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.

This study demonstrates that the deltaproteobacterium Desulfurivibrio alkaliphilus can grow chemolithotrophically by coupling sulfide oxidation to the dissimilatory reduction of nitrate and nitrite to ammonium. Key genes of known sulfide oxidation pathways are absent from the genome of D. alkaliphilus . Instead, the genome contains all of the genes necessary for sulfate reduction, including a gene for a reductive-type dissimilatory bisulfite reductase (DSR). Despite this, growth by sulfate reduction was not observed. Transcriptomic analysis revealed a very high expression level of sulfate-reduction genes during growth by sulfide oxidation, while inhibition experiments with molybdate pointed to elemental sulfur/polysulfides as intermediates. Consequently, we propose that D. alkaliphilus initially oxidizes sulfide to elemental sulfur, which is then either disproportionated, or oxidized by a reversal of the sulfate reduction pathway. This is the first study providing evidence that a reductive-type DSR is involved in a sulfide oxidation pathway. Transcriptome sequencing further suggests that nitrate reduction to ammonium is performed by a novel type of periplasmic nitrate reductase and an unusual membrane-anchored nitrite reductase. IMPORTANCE Sulfide oxidation and sulfate reduction, the two major branches of the sulfur cycle, are usually ascribed to distinct sets of microbes with distinct diagnostic genes. Here we show a more complex picture, as D. alkaliphilus , with the genomic setup of a sulfate reducer, grows by sulfide oxidation. The high expression of genes typically involved in the sulfate reduction pathway suggests that these genes, including the reductive-type dissimilatory bisulfite reductases, are also involved in as-yet-unresolved sulfide oxidation pathways. Finally, D. alkaliphilus is closely related to cable bacteria, which grow by electrogenic sulfide oxidation. Since there are no pure cultures of cable bacteria, D. alkaliphilus may represent an exciting model organism in which to study the physiology of this process.

Cable bacteria are multicellular filamentous bacteria within the Desulfobulbaceae that couple the oxidation of sulfide to the reduction of oxygen over centimeter distances via long distance electron transport (LDET). So far, none of the freshwater or marine cable bacteria species have been isolated into pure culture. Here we describe a method for establishing a stable single-strain cable bacterium culture in partially sterilized sediment. By repeated transfers of a single cable bacterium filament from freshwater pond sediment into autoclaved sediment, we obtained strain GS, identified by its 16S rRNA gene as a member of Ca. Electronema. This strain was further propagated by transferring sediment clumps, and has now been stable within its semi-natural microbial community for several years. Its metagenome-assembled genome was 93% complete, had a size of 2.76 Mbp, and a DNA G + C content of 52%. Average Nucleotide Identity (ANI) and Average Amino Acid Identity (AAI) suggest the affiliation of strain GS to Ca. Electronema as a novel species. Cell size, number of outer ridges, and detection of LDET in the GS culture are likewise consistent with Ca. Electronema. Based on these combined features, we therefore describe strain GS as a new cable bacterium species of the candidate genus Electronema, for which we propose the name Candidatus Electronema aureum sp.nov. Although not a pure culture, this stable single-strain culture will be useful for physiological and omics-based studies; similar approaches with single-cell or single-filament transfers into natural medium may also aid the characterization of other difficult-to-culture microbes.

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.

Unprecedented seabird mass mortality events (MMEs) were reported in multiple European countries in 2022 and 2023. These events were attributed to Clade 2.3.4.4b H5N1 high pathogenicity avian influenza viruses (HPAIVs). In Denmark, populations of Sandwich Terns ( Thalasseus sandvicensis) and Black-headed Gulls ( Chroicocephalus ridibundus) were significantly impacted. Sandwich Tern mortality occurred primarily between May and June of 2022 and was caused by the HPAIV genotype EA-AB. From 2022 to 2023, Danish Sandwich Tern populations decreased by 36%. Sandwich Terns commonly nest within Black-headed Gull colonies, allowing for direct contact between the species. Despite their proximity, in 2022, neither adults nor chicks of Black-headed Gulls exhibited morbidity associated with HPAIV. During the autumn of 2022 and the winter 2022/23 a novel clade 2.3.4.4b H5N1 HPAIV, genotype EA-BB, emerged in Black-headed Gulls. The new genotype caused the death of at least 3000 adult Black-headed Gulls in Denmark (i.e., >1.8% of the Danish breeding population) in the spring of 2023. Notably, in 2023 Sandwich Tern chicks, but not adults, in contact with Black-headed Gulls were affected by mass mortality. These observations suggest that adult Terns in 2023 were less susceptible to HPAIV infection, mitigating the impact of MMEs.

Microbial sulfur cycling is typically divided into an oxidative and a reductive branch, with microbes driving either sulfide oxidation or sulfate reduction distinguished by their genomic setup. Paradoxically, filamentous cable bacteria perform electrogenic sulfide oxidation but contain genes indicative of sulfate reduction, including the reductive type of dissimilatory sulfite reductase (DsrAB), whereas they apparently lack the canonical sulfur transferase DsrEFH essential for sulfur oxidation. AlphaFold2 structure prediction of conserved cable bacteria proteins with unknown functions identified a protein complex resembling canonical DsrEFH (hereafter termed DsrEFH type II). In vitro characterization of heterologously expressed DsrEFH type II confirmed its sulfur transferase function and, together with site-directed mutagenesis, verified that the conserved cysteine, Cys67, is the active sulfur transfer residue. Genes encoding the novel DsrEFH type II were found in 985 prokaryotic genomes. They typically co-occurred with genes for reductive DsrAB in microbes characterized as sulfate reducers or sulfur disproportionators. This study not only fills an important gap in the sulfide oxidation pathway of cable bacteria, but also suggests that a wide range of sulfate reducing bacteria may be more metabolically versatile than currently understood, representing a major shift in the perception of this globally significant physiological group of microorganisms.