidatus Electrothrix gigas. In the genomes of this species, we identified a gene that encodes a novel actin-like protein [denoted big bacteria protein (Bbp)]. The bbp gene was also found in a number of other giant bacteria, predominantly affiliated to Desulfobacterota and Gammaproteobacteria, indicating that there may be a genetic basis for large cell size. Thus far, mostly structural adaptations of giant bacteria, vacuoles, and other inclusions or organelles have been observed, which are employed to overcome nutrient diffusion limitation in their environment. In analogy to other actin proteins, Bbp could fulfill a structural role in the cell or potentially facilitate intracellular transport.

Cable bacteria are centimeters-long filamentous bacteria that oxidize sulfide in anoxic sediment layers and reduce oxygen at the oxic-anoxic interface, connecting these reactions via electron transport. The ubiquitous cable bacteria have a major impact on sediment geochemistry and microbial communities. This includes diverse bacteria swimming around cable bacteria as dense flocks in the anoxic zone, where the cable bacteria act as chemotactic attractant. We hypothesized that flocking only appears when cable bacteria are highly abundant and active. We set out to discern the timing and drivers of flocking over 81 days in an enrichment culture of the freshwater cable bacterium Candidatus Electronema aureum GS by measuring sediment microprofiles of pH, oxygen, and electric potential as a proxy of cable bacteria activity. Cable bacterial relative abundance was quantified by 16S rRNA amplicon sequencing, and microscopy observations to determine presence of flocking. Flocking was always observed at some cable bacteria, irrespective of overall cable bacteria rRNA abundance, activity, or sediment pH. Diverse cell morphologies of flockers were observed, suggesting that flocking is not restricted to a specific, single bacterial associate. This, coupled with their consistent presence supports a common mechanism of interaction, likely interspecies electron transfer via electron shuttles. Flocking appears exclusively linked to the electron conducting activity of the individual cable bacteria.

Cable bacteria of the family Desulfobulbaceae form centimeter-long filaments comprising thousands of cells. They occur worldwide in the surface of aquatic sediments, where they connect sulfide oxidation with oxygen or nitrate reduction via long-distance electron transport. In the absence of pure cultures, we used single-filament genomics and metagenomics to retrieve draft genomes of 3 marine Candidatus Electrothrix and 1 freshwater Ca. Electronema species. These genomes contain >50% unknown genes but still share their core genomic makeup with sulfate-reducing and sulfur-disproportionating Desulfobulbaceae, with few core genes lost and 212 unique genes (from 197 gene families) conserved among cable bacteria. Last common ancestor analysis indicates gene divergence and lateral gene transfer as equally important origins of these unique genes. With support from metaproteomics of a Ca. Electronema enrichment, the genomes suggest that cable bacteria oxidize sulfide by reversing the canonical sulfate reduction pathway and fix CO 2 using the Wood–Ljungdahl pathway. Cable bacteria show limited organotrophic potential, may assimilate smaller organic acids and alcohols, fix N 2 , and synthesize polyphosphates and polyglucose as storage compounds; several of these traits were confirmed by cell-level experimental analyses. We propose a model for electron flow from sulfide to oxygen that involves periplasmic cytochromes, yet-unidentified conductive periplasmic fibers, and periplasmic oxygen reduction. This model proposes that an active cable bacterium gains energy in the anodic, sulfide-oxidizing cells, whereas cells in the oxic zone flare off electrons through intense cathodic oxygen respiration without energy conservation; this peculiar form of multicellularity seems unparalleled in the microbial world.